Document rp23jak1knJ9d0ZpoYKbqEzR7

_ DAVIS V. DOW ATTACHMENT TO MEMORANDUM OF NOVEMBER 13, 1991 v EXHIBIT LIST Ex. # 1 2. 2a. 3. Date 10/19/44 5/7/46 1946 3/47 6/9/48 Description Report by B.K. Rowe of Biochemical Research Laboratory of Dow Chemical. Subject: "Preliminary Information Relative to the Toxicity of 2,4- Dichlorophenoxyacetic Acid". Translation from a Puerto Rican newspaper clipping entitled "Weed Killer 2,4-D Is Non-Toxic To The Human Being". Comment: This article was published in 1946 and given at an Ohio State University talk on 1/3/51 to create the thought that humans can swallow 2,4-D and no harm would be caused. If possible, getting the original copy of this would be very important; it's hard to read on the exhibit. The translation of this article would be important to obtain for us because it says "Kephart and Mitchell in the United States took doses without noxious effects!" This particular article is what got the third world countries excited about the prospects of using these new herbicides. Harrison. Jos et al. "2,4-D Toxicity-I. Toxicity Twoard Certain Species of Fish, from Am. Jour, of Pharm.. Vol. 118, pp. 442-425 (1946). Hil l . et al., "Toxicity of 2,4- Dichlorophenoxyacetic Acid For Experimental Animals", Journal of Industrial Hygiene And Toxicology. Vol. 29 pp. 85-95. Comment: They killed many animals doing these tests to determine the degree of toxicity of 2,4-D. Report by illegible name, of Biochemical Research Laboratory for the Dow Chemical Company titled: "Toxicology and Hygiene, 2,4-Dichlorophenoxyacetic Acid". Comment: This article says that 2,4-D ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 1 5. 6. 7. 8. 9. 10. lia. 12. toxicity only prevents minor hazards of systemic effects. There is no hazard of absorption through the skin. (A very misleading article by Dow Chemical.) 10/48 Evzacruirre. et al. . "Experimental Myotonia and Repetitive Phenomenon the Veratarenic Effects of 2,4-D in the Rat", publication not indicated. Comment: It shows that 2.4-D causes rat, man and goat to have nerve/muscle twitches or spasms with the development of muscular rigidity. 10/29/48 Biorn. et a l . . "Effects of 2.4-D Acid on Chicks". Science. Vol. 108. Dp. 479-480. 5/23/49 Dow report entitled "Result of Skin Irritation Tests on Various Samples of 2,4-D Taken at Various Stages in the Manufacturing Process". Comment: This says impure 2 r4-D is considerablv more irritatina than the purified product (for dermatitis). A damp product will produce more irritation than a dry one. 12/15/49 Letter to Dr. Drill of Wayne University from Rowe of Dow re: "Studies of 2,4-D and 2,4,5-T on Dogs". Apprx 1949 Rowe, et al. of Dow: "Acute Oral Toxicity of 2,4-D to Rats, Mice, Guinea Pigs and Chicks". Apprx. 1949 Rowe, et al. of Dow: "Semi-Chronic Oral Toxicity of 2,4-D to Rats". 4/11/50 Untitled chart listing ingredients of 2,4,5-T Acid and 2,4-D Acid. 8/30/50 Report to Britton of Dow from Drill of Wayne University re: "Interim Report on the Acute and Subacute Oral Toxicity of 2,4,5-T for Dogs". Comment: Says 2.4.5-T in aeneral resembles 2,4-D in toxicity and it is greater in dogs than in rats. 8/31/50 Internal Dow Memo Regarding Toxicity of 2,4-D Stating that 2,4-D is Moderately Toxic. H H ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 2 6615 13. 13a. 14. 15. 16. 17. 19. 20 11/20/50 4/52 1950 10/51 Early 1950's 1/52 4/3/52 4/3/52 4/52 French medical article on 2,4-D, no translation. English abstract of article mentioned in exhibit 13. Willard. C.J.r "Indirect Effects of Herbicides", publication not indicated. Lehman. Arnold J . . "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments Part II. Pesticides" . from The Association of Food and Drua Officials of the United States. Vol. 15 Dp. 122-129. Drill. Victor. "Acute and Sub-Acute Oral Toxicity of 2,4-D for Dogs". Comment: Three of four doas died with doses of 2,4-D. Lahman. "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments, Part II, Pesticides II, Dermal Toxicitv". from The Association of Food and Drua Officials of the United States. Vol. 16. Dp. 3-9. Internal Dow memo on "Results of Skin Irritation Tests on Current Production 2,4-D As Compared With Past Production Material". Comments: It discusses the difference in cases of skin dermatitis between the old production method and the new production method. Internal Dow memo on "Results of Range Finding Toxilogical Tests on 2,4-D Prepared By A New Method". Comment: The document discusses hazards and proper handling procedures for 2,4D. Lahman. Arnold J . . "Chemicals In Foods: A Report To The Association of Food And Drug Officials On Current Developments" from The Association of Food and Drua Officials of the Untied States. Vol. 16. pp. 47-53. CO H ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 3 6616 .2 1 22. 23. 24. 25. 26. 27. 10/52 Fall, 1952 1952 Summer, 1953 Winter, 1953 11/20/53 1953 Lahman. Arnold J. , "Chemicals In Foods: A Report To The Association of Food and Drug Officials on Current Developments, Part II, Pesticides V, Pathology", from The Association of Food and Drug Officials of the United Stat e s . Vol. 16, pp. 126132. "Harnessing Chemical Sprays to Serve the Sportsmen Through New Game Management Practices" from Down to E a r t h , pp 7-8. Comment: This is Dow hype to sell their 2,4-D and 2,4,5--T chemicals. This is their own magazine which is published quarterly to hype their products to all users. Neuns. Alva A . , "Woody Plant Control Along Roads in Shasta National Forest" from Down to E a r t h , pp. 14-15. Comment: Additional Dow hype to sell their 2,4-D and 2,4,5-T chemicals. Arend. John L. , "Controlling Scrub Aspen with Basal Sprays" from Down to E a r t h , pp. 10- 1 1 . Comment: Explains Dow's recommended applications of 2,4-D/2,4,5-T. Leonard. Oliver A . , "Chemical Control of Woody Plants in California" from Down to Ear t h . Vol. 9, pp. 2-4. Comment: Also contained in this issue is "a simple device for applying Esteron 2,4,5" by David Cook, page 5, and "Timber Stand Improvement on the W.G. Jones State Forest, Conroe, Texas", by Charles Stealey, pp. 12-14. Loustalot. A.J. et al., "2,4-D Affects Phosphorous Metabolism" from Science. Vol. 118, No. 3037, pp. 627-628. Comment: Shows 2,4-D's mode of action may be in inhibiting or interrupting the phosphate metabolism in the plant, i.e. by poisoning the enzyme or system responsible for the hydrolysis of the high energy phosphates. Dri l l . et al, "Toxicity of 2,4-D and 2,4,5-T. A Report on Their Acute and Chronic Toxicity in Dogs" from AMA ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 4 6617 28. 1953 29. Summer, 1954 30 . 10/54 31. Fall, 1954 32. 1/55 33. Winter, 1955 34. 8/57 35. 1957 Archives of Industrial Hygiene and Occupational Medicine. Vol. 7, pp. 6167. B a k e r . et al, "Suspected Poisoning of Dogs from Eating Grasses Treated with 2,4-D", from The North American Veterinarian. Vol. not indicated, p. 194. Comment: This is the poisoning of dogs by 2,4-D with resulting liver damage. This is the same type of article that appeared on the front page of USA Today in September, 1991! Walker. A.H., "Chemicals are Effective for Woody Plant Control in the South" from Down to E a r t h , pp. 10-11. R o w e . et al., of Dow: 11Summary of Toxilogical Information on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of Hazards to Livestock Associated With Their Use" from Am. J.Vet.Res. . pp. 622- 629. Comment: A very revealing article. Toxic levels of exposure are noted! Goddard. Ray E., "Killing Small Undesirable Hardwoods by the Use of Cornell Tool" from Down to E a r t h . Vol. 10, p. 5. Markauardt. Roland P. et al., of Dow: "Determination of 2,4-D in Grain and Seed" from Agricultural and Food Chemistry. Vol. 3, pp. 51-53. Lynn, G.E., "The Status of Pesticides Under the Miller Amendment to the Federal Food, Drug and Cosmetic Act", pp. 8-9. Dow Esteron 2,4,5 Label. Lynn, G.E., "Residue Tolerances for Dow Agricultural Chemicals" from Down to E a r t h . pp. 7-11. Comment: Gives registered brand name products sold by Dow in 1957 and Dow tells people their products are safe to eat when residues appear on the crops. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 5 6618 36. 37. 5/29/58 5/58 37a. 38. 6/59 1959 39. 1959 40. 2/5/60 41. 7/60 42. Hvmas of Dow, "Toxicity of Herbicides to Domestic Animals". Davis. James R. , "Basal Spray With 2,4,5T for Winter Hardwood Control in East Texas" from Journal of Forestry. Vol. 56 p. 349. Comment: This was the plaintiff in Davis v. D o w . This is an example of how 2,4,5T was used in the east Texas pines. Stranskv. John A . , "Concentrated or Diluted 2,4,5-T as a Supplement to Girdling?", from Journal of Forestry. Vol. 57, pp. 432-434. Dow Chemical Company Journal abstract regarding article on 2,4-D. Comment: Shows that the thyroid is effected from acute dose on in-house Dow memo. The comment is made "so what acute dose!" They missed the whole point that 2,4-D attacks the thyroid! Apff e l . C.A., "The Cytostatic Action of Certain Auxins: Preliminary Review", translation from the original French article. Comment: Emphasizes that the esters of 2,4-D/2,4,5-T must be purified of any contamination to be safe to users! Internal Dow memo: "Results of Range Finding Toxilogical Tests on 2,4-D Acidified With Chemically Pure HC1 and With HC1 Recovered From the Pentachlorophenol Plant". Comment: This could be a serious introduction into the 2,4-D herbicide with this hydrochloricacid that comes out of the pentachlorophenol plant. Pentachloro phenol is another producer of the deadly dioxin 2,3,7,8--TCDD! Gibbs. Carter B., "Iso-Octyl Ester of 2,4,5-T in Hardwood Control" from W e e d s . Vol. 8, pp. 462-463. Comments: This is an example of the east Texas use of this herbicide in 1960. Not used. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 6 43 . 4/62 44. 7/62 45 . 1962 45a. 1962 46. 1/15/63 47. 1/62 Abstract of article on "Nervous System Effects of a Chemical Herbicide" (2,4-D) from Industrial Hygiene Digest. Comments: Observed higher nervous system acute suffering in patient with severe damage - caution is needed in use of 2,4D. Calls for increased protection and special neurological examination of workmen in contact with 2,4-D. Florsheim. et al., "Some Effects of 2,4D on Thyroid Function in the Rat: Effects on Iodine Accumulation" from Endocrinology. Vol. 71, pp. 1-6. Foissac. Phillippe et al., "Polyneuritis After Using a Weed Killer: Containing Acid 2,4-D", from Lille Medical. Vol. 7(10), pp. 1049-51, (1962). Translation of attached French report. D e s i . et al, "New Evidence Concerning the Nervous Site of Action of Chemical Herbicide Causing Professional Intoxication" from ACTA Phvsiol. Vol. 22, pp. 72-80. Comments: Budapest study showing that 2.4-D acts on the CNS by paralyzing its function and also damages the thyroid and other organs as secondary from CNS damage. "Toxicological Properties of Terdon" prepared by Biochemical Research Laboratory of Dow Chemical. Comment: Marked "Trade Secret - contains confidential information". D e s i . et al., "Nervous System Effects of a Chemical Herbicide" from Archives of Environmental H e a l t h . Vol. 4, pp. 101108. Comment: This is a budapest study that was made on 6/20/61 where it shows central nervous system damages resulting from the use of 2,4-D. This article explains that 2.4- D is d e s i g n e d to cause disproportionate growth and quick death of certain plants (especially the dicotyledonous weeds). It shows that 2,4D has been known to cause myotonia, motor ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 7 6620 disorders, paralysis in the extremities and gastrointestinal symptoms such as vomiting and diarrhea, leukopenia, atoxia and coma by the year 1948. Here they find higher CNS activity suffered by patients with severe damage and suggest there is a need for caution in the need for 2,4D. 48. 8/15/63 Palmer. J.S., "Chronic Toxicity of 2,4D Alkanolamine Salts to Cattle" from Journal of the American Veterinary Medical Association. Voi. 143, pp. 398-399. 49. 8/63 Seaberrv. John H . , "Toxicity of 2,4-D for Man and Dog" from The Archives of Environmental Health. Voi. 7, pp. 86-93. Comment : This is about a doctor who tested dogs and also terminal human patients with 2,4-D and actually ended up killing one of the patients. 50. 1963 Dolgarrd. Mikkelsen, et al., "Toxicology of Herbicides" from The Pharmaceutical Review. Voi. 14, pp. 225-250. Comment : This document should be reviewed very carefully, it contains a variety of important revelations on the toxicity of these herbicides. 51. Jan/Feb 1964 Clark, et al., of the US Department of Agriculture. "The Fate of 2,4-D Acid in Sheep" from Agricultural and Food Chemistry. Voi. 12, pp. 43-45. 52. 3/13/64 Palazzolo. of Industrial Biotest Laboratories I nc., Report to the National Agricultural Chemicals Association. "Repeated Dermal Toxicity Studies on Three Formulations of 2,4-D". Comment : It was noted that when diesel is used with the application of 2,4-D, it will immediately penetrate the animal's skin. NOTE: This is why these herbicides are more deadly to humans through skin absorption, because of the penetrating ability of the diesel fuels that they are mixed with. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 8 6621 53 . 3/16/64 54. 1964 55. 3/15/65 56. 3/26/65 57. 6/22/65 58. 4/14/66 59. 5/66 60. 1966 Palazzolo. of Industrial Biotest Laboratories Inc., Addendum Report to the National Agricultural Chemicals Association. "Repeated Dermal Toxicity Studies on Three Formulations of 2,4-D". Comment: I only included cover page plus 1 & 45. Kirk-Othmer. Encyclopedia of Chemical Technology. pp. 258-267 and 325-338. Comment: This is a good history of the chemistry of 2,4-D and 2,4,5-T and what they were designed to do. Report to Dr. Gorden of Dow from Olson of Dow re: "The Formation of Folliculitis in the Rabbit Ear Following a Single Subcutaneous Injection of Symmetrical Tetrachlorodibenzo-dioxin in Benzene". Comment: Its all about a matter of doses! Enough will injure or kill you! Memo to Gorden of Dow from O'Hare and Olson of Dow re: "The Ineffectiveness of Washing as a Preventative Measure in the Formation of Folliculitis in the Rabbit Ear Following Repeated Applications of TCDD in Corn Oil". Comment: This shows that soap is ineffective in getting TCDD out of the exposed area! (In an insoluble solution). Dow report entitled "The Determination of 2,3,7,8-TCDD in 2,4,5-T By Gas-Liquid Chromatography". Comment: This is an admission by Dow of the health effect problems caused by TCDD. Alexander. et al of Dow: "Acute Fish Toxicity of Daxtron, Tordon and Dursban To Three Species of Fish". "2,4-D and Related Compounds" from Wildlife. Vol. 96, pp. 61-69. Comment: This is a very good review of the chemistry of this herbicide. E r n e . Kurt, "Studies on the Analytical Chemistry and Toxicology of Phenoxy Herbicides". ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 9 8622 611.. 1/23/68 61a. 1/23/68 61b. 3-4/68 Letter to Rowe of Dow from Dr. Kligman re: Experiments of Adults Receiving Small and Increasing Doses of Tetrachlorodibenzop-dioxin (TCDD). Comments: Dow is usina human cruinea piqs in a blind study to determine the effects of TCDD injected into them! Document entitled "Report to Dow Chemical Company on Group (1-6)" submitted by Clover Laboratories of Philedelphia. It appears to be the results of these laboratory tests. Comment: These are six aroups of people who are mostly blacks that were tested with TCDD on their backs and foreheads in an apparent allergy test to see how humans would react to these deadly dioxins. There is no indication any of these knew that among the different allergies they were being tested for, someone was putting the deadly dioxin directly into their bodies. Bulletin from the National Clearing House for Poison Control Centers entitled "Death from Chlorinated Phenoxyacetic Acids (2,4D; 2,4,5-T; MCPA)". Comment: 62. 10/21/68 63. 5/69 64. 12/18/69 L e n a . Marauerite of D o w . summary of an article entitled "Review on the Metabolism of Phenoxy Compounds in Plants and Animals". Table entitled "Summary of Dosages of Various Organic Herbicides that Cause Significant Weight Loss, Reduce Weight Gain, or Poisoning in Cattle, Sheep or Chickens". Production research report #106, US Department of Agriculture. Letter to Leng of Dow from Erne, Associate Professor of Vet. Science in Stockholm, Sweden regarding 2,4,5-T and 2,4-D. ATTACHMENT TO MEMORANDUM DTD 11/13/91 6623 * 65 . 12/18/69 66. 1969 67. 1969 67a. 1969 .6 8 3/9/70 69. 4/13/70 Table entitled "Phenoxy Acid Levels in Tissues of Broiler Chickens Fed 2,4-D and 2,4,5-T at 1,000 ppm in the Drinking Water For 104 Days". Medical abstract on Russian article regarding the health in workers manufacturing 2,4-D. Comment: Shows many health problems in 51.7% of all workers studied! W a y . J . M . , "Toxicity and Hazards to Man, Domestic Animals and Wildlife From Some Commonly Used Oxin Herbicides" from Residue Reviews. Vol. 26, pp. 37-62. Comment: This is the reply to the above letter. W a y . J.M., "Toxicity and Hazards to Man, Domestic Animals and Wildlife From Some Commonly Used Herbicides", from Residue Reviews. Vol. 26, pp. 37-62. Comment: This says that because man is handling the chemicals in concentrated form at all stages from manufacturing to dilution, it follows that he is at greater risk than any other organism. Points out that "Freireich in 1966 has shown that a number of drugs on a mg/kg basis are more toxic in man than in the mouse when used as a laboratory test animal bv a factor of 10 to 1 5 !" Abstract from the Health Aspects of Pesticides Abstract Bulletin. Comment: This shows that if you mix 2,4D and 2,4,5-T in esters, it can cause high mortality! Document entitled "The Analysis of 2,4- D Acid for 2,3,7,8-Tetrachlorodibenzo-p- Dioxin Content", publication not indicated. Comment: This document shows that 2,3,7,8-TCDD is in 2,4-D at Dow Chemical! They deny repeatedly throughout all the years they have been in business that the dioxin TCDD has ever appeared in their herbicide 2,4-D. This is clearly proof to the contrary. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 11 7 70. 1970 Abstract from an article in Bio Science. Comment: Page 2 shows cancellation of 2,4 ,5-T in the United States for many different uses. (This is the first time it was officially cancelled by a government agency). 71. 1970 Medical abstract on "The Effect of 2,4D on Pheasants" and attached is a Dow Chemical biology research document summarizing "The Effect of 2,4-D and Esters of the 2,4-D on Rat Embryonal, Fetal and Neonatal Growth and Development". Comment: The 2,4-D either killed or deformed alot of chickens or birds in this experiment! 72. 1970 Matsumura. Akihito, "The Fate of 2,4-D Trichlorophenoxyacetic Acid in Man" from Japanese Journal of Industrial Health. Vol. 12, October, 1970, pp. 446-451. 73 . 1970 Abstract on articles regarding "Toxic Effect of Some Herbicides on Fish and Aquatic Invertebrates" from Chemical Abstracts. Vol. 72, p. 240. Attached is a Dow memo dated 4/23/80, regarding Esteron 2,4,5 labels. Comments: Dow has started up the 2,4,5T under a different label after it was cancelled by the EPA! 73a. 1970 Rivers. et al., "Simultaneous Gas Chromatographic Determination of 2,4-D and Dicamba in Human Blood and Urine", from J . Chromatoa. Vol. 50, pp. 334-337. Comment; Suicide attempt with 2,4-D. 74. 1/23/71 Williams. C.S. of Dow, "The Status of 2,4D; 2,4,5-T; Silvex; and MCPA Herbicides", publication not indicated. Comment: This gives the chronology of the major events effecting 2,4-D and 2,4,5T from April, 1966, through January, 1971. 75. 3/71 Poland. et al., "A Health Survey of Workers in a 2,4-D and 2,4,5-T Plant With Special Attention to Chloracne, Porphyria, Cutanea, Tarda, and Psychologic Parameters". ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 12 6625 76. 3/28/71 77a. 4/27/71 77b. 4/27/71 78. 8/30/71 79. 9/1/71 80. 10/25/71 81. 1971 By "Our Washington Correspondent". Titled: "PSAC Hiccoughs Over 2,4,5-T" from Natu r e . Vol. 321, May 28, 1971, pp. 210- 211. Comment: This is an interesting article on how a private government study of 2,4,5-T was accidently released to the public and thus ended up causing the herbicide 2,4,5-T to become cancelled in April, 1970. Memo to Johnson, et al., of Dow from Gehring of Dow re: "Results of the Pilot Study on the Absorption and Excretion of 2,4-D and 2,4,5-T by Human Subjects". Comment: Here Dow is using human subjects to actually drink 2,4-D and 2,4,5-T! Gehrina. et al. of Dow, "Results of a Pilot Study on the Absorption and Excretion of 2,4-D and 2,4,5-T Following a Single Oral Dose to Human Subjects". Comment: This is referring to the same tests done in 77a. Soarschu. et al. of Dow, "A Probe Study of the Acute Oral Toxicity of 2,3,7,8Tetrachlorodibenzo-p-Dioxin in the Canine". Comment: Most of these dogs died or else had very severe health effects after these tests. Johnson. Julius, "The Public Health Implication of Wide Spread Use of the Phenoxy Herbicides in Picloran" from Bio Science. Vol. 21, pp. 899-905. Comment: This is a white wash report written by Dow! Dow report on "Eye Irritation Study Conducted on: 2,3,7,8-Tetrachlorodibenzop-Dioxin". Williams. C.S. of Dow, "The Status of 2,4D and 2,4,5-T, Silvex and MCPA Herbicides" from Down To Earth. Vol. (not indicated), pp. 12-16. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 13 82. 1971 83. 2/19/71 83a. 1971 84. Summer, 1971 85. 1970-71 86. 1/1/72 86a 1970 'S Comment: Dow describes the history of 2.4-D and 2,4,5-T development and their designed uses in this Dow Company magazine. Kolberq. et al, "The Herbicide 2,4Dichlorophenoxyacetic Acid I, Effects on L Cells" from ACTA Pharmacal et Toxicol. V o l . 29, pp. 81-86. Memo from Gehring of Dow to Gordon, et al. of Dow re: "Protocol for Consumption of 2.4- D and 2,4,5-T Study". Protocol discussed in exhibit 83 on "Human Tolerance of 2,4-D and 2,4,5-T" plus "Full Description of Analytical Standard of 2,4D". Johnson. Julius, "Safety in the Development of Herbicides" from Down to Earth. Vol. 27, pp. 1-7. Comment: This is Dow's claim of safety in their 2,4-D and 2,4,5-T. The records of shipments or sales in 1970 and 1971 of the products to the U.S. Forest Service, which were provided under court order by the plaintiffs in Davis v. Dow. Comment: Dow only produced these obscure records in the time period of 1970 and 1971, and ignored the full court order which stated they would produce all records of shipments or sales between the years 1954 and 1973. Wal k e r . et al, "Some Effects of 2,4-D and 2,4,5-T on Ehrlich Ascites Tumor Cells In Vivo and In Vitro", from Industrial Medicine. Vol. 41, No. 1, pp. 22-27. Abstracts on articles on the effect of herbicides on animals. Comment: Shows Dow's knowledge of the harmful effects of TCDD in causing cancers and many other defects! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 14 6627 877.. 2/72 88. 8/72 88a. 10/6/72 89. 10/18/72 90. 1972 91. 2/12/73 92. 4/2/73 93. Spring, 1973 94. 6/73 95. 6/73 Hussien. et a l . "Mutaaenic Effects of TCDD on Bacterial Systems". Comment: This looks at the health effects of 2,4,5-T. Kimbro. R . . "Toxicity of Chlorinated Hydrocarbons and Related Compounds" from ARCH Environ Health. Vol. 25. pp. 125131. "Summary Teratology" by B.E. Schwetz. Comment: Shows that TCDD is hiahlv toxic to the developing embryo and fetus of the rat. Lena. Marauerite of Dow. "Summary of Teratology Studies With 2,4-D". Cunninaham. et al . . "Effect of Tetrachlorodibenzo-P-Dioxin on Growth Rate and the Synthesis of Lipids and Proteins in Rats" from Bulletin of Environmental Contamination and Toxicoloav. V o l . 7. pp 45-51. Bosshardt. Hans. "Dioxins -Insidious Poisons in Our Environment", translation from and German article. Comment: Germanv bans 2.4-D at this time because of its contaminant TCDD. Bauahman, et al. . "An Analytical Method for Detecting Dioxin", presented at the conference on Dibenzo-Dioxins and DibenzoFurons. Comment: Good explanation of how TCDD works. Wilson. James. "Teratoloaical Potential of 2.4.5-T". from Down to Earth. Vol. 28. pp. 14-18. Summary of toxicology studies with 2,4D in livestock, June 1973. Summary of metabolic studies with 2,4-D and animals, June 1973. Author not indicated. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 15 6628 96. 6/73 97. 6/73 98. 9/73 99. 1973 100. 101. 1973 1973 102. 11/6/73 102a. 1/5/73 Document entitled: "Section C. Full Reports of Investigations Made With Respect to the Safety of the Pesticide Chemical". Crossland, et al., -- Title of article illegible regarding spraying of chemicals. Johnson. et al., "Chlorinated Dibenzo- Dioxins and Pentachlorophenol", from Environmental Health Perspectives. pp. 171-175. Comment: This is a good article for the defense. Dow makes improved PCP/TCDD contaminants in their product. Here they admit the older production batches of PCP/TCDD had higher values in parts per million! (This is why the actual contamination in parts per million has been so important, Dow is constantly trying to reduce the contamination level and in this case they are using a new batch.) Kociba. et al., "Results of a Two Year Chronic Toxicity and Oncogenicity Study of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin in Rats", from Toxicology and Applied Pharmacology. Vol. 46, pp. 279-303. Document untitled Excerpt Part I. Covers the periods 1965-1969 at a chemical plant producing pentachlorophenol and 2,4,5-T. Bage. et al., "Teratogenic and Embryotoxic Effects of the Herbicides Di-and Trichlorophenoxyacetic Acid", from ACTA Pharmacal et Toxicol. Vol. 32, pp. 408416. C o m m e n t : Note the conclusion on the combined effects of 2,4-D and 2,4,5-T. Crosby. et al., "Photo Decomposition of P-Chlorophenoxyacetic Acid" from Ag. Food Chem. . Vol. 21, pp. 1049-1052. C o m m e n t : Talks about 2,4-D and 2,4,5-T decomposing under sunlight or laboratory ultra violet light. Kanaga. "Environmental Impact of Herbicides", report prepared by Dow. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 16 6629 103. 1973 104. 1973 104a. 105. 1973 1/74 106. 2/74 107. 8/24/74 108. 8/28/74 109. 110. 8/28/74 8/29/74 M a y . G. "Chloracne From the Accidental Production of Tetrachlorodibenzo Dioxin", from British Journal of Industrial Medicine. Vol. 30 pp. 276-283. Y o d e r . et al., "Lymphocyte Chromosome Analysis of Agricultural Workers During Extensive Occupational Exposure to Pesticides", from Mutation Research. Vol. 21, pp. 335-340. Comment: In this test they caused mutations in the chromosomes. Karvonen. et al. of Finland: "The Health Hazards of Defoliants". Kimbrough, Renate of the Center for Disease Control, "The Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals", from CRC - Critical Reviews in Toxicology, pp. 445-498. Comment: Overall good summary of the known toxic effects at this time. Gribble. Gorden W. , "TCDD: A Deadly Molecule", from Chemistry. Vol. 47, pp. 15-18. Comment: Great history of TCDD (but the concentration level in parts per million was slanted in Dow's favor). "Illness Associated with TCDD Contaminated Soil - Missouri", from Communicable Disease Center Morbidity and Mortality Weekly Rep o r t , p. 299. New York Times article: "Death of Animals Laid to Chemical". Attached is a 2/28/80 memo to the State Department of Agriculture regarding the label for Esteron 245 Herbicide. Washington Post article: "U.S. Scientist Disputes EPA on Toxic Herbicide". Detroit Free Press article: "Herbicide Killed Missouri Animals". Comment: This article is major proof of the harm that 2,4,5-T with its contaminant TCDD can cause! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 17 6630 111. 4/9/74 112. 1974 113. 1974 114. 11/75 115. 1975 116. 1975 117. 2/9/76 Dow requests for toxilogical studies on TCDD. Noted on the request is that the sample of TCDD is extremely toxic and chloroacengenic. Comment: Dow admits TCDD is highly toxic and chloracengenic and must be handled with extreme caution. Pilinskava. N.A . , "Cytogenetic Effect of the Herbicide 2,4-D on Human and Animal Chromosomes", translation from a Russian Journal. Norris. et al., "The Toxicity of 2,3,7,8Tetrachlorodibenzo-P-Dioxin (TCDD) in Guppies", from Bulletin of Environmental Contamination and Toxicology. Vol. 12, pp. 76-80. Honoroff. Ida, article from A Report to the Consumer entitled: "Dirty Tricks". Comment: This article is very hard against Dow! It is a news letter put out to consumers to wake them up on the dangers of these herbicides. Archer. T.E., "Removal of 2,4- Dichlorophenoxyacetic Acid, (2,4,-D) Formulations from Non-Combustible Pesticide Containers", from Bulletin of E n v i r o n m e n t a l____ C o n t a m i n a t i o n ____ and Toxicology. Vol. 13, pp. 44-51. Comment: This tells how to clean used 1,5,30 and 55 gallon metal pesticide drums after use. Delvaux. et al., "Polychlorodibenzo-P- Dioxins", from Toxicology. Vol. 3, pp. 187-206. (Translation of a French article.) Comment: This shows another form of the deadly dioxin TCDD contained in a different chemical process. Abstract and attached report entitled: "The Fate of 2,4-D Following Oral Administration to Man", by Sauerhoff. et al. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 18 8.631 118. 5/5/76 119. 10/76 .1 20 1976 .1 2 1 1/24/77 121a. 5/26/77 122. 7/9/77 123. 8/77 124. 9/26/77 "Possible Toxic Effects of TCDD in Humans Listed by Dioxin Group", from Pesticide Chemical N e w s . pp. 20-23. Comment: This shows that this dioxin causes a loss of libido as one of its health effects listed. 2,4,5-T is formally banned in North Carolina. (This is where all the research is done in the United States on the effects of dioxins and especially TCDD.) Pol a n d . et a1 ., "2,3,7,8- Tetrachlorodibenzo-P-Dioxin: Environmental Contaminant and Molecular Probe", from Federation Proceedings. Vol. 35, pp.2404-2411. Courtney. K. Diane, "Mouse Teratology Studies With Chlorodibenzo-P-Dioxin", from Bulletin of Environmental Contamination and Toxicology. Vol. 16, pp. 674-681. Index and report on information on chloroacne. Comment: This is a great history of human health effects from 2,4,5-T exposure in Czechoslovakian plants from 1965-1969. Draft of EPA's dioxin working group document entitled: "Dioxin: Position D o c u m e n t ." Comment: This does major damage to Dowl C r o w . K.D., "Letter to the Editor Effects of Dioxin Exposure", from The Lancet. Vol. not indicated, p. 82. "IARC Monographs of the Evaluation of the Carcinogenic Risks of Chemicals to Man, Some Fumigants, the Herbicides 2,4-D and 2,4,5-T Chlorinated Dibenzo Dioxins and Miscellaneous Industrial Chemicals", from The World Health Organization. Comment: This contains a lot of excellent background information on the history of 2,4-D and 2,4,5-T. Abstract of Reports, Department of HEW, Division of Cancer Cause and Prevention, ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 19 6632 125. 9/28/77 126. 11/77 127. 1977 128. 6/77 129. 1977 5th Meeting of the Data Evaluation/Risk Assessment Subgroup of the Clearing house on Environmental Carcinogens. Comment: This abstract indicates that picloram is a carcinogen because of liver lesions. (Picloram contains 2,4-D.) Kociba. et a l . , "Results of a Two-Year Toxicity and Onogenicity Study of 2,3,7,8Tetrachlorodibenzo-P-Dioxin (TC D D ) in Rats", produced by the Dow Chemical Company. Comment: I only copied the first four pages which summarizes this very lengthy report. It was designed as a white wash for Dow to prove that their 2,4,5-T does not hurt anyone. V o s . et al., "TCDD Accident at a Chemical Factory in the Netherlands", publication not indicated. Comment: Shows knowledge of TCDD health effects in 1963! Chloroacne and cancer! Ahllinq. et al., "Formation of Polychlorinated Dibenzo-P-Dioxin and Dibenzofurans During Consumption of a 2.4.5- T Formulation", from Chemosphere. Vol. 8, pp 461-468. Comment: Swedish study on how you get TCDD and PCDF (furans) when manufacturing 2.4.5- T as a natural by product. Article from the Washington Post entitled: "Deadly Poison - Dioxin - Is Showing Up in Mother's Milk". Comment: Women living in or near the areas where 2,4,5-T was routinely sprayed had dioxin in their breast milk. Zetterberg. et al., "The Influence of pH on the Effects of 2,4-D on Saccharomyces Cerevisiae and Salmonella Typhimurium", from Mutation Research. Vol. 42, pp. 318. Comment: KEY DOCUMENT: Says that the toxic effects of 2,4-D are a function of a low pH levels when more of the 2,4-D is in its undissociated form. At low pH's, toxic and mutagenic effects occur. To ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 20 8633 130. 131. 132. 133. 134. 135. 136. 1977 1977 1977 1977 1977 1/7/78 1/10-11/78 stop these effects, you only have to raise the pH, such as happens when administered orally (like they do in all animal tests). T u n g , et al., HThe Problem of Mutagen Effects on the Second Generation After Exposure to Herbicides", from a Vietnamese publication. Comment: The Vietnamese made a connection between TCDD of 2,4,5-T and birth defects and a high frequency of brain defects. A l l e n . et al., "Morphological Changes in Monkey Consuming a Diet Containing Low Levels of 2,3,7,8-Tetrachlorodibenzo-PDioxin", from ED. Cosmet. Toxi c o l . Vol. 15, pp. 401-410. Comment: Caused diseases in monkeys7 bone marrow and lymphs. (This leads to lymphatic cancer and leukemia!) Abstract of article entitled "Electronacceptor Properties of Chlorinated-PDioxins, from Bull. Environ. Contam. Toxicol. Vol. 18(5), pp. 611-616. Comment: This is a good explanation of how chlorine binds TCDD together. Kenaga, Eugene of Dow, "Acute and Chronic Toxicity of 75 Pesticides to Various Animal Species". Leskowski. et al., "The Pharmacology of Experimental Myopathies", from Ann. Rev. Pharmacal Toxicol. Vol. 17, pp. 387-409. Comment: 2,4-D produces sustained contraction of muscles. Franco. Berino, "Preliminary Report on Mortality in the Ceveso Area, 1975-77". Stamped "strictly confidential". Agenda for the World Health Organization Program on Coordination of Epidemiological Studies on the Long Term Hazards of Chlorinated Dibenzodioxins and Chlorinated Dibenzofurans. Comment: This goes together with exhibit #170. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 21 137. 1/10-11/78 138. 1/10-11/78 139. 1/78 140. 1/78 140a. 5/78 141. 6/16/78 M o o r e . John A . , "Comparative Toxicity of Dibenzodioxins and Dibenzofurans", from IRAC Meeting on Dibenzodioxins and Dibenzofurans. Comment: Excellent article (goes with exhibit 163). Moore. John A . , "Comparative Toxicity of Dibenzodioxins and Dibenzofurans", presented at the IRAC Meeting on Dibenzodioxins and Dibenzofurans. Comment: This says that a result of TCDD is to attack the liver as one of its target orcrans I Same document as exhibit #137, but marked "Strictly Confidential" by Dow. "Cymptomatology Morbidity and Mortality Experience of Chlorinated Phenoxy Acid Herbicide (2,4-D; 2,4,5-T) Sprayers in Finland - A Clinical and Epidemiological Study". Working paper for IARC working group meeting. Comment: Marked "Strictly Confidential" by Dow. Poland. Allen, "Chlorinated Dibenzo-PDioxins and Dibenzofurans: Review of Toxicology and Enzyme Induction and Consideration of Mechanism of Toxicity", presented at IARC. Comment: Marked "Strictly Confidential" by Dow. D o s t . Frank, "Toxicology of Phenoxy Herbicides and Hazard Assessment of Their Use in Reforestation", U.S. Department of Agriculture Forest Service. Comment: Oregon State University study that evaluates for Region (5) in California, the hazards to human health of 2,4-D and 2,4,5-T of U.S. Forest Workers (all based on what literature and research reports have said). Dow analytical report "Determination of Phenoxy Herbicides in Blood Samples from 2,4,5-T Plant Personnel". ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 22 6635 14422.. 7/78 143. 7/28/78 144. 10/21/78 145. 5/4/77 145a. 12/13/78 146. 12/78 147. 1978 148. 1978 Yoshida. et al. . "Occuoational Exposure Measurement of an Aerial Spray Crew to a Mixed Spray of 2,4-D and 2,4,5-T", pp. l26. Dow analytical report "Determination of PHenoxy Herbicides in Urine and Blood Samples from 2,4,5-T Plant Personnel II". Rehder. et al. . "Patholoaical Embryological Investigations in Cases of Abortion Related to the Seveso Accident", from Journal of Swiss Medicine. Vol. 108. pp. 16, 17-25. Memo from Johnson of the EPA, to Dioxin Implementation Task Force re: Dioxin: Position Document. "Human Birth Defects Not Caused By 2,4,5- T or 2,4-D Exposure, Report Says, from Pesticide and Toxic Chemical News. d o . 23- 24. Comment: White wash cover uo on herbicides by Australian government. "Of Mice and Men -- The Troubles of 2,4,5T " , from Journal of Forestrv. po. 787790. Comment: Reveals the scams Dulled bv industry with the use of 2,4,5-T. Ruber. M . D . . "Carcinoaenicitv Testina of Chemicals With Particular Reference to Organochlorine Pesticides", from The Science of The Total Environment. Vol. 10. pp. 105-115. Comment: Great article - connects cancer with herbicides! Medical Article. Kreeftenbera. et al. . "Studies on 2,3,7,8-TetrachlorodibenzoP-Dioxin-Induced Immune Suppression and Decreased Resistance to Infection: Endotoxin Hypersensitivity, Serum Zinc Concentrations and Effect of Thymosin Treatment", from Toxicolocrv. Vol. 9. d d . 75-86. ATTACHMENT TO MEMORANDUM DTD 11/13/91 -23 Of! 149. 1978 150. 1978 151. 1978 152. 1978 153. 1978 153a. 1978 154. 1978-1979 Reqqiani. "Medical Problems Raised By the TCDD Contamination in Ceviso, Italy", from ARCH. Toxicol.. Vol. 40, pp. 161-188. Comment; One page only was copied which is the abstract of this very lengthy article. HEW Report: "Bioassay of Picloram For Possible Carcinogenicity", from The National Cancer Institute, Carcinogenesis Technical Report Series. #23. Comment: Pages i-viii only were copied. Working group of NIEHS and the IARC "Coordination of Epidemiological Studies on the Long Term Hazards of Chlorinated Dibenzodioxin", publication not indicated. Comment: This is a study of the 11/13/53 German accident of a chemical plant exposing workers to the deadly dioxin TCDD. Marked "Strictly Confidential" by Dow. B u s e r . et al., "Identification of Substitution Patterns in Polychlorinated Dibenzo-P-Dioxins (PCDDs) by Mass Spectrometry", from Chemosohere. pp. 199- 211. Abstract of article regarding examination of female rat liver after TCDD exposure. Comment: Discusses cytochrome (transporters of electrons to oxygen). W a s s o m . et al., "A Review of the Genetic Toxicology of Chlorinated Dibenzo-PDioxins, from Mutation Research. Vol. 47, pp. 141-160. Document entitled: "Chapter 6, Comparison of the Health Effects of Aquatic Herbicides". Also attached is Milwaukee Centennial article 11/7/80 "Pesticide Use is Still Hot Issue". Comment: This shows that 2,4-D is rapidly absorbed through membranes of the skin, lung and gastrointestinal tract and up to 32% percent of absorbed 2,4-D found in cell nuclei. Makes point that LD50 is ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 24 6637 not relevant to low-dose chronic exposure to humans. This article shows that the chemical companies perform a scam on the EPA and the public in the way that they do their tests. Published reviews of this subject are extremely unreliable, quoting other reviews with no original data and thus giving the appearance that many more studies have been done than in fact have. NOTE: Even though Dow has this document in their files, they carefully did not provide any clue as to date, author or source of publication. 155. 1978-1979 Document entitled: "Chapter 2, Health Effects of 2,4-D and its Derivatives", publication not indicated. Comment: This shows that 2,4-D is a skin carcinogen or at least a co-carcinogen with benzene which is not carcinogenic by itself in this system. This is a major break through in proving that 2,4-D causes harm and this fact has been hidden bv the chemical companies. 156. 3/79 Report entitled: "Assessment of Human Health Risk Associated With the Risk of 2,4-D in Forestry Management", from The Minnesota Department of Health. Comment: This also explains how both 2,4D and 2,4,5-T are chemically manufactured. It is a very good explanation. 156a 3/29/79 Packee, E.C., "2,4-D in the Urban and Suburban Environment in Human Health". Presented to the Nanaimo Parks and Recreation Commission. Comment: Local government speech to convince people that the key to using herbicides is to minimize one's exposure. Concludes 2,4-D is a safe and necessary substance to use in forests and parks. 157. 5/79 Attached are news articles on the ban of spraying herbicides and a 1/31/80 telegram from Dow regarding the 2,4,5-T Esteron trademark. Comment: Spraying of 2,4-D in park caused seizure of 21 month old baby in Madison, Wisconsin on 10/10/80 and a 2,4-D ban was ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 25 6638 158. after 6/4/79 159. 6/7/79 160. 6/12/79 161. 9/79 162. 9/79 163. 10/20/79 sought by the public. In another article on 10/6/80, Citizens Pesticide Control Committee in Wisconsin said "The burden of proving safety should be on the industry rather than putting the burden of proving harm on the victim". Dow production specification for 2,4,5T, mostly illegible. Comment: This shows everything in 2,4,5T, but it is very hard to read. L e n a . Marguerite of Dow, "Comparative Toxicology of Various Chlorinated Dioxins as Related to Chemical Structure", presented at the Symposium of Collaborative International Pesticide Advisory Counsel. Comment: This is a very damaging document to Dow because it shows how TCDD and PCDD cause cancer! Ruber. Melvin, "Carcinogenicity of 2,4D". Comment: Shows that 2,4-D causes cancer in rats I Lamberton. et al., "The Determination of Polychlorodibenzo-P-Dioxins in Pentachlorophenol and Wood Treatment Solutions", from Am. Ind. Hva. Assoc. J . . V o l . 4, pp. 816-822. Advertisement entitled: "Debating Scientists of Eight Nations Agree That 2.4.5- T is Safe!", from the Dow Chemical Company located in Science Magazine. Vol. 205, pp. 1330. Comment: This is the advertisement that was published after Dow Chemical Company financed and sponsored this conference. They only said 2,4,5-T was safe but failed to mention anywhere in their advertisement that it is the contaminant TCDD that makes 2.4.5- T deadly! Abstract and attached report entitled: "Reversal of Immunologic and Toxicologic Effects of a Single Exposure of 2,3,7,8Tetrachlorodibenzo-P-Dioxin in Mice". ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 26 6639 164. 165. 166. 167. 168. 169. 170. 12/7/79 1979 1979 1979 1979 1979 1979 Comment: This exhibit goes with exhibit #137. Letters to the editor: Westing of Hampshire College with rebuttal letter from Etcyl Blair of Dow Chemical. "The safety of 2,4,5-T", from Science. Vol. 206, pp. 1135-1136. Comment: One of "the top 59 world experts responds to Dow's full page add that 59 top world experts found that 2,4,5-T safe! He says that due to the TCDD, it is not safe! (And as far as he is concerned, everyone at the conference of these top 59 world experts stated the same". Abstractof article on "Study of 121 Dioxin-Exposed Workers Shows No Excess Deaths Over 30 Years". Comment: Monsanto does their own study and says TCDD won't hurt you based on findings of their 1949 chemical plant explosion. Raqqiani. "Estimation of the TCDD Toxic Potential in the Light of the Seviso Accident", from Arch. Toxicol.. Vol. 2, pp. 291-302. Hel d e r . Theo, "Effects of 2,3,7,8Tetrachlorodibenzo-P-Dioxin (TCDD) on Early Life Stages of the Pike", publication not indicated. Barsotti. et al., "Hormonal Alterations in Female Rhesus Monkeys Fed a Diet Containing 2,3,7,8-Tetrachlorodibenzo-PDioxin", from Bulletin of Environmental Contamination and Toxicology. Vol. 21, pp. not indicated. Koc i b a . et al., "Long Term Toxilogic Studies of 2,3,7,8-Tetrachlorodibenzo-PDioxin (TCDD) in Laboratory Animals", from New York Academy of Sciences. Vol. not indicated, pp. 397-404. Article entitled: "Twelve Wise Men Give 2,4-D and 2,4,5-T a Clean Bill of Health", publication not indicated. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 27 171. 1979 172. 1979 173. 1979 Comment : This is a major defense article! This goes with exhibit 136. IARC Monographs on the "Evaluation of the Carcinogenic Risk of Chemicals to Humans", Voi. 20, pp. 303, 347-353. Comment : Shows chemical and physical data of 2,4-D and 2,4,5-T with their histories. K i r k . Othmer, Encyclopedia of Chemical Technology. Third Edition, Voi. 5, pp. 797-808 and 866-867. Comment: Explains chlorophenols as applied to 2,4-D and 2,4,5-T. Gregory. Arthur on NIOSH "Occupational Exposure to Herbicides", publication not indicated. 174. 1979 (or later) 174a. 1979? 174b. Late 1970's 174c. 1979? 174d. late 1970's Comment: Great article explaining how TCDD effects you. Was s o m . et al., "A Review of the Genetic Toxicology of Chlorinated Dibenzo-PDioxins". Homberger. "The Seveso Accident, Its Nature, Extent and Consequences", publication not legible. Comment; Page 1 only was copied. Document entitled: "Section C. Toxicology-Vol. IV". Comment: It appears to be a list of toxicology studies of herbicides in birds. Poland . et al., "2,3,7,8- Tetrachlorodibenzo-P-Dioxin: Studies on the Mechanism of Action", from McArdle Laboratory for Cancer Research at the University of Wisconsin. Comment: This is a another great explanation of how TCDD gets to you. Report entitled: "Section II, 2,4-D in the Aquatic Environment", by Halter of the University of Washington. Comment: This report explains why 2,4D kills things! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 28 6641 174e. late 1970's 174f. late 1970's 175. 1/80 176. 1/80 177. 1/80 Summary prepared by O'Hare "The Effects of Repeated Applications of Symmetrical Tetrachlorodibenzo-P-Dioxin on Guinea Pig E a r s ". Comment: They killed guinea pigs in this test with TCDD! (Their response is the closest there is to humans.) Abstracts of various medical articles. Specimen Label and Attached Product Information Insert and Direction for Use on Dow Esteron 2,4,5 Herbicide. Comment: Here, Dow has started marketing Esteron 2,4,5 under another label after 2,4,5-T was banned and then reinstated by the EPA for limited use only. This is apparently Agent Orange being sold under a new Dow trademark (Esteron 2,4,5 Herbicide) in a form that is N-Butyl esters glycol butyl esters. Apparently what happened here is that Dow got caught with a large stock pile of banned Agent Orange herbicide and simply turned around and resold it under a new trade name to unsuspecting users. Zac k . et al., "The Mortality Experience of Workers Exposed to TetrachlorodibenzoP-Dioxin in a Trichlorophenol Process Accident", from Journal of Occupational Medicine. Vol. 22, pp. 11-14. Comment: This is a study of the 1949 Monsanto Chemical Plant accident. The majority of the workers at this plant were exposed directly to TCDD and contracted many ill health effects from the acute exposure. This article is an after the fact attempt to go back and cover up the seriousness of this accident. Shearer. et al., "Literature Reviews of Four Selected Herbicides: 2,4-D, Dichlobenal, Diquat and Endothall". Report by the Municipality of Metropolitan Seattle. Comment: This is an excellent study, however in the Dow production documents, they pulled many key pages. A check ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 29 178. 1/3/80 179. 1/8/80 180. 181. 182. 1/8/80 1/80 2/4/80 183. 2/8/80 184. 2/15/80 185. 2/18/80 should be made with Seattle authorities to get this publication and determine the full document. Obviously Dow does not want this released and I am sure that it would be very strong against them. Memo to all Dow 2,4,5-T and Silvex Distributors from McCoy, Dow District Sales Manager, re Esteron Brush Killer, Esteron 2,4,5 and Kuron. Comment: Here Dow is pushincr Esteron 2,4,5 (Agent Orange) after 2,4,5-T was banned. Thev bend in the interpretation from the EPA on what the term "rangeland" uses means. They decide to interpret it in their favor and sell this deadly toxin to unsuspecting users. MSDS (Material Safety Data Sheet) for Esteron 2,4,5 Brush and Weed Killer. Dow Material Safety Data Sheet on Esteron 2,4,5. Esteron 2,4,5 Herbicide Label. Memo to Cook from Bond of Dow re: "A Critical Review of: Case Control Study of Malignant Mesenchymal Soft-Tissue Tumors and Exposure to Chemical Substances". Comment: This is a defense oriented article with bias towards the Hardell Swedish studies by Dow Chemical people. Document regarding Material Safety Data Sheet on Esteron 2,4,5. Memo from Marguerite L. Leng, D.L. Costillo, et al., of Dow Chemical, regarding Esteron 2,4,5, Esteron Brush Killer and Kuron. Letter from Garner of EPA to Leng of Dow regarding revised 2,4,5-T registration. Comment: This shows how Aaent Oranae is hidden in this product (see label!). AATTTTAAPCHHMMEFMNTT TTOO MMEPMMOORDAANUDniUlMM DnTTPDi 11 11//1173//0911 -- 37 0n CC t 1 8 6 . 2/20/80 187. 4/80 188. 189. 4/22/80 4/22/80 190. 5/5/80 191. 5/16/80 192. 5/30/80 193. 6/2/80 Article from Toxic Materials News entitled "California Lawsuits Challenge State Pesticide Rules". Comment: See the 2,4-D article on the miscarriages caused in 9 out of 10 women! Article entitled: "Environmental Protection Agency Review and Conclusions Concerning Potential Health Effects of the Herbicide 2,4-D", from The Bureau of National Affairs. Inc., pp. 134-136. 2,4-D Fact Sheet. Document entitled: "2,4-D Fact Sheet", author not indicated, but appears to be Dow. Comment: This document that was produced from Dow production documents under court order states under section I (background) that 2,4,5-T causes cancer and miscarriages because of the TCDD (dioxin) manufacturing contaminant. If Dow didn't write this, then it had to be the EPA sending this to Dow outside the normal public channels telling them why they were cancelling their 2,4,5-T. In any event, it is a very damaging document to Dow. Document entitled: "Product Discontinuation - For the Product Esteron 245 Herbicide". Comment: Dow Chemical stops the use of Esteron 245 in the United States in PL and bulk sizes". Dow R&D Report: "Picloram: Results of a 32-Day Toxicity Tolerance in Feed in Mice", with attached report. J .W . H e n c k . et al., Dow R&D Report on 2,3,7,8-Tetrachlorodibenzo-P-DioxinAcute Oral Toxicity in Hamsters". This is a report summary. Letter to Stone of the EPA from Morgan of Dow re: Esteron 245 Application for Amended Registration. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 31 1 9 4 . 6/5/80 195. 6/24/80 195a. 6/24/80 196. 6/80 197. 198. 7/14/80 Dow Quality Assurance printout on Esteron 2,4,5 Brush and Weed Killer. Comment; Esteron 245 has been declared obsolete on 6/2/80. Dow form of medial information from Esteron 245. Comment; See exhibit 195a below. The back page of the document in exhibit 195 which is entitled: "Composition Listing Chemical Ingredients". Comment: This states that Esteron 245 can cause serious illness (from a Dow medical sheet). If gives all chemicals in this product and they contain no inerts 1 (In other words, nothing is hidden here in what Dow is actually putting in their product. They usually hide the deadly ingredients under inerts and then list some percentage like 35% inerts. In this case we are getting to see for the first time everything that Dow puts in their Agent Orange product.) No author, apparent summary of report "TCDD Oral and Dermal NIC Bioassav Summary" . June, 1980, no page number Comment: Concludes that TCDD is a carcinogen from both oral and skin absorption. Document entitled "Appendix E" : All registrants (by company name) with registered products containing 2,4-D. Comment: This shows the manufacturer's name and the use of these 2,4-D products. This is a copy of the previous document (exhibit 197) regarding "List of Your Company's Products Containing Some Form of 2,4-D". Comment: This is Dow's 43 different versions that they manufacture the 2,4D product with many of them containing 2.4.5-T as an active ingredient (up to a 50/50 mixture of 2,4-D and 2,4,5-T which was Agent Orange^. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 32 6645 NOTE: The EPA had already suspended 2,4,5-T at the time of this document in the United States except for specified limited uses. The point here is that you would believe that you were buying a 2,4-D product which is perfectly safe only to find it has within it a 50/50 mixture of the ingredients that make up the deadly Agent Orange. This document also lists the toxicity level of these various products and some of them are high toxic levels that would cause serious health effects. 198a. 9/23/80 199. 200. 9/8/80 9/12/80 201. 10/14/80 202. 10/23/80 Department of the Interior Transmittal sheet with the attached Pesticide Use Policy. Comment: This prohibits the use of 2,4,5Tl No explanation is given as to why they did this. Transmittal memo at Dow for information received from the EPA regarding their 2,4D study. The Carcinogen Assessment Groups Risk Assessment on 2,4,5-T, Silvex and TCDD. Comment: These are summary pages from the document giving the bottom line evaluation by this industry based group. R a m s e y . et al., "Dose Levels of 2,4-D in Forest Workers", report prepared for Dow. Comment: Dow had a slanted test conducted at the University of Arkansas on forest workers being exposed to the herbicide 2,4-D through aerial sprays. This is nothing like a real use environment where the workers were sprayed by both 2,4-D and 2,4,5-T and had their clothes completely soaked and wet and after working all day in this environment. These were merely short term exposures in a very limited environment set up to prove that 2,4-D was safe. News release from Agriculture Canada entitled: "2,4-D Tests". Comment: This shows that the Canadian government discovered a dioxin contaminant in 2,4-D and they were considering ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 33 6646 suspension of this product throughout Canada. It is a less toxic form of dioxin than the one found in TCDD, however it is still a deadly poison that his harmful to humans. 203. 10/27/80 Transcript of "Radio Noon" show entitled: "Dioxin Levels in 2,4-D and 2,4,5-T". Comment: This is an onaoina debate taking place in the news media regarding the concern of the Canadian government over the dioxin levels in these herbicides. There is a major public outcry to ban both of these products. 204. 10/80 Document regarding Wayne Omerod's press release. Comment: This was prepared by the Canadian government trying to decrease the level of alarm being felt in the public regarding the disclosure of these deadly dioxins in the herbicides 2,4-D and 2,4,5- T. 205. 10/80 Coversheet entitled: "Determination of 2,4-D Exposure Received by Forestry Applicators, Sprina. 1980" bv L a w of the University of Arkansas in cooperation with Dow. The report is not attached. 206. 11/14/80 Letter to Edwin Johnson of the EPA from John Hall of the National Forest Products Association re: The research report entitled "Determination of 2,4-D Exposure Received by Forestry Applicators, Spring 1980". 207. 11/24/80 Short article entitled: "Park Service Halts Use of 2.4-D". from C&EN Publication. 208. 1980 Van Loaten. et a l .. "Role of the Endocrine System in the Action of 2,3,7,8Tetrachlorodibenzo-P-Dioxin (TCDD) on the Thvmus". from Toxicolocrv. Vol. 15. p p . 135-144. 209. 1980 Murakami, et a l . . "Incorporation of Labelled Pesticides and Environmental 6647 ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 34 210. 1980 211. 212. 1980 1980 213 . 1980 214. 1980 215. 1980 Chemicals Into Nuclear Fraction of Cultured Human Cells", from Bulletin of E n v i r o n m e n t a l ____ C o n t a m i n a t i o n ____ and Toxicology. Vol. 24, pp. 27-30. Comment: Carefully look at the attached last page of this document from a news clipping out of Science magazine. Section from exhibit by Domenico. et al. , "Accidental Releases of 2,3,7,8Tetrachlorodibenzo-P-Dioxin (T C D D ) at Seviso, Italy", from Eco-Toxicoloov and Environmental Safety. Vol. IV, pp. 327328. This particular document is entitled "TCDD Levels in Atmospheric Particles". Newspaper article entitled "Dioxins Found in 2,4-D Alarm Canada". Various newspaper articles including one entitled: "Massive Rise in Cancer Deaths Predicted" (from the dioxin found in 2,4D in Canada). Canadian newspaper articles regarding dioxins being found in 2,4-D. Comment: Talks of a man who had his arm amputated (after the use of 2,4-D/2,4,5T) for cancer plus another article on "Doctored Chemical Tests and Wide-Ranging Dishonesty in Labs". Document from Health and Safety Alert entitled "Beware of the Weed Killer 2,4,5T and 2,4,5-TP". Comment: The Canadian Union is warning its workers to not use 2,4,5-T because it is a health hazard with increased cancer risks of the muscle and the tissue. L a m b . et a l ., "Evaluation of 2,4-D, 2,4,5T and 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) Toxicity in Mice: Reproduction and Fertility in Treated Male Mice and Evaluation of Congenital Malformations in Their Offspring", publication not indicated. Comment: This is supposed to be a study for the effects of "Agent Orange", but Dow ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 35 6648 216. 1980 217. 1980 218. 1980 219. 220. 221. 1980 - 0- 1/7/81 222. 1/7/81 provided the samples for the test and removed all TCDD impurities! Sencuzk, Watold, translation by Lilienheim in November, 1981, "Absorption of 2,4-D Through the Skin", from Warsaw Panstwowv Zakled Hiaienv. Roczniki.. Vol. 31, pp. 611-614. Comment: This shows that the period of exposure determines the amount of 2,4-D that will be absorbed through a given individuals skin, i.e. its all a question of the duration of use of this product in a given period of time. Marcel. Bickel & Stefan. Muhlebch, "Pharmacokinetics and Ecodisposition of Polyhalogenated Hydrocarbons: Aspects and Concepts", from Drug and Metabolism Reviews. Vol. 11 #2, pp. 149-190, 1980. Comment: Selected pages-- with the conclusion page showing a whole new dimension in the ecotoxicology that has been opened by the use of the herbicide 2,4,5-T with its contaminant TCDD through the food chain of the global ecosystem. Arstila. et al., "An Epidemiological Feasibility Study on the Effects of 2,3,7,8-Tetrachlordibenzo-P-Dioxin(TC D D ) on Finnish Forestry Road and Railway Workers", publication not indicated. Comment: This document is marked "strictly confidential" by Dow Chemical. Editorial entitled: "The Burial of the Two Million Dollar Teaspoon", publication not indicated. VACANT R & D report from Dow re: "Technical Grade 2,4-D: Results of a 13-Week SubChronic Dietary Toxicity Study in CDF Fisher 344 Rat", by Gorzinski. et al. A copy of the report referenced in exhibit 221. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 36 223. 1/27/81 224. 1/28/81 225. Jan/Feb 1981 226. 1/81 227. 3/81 228. 3/81 229. 4/30/81 Text of press release by Dow in Ottawa, Canada outlining measures to phase out 2,4-D herbicide products. EPA weekly report of the Office of Pesticides and Toxic Substances entitled: "Canada to Phase Out Some 2,4-D Products". Pazderova-Veiluokova. et al., "The Development and Prognosis of Chronic Intoxication By Tetrachlorodibenzo-PDioxin in M e n " , from Archives of Environmental Health. Vol. 36, pp. 5-11. S. Libich, "A Study to Assess the Occupational Exposure to 2,4-D Herbicides". Ontario Hydro Safety Services Department Booklet. Comment: This study shows "at least 35 times more 2,4-D is absorbed through the skin than is inhaled with the breathing of the air". It says to change clothes as soon as possible after using 2,4-D and wash all exposed skin areas immediately. Hayes. Mary Catherine, American Counsil on Science and Health. "The Health Effects of Herbicide 2,4,5-T". Comment: This is a white wash job done by the industry to save 2,4,5-T. Four page document including chronology of events involving 2,4,5-T and herbicide reduction statistics. Comment: This says that the 2,3,7,8 TCDD impurity in the 2,4,5-T was made known (to the public!). It also says "2,4,5-T kills plants because it interferes with the cell division (mitosis) and cell enlargement, food utilization and various other processes necessary to stabilize life. Allatair. Hay, "Secrecy at Coalite" supertitled: "Dioxin Hazards". Also abstract "Dioxin, A Sarcoma Risk", from Nature. Vol. 290, April 30, 1981, p. 729. Comment: This document connects TCDD with Sarcomas! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 37 230. 4/81 231. 232. 6/2/81 10/16/81 233 . 12/81 234. 1981 235. 1981 "Worker's Exposure to 2,4-D Study", from Research News of the Department of Agriculture. Comment: The results shown here are that absorption is directly related to the type of job, the amount of herbicide applied and the length of time it is handled. Those who mixed and loaded these herbicides have the highest total absorption, i.e. the results are based on the time of exposure, the amount of chemicals handled and the type of job. Dow Quality Assurance print out for Esteron 2,4,5 Herbicide. Document entitled: "Manufacturing Specification", apparently a routine slip, Esteron 2,4,5 BE Herbicide, handwritten notes, mostly illegible. Dow document entitled: "Medical Information Regarding Esteron 2,4,5 BE Herbicide, Esteron 2,4,5 Brush and Weed Killer and Esteron 2,4,5 Concentrate Brush and Week Killer". Report to the Council on Scientific Affairs of the AMA on "Health Effects 'Agent Orange' and Dioxin Contaminants" presented by Dr. Dolan. Comment: This is a review of how Agent Orange was made and states that TCDD is the direct and sole cause of chloracne (see page 3 of document). Abstract from the Chemical Regulation Reporter entitled: "Dioxins: Second Dow Sarcoma Case Found; Company Suggests Smoking as Factor". Comment: Four chemical workers had died of sarcoma, two were employees of Dow and were exposed to TCDD resulting in chloracne immediately. The other two workers were employees of Monsanto. They tried to white wash this exposure of their own workers who died of cancer and blame it on cigarette smoking. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 38 6 23366.. 1981 237. 4/2/81 238. 71/27/81 239 71981 240. 1/12/82 241. 3/19/82 242. 3/23/82 243. 3/24/82 Crow. K . D . . "Chloroacne and Its Potential Clinical Implications". from Clinical and Experimental Dermatolocrv (19811. Vol. 6 P pp. 243-257. Comment: "It is essential to recocmize that chloroacne appears to be the most sensitive indicator of poisoning in man". The document goes on to say that chronic exposures are the most serious to man. Two pages of handwritten notes regarding Esteron 2,4,5-T and comparing its acid equivalent with other weed killers. Mostly illegible teletype regarding 2,4D. News article entitled: "Dioxin Haunts EPA Refuse Plans". Letter from M.D. Black, California State Department of Food and Agriculture. To: R.W. Colby of Dow Chemical re: Esteron Brush Killer and Esteron 2,4,5 Herbicide and Sub-Registration to Union Carbide. Comment: It appears that Dow aot rid of its excess 2,4,5-T by subregistering it under a Union Carbide product! Letter from B.T. Buzelli to A.E. Schober, both of Dow Chemical, regarding Esteron 2,4,5 BE-TCDD analysis. Comment: THIS IS A SMOKING-GUN DOCUMENT! Here Dow knowingly sells a defective Esteron 2,4,5 BE product to consumers and hides the defects from them! This shows TCDD contamination in a 635,000 pound tank plus an additional 16,500 pound shipment that had already been packaged in 55 gallon drums. Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron 245 BE Herbicide". Letter from Authur Schober of Dow Chemical to T.E. Adamczyk of the EPA regarding Esteron 245 BE. ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 39 6652 24444.. 3/82 245. 4/8/82 246. 4/13/82 247. 4/16/82 248. 249. 5/19/82 5/21/82 250. 5/24/82 251. 8/3/82 Several letters between Dow Chemical and the EPA regarding Esteron 245 BE. Also attached is a certification statement regarding Esteron 245 BE. Comment: Dow is pullina something here! They are also selling a banned product under a different label! Dow Chemical documents, "Production Specifications Regarding Esteron 245 C Herbicide". Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron 245 BE Herbicide, Rough Draft. Document titled: "Manufacturing Specification", cover routing slips, Esteron 245 BE Herbicides, handwritten notes. Material Safety Data Sheet on Esteron 245 BE Herbicide. Memo of Dow Chemical to R.L. Eisher, et al . , from Jim Havel re: Meeting Regarding Esteron 245 BE Quality and Options. Comment: Here sludae is found in Esteron 245 BE cans. Dow concludes "There is no way to burn or dispose of the technical ester. It must be sold". This was sludae found in 55,000 gallons of their Esteron 245 BE product. Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron R 245 BE Herbicide". Comment: This is the final version of document 242 where it shows that 245 BE is the same as 2.4.5-T and is beina sold "only to deplete current stock of this technical ester" (2,4,5-T)! Report by Dow Chemical, Latin American on Esteron 245 C Herbicide. Comment: This shows that 2.4.5-T is beina distributed to Latin America after being banned in the U .S .! There is no mention made here cautioning the user of the TCDD ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 40 252. 9/82 253. 1982 254. 1982 255. 1982 256. 257. 1982 258. 1982 contaminants in this. It is noted that they make many precautionary safety statements for all of the other ingredients in this product but fail to mention the deadly TCDD! Letters between EPA and Dow Chemical regarding 2,3,7,8 TCDD and Esteron 245 BE. Comment: Dow is hidincr the TCDD results from the EPA here. (see exhibit 241) Labels for Dow Esteron 245 and Esteron 245 BE. Comment: This is the banned 2 . 4 r5-T with a changed name and slight modification of ingredients on the label to get around the EPA ruling. Corte. et al. . "2.4-D Induced Clastogenicity and Elevated Rates of Sister Chromatid Exchanges in Cultured Human Lvmohocvtes", from Journal of Heredity. Vol. 73. po. 224-226. Comment: This mav be a very sionificant document. You need to have Dr. Teitelbaum review this and determine its significance. Draper, et al. . "Applicator Exposure to 2,4-D, Dicamea and a Dicamba Isoxer", J. Environ. Sci. Heath. Vol. 4. on. 321-339. VACANT Michigan State Department of Agriculture Research Grant entitled: "Effects of Pesticides on the Kidney" by Hook and Serbia, at The Center For Environmental Toxicology at Michigan State University; East Lancing, Michigan. Comment: This is a studv of 2.4.5-T and TCDD effects. EPA Form 8570-10, November, 1974, titled: "Label Technical Data" regarding Esteron 245 BE. ATTACHMENT TO MEMORANDUM DTD 11/13/91 6654 259. 2/21/83 260. 4/22/83 261. 5/4/83 262. 5/83 Series of letters between Jim Havel and Ralph Antz of Dow Chemical regarding use directions for Esteron 245 B. Comment; This document is very damaging to Dow because it shows them selling the defective Esteron 245 BE and not wanting any of it returned to Midland, Michigan. A suggestion is made to "blend it back into a straight 245-T product at the user level". Dawn Giaradin, document titled: "Customer Request", refers to determination of 2,3,7,8 TCDD. This exhibit also contains a letter of 5/19/82 regarding Esteron 245 BE and TCDD analysis and also contains a letter from the EPA regarding TCDD analysis and a memo dated 8/5/81 headed "Distribution Emergency" referring to Esteron 245. Comment: There is something very unusual happening here, but it is hard to figure it all out! Melvin Dwane Ruber, "Carcinogenicity and Toxicity of 2,4-D", from The Science of the Total Environment. Vol. 31, 1983, pp. 203-218. Comment: This document states "2,4-D is carcinogenic in male and female rats; 2,4D is also mutenagenic and teratogenic in animals and causes poisoning in animals and human beings". (Underlining was m i n e .) Kenneth G. Torrington, M.D., letters to the editor titled: "Herbicide Exposure and Pulminary Disease", from Journal of Occupation Medicine. May 1963, Vol. 25, #5. Comment: This is an unusual exposure case; a freight train was pushing 2,4-D tank cars while performing railroad foliage clearing into a strong headwind. The spray got blown back all over the train engineer and the conductor causing serious acute health effects in them! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 42 6655 > 263. 6/83 264. 7/28/83 265. 8/83 266. 1983 267. 1983 268. 1/84 Incident report on Esteron 2,4,5 by Mrs. Gerald Grove regarding her husband; message taken by an M.S. Betty Schmidt. Document was a Dow R&D report that I only used to copy the distribution page on the back to show the worldwide offices of Dow Chemical Company. No author, "Dioxin and Human Health", apparently the full report referred to in exhibit #267. Comment; This is the full report of the white wash put out apparently by Dow to create the illusion that human health was not being affected by these dioxins. Easley. Carol Bryan, et al., "Laundering Procedures for Removal of 2,4- Dichlorophenoxyacetic Acid Ester and Amine Herbicides From Contaminated Fabrics", from Archives____ of____ Environmental Contamination and Toxicology. Vol. 12, pp. 71-76. Comment: This is a very important document in that it shows how 2,4-D and 2,4,5-T molecules are more readily absorbed through the skin, especially in wet herbicide soaked clothes! The major route of pesticide exposure in absorption is through the skin based on this study and not the respitory system because only small particles can enter the lungs, but larger more numerous particles settle on the surfaces of the skin and your clothing. Hence, attention has focused on clothing items which cover and protect skin surfaces. In this document they study the laundering procedures for the removal of 2,4-D. Bob Charlton, Dow Chemical, "Dioxin Human Health Executive Summary". C o m m e n t : Mai or Dow white wash on public presenting a distorted misstated version of the truth. and the and Libich. et al., "Occupation Exposure of Herbicide Applicators to Herbicides Used ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 43 6656 / 269. 4/84 270. 4/84 271. 1984 272. 9184 on Electrical Power Transmission Line Right of Way", from American Industrial Hygienist Association Journal. Vol. 45, pp. 56-62. Comment: This shows that exposure through the skin is the major absorption route creating up to 50 times greater than exposure than by the inhalation route when using a hand grin sprayer. During the handling and mixing of the concentrated chemical, A VERY SMALL SKIN EXPOSURE CAN BE EQUIVALENT TO A FULL DAY OF FIELD EXPOSURE. Dow report entitled: "Dioxin, Agent Orange and Human Health". Comment: This is how Dow will defend the technical issues in a lawsuit. (71 pages) It was published one month prior to the start of the Agent Orange trial litigation! Puhvel. S.M., et a l ., "Increased Epidermal Transglutaminase Activity Following 2,3,7,8-Tetrachlorodibenzo-P-Dioxin In Vivo and In Vitro Studies With Mouse Skins", from Toxicology and Applied Pharmacology. Vol. 73, pp. 311-322, 1984. Comment: This document says "in humans one of the first signs of exposure to TCDD is the development of a skin condition known as chloroacne". Hosm a n . et al., "Results From 86 Two-Year Carcinogenicity Studies Conducted By the National Toxicology Program", from The Journal of Toxicology and Environmental Heal t h . Vol. 14, pp. 621-639. Comment: This document shows how misleading many of the tests have been in showing us the true picture of these health hazards! Section from The Clinical Toxicology of Commercial Products. Williams and Wilkens publishers, 5th Edition, with a page on the chemical components of Esteron. Comment: This shows the 1984 marketing of the Dow 2,4-D and 2,4,5-T products under the trade name of "Esteron"! ATTACHMENT TO MEMORANDUM DTD 11/13/91 - 44 6657 273.. 11/15/85 274. 1985 275. 1986-1987 276. 9/7/88 277. 12/87 Elvira Spill, translated by Anita Knight and L.P. McCarthy, "The Lingering (Insidious) Contamination", subtitled: "Dioxins Have Left Their Mark on Children from Seveso Nine Years Ago". The original German article is attached. John D. Constable and Maureen C. Hatch, "Reproductive Effects of Herbicide Exposure in Vietnam: Recent Studies by the Vietnamese and Others". Tetratoaenisis Carcinoaenesis and Mutagenesis. Vol. 5, no. 231-250. Comment: This reports increases in miscarriages, still births, molar pregnancy and birth defects from the Vietnamese exposed during the war. Thompson. W.T., Agricultural Chemicals Book II - Herbicides. Section entitled: "2,4,5- T ; Dacmine, 4-T; Envert-T; Esteron 245; Phortox; Reddon; Tippon; Trioxone; Veon". Comment: This gives the chemical makeup and use applications of the various herbicides. This letter talks about research done at Texas Tech University Health Sciences Center on labels and product information for the Dow Esteron 245 (1980 time frame) and Esteron 99 concentrate (1982 time f r a m e ). Comment: This is a good background on 2,4-D and 2,4,5-T, plus their chemical makeups, the EPA cancellation of 2,4,5T and various health data sheets. Draft for Toxilogical Profile for 2,3,7,8Tetrachlorodibenzo-P-Dioxin from the U.S. Public Health Service. Comment: This document is 122 pages long and is a good overview of what TCDD is. ATTACHMENT TO MEMORANDUM DTD 11/13/91 45 nor Q THIBIT NO. IDENTIFIED (p 2 3 xy 4P W 8 W 0' r ADMITTED DATE 1978 1979 1978 8/18/80 10/12/81 6/14/73 10/13/71 00/00/00 12/18/72 10/20/79 1 DESCRIPTION Medical article. Kreeftenberg, et al. "Studies on 2,3,7,8-Tetrachlorobibenzo-pdioxin-Induced Immune Suppression and Decreased Resistance to Infection: Endotoxin Hypersensitivity, Serum Zinc Concentrations and Effect of Thymosin Treatment". Toxicology 9 (1978) 75-86. Medical article. Luster, et al. "Effects of Brief In Vitro Exposure to 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) on Mouse Lymphocytes". Journal of Environmental Pathology and Toxicology 2:956-977. Medical article. Carere, et al. "Microbiological Mutagenicity Studies of Pesticides In Vitro" Mutation Research 57 (1978) 277-286. Dow Chemical Company Certificate of Analysis of Esteron 245 Brush and Weed Killer for Customer Imperial, Inc. Computer printout entitled: "Dow Chemical Medical Department Chemical Exposure History by DR-Number" . R &D Report from Dow Chemical transmitting an attached copy of a report entitled: "Picloram-Impact on the Environment (EPA PR Notice #70-15)" by Lawrence Southwick of Dow Chemical. Report from Pierce and Ens of the Organic Chemicals Production Research Department of Dow titled: "Origin and Fate of the Chlorodioxins. Abstracts From a Symposium at the 162nd National ACS Meeting, Washington DC, September 16-17, 1971" Untitled document appearing to be the report referenced in Exhibit No. 7. Cover memo and abstract written by Marguerite Leng. Attached is report entitled: "Fate and Safe Use of Tordon in the Environment". Paper is dated July 1969 and was submitted to the EPA. Abstract and attached report entitled: "Reversal of Immunologic and Toxicologic Effects of a Single Exposure of 2,3,7,8Tetrachlorobibenzo-p-dioxin in Mice". O C K f) 8/20/79 1962 " 4/ 2/81 00/00/00 1/22/80 8/26/81 4/80 1976 1970 1/27/81 00/00/00 1979 Authored by Sharma, et al., of Dow. R & D Report abstract and attached report entitled: "Immuno-Toxicologic Studies Involving 2,3,7,8-Tetrachlorobibenzo-pdioxin (TCDD) In Mice and Rabbits" by Sharma, et al., of Dow. Translation of attached French documents entitled: "Polyneuritis After Using a Weed Killer: Containing Acid 2-4-D" by Foissac, et al., Lille Medical. Handwritten notes entitled: "Total 2,4,5T Acid Plus Estern (1982)". Two pages of hand-written notes regarding Esteron 2,4,5-T and comparing its acid equivalent with other weed killers. Information on Esteron 245 from Wolfe of Quality Assurance. Article entitled: "Important Issues Remain Unresolved in Avian Environmental Standards" from the Pesticide and Toxic Chemical News. Also included is an article entitled: "2,4-D and Spontaneous Abortions: Questions Raised by Survey". Article entitled: "Environmental Protection Agency Review and Conclusions Concerning Potential Health Effects of the Herbicide 2,4-D", from the Bureau of National Affairs, Inc., pp. 134-136. Smith, et al. "Teratogenicity of 2,3,7,8Tetrachlorobibenzo-p-dioxin in CF-1 Mice" Toxicoloav and Applied Pharmacoloav, Vol. 38. 517-523. Norris, Logan. "Degradation of Herbicides in the Forest Floor" reprinted from Tree Growth and Forest Soils, Oreaon State University Press, pp. 397-411. Text of press release by Dow in Ottawa outlining measures to phase out 2,4-D Herbicide Products. Mostly illegible teletype regarding 2,4-D. Aitio, et al. "Different Effect of 2,3,7,8-Tetrachlorobibenzo-p-dioxin On Glucuronide Congugation of Various Aglycones. Studies in Wistar and Gunn . - Rats" Toxicoloqv and Applied-, r* (P d? 26 d 27 tS*' -N .30) v_ 7 'S 12/14/70 5/19/82 1978 2/9/76 July/ August, 1973 5/29/58 00/00/00 1980 00/00/00 5/ 6/80 3/79 6661 P h a r m a c o l o g y , 47, 55-60. Dow report entitled "The Determination of 2,4,5-Trichlorophenol and 2,4,5Trichlorophenoxyacetic Acid in Blood and Urine by Gas Liquid Chromatography". Material Safety Data Sheet on Esteron 245 BE Herbicide. Somers, et al. "Reproductive Success of Hens and Cockerels Originating Sprayed with 2,4-D, 2,4,5-T and Picloram Followed by Early Performance of Their Progeny After a Comparable In Ovo Exposure". Journal name illegible. Abstract and attached report entitled: "The Fate of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Following Oral Administration to Man". By Sauerhoff, et al. Feung, et al. "Metabolism of 2,4Dichlorophenoxyacetic Acid. V. Identification of Metabolites in Soybean Callus Tissue Cultures". Agricultural and Food Chemistry, Vol. 21, pp. 637-640. Hymas of Dow. "Toxicity of Herbicides to Domestic Animals". Cook, et al., of Dow. "Mortality Experience of Employees Exposed to 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD)". Publication not indicated. Fanelli, et al. "Presence of 2,3,7,8Tetrachlorobibenzo-p-dioxin in Wildlife Living Near Seveso, Italy: A Preliminary Study" . Bulletin of Environmental Contamination and Toxicology. 24, 460463. Editorial entitled: "The Burial of the $2,000,000 Teaspoon". Publication not indicated. Bezuglyi, et al. "Clinic for Delayed Consequences of Acute Contamination From 2,4-Dichlorophenoxyacetic Acid". Translation of attached Russian Article. Abstracts from the Society of Toxicology in New Orleans. Papers entitled: "Toxicological Effects Produced in NonHuman Primates Chronically Exposed to 50 Parts Per Trillion 2,3,7,8- 1/28/81 1/23/81 00/00/00 11/24/80 8/26/80 5/ 5/80 5/ 5/80 1980 1/80 1974 8/25/76 10/76 cc o Tetrachlorobibenzo-p-dioxin (TCDD)", and "Toxicological Responses of Non-Human Primates to 2,3,4,6,7-H-Hexachlorodibenzop-dioxin". EPA Weekly Report of the Office of Pesticides and Toxic Substances entitled: "Canada to Phase Out Some 2,4-D Products". Document entitled: "Fact Sheet on 2,4-D", author and source not indicated. News article entitled: "Dioxin Haunts EPA Refuse Plans". Short article entitled: "Park Service Halts Use of 2.4-D". from C&EN Publication. Article from the Maine Times entitled: "Picloram's Turn: A Basic Philosophical Difference is Revealed". Product Discontinuation-Record of Approval Form on Esteron 245 Herbicide. Product Discontinuation-Record of Approval Form on Esteron 245 Herbicide. Gasiewicz, et al. "The Effect of Total Parenteral Nutrition on the Toxicity of 2,3,7,8-Tetrachlorobibenzo-p-dioxin in the Rat". Toxicolocrv and Applied Pharmacoloav 54, 469-488. Specimen label and attached product information insert and direction for use on Dow Esteron 245 Herbicide. Poland, et al. "Comparison of 2,3,7,Tetrachlorobibenzo-p-dioxin, A Potent Inducer of Aryl Hydrocarbon Hydroxylase, with 3-Myethylcholanthrene". Molecular Pharmacoloav, 10, 349-359. Poland, et al. "Stereospecific, High Affinity Binding of 2,3,7,8Tetrachlorobibenzo-p-dioxin by Hepatic Cytosol". The Journal of Bioloaical Chemistry, 251, 4936-4946. Poland, et al. "2,3,7,8Tetrachlorobibenzo-p-dioxin: Environmental Contaminant and Molecular Probe". Federation Proceedinas, 35, 24042411. 1979 1970 7/78 1980 1/ 7/81 00/00/00 1978 00/00/00 6/73 7/73 8/15/63 1963 1978 (2,4-D and 2,4,5-T)" Vetenskapliq Skriftservie 17, 1-24. English summary of Exhibit No. 52. Matsumura, Akihito. "The Fate of 2,4,5Trichlorophenoxyacetic Acid in Man" Japanese Journal of Industrial Health, Vol. 12, October 1970. 446-451. Yoshida, et al. "Occupational Exposure Measurement of an Arial Spray Crew to a Mixed Spray of 2,4-D and 2,4,5-T" Pages 1-26. Thunberg, et al. "Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin on the Hepatic Storage of Retinol in Rats with Different Dietary Supplies of Vitamen A (Retinol)" Arch Toxicol 45, 273-285. R & D Report from Dow re: "Technical Grade 2,4-Dichlorophenoxyacetic Acid (2,4D): Results of a 13 Week Sub-Chronic Dietary Toxicity Study in the CDF Fischer 344 Rat" by Gorzinski, et al. A copy of the report referenced in Exhibit 57. Reggiani. "Medical Problems Raised by the TCDD Contamination in Seveso, Italy". Arch Toxicol. 40, 161-188. Document entitled "Section C. ToxicologyVolume IV". It appears to be a list of toxicology study of herbicides and birds. Document entitled: "Summary of Toxicology Studies with 2,4-D in Birds". Document entitled: "Section D. The Results of Tests on the Amount of Residue Remaining, Including a Description of the Analycial Method Used". Palmer, J. S. "Chronic Toxicity of 2,4-D Alkanolamine Salts to Cattle". Journal of the American Veterinary Medical Association, Vol. 143, paae 398-399. Dolgarrd, Mikkelsen, et al. "Toxicity of Herbicides" Pharmiceutical Review 14, 225-250. HEW Report: "Bioassay of Picloram for Possible Carcinogenicity" from the 7/27/65 10/2i/65 1/15/63 National Cancer Institute, Carcinogenesis Technical Report Series, No. 23. "Report to the Dow Chemical Company Two Year Chronic Oral Toxicity of TordonBeagle Dogs" Industrial Bio-Test Laboratories. "Report to the Dow Chemical Company. Chronic Oral Toxicity of Tordon-Albino Rats" Industrial Bio-Test Laboratories, Inc. "Toxicological Properties of Tordon" prepared 10/23/64 4/18/62 2/27/63 11/ 8/63 1/28/64 12/14/64 1/ 8/80 1967 1969 12/10/69 "Results of 90 Day Dietary Feeding Studies of Triisopropanolamine Salt of 4-amino3,5,6-trichloropicolinic Acid in Rats" by Dow. Bucek, et al. of Dow. "Effect of 4-amino3,5,6-trichloropicolinic Acid and of 4amino-3,5-dichloropicolinic Acid in Growing-Fattening Swine Rations". Briscoe of Pittman, Moore Research Laboratories. "Acute Pharmacotoxicologic Tests with Tordon Acid". "The Metabolism of Tordon Herbicide by the Dog", by Dow. "The Metabolism of Tordon Herbidice by the Rat", by Dow. US Department of Interior, Fish and Wildlife Service. "Procedure for Evaluation of Acute Toxicity of Pesticides to Fish and Wildlife". MSDS for Esteron 245 Brush and Weed Killer. Youngson, et al. "Factors Influencing the Decompositions of Tordon Herbicide in Soils". Down to Earth (A Dow Chemical Company Publication) Vol. 23, pp. 3-11. Kutschinski, et al. "Residues in Various Tissues of Steers Fed 4-amino-3,5,6trichloropicolinic Acid" Publication not indicated. Could be a Dow publication. Bjerke, et al. "Residues of Picloram in Sheep Tissues" from Dow. 7b [IS/ 80 a 81 85 86 fei) 1/64 "Elimination of 2-Methyl-4Chlorophenoxyacetic Acid and 4-(2-Methyl4-Chlorophenybutyric) Acid in the Urine from Cows". Journal of Dairy Science. Vol 47, pp. 93-95. Author not indicated. 1/ 4/80 Kimble, et al. "Tetrochlorodibenzo-pdioxin Quantitation in Stack-Collected Coal Fly Ash" Science Vol. 207, pp. 59-61 1979 Raggiani. "Estimation of the TCDD Toxic Potential in the Light of the Seveso Accident". Arch Toxicol. Vol. 2, page 291-302. 6/28/79 Dialog search. 11/ 6/73 Crosby, et al. "Photo Decomposition of Pchlorophenoxyacetic Acid" Aar. Food Chem. Vol. 21, pp. 1049-1052. illegible Homberger. "The Seveso Accident, Its Nature, Extent and Consequences". Publication-- not legible. 9/23/60 Dept, of the Interior Transmittal Sheet with the attached pesticide use policy. 9/30/80 Telex to: Skinner of the Honolulu sales office. From: Ytzen. Re: prices on various Dow herbicides. 1970 Rivers, et al. "Simultaneous Gas Chromatograpic Determination of 2,4-D and Dicamba Ir. Human Blood and Urine". J . Chromatoq, 50, pp. 334-337. 1/74 Somers, et al. "Effect of External Application of Pesticides to the Fertile Egg on Hatching Success and Early Chick Performance 1. Princubation Spraying with DDT and Commercial Mixtures of 2,4-D, Picloram and 2,4-D, 2,4,5-T". Bulletin of Environmental Contamination and Toxicology, Vol. 11, pp. 33-38. 1978 German Medical Article. See Exhibit 87. 10/21/78 Rehder, et al. "PathologicalEmbryological Investigations in Cases of Abortion Related to the Seveso Accident". Journal of Swiss Medicine, Vol. 108, pp. 16, 17-25. Ef 1 9 7 8 ij Buser, et al. "Identification of Polychloronated Dibenzo-p-dioxin Isomers 1978 5/ 4/77 5/26/77 5/26/77 9/ 1/71 1976 1977 1978 12/13/78 1979 8/28/74 Found in Fly Ash". Chemosphere No. 2, pp. 165, 172. Buser, et al. "Identification of Substitution Patterns in Polychloronated Dibenzo-p-dioxins (PCDDS) by Spectrometry". Chemosphere No. , pp. 199211. Memo from Johnson of the EPA, to dioxin implimentation task force. Re: Dioxin: Position Document. Draft of EPA's dioxin working group document entitled: "Dioxin: Position Document". Last three pages of Exhibit 91. Johnson, Julius. "The Public Health Implication of Widespread Use of the Phenoxy Herbicides and Picloram" Bioscience, Vol. 21, pp. 899-905. Courtney, K. Diane. "Mouse Teratology Studies with Chlorodibenzo-p-dioxins" Bulletin of Environmental Contamination and Toxicology, Vol 16, pp. 674, 681. Alie, et al. "Chlorodibenzo-p-dioxins, and Chlorodibenzo-furans are Trace Components of Fly Ash and Flue Gas of Some Municipal Incinerators in the Netherlands". Chemosphere, No. 8, pp. 455-459. Buser, et al. "Identification of Substitution Patterns in Polychloronated Dibenzo-p-dioxins (PCDDS) by Spectrometry". Chemosphere No. , pp. 199- 211. "Human Birth Defects Not Caused by 2,4,5-T or 2,4-D Exposure, Report Says". Pesticide and Toxic Chemical News, pp. 2324. Helder, Theo. "Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) On Early Life Stages of the Pike" Publication not indicated. New York Times Article. "Death of Animals Laid to Chemical". Attached is a 2/28/80 Dow memo to the State Dept, of Agriculture regarding the label for Esteron 245 Herbicide. May-June, 1967 1969 2/13/80 Bjerke. "Determination of Residues of 4amino-3,5,6-trichloropicolinic Acid in Cereal Grains by Gas Chromatography". Aar. Food Chem., Vol. 15, pp. 469-473. Way, J. M. "Toxicity and Hazards to Man, Domestic Animals and Wildlife from Some Commonly Used Auxin Herbicides". Publication illegible. Letter from Garner of the EPA to Leng of Dow re: Revised Labelling for Various Herbicides Including Esteron 245" . ^' 1/21/80 8/24/74 1976 5/78 5/78 1/ 3/80 3/47 3/13/64 3/16/64 i r\ Letter from Leng of Dow, to Garner of the EPA regarding revised labelling. "Illness Associated with TCDD Contaminated Soil-- Missouri". Communicable Disease Center Morbiditv and Mortality Weekly Report, p. 299. Smith, et al. "Teratogenicity of 2,3,7,8Tetrachlorobibenzo-p-dioxin in CF-I Mice" Toxicoloav and Applied Pharmocoloav, 517523. Notice of research project, Tox-Tips. The project title is: "In Vitro Mutagenic Evaluation of the Herbicide 2,4-D" from the National Library of Medicine. Tox-Tips Notice of Research Project entitled: "Carcinogenesis Bioassay of Tetrochlorodibenzo-p-dioxin" from the National Library of Medicine. Letter to Garner of th EPA, from Leng, regarding: registration of various Dow Herbicides including Esteron 245. Hill, et al. "Toxicity of 2,4Dichlorophenoxyacetic Acid for Experimental Animals" Journal of Industrial Hvaiene and Toxicoloav, 29, pp. 85-95. Vol. Palazzolo of Industrial Biotest Laboratories, Inc. "Report to the National Agricultural Chemicals Association. Repeated Dermal Toxicity Studies on Three Formulations of 2,4Dichlorophenoxyacetic Acid". Palazzolo of Industrial Biotest January/ February, 1981 1/ 7/78 7/62 10/80 11/14/80 8/79 1979 1975 1966 1976 1979 Pazderova-Veglupkova, et al. "The Development and Prognosis of Chronic Intoxication by Tetrachlorodibenzo-pdioxin in Men". Archives of Environmental Health. 36. 5-11. Franco, Berrino. "Preliminary Report on Mortality in the Seveso Area, 1975-77". Stamped "Strictly Confidential" . Florsheim, et al. "Some Effects of 2,4Dichlorophenoxyacetic Acid on Thyroid Function in the Rat: Effects on Iodine Accumulation", Endocrinoloav, 71, 1-6. Cover sheet entitled "Determination of 2,4-D Exposure Received by Forestry Applicators, Spring 1980", by Lavy of the University of Arkansas in cooperation with Dow. This report is not attached. Letter to: Edwin Johnson of the EPA. From: John Hall of the National Forest Products Association. Re: The research paper entitled "Determination of 2,4-D Exposure Received by Forestry Applicators, Spring 1980" Carlstedt-Duke, Jan. "Tissue Distribution of the Receptor for 2,3,7,8Tetrachlorobibenzo-p-dioxin in the Rat". Cancer Research 39, 3172, 3176. Abstract of medical article entitled: "2,3,7,8-Tetrachlorobibenzo-p-dioxin Induction of Aryl Hydrocarbon Hydroxylase in Female Rat Liver. Evidence of De Novo Synthesis of Cytochrome". Beatty, et al. "Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) on Mammalian Cells in Tissue Cultures". Toxicoloav and Applied Pharmacoloav 31, 309,312. Erne, Kurt. "Studies on the Animal Metabolism of Phenoxyacetic Herbicides". Publication and page numbers illegible. Koschier, et al. "In Vitro Uptake of Organic Ions by Renal Cortical Tissue of Rats Treated Acutely with 2,4,5Trichlorophenoxvacetic Acid". Toxicoloav and Applied Pharmacoloav 35, 355-364. Kolmodin-Hedman, et al. "Control of Occuaptional Exposure to Phenoxy Acids /ilV 115 117 118 120 122y 6669 Laboratories, Inc. "Addendum Report to the National Agriculatural Chemicals Association. Repeated Dermal Toxicity Studies on Three Formulations of 2,4Dichlorophenoxyacetic Acid". 10/29/48 Bjorn, et al. "Effects of 2,4Dichlorophenoxyacetic Acid on Chicks" Science, Vol. 108, pp. 479-480. 00/00/00 Rowe, et al., of Dow. "Acute Oral Toxicity of 2,4-Dichlorophenoxyacetic Acid to Rats, Mice, Guinea Pigs and Chicks" 00/00/00 Rowe, et al., of Dow. "Semi-Chronic Oral Toxicity of 2,4-Dichlorophenoxyacetic Acid to Rats". 10/29/48 Bjorn, et al. "Effects of 2,4Dichlorophenoxyacetic Acid on Chicks" Science. Vol. 108, pp. 479-480. 10/54 Rowe, et al., of Dow. "Summary of Toxicological Informaton on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of Hazards to Livestock Associated with their Use". Am. J. Vet. Res, pp. 622-629. JanuaryFebruary, 1964 Clark, et al., of the US Dept, of Agriculture. "The Fate of 2,4Dichlorophenoxyacetic Acid in Sheep". Agricultural and Food Chemistry, Vol 12, pp. 43-45. 1/55 Markquardt, et al., of Dow. "Determination of 2,4Dichlorophenoxyacetic Acid (2,4-D) in Grain and Seed". Agricultural and Food Chemistry. Vol. 3, pp. 51-53 6/73 A table of contents entitled "Section C Safety and Toxicology", with a listing of studies regarding herbicides and animals. 12/63 Lisk, et al. "Elimination of 2,4-D in the Urine of Steers Fed 4-(2,4-DB) or 2,4-D". Journal of Dairy Science, Vol. 46, pp. 1435-1437. 6/73 Document entitled: "Section C. Full Reports of Investigations Made with Respect to the Safety of the Pesticide Chemical". 4/72 1T Abstract from the British Industrial Biological Research Association, Information Bulletin entitled: "Peace and Quiet for 2,4-D . . 10/18/72 Leng, Marguerite of Dow. "Summary of Teratology Studies with 2,4-D". 124 10/ 6/72 Document entitled: "Summary Teratology". 6/73 "Summary of Metabolic Studies with 2,4-D and Animals, June 1973". Author not indicated. 126 00/00/00 Document entitled: "Section C Toxicology- Volume 6", with a listing of articles on toxicology studies in livestock. 6/73 "Summary of Toxicology Studies with 2,4-D in Livestock, June 1973" 128 5/69 Table entitled: "Summary of Dosages of Various Organic Herbicides that Cause Significant Weight Loss, Reduced Weight Gain, or Poisoning in Cattle, Sheep or Chickens". Production Research Report No. 106, U.S. Dept, of Agriculture. 129 3/72 Table entitled "Summary of Dosages of Various Organic Herbicides that Cause Significant Weight Loss, Reduced Weight Gain or Poisoning in Cattle, Sheep and Chickens". Production Research Report No. 137, U.S. Dept, of Agriculture. 1973 Kociba, et al. "Results of a Two Year Chronic Toxicity and Oncogenicity Study of 2.3.7.8- Tetrachlorobibenzo-p-dioxin in Rats". Toxicology and Applied Pharmacology, 46, 279-303. 8/28/74 Article from the Washington Post entitled: "U.S. Scientist Disputes EPA on Toxic Herbicide". 132 4/72 Abstract from the British Industrial Biological Research Association, Information Bulletin entitled: "Peace and Quiet for 2,4-D . . .". 1971 Courtney, et al. "Teratology Studies with 2,4,5-Trichlorophenoxyacetic Acid and 2.3.7.8-Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology, 20, 396-403. 134 1976 Kociba, et al. "2,3,7,8- 6670 Tetrachlorobibenzo-p-dioxin (TCDD): Results of a 13 Week Oral Toxicity Study in Rats". Toxicology and Applied 1o P h a r m a c o l o g y . 35, 553-574. 1977 Allen, et al. "Morphological Changes in Monkey Consuming a Diet Containing Low Levels of 2,3,7,8-Tetrachlorobibenzo-pdioxin". Ed. Cosmet. Toxicol., Vol. 15, pp. 401-410. 136 1974 Norris, et al. "The Toxicity of 2,3,7,8- Tetrachlorobibenzo-p-dioxin (TCDD) in Guppies". Bulletin of Environmental Contamination and Toxicology, Vol. 12, pp. 76-80. 137 Sept.-Oct. Bradley, et al. "Treatment of 1974 Experimental Mammary Adenocarcinoma with Herbicides". Cancer Chemotherapy Reports. Vol. 58, pp. 745-748. 138 1972 Khera, et al. "Pre and Post Natal Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4- Dichlorophenoxyacetic Acid and their Derivatives in Rats". Toxicology and Applied Pharmacology, 22, 14-28. 139 1972 Whitehead, et al. "The Sub-Acute Toxicity of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid to Chicks". Toxicology and Applied Pharmacology, 21, 348-354. 1953 Drill, et al. "Toxicity of 2,4Dichlorophenoxyacetic Acid and 2,4,5Trichlorophenoxyacetic Acid. A Report on Their Acute and Chronic Toxicity in Dogs". AMA Archives of Industrial Hygiene and Occupational Medicine.' Vol. 7, pp. 61-67. 6/ 6/68 Letter: to: Dr. Leng of Dow, from: Donald Lisk of Cornell University's Pesticide Residue Laboratory. Re: their computations on the percent of Kuron excreted as Silvex in his cow feeding study. Attached is an article entitled: "Fate of Atrazine, Kuron, Silvex and 2,4,5-T in the Dairy Cow". Journal of Diary Science, 47, 1267-1270. 142 1977 Shadoff, et al., of Dow. "The Gas Chromatographic-Mass Spectrometric Determination of 2,3,7,8- Tetrachlorobibenzo-p-dioxin in Fat From Cattle Fed Ronnel Insecticide". Annali Pi Chimica. 67, pp. 583-592. 6671 8/17/79 "Plenty of Fuel for Agent Orange Dispute" JAMA. Vol. 242, pp. 593-597. 00/00/00 5/66 1/18/80 1967 00/00/00 12/19/79 1973 ^ 6/12/79 5/23/78 00/00/00 6/2/78 4/22/80 1978 1974 <5672 5/66 Heikes, Eugene. "Do's and Don'ts for Proper Use of Tordon Herbicides on NonCropland in Colorado" . Jackson, J. B. "Toxicologic Studies of a New Herbicide in Sheep and Cattle" . Am. J. Vet. Res., Vol. 27, oo. 821-824. Deleted order for Esteron 245. Goring, et al., of Dow. "The Effect of Tordon on Micro-Organisms and Soil Bioloaical Processes" Down to Earth, Spring, 1967, pp. 1-4. Henck, et al., of Dow. "Acnegenic Potential of 2,4-Dichlorophenoxyacetic Acid Samples". R & D Report from Dow on Exhibit 148. Yoder, et al. "Lymphocyte Chromosome Analysis of Agricultural Workers During Extensive Occupational Exposure to Pesticides". Mutation Research. 21, 335340. Reuber, Melvin. "Carcinogenicity of 2,4Dichlorophenoxyacetic Acid". Letter to: Anderson, from: Swerczek. Re: Orik, et al., v U.S. and AmChem Swerczek, T. W. "Experiments on the Toxicity of the Herbicide 2,4-D in Horses". Letter from: Anderson of the law firm of Friday, Eldredge and Clark, to: Taynor, Dow attorney. Re: Newton Countv Wildlife Association v. Larrv Hinsen. 2,4-D Fact Sheet. Reuber, M. D. "Carcinogenicity Testing of Chemicals with Particular Reference to Orqanochlorine Pesticides". The Science of the Total Environment, 10, 105-115. Grnow, W. "Metabolism of 2,4,5-T and 2,4D in Rats and Mice". Achieves of Toxicoloav. Vol. 32. d d . 217-225. Original article in German attached. "2,4-D (2,4-Dichlorophenoxyacetic Acid) and Related Compounds". Wildlife, Vol. 1/21/80 6/7/79 1972 2/74 January 10-11, 1978 00/00/00 00/00/00 1/78 11/77 00/00/00 1978 96, pp. 61-69. Letter from: Leng of Dow, to: Garner of the EPA. Re: Revised Interim Amended Registration for various herbicides including Esteron 245, with attached 5/18/80 memo from Leng to John. Leng, Margeurite, of Dow. "Comparative Toxicology of Various Chlorinated Dioxins as Related to Chemical Structure". Presented at the Symposium of Collaborative International Pesticide Advisory Council. Huston, Beverley. "Identification of Three Neutral Contaminants in Production Grade 2,4-D". J. Aar. Food Chem. Vol. 20. pp. 724-727. Gribble, Gorden W. "TCDD: A Deadly Molecule". Chemistrv, Vol. 47, pp. 15-18. Agenda for the World Health Organization Program on the Coordination of Epidemiological Studies on the Long Term Hazards of Chlorinated Dibenzo Dioxins and Chlorinated Dibenzo Furans. Arstila, et al. "An Epidemiological Feasibility Study on the Effects of 2,3,7,8-Tetrachlorobibenzo-para-dioxin (TCDD) on Finnish Forestry Road, and Railway Workers". Publication not indicated. Pocchiari, F. "Accidental TCDD Contamination in Seveso (Italy): Epidemiological Aspects". "Symptomatology, Morbidity and Mortality Experience of Chlorinated Phenoxy Acid Herbicide (2,4-D; 2,4,5-T) Sprayers in Finland. A Clinical and Epidemiological Study" Working Paper for IARC Working Group Meeting. Vos, et al. "TCDD Accident at a Chemical Factory in the Netherlands". Publication not indicated. Abstract. Greig, J. B. "NIEHS Conference on Chlorinated Dibenzo Dioxins and Dibenzo Furans Proposed Presentation". McConnell, et al. "The Comparative Toxicity of Chlorinated Dibenzo-p-dioxins in Mice and Guinea Pigs" Toxicology and Applied Pharmacology, 44, 335-356. January 10-11, 1978 Moore, John A. "Comparative Toxicity of Dibenzo Dioxins and Dibenzo Furans" IRAC Meeting on Dibenzo Dioxins and Dibenzo Furans. 171 00/00/00 Abstracts of articles on the effect of herbicides on animals. 172 1972 Cunningham, et al. "Effect of Tetrachlorobibenzo-p-dioxin on Growth Rate and the Synthesis of Lipids and Proteins in Rats". Bulletin of Environmental Contamination and Toxicology. Vol. 7, pp. 45-51. 173 8/72 Kimbrough, R. "Toxicity of Chlorinated Hydrocarbons and Related Compounds". Arch Environ Health. Vol. 25, pp. 125-131. 174 1978 Tulp, et al. "Rat Metabolism of Polychlorinated Dibenzo-p-dioxins". Chemosphere, No. 9, pp. 761-768. 175 1975 Allen, et al. "Tissue Distribution, Excretion and Biological Effects of Tetrachlorobibenzo-p-dioxin in Rats". E d . Cosmet. Toxicol.. Vol. 13, pp. 501-515. 176 1977 Nelson, et al. "2,3,7,8- Tetrachlorobibenzo-p-dioxin: In Vitro Binding to Rat Liver Microsomes". Bulletin of Environmental Contamination and Toxicology. Vol. 18, pp. 9-13. 177 11/73 Goldstein, et al. "Hepatic Porphyria Induced by 2,3,7,8-Tetrachlorobibenzo-p- dioxin in the Mouse". Research Communication in Chemical Pathology and Pharmacology, Vol 6, pp. 919-928. 178 3/1/77 Federation Proceedings Abstracts of the 61st Annual Meeting of the Federation of American Societies for Experimental Biology. 179 1976 Van Miller, et al. "Tissue Distribution and Excretion of Tritiated Tetrachlorobibenzo-p-dioxin in Non-Human Primates and Rats". Ed. Cosmet. Toxicol. Vol. 14, pp. 31-34. 1979 r*jf>yy/ Barsotti, et al. "Hormonal Alterations in Female Rhesus Monkeys Fed a Diet Containing 2,3,7,8-Tetrachlorobibenzo-p- 1978 00/00/00 1977 00/00/00 2/72 1/72 6/14/67 dioxin". Bulletin of Environmental Contamination and Toxicoloav, Vol. 21, d d . not indicated. Wassom, et al. "A Review of the Genetic Toxicology of Chlorinated Dibenzo-pdioxins" Mutation Research, 47, d d . 141160. Pitot, et al. "Promotion by 2,3,7,8Tetrachlorobibenzo-p-dioxin of Hepatocarinogenesis From Diethylnitrosamine". McArdle Laboratory for Cancer Research Digiovanni, et al. "Tumor-Initiating Ability of 2,3,7,8-Tetrachlorobibenzo-pdioxin (TCDD) in Arochlor 1254 in the Two Stage System of Mouse Skin Carcinoaenesis". Bulletin of Environmental Contamination and Toxicoloav, Vol. 18, d d . 552-557. Courtney, K. Diane. "Prenatal Effects of Herbicides: Evaluation by the Prenatal Development Index". Publication not indicated. Hussian et al. "Mutegenic effects of TCDD on bacterial systems". Walker et al. "Some effects of 2,4-D and 2,4,5-T on Ehrlich Ascites Tumor Cells In Vivo and In Vitro" Dow summary on study entitled "Residues of tordon acid in tissues from steers fed the herbaside" 1975 Vettorazi, G. "State of the art of the toxicological evaluation carried out by the joint SAO/WHO expert committee on pesticide residues. I. Ogranohalogenated pesticides used in public health and agriculture" 6-8/73 Title page "Summary sections from amendment to PP 8F0670 requesting tolerances for residues of 2,4Dichlorophenoxyacetic Acid (2,4-D)" submitted by Dow. 1980 Letter from Adamson of the Office of Science and Technology policy to: Hummell '' of Dow requesting an article on Esteron S67 6 245. 3/71 Poland, et al. "A health survey of workers in a 2,4-D and 2,4,5-T plant with special attention to Chloracne, porphyria chtanea tarda, and psychologic parameters" 1977 Coutselinis et al. "Concentration levels of 2,4-D and 2,4,5-T and forensic materials" 8/20/79 Dow R & D report on "Reversal of Immunologic and toxichologic effects of a single exposure of 2,4,7,8Tetrachlorobibenzo-p-dioxin in mice" the copy of the article is attached. 00/00/00 Rappe, Christoffer "Chemical aspects of polychlorinated dibenzo dioxins (PCDDs) and polychlorinated dibenzo furans (PCDFs)" Publication not indicated. 9/5-10/77 Reggiani of Hoffman-LaRoche and Co. "Medical problems raised by the PCDD contamination in Seeso, Italy". Presented at the fifth international conference on occupational health in the chemical industry. 5/78 Dost, Frank "Toxicology of Phenoxy herbicides and hazard assessment of their use in reforestation" U.S. of Agriculture Forest Service. 3/29/79 Packee, E.C. "2,4-D in the urban and suburban environment in human health" Presented to the Nanaimo Parks and Recreation Commission. 1973 Karvonen, et al. of Finland "The health hazards of defoliants" 00/00/00 To: illegible; from: Emerson re proported incidents of animal deaths and human illness associated with dioxin contamination. 2/78 Dost, Frank "Toxicology of Phenoxy herbicides and 2,3,7,8 Tetrachlorobibenzop-dioxin" Department of Agricultural Chemistry and Environmental Health Sciences Center at Oregon State University. 10/3/77 Dow R & D report on "Ultra-structural evaluation of liver cells from female rats maintained for two years on diets containing 2,3,7,8-tetrachlorobibenzo-pdioxin (TCDD)" The report is attached. 1/23/68 Letter to Rowe of Dow from Dr. Kligman re experiments on adults receiving small and increasing doses of Tetrachlorobibenzo-pdioxin. 202 8/22/78 Dow R & D report on "2,3,7,8- Tetrachlorobibenzo-p-dioxin: Tissue distribution and excretion following a single oral dose in female guinea pig" That study done at Dow is attached. 00/00/00 Dow form "Request for toxocnological studies on 2,3,7,8-Tetrachlorobibenzo-pdioxin" 204 7/11/79 Dow R & D report on "Urinary excretion of radio activity following oral administration of C labeled 2,3,7,8- Tetrachlorobibenzo-p-dioxin (TCDD) to female guinea pigs" That report prepared by Dow is attached. 205 2/27/78 Dow R & D report on "Three Generation reproduction study of rats ingesting 2,3,7,8-Tetrachlorobibenzo-p-dioxin" 206 1977 Rehwolbt, et al. "Investigation into the acute toxicity and some chronic effects of selected herbicides and pesticides on several fresh water fish species" Bulletin of Environmental Contamination and Toxicology. Vol. 18 pgs. 361-365. 4/27/71 Memo to Johnson et al. of Dow from Gehring of Dow re results of the pilot study on the absorption and excretion of 2,4-D and 2,4,5-T by human subjects. 4/27/71 Gehring, et al. of Dow "Results of a pilot study on the absorption and excretion of 2,4-Dichloroophenoxyacetic Acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T) following a single oral dose to human subjects" 209 11/21/73 Dow summary of "Placental transfer of 2,4- D and 2,4,5-T in pregnant rats" 210 00/00/00 Schwetz, et al. of Dow "Placental transfer of 2,4-D and 2,4,5-T in pregnant rats" 211 S77 00/00/00 Summary prepared by O'Hare "The effects of repeated applications of semetrical Tetrachlorobibenzo-p-dioxin on Guinea pig 212 0 214 215 216 217 218 219 220 221 222 223 224 225 0? 227 ears 8/25/71 Dow summary on "Excretion and tissue distribution of 2,3,7,8Tetrachlorobibenzo-p-dioxin in the rat" With the attached report. 6/21/73 Memo to Ling of Dow from Schwetz re letter report of 2,3,7,8-Tetrachlorobibenzo-pdioxin (2,3,7,8-TCDD) in guinea pigs. 5/13/75 Dow summary on "The fate of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) following single and repeated oral doses to the rat" with the attached report. 1973 Dow request for toxichological studies on 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) 9/25/73 -7 Letter to Lynn of Dow from Rose and Ramsey of Dow re "Progress Report: The Accumulation and Elimination Kinetics of 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD)" 7/2/73 Dow's request for pathology on a rat which had been given TCDD 7/2/73 Dow's request for pathology on a rat which had been given TCDD 7/2/73 D o w 's request for pathology on a rat which had been given TCDD 7/5/73 Request for pathology on a rat who had died spontaneously after receiving TCDD 7/5/85 Pathology analysis of TCDD infected rat 7/5/73 Analysis of TCDD infected rat 00/00/00 Dow's work progress sheet on a TCDD kinetic probe study 8/6/71 Medical article abstract on study of young chicken exposed to TCDD 8/6/71 Emmerson, et al. "Results of chick edema bioassay test using young chickens treated orally for twenty-one days with 2,3,7,8- ' Tetrachlorobibenzo-p-dioxin" from Dow. 2/12/73 Bosshardt, Hans "Dioxins-Insidious Poisons in our Environment" Translation from a German article. t ' 10/21/78 Rehder, et al. "Pathological-Embryological 228 229 -232^ X235/ 236 237 239 8/20/79 10/3/77 00/00/00 00/00/00 3/3/79 6/16/78 7/28/78 2/19/71 00/00/00 4/21/70 1/80 00/ 00/00 Investigations in Cases of Abortion Related to the Seveso Accident" Journal of Swiss Medicine; Vol. 108, pgs. 1617-1625. Dow R & D report on "Immunotoxological studies involving 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) in mice and rabbits" This article prepared by Dow is attached. Dow R & D report on "An ultrastructural evaluation of liver cells from female rats maintained for two years on diets containing 2,3,7,8-Tetrachlorobibenzo-pdioxin (PCDD)" With the attached report. Gregory, Arthur of NIOSH "Occupational exposure to herbicides" Publication not indicated. Poland, et al. "2,3,7,8Tetrachlorobibenzo-p-dioxin: Studies on the mechanism of action" McArdle laboratory for cancer research at the University of Wisconsin. Fanelli, et al. "Studies of TCDD levels in animals living in the Seveso area" Publication not indicated. Dow analytical report "Determination of phenoxy herbicides in blood samples from 2,4,5-T plant personnel" Dow analytical report "Determination of phenoxy herbicides in urine and blood samples from 2,4,5-T plant personnel - II" Memo from Gehring of Dow to Gordon, et al. of Dow re protocol for consumption of 2,4D and 2,4,5-T study. Protocol discussed in exhibit 234 Dow document entitled phenoxy sample of record on 2,4,5-Trichlorophenoxyacetic Acid. Zack, et al. "The mortality experience of workers exposed to Tetrachlorobibenzo-p- dioxin in a Trichlorophenol process accident" Journal of Occupational Medicine. Vol. 22, Pgs. 11-14. Williams of Dow "Fate of Tordon herbicides containing Picloram in the ecosystem" 240 1968 Moffat, R.W. "Some factors effecting the disappearance of Tordon in soil" Down to Earth, Voi. 23, Pgs. 6-10. 241 00/00/00 Goring, et al. of Dow "Residue and toxicology research" Biokemia (a Dow publication) Pgs. 2-8. 242 00/00/00 Toth, et al. "Carcinogetic bioassay of the herbiside 2,4,5-Trichlorophenoxyethanol (TCPE) with different 2,3,7,8Tetrachlorobibenzo-p-dioxin (dioxin) content in Swiss mice. Publication illegible. Pgs. 82-93. 243 9/15/79 DiGiovanni, et al. "Effects of pretreatment with 2,3,7,8- Tetrachlorobibenzo-p-dioxin on the capacity of hepatic and extra hepatic mouse tissues to convert procarcinigens to mutengens for salmonella typhimurium auxotroph" Toxicology and Applied Pharmacology 50, 229-239. 244 9/30/79 Peterson, et al. "Reversal of 2,3,7,8- Tetrachlorobibenzo-p-dioxin - induced depression of ouabain biliary excretion by pregnenolone-16-cabonitrile and spirononlactone in isolated perfused rat livers" Toxicology and Applied Pharmacology 50, 407-416. 00/00/00 Abstract of article on study of 121 dioxin exposed workers shows no excess deaths over thirty years. 246 1979 Gasiewicz, et al. "2,3,7,8- Tetrachlorobibenzo-p-dioxin tissue distribution, excretion and effects on clinical chemical parameters in guinea pigs" Toxicology and applied pharmachology 52, 329-339. 00/00/00 Notice of research project on registration of dioxin workers from the National Library of Medicine. 248 6/80 Vecchi, et al. "Effect of acute exposure to 2,3,7,8-Tetrachlorobibenzo-p-dioxin on' humoral antibody production in mice" Chem- Biol. Interactions, 30, 337-342. 249 1980 Seefeld, et al. "Blood clearance tests for detecting 2,3,7,8-Tetrachlorobibenzo-p- dioxin hepatotoxicity in rats and rabbits" Archives of the Environmental 250 2'5T) -252) (Z%2p 253 255 257 258 (P 260 261 6681 1980 1980 1/31/80 6/24/80 00/00/00 1978 00/00/00 1980 1980 1980 1980 1980 1980 Contamination and Toxicology Vol. 9, pgs. 317-327. Vanlogeton, et al. "Role of the endocrine system in the action of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) on the thymus" Toxicology 15 135-144. Magazine article "Health Hazard of Dioxins Still Uncertain" Chemical and Engineering News. Letter to the editor from Crummett of Dow to the editor of Nature Vol. 283. Dow form of medical information for Esteron 245. Document entitled composition listing chemical ingredients. Luster, et al. "Inability of passive antibodies to reverse the effect of dioxin toxcicitv" Chemosnhere No. 1 oos. 29-34. Newton, et al. "Exposure of Forest Herbivores to 2,3,7,8-Tetrachlorobibenzop-dioxin (TCDD) in areas sprayed with 2,4,5-T" Publication not indicated. Dominico, et al. "Accidental release of 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) at Seveso, Italv" Toxicology and Environmental Safetv 4, 321-326. Domenico, et al "accidental release of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) at Seveso, Italv" Eco-Toxicologv and Environmental Safetv Vol. IV, d o . 327-338. A section from the article from Exhibit 257, entitled "Environmental Persistence of TCDD in Soil". Section from Exhibit 257 entitled "TCDD Levels in Atmospheric Particles". Olson, et al. "Tissue Distribution, Excretion and Metabolism of 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) in the' Golden Syrian Hamster". Toxicology and ADDlied Pharmacology 56, 78-85. Knutson, et al. "2,3,7,8Tetrachlorodibenzo-p-dioxin: Failure to demonstrate toxicity in 23 cultured cell types". Toxicology and Applied 262 263 264 265 266 267 268 271 272 273 274 275 276 r 6682 P h a r m a c o l o g y 54, 377-383. 1978 Chemical abstracts, Voi. 89, p. 141. 9/74 1978 1978 1978 1968 1977 4/2/73 00/00/00 6/73 1972 Ilieg. Illeg. 2/75 1977 1979 Chemical abstracts on article regarding TCDD. Abstract on article regarding analysis of rat liver after exposure to TCDD. Abstract of article on study of rats exposed to TCDD. Abstract of article regarding examination of female rat liver after TCDD exposure. Abstract of article regarding exposure of TCDD in rats. Abstract of article regarding electronacceptor properties of chlorinated dibenzo-p-dioxins. Baughman, et al. "An Analytical Method for Detecting Dioxin" presented at the conference on dibenzo-dioxins and dibenzofurons. Illegible article in French on primary carcinoma of the liver in Vietnam with short English summary at the end. Crossland, et al. -- Title of article illegible regarding spraying of chemicals. Illegible graph regarding normal and abnormal fetuses of mice. Illegible medical article. French article on polychlorodibenzo-pdioxins with English summary. Oliver R. M. "Toxic Effects of 2,3,7,8Tetrachlorodibenzo-1,4-dioxin in Laboratory Workers", British Journal of Industrial Medicine, Vol. 32, pp.49-53. Miller, et al. "Increased Incidence of Neoplasms in Rats Exposed to Low Levels of 2,3,7,8-Tetrachlorodibenzo-p-dioxin" Chemosphere No. 10, pp. 625-632. Kociba, et al. "Long-Term Toxicologic Studies of 2,3,7,8-Tetrachlorodibenzo-p- dioxin (TCDD) in Laboratory Animals". New York Academy of Sciences Vol. (not indicated), pp. 397-404. 1979 Norstrom, et al. "Analysis of Some Older Scandinavian Formulations of 2,4dichlorophenoxy acetic acid and 2,4,5Trichlorophenoxy acetic acid for contents of chlorinated dibenzo-p-dioxins and dibenzo-furons. Scandinavian Journal of Work Environment and Health, Vol. V, pp. 375-378. 279 1980 Reggini, "Acute Human Exposure to TCDD in Seveso, Italy", Journal of Toxicology and Environmental Health, Vol., VI, pp. 27-43. 280 3/80 Fanelli, et al. "Presence of 2,3,7,8- Tetrachlorodibenzo-p-dioxin in Wildlife Living Near Seveso, Italy: A Preliminary Study", Bulletin of Environmental Contamination and Toxicology, Vol. 24, pp. (not indicated). 281 1/24/67 McCollister, et al. of Dow "Results of Fertility and Reproduction Studies in Rats Maintained on Diets Containing Tordon Herbicide" 1/24/77 Index and report on information on chloracne. 283 1975 Hilbig, "Studies of the Determination of Toxic Effects of Derivatives of 2,4- Dichloro and 2,4,5-Trichlorophenoxyacetic acid (2,4-D and 2,4,5-T) on Incubated Pheasant, Quail and Hen Eggs". Translation from the original German article with the original article attached. 00/00/00 Article entitled "Twelve Wise Men Give 2,4-D and 2,4,5-T A Clean Bill of Health", Publication not indicated. 285 1973 Neubert, et al. "A Survey of the Embryo Toxic Effects of TCDD in Mammalian Species". Environmental Health Perspectives, Vol. V., pp. 67-79. 285 1966 Bjorklund, et al. "Toxicological Studies of Phenoxyacetic Herbicides in Animals". 6683 Acta Vet. Scand. Vol. VII, pp. 364-390. 287 1971 Sparschu, et al. "Study of the Teratogenicity of 2,3,7,8- Tetrachlorodibenzo-p-dioxin in the Rat". 288 290 291 294 3* 297 298 299 300 6684 1973 1974 1974 1971 1975 1975 1976 8/8/73 00/ 00/00 1969 11/72 9/73 9/73 F.D. C o s m e t . T o x i c o l . V o l . 9, pp. 4 0 5 - 4 1 2 . Berndt, et a l ., "In Vitro Uptake of 2,4Dichlorophenoxyacetic Acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic Acid (2,4,5T) by Renal Cortical Tissue of Rabbits and Rats". Toxicology and ADDlied Pharmacology 26, 559-570. German medical article on 2,4,5-T and 2,4D. Fedorova, et al. "Inclusion of 2,4Dichlorophenoxyacetic Acid in Organs of Animals, Paths and Dynamics of Its Excretion" Gioiena I Sanitariia 39, d d . 105-107. Schwetz, et al. "The Effect of 2,4Dichlorophenoxyacetic Acid (2,4-D) and Esters of 2,4-D on Rat Embryonal, Foetal and Neonatal Growth and Development" F. D. Cosmet. Toxicol. Vol. 9, pp. 801-817. Delvaux, et al. "Polychlorodibenzo-pdioxins" Toxicology 3, 187-206. Translation of Exhibit 292. Rose, et al. "The Fate of 2,3,7,8Tetrachlorodibenzo-p-dioxin Following Single and Repeated Oral Doses to the Rat", Toxicology and Applied Pharmacology 36, 209-226. Letter to Sehl of Dow from Bosshardt of the Swiss Federal Research Station re: dioxin content of Dow products. Article on dioxin in German by Bosshardt. McCollister, et al. of Dow "Toxicology of Picloram and Safety Evaluation of Tordon Herbicides". Down to Earth, Vol. 25, pp. 5-10. Kerney, et al. "Persistence and Metabolism of Chlorodioxins in Soils". Environmental Science and Technology. Vol. 6, pp. 10171019. Harris, et al. "General Biological Effects of TCDD in Laboratory Animals", Environmental Health Perspectives, p. 101109. Zinkl, et al. "Hematologic and Clinical 301 >'32{p 303 305 -306y* (J07. 309 r3i) 6685 9/73 6/22/65 00/00/00 12/18/69 00/00/00 1966 1963 1966 1966 1971 1971 1975 Chemistry Effects of 2,3,7,8Tetrachlorodibenzo-p-dioxin in Laboratory Animals. Environmental Health Perspectives, pp. 111-118. Johnson, et al. "Chlorinated Dibenzodioxins and Pentachlorophenol", Environmental Health Perspectives, pp. 171-175. Dow report entitled "The Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in 2,4,5-Trichlorophenoxyacetic Acid by GasLiquid Chromatography" Document entitled "Table of Contents". Letter to Leng of Dow from Erne, Associate Prof, of Vet. Science in Stockholm regarding 2,4,5-D and 2,4,5-T Table entitled "Phenoxy Acid Levels in Tissues of Broiler Chickens Fed 2,4-D and 2,4,5-T at 1000 p.p.m. in the Drinking Water for 104 Days". Erne, Kurt "Studies on the Analytical Chemistry and Toxicology of Phenoxy Herbicides". Erne, Kurt "Detection and Determination of Chlorophenoxyacetic Acid Derivatives in Water". Acta Chemica Scandinavica 17, 1663-1676. Erne, Kurt "Determination of Phenoxyacetic Residues in Biological Materials", Acta Vet. Scand. No. 7, 77-96. Erne, Kurt "Distribution and Elimination of Chlorinated Phenoxyacetic Acids in Animals", Acta Vet. Scand. No. 7, pp. 240256. Bjorklund, et al. "Phenoxy-Acid-Induced Renal Changes in the Chicken", Acta Vet. Scand. No. 12, 243-256. Shafik, et al. "A Method for Determination of Low Levels of Exposure to 2,4-D and 2,4,5-T", Intern. J. Environ. Anal. Chem. Vol. 1, pp. 23-33. Almeida, Waldemar "Toxicological Aspects of 2,4,-D and 2,4,5-T Herbicides", publication not indicated. lp,^3 315 319 320 321 322 323 324 2/9/78 Dow R & D report on "Acute Toxicity and Chloracne Potential of Tars from the 2,4-D Plant". 0 0 /0 0 / 0 0 Wassom, et al. "A Review of the Genetic Toxicology of Chlorinated Dibenzo-dioxins" 1978 Hardell, et al. "Malignant Mesenchymal Soft-Tissue Tumors and Exposure to Phenoxy Acids of Chlorophenols", a translation from the original German article. 00/00/00 Document entitled "A Review on Swedish Epidemiologic Studies with Relation to Chlorinated Dibenzo-dioxins" 1/10- 11/78 Moore, John "Comparative Toxicity of Dibenzo-dioxins and Dibenzo-furans", presented at the IARC meeting on dibenzodioxins and dibenzo-furans. 1/78 Poland, Alan "Chlorinated Dibenzo-pdioxins and Dibenzo-furans: Review of Toxicology and Enzyme Induction and Consideration of Mechanism of Toxicity", presented at IARC. 6/72 Report on background information for Picloram. 6/72 A different version of Exhibit 319. 1966 Hardy, J. L. of Dow "Effect of Tordon Herbicides on Aquatic Chain Organisms", Down to Earth, Vol. 22, pp. 11-13. 1967 Hamaker, et al. of Dow "Prediction of the Persistence and Activity of Tordon Herbicide in Soils under Field Conditions", Down to Earth, Vol. (not indicated), pp. 30-36). 1967 Youngson, et al. of Dow "Factors Influencing the Decomposition of Tordon Herbicide in Soils", Down to Earth, Vol. 23, pp. 3-11. 1979 Seefeld, et al. "Effects of 2,3,7,8Tetrachlorodibenzo-p-dioxin on Indocyanine Green Blood Clearance in Rhesus Monkeys" Toxicology. Vol. 14, pp. 263-272. 00/00/00 6686 Berry, et al. "Studies with Chlorinated Dibenzo-p-dioxins, Polybrominated Byphenyls and Polychlorinated Byphenyls in a Two-Stage System of Mouth Skin Tumorigenesis: Potent Anticarcinogenic Effects", publication not indicated. 326 1977 Berry, et a l . "Transplacental Induction of Mixed-Function Oxygenases in Extra-Hepatic Tissues by 2,3,7,8-Tetrachlorodibenzo-p- dioxin", Biochemical Pharmacology, Vol. 26, pp. 1383-1388. 5/5/76 "Possible Toxic Effects of TCDD in Humans Listed by Dioxin Group", Pesticide Chemical News, pp. 20-23. 328 1970 Abstract on articles regarding toxic effect of some herbicides on fish and aquatic invertebrates from Chemical Abstracts, Vol. 72, p. 240. Attached is a Dow memo dated 4/23/80, regarding Esteron 2,4,5 labels. 329 00/ 00/00 Handwritten notes on Esteron 2,4,5. 330 1965 Goring, et al. of Dow "Tordon Herbicide . . . Disappearance from Soils" Down to Earth, Vol. 20. Magazine also contains other articles on Tordon. 2/18/80 Letter from Garner of the EPA to Leng of Dow regarding revised 2,4,5-T registration. 332 2/20/80 Article from Toxic Materials News entitled "California Lawsuits Challenge State Pesticide Rules" 8/31/50 Internal Dow memo regarding toxicity of 2,4,-D stating that 2,4,-D is moderately toxic. 4/10/52 Internal Dow memo on "Results of Range Finding Toxicological Tests on 2,4Dichlorophenoxyacetic Acid Prepared by a New Method". The document discusses hazards and proper handling procedures for 2,4-D. 4/10/72 Internal Dow memo on "Results of Skin Irritation Tests on Current Production 2,4-Dichlorophenoxyacetic Acid as Compared with Past Production Material". It discusses the difference in cases of skin' dermatitis between the old production method and the new production method. 2/5/60 6687 Internal Dow memo "Results of Range Finding Toxicological Tests on 2,4Dichlorophenoxyacetic Acid Acidified with Chemically Pure HC1 and with. HC1 Recovered from the Pentachlorophenol Plant 337 00/00/00 Drill, Victor "Acute and Subacute Oral Toxicity of 2,4-Dichlorophenoxyacetic Acid for Dogs" 1/3/80 Memo to all Dow 2,4-T and Silvex distributors from McCoy, Dow District Sales Manager Re Esteron Brush Killer, Esteron 245 and Kuron. Copy of Exhibit 338. 340 1978 Newton, et al., "Exposure of Forced Herbivores to 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) an Area Sprayed with 2,4,5-T" . Bulletin of Environmental Contamination and Toxicology, Vol. 20, pp. 743-750. 341 5/5/80 Document entitled "Produce Discontinuation - Record of Approval for the Product Esteron 245 Herbicide. 342 6/2/80 Letter to Stone of the EPA from Morgan of Dow re Esteron 245 application for amended registration. 343 1/80 Esteron 245 herbicide label. 344 1971 Hansen, et al. "Chronic Toxicity of 2,4- Dichlorophenoxyacetic Acid in Rats and Dogs". Toxicology and Applied Pharmacology, 20, 122-129. 345 1970 Medical abstract on the effect of 2,4-D on pheasants and attached is a Dow chemical biology research document summarizing "The Effect of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Esters of the 2,4-D on Rat Embryonal, Fetal and Neonatal Growth and Development" 346 3/56 Rogoff, et al. "Bacterial Decomposition of 2,4-Dichlorophenoxyacetic Acid" Journal of Bacteriology, Vol. 71, pp. 303-307. 8/29/74 Newspaper article from the Detroit Free Press "Herbicide Killed Missouri Animals"'. 348 1977 Mahle, et al. "Search for the Presence of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in 6688 Bovine Milk". Bulletin of Environmental Contamination Toxicology. Vol. 18, pp. 123-130. 349 9/4-7/78 Bisanti, et al. "Experience of the Accident of Seveso". 6th Conference of the European Teratology Society. 350 1965 Lynn, G. E. "A Review of Toxicological Information on Tordon Herbicides". Down to Earth (pp. and Vol. not indicated). 1/79 Homberger, et al. "The Seveso Accident: Its Nature, Extent and Consequences", publication not indicated. 352 1980 Olson, et al. "Toxicity of 2,3,7,8- Tetrachlorodibenzo-p-dioxin in the Golden Syrian Hamster". Toxicology and Applied Pharmacology. 55, 67-78. 353 1980 Jones, et al. "Dependents of the Porphyrogenic Effect of 2,3,7,8- Tetrachlorodibenzo-p-dioxin upon Inheritants of Aryl Hydrocarbon Hydroxylase Responsiveness". Toxicology and Applied Pharmacology, 53, 42-49. 354 1978 Aitio, et al. "Organ Specific Induction of Drug Metabolizing Enzymes by 2,3,7,8- Tetrachlorodibenzo-p-dioxin in the Rat". Toxicology and Applied Pharmacology, 44, 107-114. 1/80 Shearer, et al. "Literature Reviews of 4 Selected Herbicides: 2,4,-D, Dichlobenal, Diquat and Endothall". Report by the Municipality of Metropolitan Seattle. 356 1977 Shadoff, et al. "The Gastromatagraphic- Mass Spectrometric Determination of 2.3.7.8- Tetrachlorodibenzo-p-dioxin in Fat from Cattle Fed Ronnel Insecticide", Annali Pi Chimica. 67, 583-592. 9/28/77 Kociba, et al. "Results of a Two-Year Toxicity and Oncogenicity Study of 2.3.7.8- Tetrachlorodibenzo-p-dioxin (TCDD) in Rats" produced by Dow Chemical Company. 358 3/15/65 Report to Dr. Gordon of Dow from Olson of Dow re "The Formation of Folliculitis in the Rabbit Ear Following a Single Subcutaneous Injection of Symmetrical Tetrachlorodibenzo-dioxin in benzene. 359 00/00/00 Memo from O'Hare of Dow re: same topic as Exhibit No. 358. 360 6/65 Memo to Gordon of Dow from O'Hare and Olson of Dow re: "The Ineffectiveness of 3 Washing as a Preventative Measure in the Formation of Folliculitis in the Rabbit Ear Following Repeated Applications of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Corn Oil". 361 5/18/70 Sparschu, et al. of Dow "Teratogenic Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat". 00/00/00 Document entitled "Report to Dow Chemical Company on Group (1-6)" submitted by Clover Laboratories of Philadelphia. It appears to be the results of animal laboratory tests. 363 8/30/71 Sparschu, et al. of Dow "A Probe Study of the Acute Oral Toxicity of 2,3,7,8- Tetrachlorodibenzo-p-dioxin in the Canine" 364 3/22/71 Dow report on "Acute Oral Lethality Studies Conducted on 2,3,7,8- Tetrachlorodibenzo-p-dioxin Using Guinea Pigs" 10/25/71 Dow report on "Eye Irritation Study Conducted on: 2,3,7,8Tetrachlorodibenzo-p-dioxin" 366 5/30/74 Kociba, et al. of Dow "2,3,7,8- Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-Week Oral Toxicity Study in Rats" 367 1972 Zitko, V. "Absence of Chlorinated Dibenzo Dioxins and Dibenzo Furans from Aquatic Animals", Bulletin of Environmental and Toxicology, Vol. 7, pp. 105-110. 368 00/00/00 Dow production specification for 2,4,5-T, mostly illegible. 1971 Collins, et al. "Teratogenic Studies with 2,4,5-T and 2,4-D and the Hamster", Bulletin of Environmental Contamination and Toxicology, Vol. 6, pp. 559-567. 370 1973 Bage, et al. "Teratogenic and Embryotoxic Effects of the Herbicides Di- and Trichlorophenoxyacetic acid", Acta Pharmacol, et Toxicol., Vol. 32, pp. 408- 416. 6690 5/80 DiGiovanni, et al. "Time-Dependent Inhibition by 2,3,7,8-Tetrachlorodibenzop-dioxin of Skin Tumorigenesis with Polycyclic Hydrocarbons" Cancer Research, 372 \ r) 73/ 374 377 ''V-., ^378) {%Qo) 381 382 ;3 8 y 384 1973 10/14/80 00/00/00 1/5/73 1/5/73 7/71 4/9/74 00/00/00 4/13/70 4/11/75 3-4/68 00/ 00/00 6691 40, 1580-1587. Poland, et al. "Chlorinated Dibenzo-pdioxins: Potent Inducers of Aminolevulinic Acid Synthetase and Aryl Hydrocarbon Hydroxylase", Molecular Pharmacology. 9, 736-747. Ramsey, et al. "Dose Levels of 2,4-D in Forced Workers" report prepared for Dow. Stevens, et al. "Effects of Plant Growth Regulators and Herbicides on Metabolism of C-Labelled Acetate in Pea Root Tissues", publication not indicated. Dow summary of report entitled "Environmental Impact of Herbicides". Kenaga "Environmental Impact of Herbicides", report prepared by Dow. Author not indicated, "Residual Characteristics of Picloram in Grassland Eco-Systems". Dow requests for toxicological studies on TCDD. Noted on the request is that the sample of TCDD is extremely toxic and chloracengenic. Report entitled "Section II, 2,4-D in the Aquatic Environment" by Halter of the University of Washington. Report to Lynn of Dow from Dunn of Dow re results of chloracne studies conducted on assorted 2,4-D, 2,4,5-T, Silvex samples and their esters, orange and diamonds crop rider amine 4-T-2. Same as Exhibit 380 except with different handwritten markings. Dow report entitled "An Improved Method for the Determination of 2,4Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic acid (2,4,5T) Concentrations in Plasma and Urine" Bulletin from the National Clearing House for Poison Control Centers entitled "Death from Chlorinated phenoxyacetic acids (2,4D; 2,4,5-T; MCPA)" Translation from a Puerto Rican newspaper clipping entitled "Weed Killer 2,4-D is (zos) 387 391 393 394 395 395 397 398 6692 1950? 11/72 00/00/00 10/ 21/68 00/00/00 11/65 1979 1978 00/00/00 8/78 00/00/00 11/76 00/00/00 12/1/75 Non-Toxic to the Human Being Willard, C. J. "Indirect Effects of Herbicides", publication not indicated. Abstract of article entitled "No Herbicide Residues In Meat" from the Health Aspects of Pesticides Abstract Bulletin from the EPA. Drill, Victor "Acute and Subacute Oral Toxicity of 2,4,5-Trichlorophenoxyacetic Acid for Eggs" Leng, Marguerite of Dow, summary of an article entitled "Review on the Metabolism of Phenoxy Compounds in Plants and Animals" Kenaga, Eugene of Dow, "Acute and Chronic Toxicity of 75 Pesticides to Various Animal Species" Kay, et al. "Subacute Dermal Toxicity of 2,4-D", Archives of Environmental Health, Vol. 11, pp. 648-651. Medical article with both author and title illegible. Gehring, et al. "Phenoxy Acids: Effects and Fate in Mammals", Ecol. Bull. 27, 122133. Abstracts of medical articles on pesticides. Hilbig, V. "Studies to Determine the Toxic Effects of Derivatives of the 2,4-Dichlor and 2,4,5-Trichlorphenoxyacetic Acids (2,4-D and 2,4,5-T) on Eggs for Incubation of Pheasants, Quail and Chickens", translated from the original German. Abstracts of medical articles. Koschier, et al. "Studies on the Mechanism of Renal Transport Depression Caused by Pretreatment with 2,4,5Trichlorophenoxyacetate", Journal of Toxicology and Environmental Health, Vol. 2, pp. 323-334. Abstracts of medical articles. One-page section from Toxic Materials News. 399 00/00/00 Abstract of medical articles. 400 10/72 Abstracts from the news letter of the Environmental Mutagen Society. 401 00/00/00 Abstracts from the Information Bulletin of the British Industrial Biological Research Association. 402 1970 Abstract from an article in Bioscience. 403 3/9/70 Abstract from the Health Aspects of Pesticide Abstract Bulletin. 404 4/52 Lehman, Arnold J. "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments" from The Association of Food and Druo Officials of the United States, Vol. 16, pp. 47-53. 405 10/51 Lehman, Arnold J. "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments -- Part II, Pesticides" from The Association of Food and Drua Officials of the United States, Vol. 15, pp. 122-129. 406 8/30/50 Report to Britton of Dow from Drill of Wayne University re: Interim Report on the Acute and Subacute Oral Toxicity of 2,4,5-Trichlorophenoxyacetic Acid for Dogs. 407 12/15/49 Letter to Dr. Drill of Wayne University from Rowe of Dow re: Studies of 2,4-D and 2,4,5-T on Dogs. 408 00/00/00 Schwetz of Dow "The Effect of 2,4- Dichlorophenoxyacetic Acid (2,4-D) and Esters of 2,4-D on Rat Embryonal, Fetal and Neonatal Growth and Development" 409 00/00/00 Abstract of reports. 410 00/00/00 Abstract of reports. 411 7/71 Summary of article entitled "Residual Characteristics of Picloram and Grassland Eco-Systems" 412 various Dow summaries of research reports on herbicides and their toxicity. 6693413 4/14/66 Alexander, et al. of Dow "Acute Fish Toxicity of Daxtron, Tordon, and Dursban to Three Species of Fish" 414 00/00/00 Summary of report on Tordon. 415 11/22/65 Alexander, et al. of Dow "Acute Fish Toxicity of Daxtron, Tordon and Dursban to Three Species of Fish" 416 2/10-13/69 McCollister of Dow "Toxicity and Safety Evaluation of 4-Amino-3,4,6Trichloropicolinic Acid (Picloram)" presented to the Reed Science of America Meeting. 417 5/9/67 Summary of report entitled "The Development of Certain Tordon Formulations in Argentina and Chile", also attached are other summaries of articles concerning herbicides. 418 8/24/65 Summary of article entitled "A Game Bird Toxicology Study -- Acute Dietary Feeding of Tordon in Bobwhite Quail", attached is the actual article. 419 12/7/62 Report from Dow entitled "Results of Food Acceptance Tests for Rodent Repellency on Certain Pyridine and Iodonium Derivatives and Pentachlorophenol" 420 1/16/63 Dow report entitled "Results of Range Finding Toxicological Tests on 4-Amino- 3.4.6- Trichloropicolinic Acid (Tordon -- trade name). 421 11/15/62 Dow report entitled "Results of 90-Day Dietary Feeding Studies of 4-Amino-3,5,6- Trichloropicolinic Acid in Rats". 422 6/1/61 Dow report entitled "Results of Range Finding Toxicological Tests on 4-Amino- 3.5.6- Trichloro Picolinic Acid". 423 1/74 Report entitled "Picloram: The Effects of Its Use As A Herbicide on Environmental Quality", from the Associate Committee on Scientific Criteria for Environmental Quality. 424 00/00/00 Dow R and D report on an article entitled "The Cytogenetic Effects of Picloram on the Bone Marrow Cells of Rats" and that report by Mensik of Dow is attached. 425 00/00/00 A report on the toxicity of herbicides to fish and other aquatic organisms. Author and title are not listed. 426 427 428 430 431 432 433 434 (J3* & 6695 2/27/69 8/18/64 Illeg. 11/75 10/31/63 1976 1972 8/78 1977 00/00/00 1975 1978 Dow summary on the article entitled "The Toxicity of 2,4-D, 2,4,5-T, Silvex, MCPA, Dalapon, Erbon, TCA, Picloram, DNBP; and Their Derivatives and Formulations to Fish and Other Aquatic Organisms". Dow report entitled "Results of Human Irritation and Sensitization Studies Conducted on Tordon 101 Mixture (5% Aqueus Solution)" by Hilltop Research Institute, Inc. Somers, et al. "The Effect of External Application of Pesticides to the Fertile Egg on Hatching Success and Early Chick Performance 3. Consequences of Combining 2,4,-D with Picloram and Extremes in Contamination", Bulletin of Environmental Contamination and Toxicology, pp. 511-516. Honoroff, Ida article from A Report to the Consumer entitled "Dirty Tricks". Dow report entitled "Results of Human Skin Irritation and Skin Sensitization Tests on M-2431 (A 10% Aqueus Solution of Potassium-4-Amino-3,5,6-Trichloro Picolinate) and Tordon 22K (M-2477)" Pegg, et al. "Effect of 2,3,7,8Tetrachlorodibenzo-p-dioxin on Renal Function in the Rat", Journal of Toxicology and Environmental Health. Vol. 2, pp. 55-65 Heath, et al. "Comparative Dietary Toxicities of Pesticides to Birds", special scientific report (Wildlife No. 152 Bureau of Sport Fisheries and Wildlife, U.S. Department of the Interior, pp. 1-28). Tuchmann-Dupleisis, H. "Pollution of the Environment and Offspring Apropos of the Accident of Seveso". Medecine et Hygiene, Vol. 36, pp. 1758-1766. Tuchmann, H. "Embryological Problems Posed By The Accident At Seveso", Concours Medical. Vol. 44, pp. 6869-6897. Pesticide abstract on article regarding 2,4,5-T. Abstract on article regarding TCDD. Abstract on article regarding TCDD from 438 439 440 441 442 443 444 : 445/ /' V__ - 446 448 449 450 6696 1977 1978 1979 1979 1979 00/00/00 5-5/79 1977 00/00/00 1977 10/77 7/77 00/00/00 Chemical Abstracts. Vol. 88. Mostly illegible abstract on pesticide article. Abstract on article regarding TCDD. Abstract on articles regarding TCDD. Abstract on article regarding TCDD. Abstract on article regarding TCDD in the rat. Poland, et al. "2,3,7,8Tetrachlorodibenzo-p-dioxin: Segregation of Toxicity with the AH Locus", publication not indicated. Manis, et al. "Introduction of Iron Transport by A Potent Inducer of Aryl Hydrocarbon Hydroxylase, 2,3,7,8Tetrachlorodibenzo-p-dioxin", Archives of Environmental Health. Vol. 34, pp. 141145. Author not indicated, "Chloracne Due to Tetrachloro-2,3,7,8-dibenzo-p-dioxin in Children", Annals Dermatologic Venereologic. Vol. 104, pp. 825-829. Fanelli, et al. "Degradation of 2,3,7,8Tetrachlorodibenzo-p-dioxin in Organic Solvents by Gamma Ray Irradiation", publication not indicated. Van Miller, et al. "Increased Incidents of Neoplasms in Rats Exposed to Low Levels of 2,3,7,8-Tetrachlorodibenzo-p-dioxin", publication not legible. Notice of research project on the toxic effects of 2,3,7,8-Tetrachlorodibenzo-pdioxin in Rhesus monkeys. Morita, et al. "Clearance and Tissue Distribution of Polychlorinated Dibenzofurons in Mice", Bulletin of Environmental Contamination and Toxicology, Vol. 18, pp. 61-55. Document entitled Critique of Manuscript No. 5482, "The Comparative Toxicity of Chlorinated Dibenzo-P-Dioxin Isomers in Mice and Guinea Pigs" by E. E. McConnell, et al., attached is a document dated 4/30/77 that contains more of the critique 451 452 453 454 455 456 457 V4S& 460 461 i 6697 00/00/00 2/26/78 00/00/00 10/79 1980 1976 1978 8/73 1974 1973 1/52 of this article. Report reference in Exhibit 450. Report of the consultative Council on Congenital Abnormalities in the Yarram District (Canada). Rappe, et al. "Formation of Polychlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurons (PCDFs) by Burning or Heating Chlorophenates", Chemosphere (no Volume listed). Colon, et al. "Anticarcinogenic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Benzo (a) Pyrene and 7,12-Dimethylbenz(a) Anthracene Tumor Initiation and Its Relationship to DNA Binding", Cancer Research, Vol. 39, pp. 4027-4033. Murakami, et al. "Incorporation of Labelled Pesticides and Environmental Chemicals into Nuclear Fraction of Cultured Human Cells", Bulletin of Environmental Contamination and Toxicology, Vol. 24, pp. 27-30. Woodward, D. F. "Toxicity of the Herbicides Dinoseb and Picloram to Cutthroat (Salmo Clarki) and Lake Trout" J. Fish. Res. Board Can. 33, 1671-1676. Somers et al. "Hatching Success and Early Performance of Chicks from Eggs Sprayed with 2,4-D, 2,4,5-T and Picloram at Various Stages of Embryonic Development", Bulletin of Environmental Contamination and Toxicology. Vol. 20, pp. 289-293. Lichtenstein, et al. "Synergism of Insecticides by Herbicides", Science, Vol. 181, pp. 847-849. Pilinskaya, M. A. "Cytogenetic Effect of the Herbicide 2,4-D on Human and Animal Chromosomes", translation from a Russian journal. Whitehead, C. C. "Growth Depression of Broilers Fed on Low Levels of 2,4Dichlorophenoxyacetic Acid", Br. Poult. Sci.. Vol. 14, pp. 425-247. Lehman "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments, Part II, 462 463 f4> 465 466 467 468 469 TCP 471 472 473 6698 10/52 1973 5/1/80 19 73 ;75 1/8/80 2/8/80 1/80 1/80 1973 1979 1979 5/31/79 Pesticide Section II, Dermal Toxicity", Association of Food and Drua Officials of the United States. Vol. 16, d o . 3-9. Lehman, Arnold J. "Chemicals in Foods: A Report to the Association of Food and Drug Officials on Current Developments, Part II, Pesticide Section V, Pathology", Association of Food and Drua Officials of the United States, Vol. 16, d p . 126-132. Flick, et al. "Studies of the Chick Edema Disease: Toxicity of Chick Edema Factors in the Chick, Chick Embryo, and Monkey", Poultrv Science, Vol. 52, d d . 1637-1641. Report entitled "2,4-Dichlorophenoxyacetic acid (2,4-D) Evaluation of the Human Health Hazards", prepared by the Department of Industrial Relations. Abstracts on articles regarding pesticides. Dow material safety data sheet on Esteron 2,4,5. Document regarding material safety data sheet on Esteron 2,4,5. Printout on Esteron 2,4,5. Printout on Esteron 2,4,5. May, G. "Chloracne from the Accidental Production of Tetrachlorodibenzo Dioxin", British Journal of Industrial Medicine, Vol. 30, pp. 276-283. Kociba, et al. "Toxicologic Studies of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Rats", Toxicoloav and Occupational Medicine, d d . 281-287. Murray, et al.."Three Generation Reproduction Study of Rats Given 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) in the Diet", Toxicoloav and Applied Pharmacoloav, Vol. 50, d d . 241-252. Sharma, et al. "Effects of 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) on Splenic Lymphocyte Transformation in Mice after Single and Repeated Exposures", Annals of the New York Academv of Sciences, Vol. 320, pp. 487-497. 4/13/70 Document entitled "The Analysis of 2,4-D Acid for 2,3,7,8-Tetrachlorodibenzo-pdioxin Content", publication not indicated. 1977 Tung, et al. "The Problem of Mutagen Effects on the Second Generation after Exposure to Herbicides", from a Vietnamese publication. 8/80 Cook, et al. "Mortality Experience of Employees Exposed to 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD)", Journal of Occupational Medicine, Voi. 22, pp. 530-532. 477 5/79 Woodward, D. F. "Assessing the Hazard of Picloram to Cutthroat Trout", Journal of Range Management, Voi. 32, pp. 230-232. Also attached are news articles on the ban on spraying herbicides and a 1/31/80 telegram from Dow regarding the 2,4,5-T Esteron trademark. 478 1978 Abstract on article regarding TCDD. 479 1978 Abstract on article regarding pesticides. 480 1978 Abstract on article regarding TCDD. 481 1978 Abstract on article chicken egg sprayed with herbicides. 482 1978 Abstract of article regarding TCDD. 483 1978 Abstract of article regarding TCDD. 484 1977 Abstract of article regarding dioxin, 485 1977 Abstract of article on TCDD exposure in primates. 486 1977 Abstract of article on renal effects of TCDD. 487 1975 Abstract of article on effect of TCDD on pregnant rats. 488 1977 Zetterberg, et al. "The Influence of PH on the Effects of 2,4-D on Saccharomyces Cerevisiae and Salmonella Typhimurium", Mutation Research, 42, pp. 3-18. 6/77 Article from the Washington Post entitled "Deadly Poison -- Dioxin -- Is Showing up in Mother's Milk". 390 ^91 416-491 492 (3p> j3 & 6 ? 498 ^y' 5/30/74 5/30/74 Kociba, et al. "2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-Week Oral Toxicity Study in Rats" by Dow Chemical. Dow summary of article referenced in Exhibit 490. 6700 00/00/00 00/00/00 11/8/80 11/7/80 11/7/80 Clegg et. al. "Teratogenicity of pesticides, their Metabolities and Contaminants" publication not indicated. Document entitled "Order and Notice" addressed Dear Registrant from Johnson, Deputy Assistant Administrator for pesticide programs regarding the EPA review of available scientific information on the potential health effects of 2,4-D. Newspaper article from the Milwaukee Sentinel, "More Pesticide Protection Uraed For Migrant Farmworkers In State." Newspaper article from The Daily Tribune of Wisconsin Rapids, "Proposed Pesticide Control Rules Debated Again." Newspaper article from The Milwaukee Sentinel, "Cost Of Pesticide Rule Chancres Feared." 11/11/80 & 11/13/80 Newspaper articles from The Milwaukee Sentinel entitled, "Rules on Pesticide Use Disputed" and "Pesticide Testimony Clashes." 11/12/80 Newspaper article from The Milwaukee Sentinel, "Green Bav Official Sees Problem with Pesticide Proposal." 09/8/80 Transmittal sheet indicating receipt of information from the EPA. 00/00/00 Document entitled "Chapter Six, Comparison of the Health Effects of Aquatic Herbicides." Also attached is Milwaukee ' Sentinel article 11/7/80 "Pesticide Use is Still Hot Issue." 00/00/00 Document entitled "Chapter 2, Health Effects of 2,4-D (2,4Dichlorophenoxyacetic Acid) and its derivatives" Publication not indicated. 1980 1980 A variety of newspaper articles regarding Dioxins found in 2,4-D in Canada. Various newspaper articles including one entitled massive rise in cancer deaths predicted. 10/27/80 Transcript from CBS radio show regarding dioxin levels in 2,4-D and 2,4,5-T. 1980 Canadian newspaper articles regarding dioxins being found in 2,4-D. 07/04/80 Document entitled, "Appendix A, list of your companies products containing some form of 2,4-D". 00/00/00 Document entitled, Appendix B and Appendix C, it appears to be authored by the EPA. 07/11/80 00/00/00 Document entitled, "Appendix D. List of your company's products containing some form of 2,4-D formulated in a liquid form or emulsifiable concentrate." Document entitled Appendix G all registrants (by company name) with registered products containing 2,4-D. 03/79 Report entitled assessment of human health risk associated with the risk of 2,4-D in forestry management from the Minnesota Department of Health. 1964 Wiltse of Dow "Tordon herbicide as a soil treatment for brush control" Down To Earth Volume 19, pages 3-6. 1971 Goring et. al. of Dow "The Degradation and Movement of Picloram in soil and water" Down To Earth Volume 27 paqes 12 throuqh 15. 4/22/80 Document entitled "2,4-D Fact Sheet" author not indicated, appears to be Dow. 514 7/14/80 Copy of previous document regarding "List of your Company's products containing some form of 2,4-D." 9/08/80 Transmittal memo at Dow for information received from the EPA regarding 2,4-D study. 02/04/80 Memo to Cook from Bond of Dow re: A Critical Review of: "Case Control Study of Malignant Mesenchymal Soft Tissue Tumors and Exposure to Chemical Substances". 517 01/74 Kimbrough, Renate of the Center for Disease Control "The Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals" CRC Critical reviews in Toxicology pages 445-498. 05/18 March/April 1977 Kimbrough, et. al. "Epidemiology and pathology of a tetrochloral dibenzo dioxin poisoning episode" Archives of Environmental Health, pages 77-85. 519 00/00/00 Dow label for Tordon 10K pellets. 00/00/00 Lamb et. al. "Evaluation of 2,4520 Dichlorophenoxyacetic Acid (2,4-D), 2,4,5- Trichlorophenoxyacetic Acid (2,4,5-T), and 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) toxicity in mice: reproduction and fertility in treated male mice and evaluation of congenital malformations in their offspring", publication not indicated. 1981 Ericksson et. al. "Soft Tissue sarcomas and exposure to chemical substances: a case referent study". British Journal of Industrial Medicine Volume 38, pages not indicated. 522 9/73 Moore et. al. "Postnatal effects of 6702 maternal exposure to 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)" Environmental Health Perspectives pages 81 through 85. /(52 / 52a/ 53 535 1965 Nielsen et. al. "Fatal poisoning in man by 2,4-Dichlorophenoxyacetic Acid (2,4-D) Determination of the agent in forensic materials" Acta Pharmacol, et toxicol. Volume 22, pages 224-234. 1969 Medical abstract on Russian article regarding the health in workers manufacturing 2,4-D. 05/09/70 Berwick, Philip "2,4-Dichlorophenoxyacetic Acid poisoning in man" JAMA Volume 214 pages 1114 through 1117. 1970 Medical abstract of article regarding 2,4D poisoning in man. 00/00/00 Computer information search request entitled 2,4-D. 00/00/00 Computer search entitled Dioxin. Continuation of Exhibit 528. Continuation of Exhibit 528. Continuation of Exhibit 528. 00/00/00 Computer search entitled Dioxin, 2,4-D, 2,4,5-T. Continuation of Exhibit 532. 1970 Paper from the Pesticide Symposia entitled "2,4,5-T in the rat: Excretion Pattern, Serum Levels, Placental Transport, and Metabolism" by K.D. Kourtney. 1962 Desi et. al. "New evidence concerning the nervous site of action of a chemical herbicide causing professional intoxication" ACTA Physiol Volume 22, pages 72-80. 05/77 Taylor e t . al. "Chloracne from manufacture of a new herbicide" ARCH Dermatol 113: 616-619. 1972 Jones, et. al. "A technic for testing acnegenic potency in rabbits, applied to the potent acnegen 2,3,7,8Tetrachlorobibenzo-D-dioxin. The Journal of Investiaative Dermatoloav. Volume not indicated, pages 511-517. 09/73 Schwetz, et. al. "Toxicology of Chlorinated Dibenzo-P-Dioxins" Environmental Health PersDectives, 87-93. Daoe Continuation of Article reference in Exhibit 538. 11/80 Huff, et. al. "Long Term Hazards of Polychlorinated Dibenzodioxin and Polychlorinated Dibenzofurans" Environment Health Persoectives Volume 36, page 221-240. 1974 Greig, et. al. "Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin on stimulated DNA Synthesis in the Liver and Kidney of the Rat." Chem-Biol. Interactions Volume 8 pages 31-39. 04/11/80 1980 Telegram from Charlton of Dow entitled "2,4,5-T Update". Dominico, et. al "Accidental Release of 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) at Seveso, Italy. II TCDD distribution in the soil surface layer" Echotoxicoloav and Environment Safety Volume IV oaoe 298320. 1980 Part 4 of article began in Exhibit 543 entitled "Vertical distribution of TCDD in soil." 05/06/81 Dow customer requests from Midland Analytical Laboratories regarding TCDD in E s teron 245. 03/06/80 Dow R & D report summarizing article entitled "2,4-D formulations containing specific amounts of 2,4,5,7Tetrachloroxanthone: chloracnegenic potential" with the attached article. 1977 Guarino et.al. "Fate in Distribution of the Herbicides Dichloral phenoxicedic acid (2,4-D) and 245 trichloral phenoxiacidic acid (145T) in the dogfish shark" Xenobiotica, Volume 7 pages 623-631. 12/23/80 News release "Dow Study Show No Correlation Between Dioxin Exposure and Pregnancy Results". 1973 Huff, et. al. "Hazardous Contaminants: Chlorinated Dibenzodioxins and Chlorinated Dibenzofurans" Second Annual Conference of Environment Chemicals Human and Animal Health, pactes 175-197. CO/OO/OO Poland, et. al. "An Estimate of The Maximum In Vivo Covalent Binding of 2,3,7,8-Tetrachlorobibenzo-p-dioxinto rat liver protein RNA and DNA" publication not indicated. 8/1976 Poland, et. al. "Stereospecific, High Affinity Binding of 2,3,7,8Tetrachlorobibenzo-p-dioxin by Hepatic Cytosol: Evidence that the binding species is the receptor for the induction of aryl hydrocarbon hydroxylase" publication not indicated. 1978 Viviani, et. al. "Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclia and microsomes by phnobarbital, 3-methylcholanthrene, 2,3,7,8-Tetrachlorobibenzo-p-dioxin, bieldrin and other inducers" Biochemical Pharmacoloav, Volume 27, paqes 2103 through 2108. 1975 Nebert, et. al. "Genetic Expression of 554 555 556 558 560 561 Ayrol hydrocarbon hydroxylase activity in the mouse" J.Cell. physiol. 85, pages 393-414. 1980 Poland, et. al. "2,3,7,8Tetrachlorobibenzo-p-dioxin" segregation of toxicity with the AH locus" Molecular Pharmacology volume 17 pages 86-94. 1978 Gellert, et. al. "Topical Exposure of Neonates to Hexachlorophene: long standing effects on mating behavior and prostatic development in rats" Toxicology and Applied Pharmacology Volume 43 pages 339 through 349. 1977 Yang, et. al. "Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin on plasma disappearance and biliary excretion of foreign compounds in rats" Toxicology and Applied Pharmacology Volume 40 pages 485496. 3/80 Dow summary on reports entitled "Histopathological and Ultrastructural studies of Liver Tissue from TCDD-exposed beach mice" 1979 Abstract entitled "The Dioxin Saga-Monkey Reproduction" 1981 Cantoni, et. al. "Porphyrogenic Effect of Chronic Treatment with 2,3,7,8Tetrachlorobibenzo-p-dioxin in Female Rats. Dose-effect relationship following urinary excretion of porphyrins" Toxicology and Applied Pharmacology. Volume 57, pages 156-163. 00/00/00 1980 Khera, et. al. "Polychlorodibenzo-Pdioxins: Perinatal Effects and the Dominant Lethal Test in Wistar Rats", Publication not indicated. Van Logten, et. al. "Role of The Endocrine system in the action of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) on the thymus" Toxicology Volume 15, pages 135144. 1979 Niensedt, et. al. "Effect of 2,3,7,8Tetrachlorobibenzo-p-dioxin in the Hepatic Metabolism of Testosterone in the Rat." Toxicoloqv Volume 13 paces 233-236. 11/16/68 1978 Higginbotham, et. al. "Chemical and Toxicological Evaluations of isolated and Synthetic Chloro Derivatives of Dibenzo-Pdioxin" Nature, Volume 220, Dates 702-702. McDonnel, et. al "The Toxico Pathology of TCDD" Publication not indicated. 07/09/77 00/00/00 Crow, K.D. "Letter to the Editor - Effects of Dioxin exposure" The Lancet, Volume not indicated, page 82. Working group of the NIEHS and the IARC "Coordination of Epidemiological Studies on the Long Term Hazards of Chlorinated Dibenzodioxin" Publication not indicated. 1978 Report entitled "Report of The Consultative Counsel on Congenital Abnormalities In the Yarram District" presented to both Houses of Parliament in Victoria, Australia. 11/7/78 Letter to Houghton, Minister of Health in Australia from Schroeter, California Attorney re: report of 2,4-D and 2,4,5-T investigation. 11/14/78 Letter to Schroeter of Los Angeles from Houghton of Australia transmitting copies of the Australian report. 08/77 "IARC Monographs on the Evaluation of the Carcinogenic Risks of Chemicals to Man. Some fumigants, the Herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals" from The World Health Organization. 1975 Archer, T. E. "Removal of 2,4Dichlorophenoxyacetic Acid (2,4-D) Formulations from Non-combustible Pesticide Containers" Bulletin of 1980 1973 5/4/81 1973 8/63 1980 00/ 00/00 1972 7/70 7/17/70 Environmental Contamination and Toxicology Volume 13 pages 44-51. Continuation of Article Referenced in Exhibit 570. Abstract of article regarding TCDD and toxicity. Fishbein, Laurence "Mutations and Potential Mutations in the Biosphere. 1. DDT and Its Metabolites, Polychlorinated Biphenyls, Chlorodioxins, Polycyclic Aromatic Hydrocarbons, Haloethers", The Science of the Total Environment, Vol. 4, pp. 305, 340. Letter to the editor entitled TCDD and Hexachlorophene, Chemical and Engineering News, pp. 2 and 58. Schwetz, et al. "Toxicology of Chlorinated Dibenzo-p-dioxins", Advances in Chemistry Series. No. 120, pp. 55-69. Seabury, John H. "Toxicity of 2,4Dichlorophenoxyacetic Acid for Man and Dog", Archives of Environmental Health. Vol. 7, pp. 86-93. Outline and abstract of article. Townsend, J. C. "Survey of Reproductive Events of Wives of Employees Exposed to Chlorinated Dioxins" Lamb, et al. "Evaluation of 2,4Dichlorophenoxyacetic Acid (2,4-D), 2,4,5Trichlorophenoxyacetic Acid (2,4,5-T), and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Toxicity in Mice: Reproduction and Fertility in Treated Male Mice an Evaluation of Congenital Malformations in Their Offspring" Epstein, Samuel S. "Teratological Hazards Due to Phenoxy Herbicides and Dioxin Contaminants", Environmental Science Research. Vol. 2, pp. 708-729. Article from the Ontario Department of Health entitled "Revised Registration Status of 2,4-T in Canada" Dow summary of article entitled "Results of Tolerance Studies of 4-Amino-3,4,6Trichloropicolinic Acid (Picloram) in Pregnant Rats" 582(a) 583 584 585 586 (^88^ 589 590 <3 592 (593' 6709 1981 1/20/71 10/14/80 5/5-7/81 3-4/78 00/00/00 1971 Various 00/00/00 10/14/80 1980 1979 Tondeur, et al. "New Screening Methods for Acetic Toxic Substances Using Negative Chemical Ionization Mass Spectrometry. Tetrachloroterephalate in Human Urines", Environmental Science and Technology. Vol. 15, pp. 216-219. Report entitled "Determination of 2,4Dichlorophenol and 2,4-Dichlorophenoxyacetic Acid in Human Blood by Gas Liquid Chromatography", author not indicated. Langghorst, M. L. of Dow "Monitoring Esteron 245 in Air" Kociba, et al. 'of Dow "A Review of the Toxicity of 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) with a Comparison of Other Chlorinated Dioxin Isomers", presented at a meeting of the Central States Association of Food and Drug Officials. Danon, et al. "Subacute Skeletal Myopathy Induced by 2,4-Dichlorophenoxyacetate in Rats and Guinea Pigs", Muscle and Nerve, Vol. 1, pp. 89-102. Report to the Council on Scientific Affairs of the AMA on "Health Effects of 'Agent Orange' and Dioxin Contaminants", presented by Dr. Dolan. Williams, C. S. of Dow "The Status of 2,4D, 2,4,5-T, Silvex and MCPA Herbicides", Down to Earth. Vol. (not indicated), pp. 12-16. Abstracts of articles on TCDD. Abstract of article on 2,4,5-T, 2,4-D and the fetus. Ramsey, et al. of Dow "Dose Levels of 2,4D in Forced Workers" Bradlaw, et al. "Comparative Induction of Aryl Hydrocarbon Hydroxylese Activity In Vitro by Analogues of Dibenzo-p-dioxin", Ed. Cosmet. Toxicol., Vol. 18, pp. 627635. Greig, J. B. "The Toxicology of 2,3,7,8Tetrachlorodibenzo-p-dioxin and Its Structural Analogues", Ann. Occup. Hva., Vol. 22, pp. 411-420. 00/00/00 10/48 1/62 1977 6/16/80 7/11/80 9-10/66 4/11/80 1971 3-4/80 00/ 00/00 1981 Sauerhoff, et al. "The Fate of 2,4Dichlorophenoxyacetic Acid (2,4-D) Following Oral Administration to Man", from Toxicology (no volume or page numbers listed). Eyzaguirre, et al. "Experimental Myotonia and Repetitive Phenomenon the Veratrinic Effects of 2,4-Dichlorophenoxyacetate (2,4-D) in the Rat", publication not indicated. Defi, et al. "Nervous System Effects of a Chemical Herbicide", Archives of Environmental Health. Vol. 4, pp. 101-108 Leskowski, et al. "The Pharmacology of Experimental Myopathies", Ann. Rev. Pharmacol. Toxicol.. Vol. 17, pp. 387-409. Report by the Hazard Alert System Epidemiological Studies Laboratory of the State of California on "2,4Dichlorophenoxyacetic Acid (2,4-D) Evaluation of the Human Health Hazards". Document entitled "Appendix D - List of Your Company's Products Containing Some Form of 2,4-D Formulated in a Liquid Form or Emulsifiable Concentrate" Khanna, et al. "Metabolism of C-Labelled 2,4-Dichlorophenoxyacetic Acid in Rats", J. Aar. Food Chem., Vol. 14, pp. 500-503. Letters to the editor entitled "Agent Orange and Skin Rash -- A Different Experience" and "Agent Orange and Spontaneous Abortions"., JAMA, Vol. 243, pp. 1422-1423. Abstract translation of an article regarding 2,4-D and health workers from The Health Aspects of Pesticide, Vol. 4, pp. 139-140. De Beer, et al. "Suicidal Poisoning by MCPA and Parathion", Journal of Analytical Toxicology, Vol. 4, pp. 91-98. Lucier, et al. "Effects of Environmental Agents on Male Reproduction" from the Environmental Toxicology Branch of the National Institute of Environmental Services. Abstract from the Chemical Regulation 606 0? (gp (pSP 612 614 615 616/) 6711 1980 10/27/80 10/23/80 00/00/00 10/24/80 00/00/00 11/75 2/1/80 6/69 9/73 1962 1971 Reporter entitled "Dioxins: Second Dow Sarcoma Case Found; Company Suggests Smoking as Factor" Abstract of article entitled "Dioxin: Induction of Xenobiotic Metabolism -- II" Transcript of Radio Noon Show entitled "Dioxin Levels in 2,4-D and 2,4,5-T" News release from Agriculture Canada entitled "2,4-D Tests" Document regarding Wayne Ormerod's press release. Memo from Chevalier, Executive Director of the Canadian Agricultural Chemicals Association re the 2,4,-D issue. Document entitled "Health and Safety Alert -- Beware of the Weed Killer 2,4-T and 2,4,5-TP" Vettorazzi, G. "Toxicological Decisions and Recommendations Resulting from the Safety Assessment of Pesticide Residues in Food", CRC Critical Reviews in Toxicoloav, pp. 125-183. Article in the Chemical Reaulation Reports, "Dioxins: VA Study Finds Trace Contamination in Exposed Veterans Control Group" Innes, et al. "Bioassay of Pesticides and Industrial Chemicals for Tumorigenicity in Mice: A Preliminary Note", J. Nat. Cancer Inst. 42. pp. 1101-1114. Bowes, et al. "The Search for Chlorinated Dibenzofurons and Chlorinated Dibenzodioxins in Wildlife Populations Showing Elevated Levels of Embryonic Death", Environmental Health Perspectives, pp. 191-198. Jones, et al. "A Technic for Testing Acnegenic Potency in Rabbits, Applied to the Potent Acnegen 2,3,7,8Tetrachlorodibenzo-p-dioxin", The Journal of Investiaative Dermatolocrv, Vol. 39. pp. 511-518. Kolberg, et al. "The Herbicide 2,4Dichlorophenoxyacetic Acid I Effects on L Cells". Acta Pharmacol, et Toxicol., Vol. (69> /62V 625 626 627 628 629 630 / 632 633 6712 4/73 1969 1959 12/7/73 5/23/49 4/81 6/2/81 00/00/00 5/29/81 2/80 6/5/80 6/4/79 1/22/80 1/5/81 00/00/00 9/80 29, pp. 81-86. Erne, et al. "Toxicity Studies with Phenoxy Herbicides in Reindeer", translation from Swedish article. Way, J. M. "Toxicity and Hazards to Man, Domestic Animals and Wildlife from some Commonly Used Auxin Herbicides" Residue Reviews, Vol. 26, pp. 37-62. Apffel, C. A. "The Cytostatic Action of Certain Auxins; Preliminary Review", translation from the original French article. Dow R & D report on "Wipe Tests at the Trichlorophenol Plant, 804 Building" Dow Report entitled "Result of Skin Irritation Tests on Various Samples of 2,4-D Taken at Various Stages in the Manufacturing Process". "Worker's Exposure to 2,4-D Studies" from Research News of the Department of Agriculture. Dow quality assurance printout for Esteron 245 Herbicide. Abstract of Article entitled "Survey of Reproductive Events of Wives of Employees Exposed to Chlorinated Dioxins". Dow quality assurance printout on Esteron 245 herbicide. Dow quality assurance printout on Esteron 245 brush and weed killer. Dow quality assurance printout on Esteron 245 brush and weed killer. Dow quality assurance printout on Esteron 245 brush and weed killer. Dow quality assurance information on Esteron 245. EPA pesticide establishment report worksheet on Esteron 245. Esteron 245 herbicide label, Dominico, et al. "Accidental Release of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 634 635 3 637 638 639 640 @> 642 643 644 645 6713 00/00/00 1978 00/00/00 00/00/00 Various 5/3/79 6/29/81 1979 9/79 00/00/00 at Seveso. Italv". Ecotoxicoloav and Environmental Safety, Vol. 4, pp. 283-297. A continuation of the article in Exhibit 633. Continuation of the article referenced in 633. Rappe, et al. "Identification and Quanitification of Polychlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurons (PCDFs) in 2,3,5-Ester Formulation in Herbicide Orange". Chemosphere (no volume or paae numbers indicated) Buser,.et al. "Formation of Polychlorinated Dibenzofurans (PCDFs) from the Pvrolvsis of PCBs", Chemosphere, 1, pp. 109-119. Buser, et al. "Polychlorinated Dibenzofurans (PCDFs) Found in Yusho Oil and in Used Japanese PCB", Chemosphere (Vol. and page numbers not indicated) Buser, et al. "Identification of Polychlorinated Dibenzofuran Isomers in Flv Ash and PCB Pvrolvses", Chemosphere, Vol. and page numbers not indicated. Abstracts from articles on TCDD. Article from Chemical Week entitled "Dioxin Is Found In Clean-up Workers' Blood" Dagani, Ron "Seveso: Five Years Late, Questions Remain", C & EN pp. 18-20. Albro, et al. "A Radio Immuno Assay for Chlorinated Dibenzo-p-dioxins", Toxicolocrv and Applied Pharmacoloav, Vol. 50, pp. 137-146. Lamberton, et al. "The Determination of Polychlorodibenzo-p-dioxins in Pentachlorophenol and Wood Treatment Solutions", Am. Ind. Hva. Assoc. J . , Vol. 40, pp. 816-822. Document entitled "Procedure For Interim Assessment of Risk Associated with the Emission of Tetrachlorinated Dioxins (TCDDs) from Municipal Waste Combustors", author not indicated. 646 647 648 649 650 (fp 652 653 654 655 656 ,o r ssi, 658 659 660 661 n < m. n ( i^: 9/29/77 2/27/88 10/9/81 10/9/81 1974 1974 1976 1973 1976 1976 1974 1974 11leg. 7/74 1976 1975 Dow R & D report on the study "Toxicity of 2,4-Dichlorophenoxyacetic Acids to Daphnids", with the article attached. Dow R & D report on "Three Generation Reproduction Study of Rats Ingesting 2,3/7,8-Tetrachlorodibenzo-p-dioxin" with the attached article by Murray et al. of Dow. Article entitled "2,4-Dichlorophenoxy acetic Acid, Technical" by the International Research and Development Corporation. Article entitled "Determination of Acute Dermal LD-50 in Rabbits" by the International Research and Development Corporation. Abstracts of articles on 2,4-D Arkhipov, et al. "Carcinogenic Properties of Herbicide 2,4-D Amine Salt", translated from the original Russian article. Abstract of article on 2,4-D. Abstract of article on 2,4-D. Abstract of article on 2,4-D. Amma, et al. "Effect of 2,4Dichlorophenoxyacetic Acid on Rabbit Duodenum", publication not indicated. Abstract on article regarding dermatitis after exposure to 2,4-D. Abstract from article on herbicide exposure, mortality and tumor incidents for Swedish railroad workers. Illegible abstract of articles. Abstract of article on 2,4-D and 2,4,5-T. Kostantinova, et al. "Embryotropic Effect of the Products from the Decomposition of Herbicides Based on 2,4-Dichlorophenoxyacetic Acid", translation from the original Russian article. Haag, et al. "The Influence of NonCytotoxic Concentrations of the Herbicide 2,4-Dichlorophenoxyacetic Acid on the DNA 662 663 664 666 667 668 01 1) 673 I 1975 00/00/00 00/00/00 1985 1972 10-11/82 1982 1982 6/26/82 1982 1982 1972 Synthesis in Cultured Vertebrate Cells", Archives of Toxicology, Vol. 33, pp. 91- 102. Winell, Margaret "An International Comparison of Hygienic Standards for Chemicals in the Work Environment", publication not indicated -- article mostly illegible. Computer search for article on pesticides. Computer search for articles on 2,4-D and 2,4,5-T. Literature search for articles on mutagenesis and 2,4-D. Epstein, et al. "Detection of Chemical Mutagens by the Dominant Lethal Assay in the Mouse", Toxicology and Applied Pharmacology. Vol. 23, pp. 288-325. Cushman, et al. "Allergic Hypersensitivity to the Herbicide 2,4-D in Balb/C Mice", Journal of Toxicology and Environmental Health. Vol. 10, pp. 729-741. Corte, et al. "2,4-D Induced Clastogenicity and Elevated Rates of Sister Chromatid Exchanges in Cultured Human Lymphocytes", Journal of Heredity, Vol. 73, pp. 224-226. Taskar> et al. "Measurement of 2,4Dichlorophenoxyacetic Acid (2,4-D) after Occupational Exposure", Bulletin of Environmental Contamination and Toxicology. Vol. 29, pp. 586-591. Milham, Samuel "Herbicides, Occupation and Cancer" The Lancet, Vol. 1, pp. (not indicated). Draper, et al. "Applicator Exposure to 2,4-D, Dicamea and a Dicamba Isoxer", J. Environ. Sci. Health. Vol. 4, pp. 321, 339. Nash, et al. "Applicators Exposure to 2,4D", Pesticide, Residues and Exposure, part of the American Chemical Society Symposium Series. Buslovich, et al. "Distribution of Herbicides -- Phenoxy-acids, Chloroderivatives in the Body of Albino 674 676 677 678 679 680 681 682 683 684 "''" N 685 Rats", translation from the original Russian article. O n 1 C /iD 00/00/00 1975 1975 1984 MarchApril, 1984 1984 00/00/00 1972 1972 5/72 00/00/00 1984 1985 Untitled document, apparently bibliography of various articles. Kohli, J. D., et al. "2874. Elimination of 2,4-D in Man". Food and Cosmetics Toxicology, Vol. 13, 1975. Abstract by Senczuk and Pogorzelka. "The Course of Excretion of 2,4Dichlorophenoxyacetic Acid with Urine". From Rocz Panstw. Zakl. Hiq. 26(2):217222, 1974. Polish. Kaije, Linnainmaa. "Induction of Sister Chromatid Exchanges by Tier Peroxisome Proliferators 2,4-D, MCPA and Clofibrate In Vivo and In Vitro". Carcinogenesis. Vol. 5, No. 6, pp. 703-707, 1984. Abstract of article, J. Dougherty, et al. "Effects of 2,4-D on Schedule Controlled Behavior". Neurobehaviorial Toxicology and Teratology, Vol. 6, pp. 177-179, 1984. Abstract by Toyoshima, et al. "Does 2,4Dichlorophenoxyacetic Acid (2,4-D) Cause Polyneuropathy?". Several basically illegible pages of abstracts. "Fatal Human Ingestions of 2,4-D, A Common Herbicide". No author indicated. Reference Arch. Pathol. 94(3):27-275, 1972. 3 pages of illegible articles. Illegible abstracts from Health Aspects of Pesticides Abstract Bulletin, June, 1972, Vol. 5, N o . 6. Illegible page from Toxicology and Applied Pharmacology. Table of Contents from booklet titled: "Environmental Health Criteria 29, 2,4Dichlorophenoxyacetic Acid (2,4-D)". No author is legible. Frank, R. A. Campbell et al. "Forestry Workers Involved in Aerial Application of 2,4-Dichlorophenoxyacetic Acid, 2,4-D: 687 688 689 690 691 692 693 (i 695 Exposure and Urinary Excretion" . From Archives of Environmental Contamination and Toxicology, Vol. 14, 427-435, 1985. 10/85 Knopp, et al. "Radioimmunoassay for 2,4Dichlorophenoxyacetic Acid". Archives of Toxicology. 1984, Vol. 58, pp. 27-32 5/83 Kenneth G. Torrington, MD. Letters to the editor titled: "Herbicide Exposure and Pulmonary Disease" Journal of Occupational Medicine, May 1983, Vol. 25, No. 5. 00/00/00 Abstract by Linnainmaa and H. Vainio, "SCE's Among Herbicide Sprayers Exposed to 2,4-D and MCPA". Mutation Research, Vol. illegible. 1983 Linnainmaa, K. "Sister Chromatid Exchanges Among Workers Occupationally Exposed to Phenoxy Acid Herbicides 2,4-D and MCPA". Teratoqenesis Carcinogenesis and Mutagenesis, Vol. 3, pp. 269-279, 1983. 1983 Squibb, et al. "Neurobehavioral Assessment of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Rats". Neurobehavioral Toxicology and Teratology, Vol. 5, pp. 331-335, 1983. 1983 Abstract titled: "Toxicologic Studies in a Fatal Overdose of 2,4-D, MCPP and Chlorpyrifos". Author and publication illegible. Abstract illegible. May/June, 1983 Osterloh, et al. "Toxicologic Studies in a Fatal Overdose of 2,4-D, MCPP and Chlorpyrifos". Journal of Analytical Toxicology, Vol. 7, May/June, 1983. ^ 1983 Melvin Dwaine Reuber. "Carcinogenicity and Toxicity of 2,4-Dichlorophenoxyacetic Acid". The Science of the Total Environment. Vol 31, 1983, pp. 203 to 218. 1972 Abstract by Anna Gel-Dyevaag, and Antipova, L. N. "Change in Some Indexes of Protein Metabolism During the Action of Magnesium Chlorate on Uninjured Skin". Zdravookhr. Turkn 1971, together with abstract by Lusoshkina, et al. Titled: "Lipid-Protein-Carbohydrate Metabolism in Workers During 2,4-Dichlorophenoxyacetic Acid Production". Tr. Azerb. Nauch.Issled. Inst. Tr. Profzabol, 1970. Chemical Abstracts, Vol. 77, 1972. 696 697 /698y /'7og/ 702 703 704 705 9/15/83 10/83 1983 1982 1980 ^ 12/19/79 1980 00/00/00 1979 6718 1985 Harrivainio, et al. "Hpyolipidemia and Peroxisome Proliferation Induced by Phenoxy Acetic Acid Herbicides in Rats". Biochemical Pharmacology/ Vol. 32, No. 18, pp. 2775-2779, 1983. P. J. Dierickx. "Interaction of Chlorophenoxyalkyl Acid Herbicides with Rat Liver Glutathione S-transferases". Food and Chemical Toxicology, Vol 21, No. 5, pp. 575-579, 1983. Carol Brian Easley, et al. "Laundering Procedures for Removal of 2,4Dichlorophenoxyacetic Acid Ester and Amine Herbicides from Contaminated Fabrics". Archives of Environmental Contamination and Toxicology, Vol. 12, 71-76, 1983. Terrie L. Lavy, et al. "(2,4Dichlorophenoxy)acetic Acid Received By Aerial Application Crews During Forest Spray Operations". Journal of Agricultural Food Chemistry, 1982, Vol. 30, pp. 375-381. Sencuzk, Watold. Translation by Lillian Heim on November, 1981. "Absorption of 2,4-Dichlorophenoxyacetic Acid Through the Skin". Warsaw Panstwowv Zaklad Higieny. Roczniki., Vol. 31, No. 6, 611-614. R & D Report from Dow Chemical transmitting report titled: "Acnegenic Potential of 2,4-Dichlorophenoxyacetic Acid Samples" by J. W. Henck, K. S. Rao, and J. C. Jersey of Dow Chemical. Sencuzk, Watold. Translation by Lillian Heim on November, 1981. "Absorption of 2,4-Dichlorophenoxyacetic Acid Through the Skin". Warsaw Panstwowv Zaklad Higienv. Roczniki., Vol. 31, No. 6, 611-614. Illegible pages of abstracts. Heikki A. Elo and Pauli Ylitalo. "Distribution of 2-methyl-4chlorophenxoyacteic Acid and 2,4Dichlorophenoxyacetic Acid in Male Rats: Evidence for the Involvement of the Central Nervous System in Their Toxicity". Toxicology and Applied Pharmacology. Vol. 51, pp. 439-446, 1979. E. Toyoshima, et al. "2,4- 1983 00/00/00 1973 10/16/81 1960 1978 1978 1977 1978 1977 and 1978 1978 Dichlorophenoxyacetic Acid (2,4-D) Does Not Cause Polyneuropathy in the Rat" Journal of the Neurological Sciences. 1985, Vol. 70, 225-229. Birgetta Colmodin-Hedman, et al. "Studies on Phenoxy Acid Herbicides Field Study". Archives of Toxicology, 1983, Vol. 54, 256-265. Illegible abstract of "Absorption and Excretion of 2,4-Dichlorophenoxyacetic Acid in Man" by Kohli. Illegible article. R & D Report from Dow Chemical transmitting attached copy of abstract and report titled: "Purified 2,4Dichlorophenoxyacetic Acid (2,4-D): Results of a 13 Week Sub-Chronic Dietary Toxicity Study in the CSF Fischer 344 Rat", by S. J. Gorzinski, et al., of Dow Chemical. Abstract of Report by Schillenger, J. I. "Hygienic Evaluation of Agricultural Production with the Use of Herbicide". J . Hyq. Epidemiol. Microbiol, and Immunology, 1960, Vol. 4, No. 2, 243-252. Untitled document, apparently bibliography. Abstract and article by T. K. Konstantinova, et al. "Data for the Hygienic standardization of 2,4-D Herbicides and Environmental Substances". Giqieana I Sanitariia, No. 11, 9-13 (1978). Abstract of medical article titled: "Genetic Effects of Phenoxy Acids on Microorganisms". Francis J. Koschier, Peggy R. Gerrard, and Suk Ki Hong. "Transport of 2,4Dichlorophenoxyacetate by Rat Renal Cortical Slices". Toxicology and Applied Pharmacology, 45, pp. 883-849. 1978. Illegible abstracts. Kaskevich and Soboleva. "A Case of Acute Poisoning with Herbicide 2,4-D (Late Aftereffects)" Hygiene of Labor and Occupational Diseases. Vol. 10, 1978, Medicine Moscow 26-31. 717 1977 Apparent bibliography. 718 10/77 Erno Dux, et al. "The Possible Cellular Mechanism of 2,4-Dichlorophenoxyacetate Induced Myopathy". FEBS Letters. Vol. 82, No. 2, 10/77, pp. 219-222. 719 1977 Heikki Elo and Pauli Ylitalo. "Substantial Increase in the Level of Chlorophenoloxyacetic Acids in the CNS of Rats as a Results of Severe Intoxication". Letters to the Editor, ACTA Pharmacol. Et Toxicol. 1977, Vol. 41, p. 280-284. 720 1966 Bibliography. 721 1/74 J. D. Somers, E. T. Moran, Jr., and B. S. Reinhart. "Effect of External Application of Pesticides to the Fertile Egg on Hatching Success and Early Chick Performance, 3 Consequences of Combining 2,4-D with Picloram and Extremes in Contamination". Bulletin of Environmental Contamination and Toxicology, Vol. 11, No. 1. January, 1974, pp. 511-516. 722 1974 Abstracts from Chemical Abstracts, Vol. 80, 1974. 11/8/71 Report to Dow chemical Company titled: "Teratogenic Study with 2,4-D Acid in Swiss Webster Mice". By Sandra Haley of Industrial Biotest Laboratories, Inc. 6/81 Dick Bieler. Note under "Tellig It Like it Is" in Agra Chemical A g e , June 1981. 725 7/62 Abstracts from Occupational Diseases. Vol. 37, No. 7, p. 713 and 714. July 1962. 726 3/24/54 Patent specification for anti-viral compounds comprising phenoxy-pyrimidine derivatives and their manufacture, by the Wellcome Foundation. 727 11/20/53 A. J. Loustalot and others. Titled: "2,4-D Effects Phosphorous Metabolism". Science. Vol. 118, No. 3037, pp. 627-628. 1953. 728 4/62 Abstract of article on nervous system Db '1*0k effects from Industrial Hygiene Digest. 729 1961 Abstracts of articles in Occupational Diseases, Vol. 36, No. 3, p. 245. 1959 Dow Chemical Company Journal abstract regarding article on 2,4-D. 731 1979 Walter Nienstedt, et al. "Effect of 2.3.7.8- Tetrachlorobibenzo-p-dioxin on the Hepatic Metabolism of Testosterone in the Rat". Toxicology, Vol. 13, 233-236. 1979 732 1981 K. G. Jones, F. M. Cole and G. D. Sweeney. "The Role of Iron in the Toxicity of 2.3.7.8- Tetrachlorobibenzo-p-dioxin (TCDD)". Toxicology and Applied Pharmacology, 61, pp. 74-88. 1981. 733 1980 Alessandro Di Domenico, et al. "Accidental Release of 2,3,7,8- Tetrachlorobibenzo-p-dioxin (TCDD) at Seveso, Italy". Eco Toxicology and Environmental Safety. 4, pp. 283-297, 1980. 1984 S. M. Puhvel, D. C. Ertl, and C. A. Linberg. "Increased Epidermal Transglutaminase Activity Following 2.3.7.8- Tetrachlorobibenzo-p-dioxin: In Vivo and In Vitro Studies with Mouse Skins". Toxicology and Applied Pharmacology, Vol. 73, pp. 42-47 (1984). 735 1984 Richard P. Tkoshakji, Raymond D. Harbison, and Milton T. Bush. "Studies on the Metabolic Fate of [14c]2,3,7,8- Tetrachlorobibenzo-p-dioxin (TCDD) in the Mouse". Toxicology and Applied Pharmacology, Vol. 73, pp. 69-77 (1984) 736 1984 Mark S. Seefeld, Steven W. Corbett, Richard E. Keesey and Richard E. Peterson. "Characterization of the Wasting Syndrome in Rats Treated with 2,3,7,8- Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology. Vol. 73, pp. 311-322 (1984). 737 1982 J. G. Vos, F. X. R. Van Leeuwen, and P. Be Jong. "Acnegenic Activity of 3- Methylchlolanthrene and Benzo[a]pyrene and a Comparative Study with 2,3,7,8- Tetrachlorobibenzo-p-dioxin in the Rabbit and Hairless Mouse". Toxicology, Vol. 23, 6721 (1982), pp. 187-196. 738 1984 M. R. Lovati, et al. "Increased Plasma and Aortic Triglycerides in Rabbits After Acute Administration of 2,3,7,8- 739 '740 741 744 747 6722 1984 1984 1984 12/9/83 1984 1984 1984 1984 1984 1984 Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology, Vol. 75, pp. 9197 (1984). J. B. Greig, et al. "Incomplete Correlation of 2,3,7,8-Tetrachlorobibenzop-dioxin Hepatotoxicity with AH Phenotype in Mice". Toxicology and Applied Pharmacology, Vol. 74, pp. 17-25 (1984). John F. Gierthy, and Debra Crane. "Reversible inhibition of In Vitro Epithelial Cell Proliferation by 2,3,7,8Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology, No. 74, pp. 9198 (1984). Friedman. "Does Agent Orange Cause Birth Defects". Teratology, Vol. 29, pp. 193221 (1984). From Proceedings of the Second Annual G. O. Broun Senior Symposium titled: "Dioxin: Lessons From the Missouri Experience". Bulletin of Environmental Contamination and Toxicology, Vol. 33, No. 6 (Dec. 1984). Robert L. Powell. "Dioxin in Missouri: 1971-1983". Bulletin of Environmental Contamination and Toxicology. Vol. 33, pp. 648-654. Carl Reko. "The Pscyo-Social Impact of Environmental Disasters". Bulletin of Environmental Contamination and Toxicology. (1984), Vol. 33, pp. 655-661. Karen B. Webb. "The Pilot Missouri Health Effect Study". Bulletin of Environmental Contamination and Toxicology (1984), Vol. 33, pp. 662-672. Allen P. Knutsen. "Immunological Effects of TCDD Exposure in Humans". Bulletin of Environmental Contamination and Toxicology (1984), Vol. 33, pp. 673-781. Irving J. Selikoff. "Lessons for Living in a Chemical World". Bulletin of Environmental Contamination and Toxicology (1984), Vol. 33, pp. 682-695. Alvin L. Young, USAF. "Determination and Measurement of Human Exposure to Dibenzop-dioxins". Bulletin of Environmental Contamination and Toxicology (1984), Vol. 749 750 751 752 753 (S> 755 756 757 760 } 1984 1984 00/00/00 5/17/83 9/12/83 11/82 7/74 00/00/00 10/11/50 4/11/50 3/17/50 6723 33, pp. 702-709. Patricia Moteleone. "Searching for Genetic Effects of Toxic Chemicals: Chromosomal Aberration and Sister Chromatid Exchange". Bulletin of Environmental Contamination and Toxicology (1948), Vol. 33, pp. 710-725. Robert Schreiber. "The Clean-Up Begins". Bulletin of Environmental Contamination and Toxicology (1984), Vol. 33, pp. 726734. Bibliography titled: "References". No article attached. Report by WIL Research Laboratories titled: "A Range Finding Teratology Study in Fischer 344 Rats with 2,4Dichlorophenoxyacetic Acid", by Stanley E. Kopp, et al. Report by Hazelton Laboratories America, Inc. titled: "Sub-Chronic Toxicity Study in Rats 2,4-Dichlorophenoxyacetic Acid 2,4-D", by David G. Seroda, Ph.D., et al. Report from the United States Environmental Protection Agency titled: "2,4-D Fact Sheet". November, 1984. Jerry A. Burton, MD, PhD, et al. "Absorption of Herbicides From the Rat Lung". Archives of Environmental Health, Vol. 29, July, 1974. Abstract from Toxicology and Pharmacology. No exhibit. Report by Biochemical Research Department of Dow Chemical. Subject: Results of Skin Irritation and Skin Sensitization Tests Conducted on Human Subjects with Old and New Preparations of 2,4Dichlorophenoxyacetic Acid. Untitled chart listing ingredients of 2,4,5-T Acid and 2,4-D Acid. Request for Biological Test to Biochemical Research Laboratory for 2,4Dichlorophenoxyacetic Acid Skin Irritation. Probably refers to Exhibit 758. 761 8/31/50 Biochemical Research Dept, of Dow Chemical. Report by V. K. Rowe titled: "Results of Semi-Chronic Oral Toxicity Studies on 2,4-Dichlorophenoxyacetic Acid". 762 6/25/47 Biochemical Research Laboratory of Dow Chemical Co. Report titled: "Range Finding Toxicological Tests on Chickens with 2,4-Dichlorophenoxyacetic Acid by D. R. Mussel", report by V. K. Rowe. 6/12/47 Report by Biochemical Research Laboratory of the Dow Chemical Company. Report by V. K. Rowe, et al. Subject: "The toxicological properties of 2,4Dichlorophenoxyacetic Acid and Its Sodium and Alkanolamine Salts as Revealed by Experiments on Laboratory Animals." 764 00/00/00 Report titled: "Acute Oral Toxicity of 2,4-Dichlorophenoxyacetic Acid to Rats, Mice, Guinea Pigs and Chicks", by V. K. Rowe, et al., of Biochemical Research Laboratory, Dow Chemical Company. 10/19/44 Report by V. K. Rowe of Biochemical Research Laboratory of Dow Chemical. Subject: "Preliminary information relative to the toxicity of 2,4Dichlorophenoxyacetic acid." 766 6/25/47 Report by V. K. Rowe of Dow Chemical. Subject: "Range finding toxicological tests on chickens with 2,4- Dichlorophenoxyacetic acid." 767 1985 T. A. Antonenko, et al. "Certain Aspects of the Mechanism of Embryotropic Effects of 2,4-Dichlorophenoxyacetic Acid". Giqiena I Sanitaria. Vol. 10, pp. 76-77 (1985). 2/ 26/86 R & D Report of Dow Chemical. Abstract with report attached titled: "2,4-D Acid. Acute Toxicologic Properties", by J. M. Wall. 769 00/00/00 Abstract of study-- illegible. 770 1985 Vivanna A. Rivarola, et al. Titled: "DNA and Protein Synthesis Inhibition in 6724 Chinese Hamster Ovary Cells by Dichlorophenoxyacetic Acid". Toxicology Letters, Vol. 29 (1985), pp. 137-144. ^ 2/86 Kirstin Wiklund, and Lars Eric Holn. (0P <$p 774 111 778 ' 779 ^ 4,780/ 781 1/81 6/9/48 3/2/83 5/26/83 9/12/83 1/21/85 12/81 3/29/82 1982 00/00/00 ^ "Soft Tissue Sarcoma Risk in Swedish Agricultural and Forestry Workers". Journal of the Nation Cancer Institute. Feb. 1986, Vol. 76, No. 2, pp. 229-234. S. Libich. "A Study to Assess the Occupational Exposure to 2,4-D Herbicides". Ontario Hvdro Safety Services Department Booklet. Report by illegible name, of Biochemical Research Laboratory for the Dow Chemical Company titled: "Toxicology and Hygiene, 2,4-Dichlorophenoxyacetic Acid". Dean E. Rodwell, of WIL Research Laboratories, Inc. Final report: "A Teratology Study in Fischer 344 Rats with 2,4-Dichlorophenoxyacetic Acid". Memo to technical members of 2,4-D task force, from J. D. Connor, Jr. of McKenna, Connor and Kunyeld, re: transmittal to EPA of report on teratology study in Fischer 344 rats with 2,4Dichlorophenoxyacetic Acid. David G. Seroda, et al., of Hazelton Laboratories America, Inc. Final report: "Sub-Chronic Toxicity Study in Mice 2,4Dichlorophenoxyacetic Acid, 2,4-D, Submitted to Industry Task Force on 2,4-D research data." David G, Seroda and Samuel Machotka. "Addendum to Final Report on Sub-Chronic Toxicity Study in Rats 2,4Dichlorophenoxyacetic Acid, 2,4-D". Sales specification of Dow Chemical Company for Esteron 245 BE Herbicide. Dow Chemical Company Quality Assurance Sales Specifications for Esteron (R) 2,4,5-BE Herbicides. R. J. Kociba, and B. A. Schwetz of Dow Chemical. Titled: "Toxicity of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)". Druo Metabolism Reviews, Vol. 13, No. 3, d p . 387-406 (1982). Nels Mahle, et al. "The Gas Chromatographic-Mass Spectrometric Determination of 2,3,7,8Tetrachlorobibenzo-p-dioxin in Fat from Cattle Fed Ronnel Insecticide". 778822 September- Daniel O. Hryhorczuk, et al. Titled: "A October, Wire Reclamation Incinerator as a Source 1981 of Environmental Contamination with Tetrachlorodibenzo-p-dioxin and tetrachlorodibenzofurans". Archives of Environmental Health, Sent./Oct. 1981, Vol. 36, No. 5, pp. 228-234. 783 12/9/80 R & D Report of Dow Chemical. Abstract of toxicology report titled: "Pharmacokinetics of 2,4- Dichlorophenoxyacetic Acid in Fischer 344 Rats", by F. A. Smith, et al. 784 3/7/84 Document of Dow Chemical Company by Enrice C. Falla. Titled: "Certificaldo". Cover letter is in Spanish, pages following in English. On level of 2,3,7,8- Tetrachlorobibenzo-p-dioxin in Esteron 245-C.; 785 12/9/80 Report by F. A. Smith, et al., of Dow Chemical, titled: "Pharmacokenticis of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Fischer 344 Rats". 786 10/24/80 R. R. Bumb, et al. "Trace Chemistry of Fire: A Source of Chlorinated Dioxins". Science, Vol. 210, 24 October, 1980, pp. 385-390. 787 1982 Kathlene T. Shiverick and Thomas S. Muther. "Effects of 2,3,7,8- Tetrachlorobibenzo-p-dioxin on Serum Concentrations on the Uterotrophic Action of Exoaenous Estrone in Rats". Toxicoloav and Applied Pharmacoloav, Vol. 65, pp. 170-176 (1982). 788 3/4/82 Dow Chemical memo from: Conrad D. Woods. To: E. M. Ross. Re: Esteron 245BE Herbicides. 789 1982 Report by Harri Zainio, et a l . Titled: "Phenoxy Acid Herbicides Caused Peroxisome Proliferation in Chinese Hamsters". Institute of Occupational Health in Helsinki, Finland. 790 1/82 Letter from: Richard F. Mountfort, of U.S. EPA. To: Arthur Schober of Dow. Re: Esteron 245BE. 791 1/12/82 Letter from: M. D. Black, California 6726 State Department of Food and Agriculture. To: R. W. Colby of Dow Chemical. Re: 792 793 794 795 ^79j) * 798 JP 800 801 302 5/21/82 3/17/83 3/7/83 4/29/82 1/80 1982 1973 3/28/71 1981 1985 6/84 8727 CO Esteron Brush Killer and Esteron 245 Herbicide, and sub-registration to Union Carbide. Memo of Dow Chemical to: R. L. Eisher, et al. From: Jim Havel. Re: meeting regarding Esteron 245BE quality and options. Dow Chemical Latin America Report, by Jorge Roldan. Titled: "Esteron 245C Herbicide Validation of Assay" . R & D Report of Dow Chemical by H. C. Alexander, et al. Titled: "Acute Toxicity of 2,4-Dichlorophenoxyacetic Acid to Representative Aquatic Organisms". R & D Report of Dow Chemical Company by D. C. Kaufman. Titled: "Validation of the Method for the Assay of Esteron 245BE". T. L. Lavy, et al. "Field Worker Exposure in Helicopter Spray Pattern of 2,4,5-T", Bulletin of Environmental Contamination and Toxicology, Vol. 24, Issue 1, January 1980, pp. 90-96. M. L. Leng, et al. Review of studies with 2.4.5- Trichlorophenoxyacetic Acid In Humans, Including Applicators Under Field Conditions". Pesticide Residues and Exposure, 1982. Chemical abstract from Toxicology, Vol. 78 (1973) . By "Our Washington Correspondent". Titled: "PSAC Hiccoughs Over 2,4,5-T" Nature. Vol. 321, May 28, 1971, pp. 210211. J. D. Young, et al. "Pharmacokinetics of 2.4.5- TPGBE Ester Applied Dermally to Rats". Journal of Toxicology and Environmental Health. Vol. 8, pp. 401-408 (1981) . Abstract of Teratology Society Annual Meeting, July 7-11, 1985. Kamal, A. Rshid, et al. "Testing of 2.4.5- T Amino Acid Conjugates for Mutagenic Activity in Salmonella Typhimurium Strains". Mutation Research, Vol. 136 (1984), 217-221. 880033 804 805 806 807 808 809 810 811 812 813 814 i 6728 12/82 1982 Abstract and comment from Food and Chemical Toxicoloav, Da. 973, Vol. 20, No. 6, December 1982. Abstract of a chemical report on ingestion of 2,4,5-T by humans. 1982 By David L. Eaton, Biliary Excretion of 2,4,5-Trichlorophenoxyacetic Acid in the rats. Toxicoloav Letters Volume 14, 1982 pages 175 to 181. 09/81 K. Diane Courtney and Marian T. Ebran, "2,4,5-T effects on Cardiac and Serum Lactic Dehydrogenase (LDH and Creatine Kinase (CK) Isozymes I Maternal Enzyme activities and Isozvmes Drofiles" Archives of Environmental Contamination and Toxicoloav Volume 10, oaaes 571-581, 1981. 09/81 1981 K. Diane Courtney and Marian T. Ebran, "2,4,5-T Effects on Cardiac and Serum Lactic Dehydrogenases (LDH) and Creatine Kinase (CK) Isozymes II: Neonatal enzyme activities and isozyme profiles" from Archives of Environmental Contamination and Toxicoloav Volume 10, oaaes 583-595, 1981. Abstract of Article "2,4,5-T in birth defects." 2/81 Sydney L. Beck, "Assessment of Adult Skeletons to detect prenatal exposure to 2,4,5-T of triflurolin in mice" Teratoloav, Volume 23. No. 1. Februarv 1981, pages 33-55. 1971 Chemical Abstracts illegible. 1973 Article Abstract on 2,4-D and 2,4,5-T in bovine albumin. 1973 Illegible abstract. 1971 Illegible abstract. 1972 *7r\ Illegible abstracts from Toxicoloav and Pharmacology. 3/81 Marv Katherine Haves, American Council on Science and Health, The Health Effects of Herbicide 2,4,5-T. 00/00/00 Four page document including chronology of events involving 2,4,5-T and herbicide reduction statistics. 8/31/50 V .A . Drill, Wayne University, Report for Dow Chemical Company, "Interim Report on The Acute and Subacute Oral Toxicity of 2,4-Dichlorophenoxyacetic Acid for Dogs. 1976 Abstract of Russian Article on Embryotoxic effect of herbicides. 9/72 Illegible abstracts from Health Aspects of Pesticides Abstract Bulletin, Volume 5 No. 9. 00/00/00 Unidentified report on odor and taste thresholds. 11/07/59 Norman P. Goldstein, et. al. "Peripheral Neuropathy after Exposure to an Ester of Dichlorophenoxvacetic Acid, Journal of American Medical Association, November 7, 1959 pages 1306 to 1309. 01/31/59 C.A. Apffel, "Cytostatic effects of certain Auxins", translation is attached from La Press Medical, 1959 Volume 67, pages 207-209. 12/09/80 01 & 02/ 1964 R & D report of Dow Chemical Company by F.A. Smith, et. al. "Pharmakinotics of 2,4-Dichlorophenoxyacetic Acid in Fisher 344 Rats. Routing slip and report by Donald E. Clark, et. al. of U.S.D.A. "The Fate of 2,4-Dichlorophenoxyacetic Acid in Sheep." Agricultural and Food Chemistry, Volume 12, No. 1, page 43, Jan/Feb. 1964. 825 1976 Bibliography. 826 03/82 Harry Vainio, et. al. "Phenoxy Acid Herbicides cause Peroxisome Proliferation in Chinese Hamsters" Scandinavian Journal of Work Environment and Health. Volume 8 Number 1, March 1982, pages 70 - 73. 827 04/72 Illegible abstracts from Health Aspects of Pesticides Abstract Bulletin, Volume 5 No. 4 April, 1972. 828 00/00/00 Illegible abstract. 829 1977 John F. Young and Thomas J. Haley, "Pharmacokinetic study of a Patient Intoxicated with 2,4-Dichlorophenoxyacetic Acid and 2 methoxy-36-dichlorobenzoic acid" Clinical Toxicology, Volume 11, number 5, pages 489 to 500, 1977. 830 00/00/00 "Comments on the Improbability of 2,4-D as a Cause of Peripheral Neuropathy", no author. 04/22/80 EPA Fact Sheets on 2,4-D, no author. 01/23/81 Fact sheet on 2,4-D, no author. 10/82 Document titled 2,4-D Fact sheet from U. S. Environmental Protection Agency, no author. 02/85 Table of Contents and Introduction to Report from Japan, U.S. Joint Seminar, "Toxicity of Chlorinated Byphenals Dibenzofurans, Dibenzodioxins and Related Compounds" Environmental Health Perspectives. Volume 59, February 1985. 835 1985 Shu-Tao Shu, et. al, "Discovery and 6730 Epidemiology of PCB Poisoning in Taiwan, a four year follow-up" Environmental Health Perspectives . Volume 59, pages 5 to 10, 1985. 1985 1985 1985 1985 1985 1985 1985 1985 Harukini Urabe and Masakazu Asahi, "Past and Current Dermatological Status of Yusho patients." Environmental Health Perspectives. Volume 59, pages 11-15, 1985. Yau-Chin Lu, "Clinical Findings in Immunological Abnormalities in Yu-Cheng patients" Environmental Health Perspectives. volume 59 pages 17-29, 1985. Yoichi Nakanishi, et. al., "Respiratory Involvement and Immune Status in Yusho patients" Environmental Health Perspectives, Volume 59 pages 31 to 36, 1985. Kimi Hiro Akagi and Makoto Okumura, "Association of Blood Pressure and PCB level in Yusho patients", Environmental Health Perspectives, Volume 59, pages 37 - 39, 1985. Fumio Yamashita and Masao Hayashi, "Fetal PCB Syndrome: Clinical Features Intrauterine Growth Retardation and Possible Alteration in Calcium Metabolism" Environmental Health Perspectives, Volume 59, pages 41 to 45, 1985. Takesumi Yoshimura and Hitomi Hayabuchi, "Relationship Between The Amount of Rice Oil Ingested by Patients with Yusho and Their Subjective Symptoms" Environmental Health Perspectives. Volume 59 pages 4751, 1985. Yoshito Masuda, et. al., "PCB and PCDF Congeners in the Blood and Tissue of Yusho and Yu-Chang Patients" Environmental Health Perspectives Volume 59, pages 5358, 1985. Paul H. Chen, et. al., "Polychlorinated Biphenyls Dibenzofurans and Quaterphenyls in Toxic Rice-Bran Oil and in the Blood 844 845 846 847 848 849 850 1985 1985 1985 1985 1985 1985 1985 and Tissue of Patients with PCB poisoning (Yu-Chang) in Taiwan" Environmental Health Perspectives. Volume 59, pages 59-65, 1985. Hideaki Miyapa, et. al. "PCB's, PCQ's and PCBf's In Tissues of Yusho and Yu-Chang Patients from Environmental Health Perspectives. Volume 59, pages 67-72, 1985. Takashi Kashimoto, et. al., PCB's, PCQ's PCDf's in Blood of Yusho and Yu-Chang patients" Environmental Health Perspectives. Volume 59, pages 73-78, 1985. Nobuharu Kunita, e t . al., Biological Effect of PCB's, PCQ's PCDF's Present in the Oil Causing Yusho and Yu-Chang" Environmental Health Perspectives, Volume 59, pages 79 to 84, 1985. Achiro Hara, "Health Status and PCB's in Blood of Workers Exposed to PCB's and of Their Children" Environmental Health Perspectives, Volume 59, pages 85-90, 1985. Makoto Takamatsu, et. al. "Surveys of Workers Occupationally Exposed to PCB's and of Yusho Patients", Environmental Health Perspectives. Volume 59, pages 9197, 1985. Renate D. Kinbrough, "Laboratory and Human Studies on Polychlorinated Bifenal, (PCB's) and Related Compounds", Environment Health Perspectives, Volume 59, pages 99-106, 1985. Junya Nagayama, et. al, "Inducing Potency . of Aryl Hydrocarbon Hydroxylase Activity In Human Lymphoblastoid Cells and Mice by Polychlorinated Dibenzofuran Ponegeners", Environmental Health Perspectives, Volume 59, pages 107 to 112, 1985. 74 851 852 853 854 855 856 857 858 1985 1985 1985 1985 1985 1985 1985 6733 1985 Hidetoshi Yoshimura, et. al., "Inductive Effect on Hepatic Enzymes and Toxicity of Congeners of PCB's and PCDF's" Environmental Health Perspectives. Volume 59, pages 113 to 119, 1985. Allison E. N. Vickers, et. al. "Mechanism of Action and Toxic Halogenated Aeromatics" Environmental Health Perspectives, Volume 59, pages 121 to 128, 1985. Takaya Mio and Kimiaki Sumino, "Mechanism of Biosynthesis of Methylsulfones from PCB's and related compounds" Environmental Health Perspectives, Volume 59, pages 129 to 135, 1985. Sano S. Kawanishi and Y. Seki, "Toxicity of Polychlorinated Byphenyl with Special Reference to Porphyrin Metabolism" Environmental Health Perspectives, Volume 59, pages 137 to 143, 1985. Thomas O. Tiernan, et. al, "Sources and Fate of Polychlorinated Dibenzodioxins, Dibenzofurans and Related Compounds in Human Environments" Environmental Health Perspectives, Volume 59 pages 145-158, 1985. Tadaaki Wakimoto and Ryo Tatsukawa, "Polychlorinated Dibenzo-P-dioxins and Dibenzofurans in Fly Ash and Cinders collected from Several Municipal Incinerators in Japan", from Environmental Health Perspectives, Volume 59, pages 159 to 162, 1985. Warren'T. Piver and F. Thomas Lindstron, "Waste Disposal Technology for Polychlorinated Biphenyls" Environmental Health Perspectives, Volume 59, pages 163 to 177, 1985. Norton Nelson, "Perspectives On Polyhalogenated Aeromatic Compounds", Environmental Health Perspectives, Volume 59 pages 179 to 181, 1985. 1977 Abstract of Article from Chemistry Biological on Metabolism of 2,3,7,8-T. 1977 Chemical Abstract 2,3,7,8-T in Monkeys Chemical Abstracts. Volume 88, 1978. 03/01/80 Andrea Poli, et. al., "Increased Total and High Density Lipoprotein Cholesterol with Apoprotein Changes resembling Streptozotocin Diabetes in Tetrachlorodibenzodioxin (TCDD) treated rats", Biochemical Pharmacology, Volume 29, No. 5, March 19, 1980, pages 835 to 838. 05/79 David L. Berry, e t . al. "Studies with Chlorinated Dibenzo-P-dioxin polybrominated biphenyls and polychlorinated biphenyls in a two stage system of mouse skin tumorigenesis: potent anticarcinogenic effects" Annals New York Academy of Sciences, Volume 320, May 1979, pages 405-414. 10/79 David L. Eaton and Curtis E. Claassen, "Effects of 2,3,7,8-Tetrachlorobibenzo-pdioxin, Kepone and polybrominated bifenals on transport system in isolated rat hepatocytes" Toxicology and Applied Pharmacology, Volume 51, 137 to 144, 1979. 10/24/79 Summary of Study titled "Death Among TCDD Exposed Workers Found Not to Exceed National Average", Pesticide and Toxic Chemical News, page 21. 1980 Marcel Bickel and Stefan Muhlebach, "Pharmacokinetics and Echodisposition of Polyhalogenated Hydrocarbons: Aspects and Concepts" Drug and Metabolism Reviews. Volume 11, Number 2, pages 149 to 190, 1980. 1979 Abstract from Chemical Abstracts. Volume 90, No. 867, June 20, 1980, One page report on TCDD from publication, the Bureau of National Affairs, Inc., no author, may be summary or abstracted report. 867 868 869 870 871 872 873 875 6735 1984 06/80 6/80 10/84 1981 1982 1978 1984 1982 R. P. Sharma of Utah State, R. J. Kociba, et al., of Dow Chemical "Immunotoxicologic Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) in rabbits" Jepto Volume 5, pages 321 to 328, 1984. No author, apparent summary of report "TCDD oral and dermal NIC Bioassav Summary. June 1980, no page number. NCI Bioassay Summary. R. J. Kociba and O. Cabey of Dow Chemical. Report titled: "Comparative Toxicity and Biologic Activity of Chlorinated Bibenzop-dioxins and Furans Relative to 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)" for presentation at Fourth International Symposium of Chlorinated Dioxins and Related Compounds. J. M. Henck, et al. "2,3,7,8- Tetrachlorobibenzo-p-dioxin: Acute Oral Toxicity in Hamsters". Toxicology and Applied Pharmacology, Vol. 59, pp. 405-407 (1981) . R. J. Kociba and B. A. Schtwez of Dow. "A Review of the Toxicity of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) With a Comparison to the Toxicity of Other Chlorinated Dioxin Isomers". Association of Food and Drug Officials Quarterly Bulletin, Vol. 46, No. 3, pp. 168-183 (1982) . R. C. Sharma, R. J. Kociba, and P. J. Goering. "Reversal of Immunologic and Toxicologic Effects of a Single Exposure of 2,3,7,8-Tetrachlorobibenzo-p-dioxin in Mice". Trace Substances and Environmental Health. XII (1978). Richard Kociba. "Evaluation of the Carcinogenic and Mutagenic Potential of 2,3,7,8-TCDD and Other Chlorinated Dioxins". From Banbureri Report. No 18, Biological Mechanisms of Dioxin Action, pp. 73-84 (1984). H. Weber, et al. "Acute Oral Toxicity of TCDD-Metabolites in Male Guinea Pigs" Toxicology Letters, Vol. 14 (1982) pp. 117-122. 876 12/82 A. M. Rogers, M. E. Andersen and K. C. Back. "Mutagenicity of 2,3,7,8- Tetrachlorobibenzo-p-dioxin and Perfluoro- n-decanoic Acid in L5178Y Mouse-Lymphoma Cells". Mutation Research, Vol. 105, No. 6, pp. 445-449, December, 1982. 877 1982 Johan Lund, et al. "Cytosolic and Nuclear Binding Proteins for 2,3,7,8- Tetrachlorobibenzo-p-dioxin in the Rat Thymu" Foreign Language Publication, Vol. 716 (1982) pp. 16-23. 878 8/82 Joyce Knutson and Alan Poland. "Response of Murine Epidermis to 2,3,7,8- Tetrachlorobibenzo-p-dioxin Interaction of the Ah and Hr Loci". Cell, Vol. 30, pp. 225-234, August, 1982. 879 1982 Revinder N. Kuri, et al. "Differential Effect:of 2,3,7,8-Tetrachlorobibenzo-p- dioxin;on Nuclear RNA Polymerase Activity in the Rat Liver and Thymus". Publication illegible, Vol. 31, No. 15, pp. 2459-2462 (1982). 880 8/82 Joyce Knutson and Allen Poland. "Response of Murine Epidermis to 2,3,7,8- Tetrachlorobibenzo-p-dioxin Interaction of the Ah and Hr Loci". Cell, Vol. 30, pp. 225-234, August, 1982. 881 8/82 J. C. Ramsey, et al. "The In Vivo Biotransformation of 2,3,7,8- Tetrachlorobibenzo-p-dioxin (TCDD) in the Rat". Toxicology and Applied Pharmacology. Vol. 65, No. 1, August 1982, pp. 180-184. 12/82 W. P. McNulty and K. A. Nielson-Smith. "Letters to the Editor: Persistence of TCDD in Monkey Adipose Tissue". Food and Chemical Toxicology, Vol. 20, No. 6, December, 1982, pp. 985-986, with attached abstracts from The Toxicologist. Vol. 2 (1982). 883 1/82 Report abstract on TCDD from The Toxicologist. Vol. 2. J84 1981 G. Reggiani. "Medical Survey Techniques in the Seveso TCDD Exposure". Journal of Applied Toxicology, Vol. 1, No. 6, 1981, pp. 323-331. 6736 78 885 886 887 888 889 890 891 892 893 I 1984 1985 1985 1985 3/85 3/15/85 1985 4/85 2/85 79 Mortelmans, et al. "Mutagenicity Testing of Agent Orange Components and Related Chemicals". Toxicology and Applied Pharmacology, Vol. 75, pp. 137-146 (1984). K. Hemminki and P. Vineis. "Extrapolation of the Evidence on Teratogencity of Chemicals Between Human and Experimental Animals. Chemicals Other than Drugs". Teratogenesis, Carcinogenesis and Mutagenesis. Vol. 5, pp. 251-318 (1985). N. Isaacson Kerkvliet, et al. "Humoral Imunotoxicity of Polychlorinated Diphenyl Esters, Phenoxyphenols, Dioxins and Furans Present as Contaminants of Technical Grade Pentachlorophenol". Toxiclogy, Vol. 36 (1985) pp. 307-324. Dennis E. Chapman and Carol M. Schiller. "Dose Related Effects of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) in C57BL/6J and DBA/2J Mice". Toxicology and Applied Pharmacology, Vol. 78, pp. 147-157 (1985) . Rosemary Osborn, and William F. Greenlee. "2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) Enhances Terminal Differentiation of Cultured Human Epidermal Cells". Toxicology and Applied Pharmacology, Vol. 77, pp. 434-443 (1985). R. Walden and C. M. Schiller. "Short Communications Comparative Toxicity of 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) in 4(sub) Strains of Adult Male Rats". Toxicology and Applied Pharmacology, Vol. 77, pp. 490-495 (1985). Robert W. Morre, et al. "Androgenic Deficiency in Male Rats Treated with 2,3,7,8-Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology. Vol. 79, pp. 99-111 (1985). L. W. Robertson, et al. "Absence of Lipid Peroxidation as Determined by Ethane Exhalation in Rats Treated with 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)". Archives of Toxicology (1985) Vol. 57, pp. 13-16. Junya Nagayama, et al. "Genetically Mediated Induction of Aryl Hydrocarbon Hydroxylase Activity in Mice of Polychlorinated Dibenzo Furan Isomers in (dsi 'y:y 895 896 897 898 899 901 902 6738 2,3,7,8-Tetrachlorobibenzo-p-dioxin". Archives of Toxicology. Vol. 56, No. 4, February 1985, pp. 226-229. 1985 William F. Greenlee, et al. "Evidence for Direct Action of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) on Thymic Epithelium". Toxicology and Applied Pharmacology, Vol. 79, pp. 112-120 (1985) i. 3/85 William P. McNulty. "Toxicity and Fetotoxicity of TCDD, TCDF, and PCB Isomers in Rhesus Macaques (Macaca Mulatta)". Environmental Health Perspectives, Vol. 60, pp. 77-88, 1985. July 7 and Abstracts of report from annual meeting 8, 1985 Behavioral Teratology Society. 1985 John D. Constible and Maureen C. Hatch. "Reproductive Effects of Herbicide Exposure in Vietnam: Recent Studies by the Vietnamese and Others". Teratogenesis. Carcinogenesis and Mutagenesis, Vol. 5, pp. 231-250. 00/00/00 R. L. Jansing and W. Shain. "Aryl Hydrocarbon Hydroxylase Induction in Adult Rat Hepatocytes In Primary Culture by Several Chlorinated Aromatic Hydrocarbons Including 2,3,7,8-Tetrachlorobibenzo-pdioxin". Fundamental and Applied Toxicology. Vol. 5, 713-720 (1985). 1985 Christopher K. Kelling, et al. "Hypophagia-Induced Weight Loss in Mice, Rats and Guinea Pigs Treated with 2,3,7,8Tetrachlorobibenzo-p-dioxin". Fundamental and Applied Toxicology. Vol. 5, pp. 700712 (1985). 11/15/85 Elvira Spill, translated by Anita Knight and L. P. McCarthy. "The Lingering Insidious Contamination", subtitled "Dioxins Have Left Their Mark on Children From Seveso Nine Year Ago". Original German article is attached. 1985 1985 Michael I. Luster, et al. "Acute Myelotoxic Responses in Mice Exposed to 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD)". Toxicology and Applied Pharmacology. Vol. 81, 156-165 (1985). Victor:J. Wroblewski and James R. Olson. "Hepatic Metabolism of 2,3,7,8- RD 903 OS(9 ' 906 907 908 909 910 911 6/6/85 1982 2/24/82 1982 9/80 12/81 3/85 2/85 1985 81 Tetrachlorobibenzo-p-dioxin (TCDD) in the Rat and Guinea Pig". Toxicology and Applied Pharmacology, Vol. 81, pp. 231-240 (1985) . Carol M. Schiller, et al. "Effect of 2,3,7,8-Tetrachlorobibenzo-p-dioxin (TCDD) and Fasting on Body Weight in Lipid Parameters in Rats". Toxicology and Applied Pharmacology, Vol. 81, pp. 356-361 (1985) G. May. "Tetrachlorodibenzodioxin: A Survey of Subjects Ten Years After Exposure". British Journal of Industrial Medicine (1982) Vol. 39, pp. 128-135. Abstract of study NTPT Tests on TCDD Show Cancer Causing Properties for Animals, from Toxic Materials News, p. 60 (1982). G. Ideo, et al. "Increased Urinary DGlucaric Acid Excretion by Children Living in an Area Polluted with Tetrachlorodibenzoparadioxin (TCDD)". Clinica Chimica Acta. Vol. 120 (1982) pp. 273-283. Francesco Pocchiari. "Forward to Papers 1-6: Accidental Release of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) at Seveso, Italy". Ecto Toxicology and Environmental Safety, Vol. 4, p. 282 (1980) . G. Reggiani. "Medical Survey Techniques in the Seveso TCDD Exposure". Journal of Applied Toxicology. Vol. 1, No. 6 (1981) pp. 323-331. Abstract of report on dioxin from The Toxicologist, Vol. 5, No. 1 (1985), pg. 481. Junya Nagayana, et al. "Genetically Mediated Induction of Aryl Hydrocarbon Hydroxylase Activity in Human Lymphoblastoid Cells by Polychlorinated Dibenzofuran Isomers and 2,3,7,8Tetrachlorobibenzo-p-dioxin" . Archives of Toxicology, Vol. 56, No. 4 (1985) pp. 230235. L. S. Birnbaum, et al. "Toxic Interaction of Specific Polychlorniated Biphenyls and 2,3,7,8-Tetrachlorobibenzo-p-dioxin: Increased Incidence of Cleft Palate in Mice". Toxicology and Applied Pharmacology. Voi. 77, pp. 292-302 (1985). it 1981 Allatair Hay. "Secrecy at Colwaite", supertitled "Dioxin Hazards". Also abstract "Dioxin, A Sarcoma Risk". From Nature, Voi. 290 (April 30, 1981), p. 729. 913 1985 Lori G. Hudson, et al. "Regulation of Epidermal Growth Factor Binding in a Human Keratinocyte Cell Line by 2,3,7,8- Tetrachlorobibenzo-p-dioxin". Toxicology and Applied Pharmacology, Voi. 77, pp. 251-259 (1985). 914 1980 C. Rappe. "Chloroaromatic Compounds Containing Oxygen", subtitled "Phenols, Diphenol Ethers, Dibenzo-p-dioxins and Dibenzo Furans", from Handbook of Environmental Chemistry. Voi. 3 (1980) pp. 157-179. 915 1983 Abstracts and chemical diagrams attached to article by C. Rappe, et al., titled: "Identification of Polychlorinated Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Human Samples Occupational Exposure and You Show Patients". Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, pp. 241-253 (1983). 916 1982 Joyce A. Goldstein, Patricia Linko and Hinda Bergman. "Induction of Porphyria in the Rat by Chronic Versus Acute Exposure to 2,3,7,8-Tetrachlorobibenzo-p-dioxin". Biochemical Pharmacology. Voi. 31, No. 8, pp. 1607-1613 (1982). 917 5/17/78 English summary attached to article by Creso E., et al. "Efftti Neuropsicofarmacologici Della TCDD". From Italian publication, Voi. 56 (1978) pp. 1592-1595. 918 1977 Abstract of studies on 2,3,7,8-T 919 1979 Abstract on dioxin exposed workers. 1981 K. D. Crow. "Chloracne and Its Potential Clinical Implications". Clinical and Experimental Dermatology (1981), Voi. 6, pp. 243-257. 921 1976 Abstracts on studies of TCDD. 922 6 7 4 0 1978 Abstracts on tables attached to article by Philip W. Albro, et al. "Effects of 82 00/00/00 1977 1982 6/28/80 1983 11/4/81 1982 1975 11/16/68 2,3,7,8-Tetrachlorobibenzo-p-dioxin on Lipid Profiles in Tissue of the Fischer Rat". Chemico Biological Interactions. Vol. 23 (1978) pp. 215-3330. Illegible front page, tables attached to report by Louise M. Vail and Rajendra S. Chhabra. "Intestinal Absorption of Nutrients in Rats Treated with 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)". Journal of Toxicoloav and Environmental Health. Vol. 8, pp. 629-638 (1981). Illegible pages attached to report by C. H. Bastomsky. "Enhanced Thyroxine Metabolism in High Uptake Goiters in Rats After a Single Dose of 2,3,7,8Tetrachlorobibenzo-p-dioxin". Endocrinoloav, Vol. 101, No. 1, d o . 292296 (1977). S. M. Puhvel, et al. "Hairless Mice as Models for Chloracne: A Study of Cutaneous Changes Induced by Topical Application Chloracneaens". Toxicoloav and Applied Pharmacoloav, Vol. 64, pp. 492-503 (1982). Abstract of report: "Dioxin and the Heart" from Lancet, H. Poiger and C. H. Schlatter. "Animal Toxicology of Chlorinated Dibenzo-pdioxins". Chemosphere Vol. 12, No. 4/5, pp. 453-462 (1983). R & D Report of Dow Chemical by J. C. Ramsey et al. "The In Vivo Biotransformation of 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD) in the R a t ." Thomas 0. Tiernan, "Chlorodibenzodioxins and Chlorodibenzofurans: An overview", Detoxication of Hazardous Waste paaes 243 to 260, 1982. There are additional tables and chemical diagrams attached to this. Abstract of various Articles. G.R. Higgenbotham, et al., of FDA, "Chemical and Toxicological Evaluations of Isolated and Synthetic Chloro Derivatives of Dibenzo-P-dioxin". Nature, Volume 220, pages 702, 703, 1968. Abstract of Article on Carcinogenicity, French publication, page 1447. 933 1972 Abstract of Article on "Chlorodbenzodioxins in the Liver" Information Bulletin BIBRA Volume II, Number 9, 1972. 934 04/72 Several abstracts of articles mostly illegible, apparently German language publication, Volume 59, pages 173 to 174. 935 1975 Abstracts from Chemical Abstracts, Volume 82, 1975. 936 1973 Abstracts from publication Chemical Abstracts, Volume 79, mostly illegible, number 937, 1973. Unidentified list of references, bibliography. 937 00/00/00 Bibliography of articles. 938 08/20/74 Abstracts of Articles form several different publications. 939 1982 Lorris G. Cockerham and Alvin L. Young, "The Absence of Hepatic Cellular Anomalies In TCDD Exposed Beach Mice - A Field Study", Environmental Toxicology and Chemistry. Volume 1, pages 299 to 308, 1982. 07/82 Abstract of Article and separate article by C.S. Calder and M. Pollack and R. E. Lovelace, "Failure of Dioxin to Cause of Polyneuropathy in Rats", University of Otago Medical School. Volume 60, No. 2, July 1982. 941 1979 Abstracts of articles from Clinical Toxicology. Volume 15, page 478, 1979. 942 1981 Bibliography attached to report by Lavinia Cantoni, et. al. "Induction of Mixed Function Oxidase by chronic treatment with 2,3,7,8-Tetrachlorobibenzo-p-dioxin in female rats". Toxicology, Volume 21, 1981, 6742 pages 159 to 167. 945 946 947 950 1982 Bibliography attached to article by Caramachi, et. al. "Analysis of Exposure to Environmental Contamination by TCDD in individuals effected by dermatological lesions" from L'loiene Moderna, 1982, Volume 77, pages 681 to 706. 09/73 Abstract and bibliography attached to article by Edward J. Burger, Jr. "Summary: Conference on Dibenzo dioxins and dibenzofurans, National Institute of Environmental Health Services", Environmental Health Services, September, 1973, pages 279 to 282. 1983 1982 1981 Abstract and diagrams attached to report by C.E. Blank, Patricia Cooke and A.N. Potter, "Investigations for Genotoxic effects after exposure to crew, 2,4,5Trichlorophenol" British Journal of Industrial Medicine, 1983, Volume 40, pages 87 to 91. Abstract of articles from annual meeting of the American Pediatric Society and Society for Pediatric Research. Abstract and diagrams attached to article by David R. Bickers, M.D. "Treatment of Porphyrias: Mechanisms of Action", Journal of Investigative Dermatology, Volume 77, pages 107 to 113, 1981. 1983 G. G. Bond, et. al. "Medical and Morbidity Surveillance, findings among employees potentially exposed to TCDD", British Journal of Industrial Medicine. 1983, Volume 40 page 318 to 324. 11/16/68 G.R. Higgenbothem, et.al. of the FDA, "Chemical and Toxicological Evaluations of Isolated and Synthetic Chloro derivatives of dibenzo-P-dioxin", Nature, Volume 220, November 1968. page 702 and 703. 1986 6743 Carl L. Potter, et. al. "Relationship of Alterations in Energy Metabolism to Hypophagia in Rats Treated With 2,3,7,8Tetrachlorobibenzo-p-dioxin" Fundamental and Applied Toxicology, Volume 6, page 89 1045 1046 1048 1049 1050 1051 1052 1053 1054 1055 03/19/82 1982 Letter from Thomas Adamczyk from EPA to Arthur Schober of Dow regarding Esteron 245 BE. Several letters between Dow Chemical and EPA regarding Esteron 245 BE. Also attached is certification statement regarding Esteron 245 BE. 00/00/00 EPA form 8570-10, November 1974 titled "Label Technical Data" regarding Esteron 245 BE. 00/00/00 Labels for Dow Esteron 245 and Esteron 245 BE. 00/00/00 F. Safe, et. al. Report titled "PCBB's and related compounds: Metabolism and Biochemistry. 06/83 Incident report on Esteron 245 by Mrs. Gerald Grove regarding her husband taken by Betty Schmidt. 12/81 Dow document titled "Medical Information Regarding Esteron 245BE herbicide, Esteron 245 brush and weed killer and Esteron 245 concentrate brush and weed killer." 08/81 Documents, including a report of telephone inquiries involving Esteron 245, medical information, incident report, data sheet of properties, health hazards and precautions for safe handling of materials and material safety data sheet. 08/27/82 03/82 Emergency response information sheet regarding Esteron 245 brush killer, also attached is Interoffice memo from Dow Chemical referring to attached document, State of Alabama, notice of suspension of sale. Dow specimen label for Esteron 245 BE. 04/08/82 Dow chemical documents, "Production Specifications Regarding Esteron 245 C 1056 1057 1058 1061 1062 1063 1064 1065 .066 1067 Herbicide. 00/00/00 Report by H. Poiger and H. R. Buser, "Structure Elucidation of Manillion TCDD Metabolites". 03/24/82 Letter from Arthur Schober of Dow Chemical to T.E. Adamczyk of the EPA regarding Esteron 245 BE. 03/82 1978 Copy of specimen label for Dow Esteron 245 BE. Lennart Hardell and Anita Sandstrom, translation of an article titled "Malignant Mesenthyne soft tissue tumor when exposed to phenoxy acid or Cholorphenolo" Lakartibningen. Volume 75, number 40, 1978. -I|f 02/15/80 1982 5/24/82 1982 10/16/81 4/82 P'*? A i HI >' 9/23/80 1984 Memo from Marguerite L. Ling, G. L. Costillo, et al., of Dow Chemical, regarding Esteron 245, Esteron Bush Killer and Kuron. Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron R 245BE Herbicide. Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron R 245BE Herbicide". Document titled: "Dow Chemical Company Quality Assurance Production Specification for Esteron R 245BE Herbicide", rough draft. Document titled: "Manufacturing Specifications", apparently a routing slip, Esteron 245BE Herbicide, handwritten notes, mostly illegible. Document titled: "Manufacturing Specifications", cover routing slips, Esteron 245BE Herbicides, handwritten notes. Document titled: "Production Specification Dow Chemical Esteron 245BE Herbicide", rough draft. Report "The Pharmacokinetic Evaluation of 98 1068^ 1069 /fO'70^ /071/ L. ,, ( 1072^ 1073 107.4.^ 1075 (f077 ' - vl.078 , 10795 (1080 : C'-yfin 7/22/85 7/26/85 4-6/52 1948 1983 6/25/85 1953 8/26/50 11/20/50 4/63 10/71 1970 C-2,4-Dichlorophenoxyacetic Acid (2,4-D) in the Mouse", prepared by the Office of Quality Assurance of Hazleton Laboratories and Submitted to the Industry Task Force on 2,4-D research data in Midland, Michigan. Dow R & D report on the article "Molten 2.4- D Acid: Dermal Sensitization Study in Hartley Guinea Pigs" with the report attached. Final report "A Dietary Two-Generation Reproduction in Fischer 344 Rats with 2,4Dichlorophenoxyacetic Acid" prepared for the Industry Task Force on 2,4-D research data. Zimmerman, et al. "Detection and Determination of 2,4-D as a Contaminant by Biological Methods", publication not indicated. French article on 2,4-D, no translation. Reuber, Melvin "Carcinogenicity and Toxicity of 2,4-Dichlorophenoxyacetic Acid", The Science of the Total Environment. Vol. 31, pp. 203-218. Report from Hazelton Laboratories, "Oncogenicity Study in Mice 2,4Dichlorophenoxyacetic Acid 52 Week Progress Report" submitted to the Industry Task Force on 2,4-D research data. Baker, et al. "Suspected Poisoning of Dogs from Eating Grasses Treated with 2,4-D", The North American Veterinarian. Vol. not indicated, p. 194. Audus, L. J. "Biological Detoxication of 2.4- Dichlorophenoxyacetic Acid in Soils: Isolation of an Effective Organism" Nature, Vol. 166, p. 356. French medical article on 2,4-D, no translation. English abstract of article mentioned in . Exhibit 1076. Abstract of article regarding 2,4-D. Abstract of articles on 2,4-D. Abstract of article on 2,4-D. 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 3/14/47 1946 9/19/85 4/2/84 12/13/83 12/18/83 00/00/00 00/00/00 Various 1/69 Various 3-4/68 Various 11/26/80 12/74 Hsueh, et al. "Effects of 2,4-D on Seed Germination and Respiration", Science. Vol. 105, pp. 283-285. Abstract of article on 2,4-D Toxicity and Fish. Dow R & D report on "Effect of Dissolved Oxygen Concentration on the Biodegradation of 2,4-Dichlorophenoxyacetic Acid", this article is attached. Dow R & D report on "Substrate Specificity of a Mixed Microbial Culture Isolated on 2,4-Dichlorophenoxyacetic Acid", report is attached. Dow R & D report on "High Performance Liquid Chromatography Determination of 2,4-Dichlorophenoxyacetic Acid in Waste Treatment Effluents", report is attached. Dow R & D report on "Effect of Bioaugmentation on the Removal of 2,4Dichlorophenoxyacetic Acid in Waste Water Treatment" with attached report. Illegible document. Illegible document. Abstracts of different articles, some are illegible. Fullerton, Pamela "Toxic Chemicals and Peripheral Neuropathy", publication illegible. Abstract of articles on herbicides. "Effect of 2,4-D on Incorporation of C into Urinary Metabolites from Rats Receivinq C-Labelled Acetate", J. Aar. Food Chem., Vol. 16. d d . 358-360. Abstracts of articles on 2,4-D. "National Park Service Urged By AFBF, NFPA, NCA to Discontinue 2,4-D Use", Pesticide and Toxic Chemical News, d o . 19. 18- Schoemaker, et al. "Direct Injection of Experimental Mammary Adenocarcinoma with Picloram", The American Journal of the Medical Sciences, Vol. 268, pp. 333-335. 1096 1097 1098 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 'y 4 `i LJ 00/00/00 6/29/67 00/00/00 9/26/77 1969 Various 9/70 2/72 2/4/80 1983 7/78 1976 11/16/82 4/30/82 Reuber, Melvin "Carcinogenicity of Picloram", publication not indicated. Dow summary on "Residues of Tordon Acid in Milk from Cows Fed the Herbicide", the report is not attached. Dow research report "Results of Additional Acute Oral Tests and Skin Absorption Studies on 4-Amino-3,5,6-Trichloro Picolinic Acid" Document entitled "Review of the Bioassay of Picloram for Carcinogenicity by the Data Evaluation/Risk Assessment Subgroup of the Clearing House on Environmental Carcinogens". Abstract on article regarding herbicide residues in milk from cows. Abstracts of articles regarding Picloram. Weimer, et al. "Toxicological Studies on the Herbicide 'White' in Animals", Dept. of the Army. Edgewood Arsenal Research Laboratories. Heath, et al. "Comparative Dietary Toxicities of Pesticides to Birds", from the Division of Wildlife Research Bureau of Sport Fisheries and Wildlife. Dow R & D report on "Kinetics of CLabelled Picloram in Male Fischer 344 Rats" with attached report. Woodruff, et al. "Pesticide-Induced Complete Impartial Chromosome Loss in Screens with Repair Defective Females of Drosophila Melanogaster", Environmental Mutagenesis. V o l . 5, pp. 835-846. Abstract of article on Pesticides and Rats. Guerzoni, et al. "Mutagenic Activity of Pesticides", translation from original Italian article. Dow R & D report on "The Toxicity of Picloram (4-Amino-3,5,6-Trichloro Picolinic Acid to Representative Fresh Water Organisms. Dow R St D report on "Picloram: Results of Ilio 1111 1112 1113 1114 1115 1117 1118 1119 1120 1121 674 9 6/9/82 7/29/82 1983 7/28/83 8/19/83 10/10/83 1984 3/12/73 2/84 1984 8/16/84 8/13/84 A Two-Week Dietary Probe Study in CDF Fischer 344 Rats", with attached report. Dow R & D report "Results of a Six-Month Dietary Toxicity Study of Picloram (4Amino-3,5,6-Trichloro Picolinic Acid) Administered in the Diet to Male and Female Beagle Dogs" with attached report. Dow R & D report on "Picloram: The Influence of Hexachlorobenzene Concentration on Picloram-Induced Liver Toxicity in Fischer 344 Rats" with attached report. Borzelleca, Joseph F. "A Review of Volatile Organic Contaminant Data", publication not indicated. Dow R & D report on "Picloram: Quantitative Determination in Human Blood and Urine" with attached report. Dow R & D report on "Picloram: Pharmacokinetics in Human Volunteers" with attached report. Dow R & D report "An Acute Oral Toxicity Study in the Mallard with Picloram Technical" with attached report. Libich, et al. "Occupational Exposure of Herbicide Applicators to Herbicides Used Along Electric Power Transmission Line Right of Way", American Industrial Hygienist Association Journal, Vol. 45, pp. 56-62. Suschetet, et al. "Physiopathological and Nutritional Effects of 4-Amino-3,5,6Trichloro Picolinic Acid (Picloram) on the Rat" from a French article. National Toxicology Program annual Plan for fiscal year 1984 from the Department of Health and Human Services. Nolan, et al. "Pharmacokinetics of Picloram in Male Volunteers", Toxicology and Applied Pharmacology, Vol. 76, pp. 264-269. Dow R & D report "Biological Mechanisms Involved in the Toxicity of Picloram Herbicide in Rats" with attached report. Dow R & D report "The Toxicity of 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1133 /^ 1 3 4 / / ^ 6750 6/18/84 6/12/84 4/9/85 00/00/00 1985 3/86 1/9/86 1/6/86 11/20/50 1963 00/00/00 1962 4/13/70 103 Technical Picloram to the Embryo, Larval, and Juvenile Stages of the Rainbow Trout", with attached report. Dow R & D report "The Acute and Chronic Toxicity of Technical Picloram (4-Amino3,5,6-Trichloro Picolinic Acid) to Daphnia Magna Straus" with attached report. Dow R & D report "The Determination of 4Amino-3,5,6-Trichloro Picolinic Acid in Water by High Performance Liquid Chromatography During Aquatic Toxicity Studies" with attached report. Dow R & D report "Evaluation of Picloram in the Mouse Bone Marrow Micronucleus Text", with attached report. Mullison, Wendell R. of Dow "A Toxicological and Environmental Review of Picloram", publication not indicated. John-Greene, et al. "Teratological Evaluation of Picloram Potassium Salt in Rabbits", Fd. Chem. Toxic., Volume and page numbers not indicated. Abstract of article on Picloram. Dow R & D report "Picloram: A Two-Year Dietary Chronic Toxicity-Oncogenicity Study in Fischer 344 Rats", with attached report. Dow R & D report "Evaluation of the Toxicity of Technical Picloram to Fresh Water Green Alga", with attached report. French article on 2,4-D. Citation of article on 2,4-D. Shafik, et al. "A. Method for Determination of Low Level of Exposure to 2,4-D and 2,4,5-T", from the Public Health Service Division of Pesticide, Chemistrv and Toxicoloov. Cite on article on 2,4-D. Memo from Dunn of Dow to Lyn of Dow re results of Chloracne studies conducted on assorted 2,4-D, 2,4,5-T, Silvex samples and their esters, orange and diamonds crop rider amine 4-T-2. 113"5^ 1136 1137 1138 1139 1140 1141 1143 1144 1145 i 2/13/70 Dow requests for preliminary toxicological studies on 2,4-D. 00/00/00 Dow report entitled "Chlorophenoxy Herbicides Drinking Water Limits" 1970 Abstract on article regarding pesticide residues in natural waters. 10/84 Rubinstein, et a l . "Inhibition of Intercellular Communication in Cultures of Chinese Hamster V-79 Cells by 2,4Dichlorophenoxyacetic Acid and 2,4,5Trichlorophenoxyacetic Acid", Fundamental and Applied Toxicology, Vol. 4, pp. 731739. 00/00/00 Cortney, K. D. "2,4,5-T in the Rat: Excretion Pattern, Serum Levels, Placental Transport and Metabolism", Pesticides Symposia, pp. 277-283. 8/84 Mortelmans, et al. "Muted Genicity Testing of Agent Orange Components and Related Chemicals", Toxicology and Applied Pharmacology, Vol. 75, pp. 137-146. 1985 Singh, et al. "Inhibition of Human Glutathione S-Transferases by 2,4Dichlorophenoxyacetate (2,4-D) and 2,4,5Trichlorophenoxyacetate (2,4,5-T)", Toxicology and Applied Pharmacology, Vol. 81, pp. 328-336. 3/82 Riihimaki, et al. "Mortality of 2,4Dichlorophenoxyacetic Acid and 2,4,5Trichlorophenoxyacetic Acid Herbicide Applicators in Finland", Scandinavian Journal of Work Environment and Health. Vol. 8, pp. 37-42. 1/15/84 Kawashima, et al. "Effects of 2,4Dichlorophenoxyacetic Acid and 2,4,5Trichlorophenoxyacetic Acid on Peroxisomal Enzymes in Rat Liver", Biochemical Pharmacology, Vol. 33, pp. 241 through 245. 1984 Vessey, et al. "Differential Activation and Inhibition of Different Forms of Rat Liver Glutathione S-Transferase by the Herbicides 2,4-Dichlorophenoxyacetic Acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T)", Toxicology and Applied Pharmacology, Vol. 73, pp. 492-499. f 1 4/73 Abstract of article on 1 DA 1146 1147 1148 1149 1151 1152 1153 1154 1155 1156 1157 1158 a 1975 11/75 1984 1/23/84 11/84 5/16/80 1/30/82 2/7/78 8/23/65 1/31/63 4/1/63 12/30/65 1979 6 7 5 2 1977 Chlorodibenzodioxins in the liver. Abstract of article on dioxins. Citation of article on the effects of TCDD in rats. Haseman, et al. "Results From 86 Two-Year Carcinogenicity Studies Conducted by The National Toxicology Program". Journal of Toxicology and Environmental Health. Vol. 14, pp. 621-639. Niosh. Current Intelligence Bulletin. No. 40. "2,3,7,8-Tetrachlorobibenzo--p-dioxin (TCDD 'Dioxin')". Dow report entitled: "Dioxin, Agent Orange and Human Health". Dow R & D Report. "Picloram: Results of a 32 Day Toxicity Tolerance in Feed in Mice", with attached report. Dow R & D Report. "Comparative Toxicity of Three Samples of Technical Picloram Containing Various Amounts of N-(2,3,7,8Tetrachloro-2-pypridinyl)guanidine 2 Bluegill", with attached report. Health, Education and Welfare Release regarding a report on animal tests of Picloram for carcinogenicity. Dow report. "A Game Bird Toxicology Study-- Acute Dietary Feeding of Tordon to Wild Type Mallard Ducklings", with attached report. Dow report. "Fish Toxicity of Lili", with attached report. Dow Report. "Toxicology Evaluation of K38, 232 (Picolinic Acid: 4-amino-3,5,6trichloro)", with attached report. Dow report. "Toxicological Properties and Industrial Handling Hazards of 11-Tordon Process Samples". Morpurgo, et al. "Analysis of Mitotic Non-disjunction with Aspergillus Nidulans". Environmental Health Perspectives, Vol. 31, pp. 81-95. Bignami, et al. "Mutagenic and Recombinogenic Action of Pesticides in 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 Various 1965 1974 1974 7/30/80 3/20/81 00/00/00 3/20/81 5/81 Various 1984 7'<o K o & iD 1 Aspergillus Nidulans". Mutation Research Vol. 46, pp. 395-402. Abstracts of various articles on herbicides. Cope, F. G. "Toxicity of Tordon 101 to Rainbow Trout". Pesticide Proa, Vol. 3. pp. 89-90. Somers, et al. "Effect of External Application of Pesticides to the Fertile Egg on Hatching Success and Early Chick Performance. No. 2. CommercialHerbicides Mixtures of 2,4-D with Picloram or 2,4,5-T Usina the Pheasant". Bulletin of Environmental Contamination and Toxicoloav, Vol. 11, d o . 339-342. Suschetet, et al. "Toxicity and Nutritional of Two Herbicides in the Rat: Picloram (4-amino-3,5,6-trichloropicolinic Acid and Atrazine 2-chloro-4ethylamino-6-isopropylamino-s-triazine)" . Translated from the French article. "Picloram: Results of a 13 Week Toxicity Study in Mice", with attached report. Dow R & D report. "Acute Toxicological Properties and Industrial Handling Hazards of Tordon Acid: 4 and 6 Isomers with Impurities, Not Washed". Dow data sheet of properties, health hazards and precautions for safe handling of materials for Tordon Acid. Dow R & D report. "Acute Toxicological Properties and Industrial Handling Hazards of Tordon Acid: 4 and 6 Isomers with Impurities, Not Washed". Reuber, Melvin. "Carcinogenicity of Picloram". Journal of Toxicoloav and Environmental Health, Vol. 7, d o . 207-222. Various abstracts of articles on herbicide used, including one entitled: "Exposure of Forest Workers Using Herbicides Measured". Libich, et al. "Occupational Exposure of Herbicide Applicators to Herbicides Used Along Electric Power Transmission Line Riaht of Wav". American Industrial Hvaienists Association Journal. Vol. 45, 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1182 Q i9 00/00/00 00/00/00 1972 4/88 6/83 5/22/88 1/89 9/88 2/15/79 7/5/88 00/00/00 11/5/82 3/73 9/79 im pp. 56-62. Illegible article. Document with handwritten title: "Picloram Snarl" and printed list of participants. Abstract of article on Picloram. Draft Environmental Impact Statement: "Vegetation Management in the Coastal Plain-- Piedmont", by the USDA Forest Service. Draft Environmental Impact Statement on Vegetation Management for Reforestation, from the USDA Forest Service. Supplement Draft Environmental Impact Statement on Vegetation Management for Reforestation, USDA Forest Service. Final Draft Environmental Impact Statement on Vegetation Management in the Coastal Plains/Piedmont, USDA Forest Service. Review draft record of decision for USDA Forest Service Specific Northwest Region Environmental Impact Statement on Managing Competing and Unwanted Vegetation. "The Biologic and Economic Assessment of 2,4,5-T", submitted to the Environmental Protection Agency by USDA. It includes the supplement report. Twenty photographs of the Johnson Wash project. Computer printout of product information for Esteron 99, Esteron 99 concentrate and Formula 40 Herbicide. Document entitled: "2,4-D Herbicide", giving information about the product, prepared by Donald Reed of the FDA. Lindenmuth and Davis (plaintiffs' decedent). "Predicting Fire Spread in Arizona's Oak Chaparral", USDA Forest Service research paper. Advertisement entitled: "Debating Scientists of Eight Nations Agree Pesticide 2,4,5-T is Safe!", by the Dow Chemical Company, located in Science 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 6755 M a g a z i n e , Vol 205, pp. 1330. 9/3/54 Davis, James R. "Comments on Summer's Work, 1954", prepared at the Ft. Valley Research Center. 10/1/47 Job duty description for position of research forester at the East Texas Research Center. 1/20/48 Job duties for position of research forester (general) for the East Texas Research Center. 00/00/00 Description of duties for research forester (general). 10/24/57 Description of duties for research forester (general). 3/2/64 Tasks and performance levels for research forester (forest fire). 00/00/00 Document entitled: "Operating Plan Johnson Wash Prescribed Fire Experimental Area, Prescott National Forest". 00/00/00 Document entitled: "Memorandum of Understanding for Research Burning in the Southwestern Region". 9/69 Fact sheet: Chaparral treatment and management on the Tonto National Forest. 8/10/70 USDA transmittal of the operating plan for the Johnson Wash Chaparral Study area. Various Series of work plans for James R. Davis. dates in the 1950's MarchApril, 1989 Alavanja, et al. "Mortality Among Forest and Soil Conservationists". Archives of Environmental Health, Vol. 44, pp. 94-101. 1984 Sections from The Clinical Toxicology of Commercial Products, Williams and Wilkens Publishers, 5th Ed., with a page on the chemical components of Esteron. 3/56 Davis, James R. "2,4,5-T-- Low Volatile Esters Effective". From the Southern Forestry Notes of the USDA Forest Service, page number not indicated. 5/58 Davis, James R. "Basal Spray with 2,4,5-T for Winter Hardwood Control in East 1199 1200 1201 1202 1203 1204 1205 1207 1208 1209 1210 11/59 6/62 1959 7/60 6/59 7/22/54 1959 7/58 1955 1968 1968 1968 6756 Texas". Journal of Forestry. Voi 56, p. 349. Davis, James R. "Low-Volatile 2,4,5-T Effective as Basal Spray". Journal of Forestry. Vol. 57, p. 851. Lindenmuth and Davis, James R. "Chemical Treatment Helps Burn Brush Safely in Arizona". The Timberman. Vol. 63, p. 9. Gibbs, Carter B. "Amines of 2,4-D Hold Promise for Hardwood Control". Down to Earth, Vol 15, p. 6. Gibbs, Carter B. "Iso-Octyl Ester of 2,4,5-T in Hardwood Control". Weeds, Vol. 8, pp. 462-463. Stransky, John J. "Concentrated or Diluted 2,4-D as a Supplement to Girdling?". Journal of Forestry. Vol. 57, pp. 432-434. Harrington, et al. "Work Plan, a Study of the Effectiveness of Axe and Machine Girdles in Conjunction with 2,4,5-T". Stevenson, et al. "Selective Control of Cull Hardwoods in East Texas". From the Southern Forest Experiment Station, Forest Service, USDA. Davis, James R. "Diluted 2,4,5-T More Lethal than Undiluted in East Texas". Journal of Forestry. Vol. 56, p. 516. Davis, James R . , et al. "Quick Bunyan, the Needle!, Tree Injector Kills Cull Hardwoods" Southern Lumberman, pp. 171172. Davis, James R., et al. "A Fire Prescription for Consuming Ponderosa Pine Duff". USDA Forest Service Research Note, pp. 1-4. Davis, James R. "Freeze Drying for Preparing Arizona Chaparral Plants for Energy Analysis". USDA Forest Service Research Note, pp. 1-2. Ffolliott, Clary, and Davis (James R . ) "Some Characteristics of the Forest Floor under Ponderosa Pine in Arizona". USDA Forest Service Research Note, pp. 1-4. 1211 1212 1213 1214 1215 1215 1217 1218 1219 1220 1221 1222 1223 1224 6757 1970 3/65 Various 3/65 6/59 6/65 7/58 00/00/00 Various 3/13/85 Various 11/23/76 and 11/9/76 Various Various i nn Lindenmuth and Davis, James R. "Foliar Moisture Content of Chaparral in Arizona, Accounting for Its Variation and Relating it to Prescribed Fires". USDA Forest Service Research Note, p. 1-8. Davis, James R. "A Survey of an Intentional Burn in Arizona Ponderosa Pine". USDA Forest Service Research Note, pp. 1-2. Various short articles by James R. Davis, previously cited. Davis, James R. "A Survey of an Intentional Burn in Arizona Ponderosa Pine". USDA Forest Service Research Note. pp. 1-2. Stransky, John J. "Concentrated or Diluted 2,4-D as a Supplement to Girdling?". Journal of Forestry, Voi. pp. 432-434. 57, Worley, David P. "The Beaver Creek Pilot Watershed for Evaluating Multiple-Use Effects of Watershed Treatments". USDA Forest Service Research Paper, pp. 1-12. Shiue, et al. "Aspen Debarking wit 2,4,5T " . Journal of Forestry, Voi. 56, pp. 503-507. List of publications by James R. Davis. Discovery papers from the Agent Orange Case (MDL 381) from the U. s. District Court, Eastern District of New York. Transmittal and documents of the personnel record of James R. Davis, from the US Office of Personnel Management. Additional personnel records of James R. Davis, including USDA-Forest Service Performance Rating Sheets. Funeral bills for James R. Davis. Social Security Benefit Statement, Award Certificate and Medi-Care Information provided to Ruth Davis. Retirement benefit information for Ruth Davis. 1225 1226 1227 1228 1229 1230 1231 1232 1234 1235 1236 1237 1238 1239 1240 1241 1242 6758 Various Various 10/76 7/26/88 4/19/88 1/6/89 7/29/88 Various 2/16/86 Various 1957 00/00/00 7/31/56 7/5/57 6/5/57 3/18/57 3/18/57 5/24/57 Tax information for James and Ruth Davis. Medical records for James R. Davis. Death Certificate for James R. Davis. Exhibits to the deposition of Dr. Ozonoff. Exhibits to the deposition of Ruth Davis. Exhibits to the deposition of Dr. Marvin Legator. Exhibits to the deposition of Peter Formuzis, Ph.D. 10 family photographs. Article from Times-Colonist Newspaper: "Dow Chemical's Works at Polishing Tarnished Image". Employment information for Ruth Davis. Document entitled: "Annual Report 1975-- Second Study of Concentrated 2,4,5-T in Hardwood Sprout Control", showing assignment of project to James Davis. Document entitled: "What to See in the Experimental Area", with attached fire fact sheets. Davis, James R., et al. "Work Plan at Establishment Report: Second Study of Concentrated 2,4,5-T in Hardwood Sprout Control". Davis, James R., et al. "Final Office Report: Second Study of Concentrated 2,4,5-T in Hardwood Sprout Control". Davis, James R., et al. "Establishment Report: 2,4-D Amine in Little Tree Injector Wounds in Hardwood". Davis, James R. "Work Plan: 2,4-D Amine in Little Tree Injector Wounds in Hardwoods". Davis, James R. "Work Plan: Effectiveness of Isooctyl Ester of 2,4,5-T Used in Hardwood Control with the Little Tree Injector. Davis, James R. "Establishment Report: 1243 1244 f1245' 1247 1248 1249 1250 253' ,1254 6759 4/30/58 1977 3/18/88 1979 1964 1979 00/00/00 1977 1/80 8/80 3/71 Effectiveness of Isooctyl Ester of 2,4-D Used in Conjunction with the Little Tree Injector". Gibbs, Carter B. "Progress Report: 2,4-D Amine in Little Tree Injector Wounds on Hardwoods". Van Miller, et al. "Increased Incidence of Neoplasms in Rats Exposed to Low Levels of 2,3,7,8-Tetrachlorobibenzo-p-dioxin". Chemosphere, No. 9, pp. 537-544. Kahn, et al. "Dioxins and Dibenzofurans in Blood and Adipose Tissue of Agent Orange-- Exposed Vietnam Veterans and Matched Controls". JAMA, Vol. 259, pp. 1661-1667. IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 20, pp. 303, 347-353. Kirk-Othmer. Encyclopedia of Chemical Technology, pp. 258-267 and 325-338. Kirk-Othmer. Encyclopedia of Chemical Technology, 3rd Ed., Vol. 5, pp. 797-808 and 866-867. IARC Monographs, Vol. 20, pp. 354-367. No exhibit. Ahlling, et al. "Formation of Polychlorinateddibenzo-p-dioxins and Dibenzofurans During Consumption of a 2.4.5-T Formulation". Chemosphere, No. 8, pp. 461-468. Ott, et al. "A Mortality Analysis of Employees Engaged in the Manufacture of 2.4.5- Trichlorophenoxyacetic Acid". Journal of Occupational Medicine, Vol. 22, pp. 47-50. Cook, et al. "Mortality Experience of Employees Exposed to 2,3,7,8Tetrachlorobibenzo-p-dioxin (TCDD)". Journal of Occupational Medicine, Vol. 22, pp. 530-532. Poland, et al. "A Health Survey of Workers in a 2,4-D and 2,4,5-T Plant". Archives of Environmental Health, Vol. pp. 316-327. 22, 1257 1258 ; 12>59-- > 1260 1262" 1264 1265 1266 1/31/81 3/14/81 1978 9/73 8/77 12/7/79 12/78 5/88 May 28 through 30, 1980 Various 00/00/00 1986-1987 6760 Honchar, et al. "2,4,5-T Trichlorophenol and Soft Tissue Sarcoma". The Lancet. pp. 268-269. Cook, Ralph R. of Dow. "Dioxin, Chloracne, and Soft Tissue Sarcoma". Lancet, pp. 618-619. The Kociba, et al., of Dow. "Results of a Two Year Chronic Toxicity and Oncogenicity Study of 2,3,7,8-Tetrachlorobibenzo-pdioxin in Rats". Toxicology and Applied Pharmacology, Vol. 46, pp. 279-303. Langer, et al. "Formation of Dibenzo Dioxins and Other Condensation Products from Chlorinated Phenols and Derivatives" . Environmental Health Prespectives, pp. 37. IARC monographs on the evaluation of the carcinogenic risk of chemicals to man. Some fumigants, the herbicides 2,4-D and 2.4.5-T, Chlorinated Dibenzo Dioxins and miscellaneous industrial chemicals. Vol. 15. Blair, Etcyl, of Dow. "The Safety of 2.4.5-T. Science, Vol. 206, pp. 11351136. "Of Mice and Men-- The Troubles of 2,4,5T " . Journal of Forestry, pp. 787-790. Breslin, et al. "Proportionate Mortality Study of U.S. Army and U.S. Marine Corp. Veterans of the Vietnam War". Journal of Occupational Medicine, Vol. 30, pp. 412419. Kimbrough, Renate D., of the Center for Disease Control. "2,3,7,8Tetrachlorobibenzodioxin (TCDD)-- Toxicity in Animals Relevance to Human Health, With Notes on 2,4,5-T, Picloram, Cacodylic Acid, and 2,4-D". Labels and product information for Dow Esteron 245, and Esteron 99 Concentrate. Hayes, Wayland J. "Pesticides Studied in Man". Publication not indicated. Thomson, W. T. "Agricultural Chemicals Book II-Herbicides". Section entitled: "2,4,5-T, Dacamine 4-T, Envert-T, Esteron 245, Phortox, Reddon, Tippon, Trioxone, 1267 ,'1268/ 1271 12722 (1274/ 1275 '1 2 7 8 / Veon". 00/00/00 "Clinical Toxicology of Agricultural Chemicals", section 2 on Phenoxy herbicides. 8/87 "2,4,5-Trichlorophenoxyacetic Acid", a health advisory from the EPA. 4/17/81 Hanify, et al. "Aerial Spraying of 2,4,5T and Human Birth Malformations: An Epidemiological Investigation". Science, Vol. 212, pp. 349-351. $L ?/86 EPA pesticide fact sheet on 2,4-D, including a March, 1987 update. 00/00/00 Gosselin, et al. "Clinical Toxicology of Commercial Products", 5th Edition, with a section on 2,4-D. 00/00/00 Hayes, Wayland, J. "Pesticides Studied in Man", with a section on 2,4-D. 7/86 Mullison, Wendell. "An Interim Report Summarizing 2,4-D Toxicological Research Sponsored by the Industry Task Force on 2.4- D Research Data and a Brief Review of 2.4- D Environmental Effects". 9/78 "Occupational Health Guideline for 2,4-D", from the U.S. Dept, of Health and Human Services and the U.S. Dept, of Labor. 1986-1987 Thomson, W. T. 1986 to 1987 revision on section regarding 2,4-D from the "Agricultural Chemicals Book IIHerbicides". 9/5/86 Hoar, et al. "Agricultural Herbicide Use and Risk of Lymphoma and Soft Tissue Sarcoma" . JAMA, Vol. 256. 7/17/79 Portion of Federal Register. "Preliminary Determination Concerning the Rebuttable Presumption Against Registration of Certain Uses of Pesticide Products Containing 2,4,5-Trichlorophenol (2,4,5T); Hearing; Availability of Position Document". Vol. 44, pp. 41531-41543. 12/13/79 Portion of Federal Register. "Environmental Protection Agency-- Final Determination Concerning the Rebuttable Presumptions Against Registration for Certain Uses of Pesticide Products Containing 2,4,5-T and Silvex and Notice f'lilSy 1281 1282 1283 ^1284/ 1285 1286 1287 1288 1289 1290 1291 of Intent to Hold a Hearing", pp. 7232872341. 10/18/83 Federal Register. "Environmental Protection Agency-- 2,4,5-T and Silvex Products; Intent to Cancel Registration of Pesticide Product Containing 2,4,5-T and Silvex; Revocation of Notices of Intent to Hold a Hearing to Determine Whether Certain Uses of 2,4,5-T or Silvex Should be Cancelled", Vol. 48, pp. 48434-48436. 9/12/80 The Carcinogen Assessment Group's Risk Assessment on 2,4,5-T, Silvex and TCDD. Fall, 1952 "Harnessing Chemical Sprays to Serve the Sportsmen Through New Game Management Practices". Down to Earth, pp. 7-8. 6762 1952 1957 00/00/00 1971 1973 1953 1954 1955 1959 Various 115 Neuns, Alva. "Woody Plant Control Along Roads in Shasta National Forest". Down to Earth, pp. 14-15. Lynn, G. E. "Residue Tolerances for Dow Agricultural Chemicals". Down to Earth, pp. 7-11. Williams, C. S., of Dow. "The Statue of 2,4-D, 2,4,5-T, Silvex and MCPA Herbicides". Publication not indicated. Johnson, Julius. "Safety in the Development of Herbicides". Down to Earth,:V o l . 27, pp. 1-7. Wilson, James. "Teratological Potential of 2,4,5-T". Down to Earth, Vol. 28, pp. 14-18. Arend, John L. "Controlling Scrub Aspen with Basal Sprays". Down to Earth, pp. 10- 1 1 . Walker, A. H. "Chemicals are Effective for Woody Plant Control in the South". Down to Earth, pp. 10-11. Lynn, G. E. "The Status of Pesticides Under the Miller Amendment to the Federal . Food, Drug and Cosmetic Act". pp. 8-9. Gibbs, Carter. "Amines of 2,4-D Hold Promise for Hardwood Control". Publication not indicated. Photographs of James R. Davis at work and MOYER SHIPPING CONTENTS INVENTORY LARGE WHITE BOX (18" X 11 1/2" X 10") 45 Pounds 1 Davis v. Dow Exhibits # 1 - # 277 (+25 sub-parts) 302 total exhibits; 2948 pages total. 2. Davis v. Dow Attachment to Memorandum of November 13, 1991EXHIBIT LIST OF DOCUMENTS (45 Pages). 3. Davis v . Dow Legal Documents, Exhibits # 1 . - XVI. (203 p a g e s ). 4. Davis v. Dow Attachment to Memorandum of November 13, 1991SUPPLEMENTAL LIST OF LEGAL DOCUMENTS, (3 pages). 5. Keister v. Dow Exhibits # 1 - # 49 (+6 sub-parts) 758 total pages. 6- Keister v. Dow Memorandum of November 28, 1991 on Review of Documents (3 pages). 7. Keister v. Dow Attachment to Memorandum of November 28, 1991 EXHIBIT LIST OF DOCUMENTS (13 pages). 8. Newman v . AT&T Exhibits # 1 - # 20 (196 pages). 9- Newman v. AT&T Memorandum of 29, 1991 on Review of Documents (3 pages). 10. Newman v. AT&T Attachment to Memorandum of November 29, 1991 EXHIBIT LIST OF DOCUMENTS (6 pages). FEDEX PAK (12" x 15 1/2") 1- Davis v. Dow Exhibits # 1 - # 25 on Dow's Publications and Hype on Herbicides; Supplement to Memo of November 13, 1991 (102 pages). 2. Davis v . Dow Supplemental List of Exhibits on Dow's Publications and Hype on Herbicides (2 pages). 3. Memorandum of November 30, 1991 on 2,4-D and 2,4,5-T Chemical Formulas and Key Epidemiological Studies with listing of Documents #1 - # 10 (3 pages) 6764 6767  c ; Kou>*> j . #DOW748556;h From the L ab oratory o flT crato logy , K arolin sk a Institute!, OO'' Orv.' S-104 01 Sto ck h o lm , Sw eden .6> Teratogenic and Embrotoxic Effects of the A . S d J - ) Herbicides Di- and Triclilorophenoxyacetic Acids >o FJb:J.mo (2 ,4-D and 2 , 4 , 5-T) XH, By Gertrud B 3ge, E ra C ekanora and K . S . Larsson (R eceived N ovem ber 28, 1972; A ccepted D ecem ber 13, 1972) Abstract: C om m ercial solutions o f phenoxyacetic acid s w ere tested fo r terato genic effects in N M R I-m ice. T h e Sw edish p ro d u ct H o rm o sly r 500-T con tain ed only 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) w hile H o rm osly r 64 w as a m ix ture o f 2,4-dichlorophenoxyacetic acid (2,4-D ) and 2,4,5-T (2:1). Subcutan eous injections w ere giVcn fro m d a y 6 th rou gh d a y 14 o f pregn an cy an d the an im als were sacrificed on day 18. T h e num ber o f resorbed em bryos, living em bryos with gross m alform ations, as w ell as internal and skeletal m alform ation s w ere re corded. It w as found that both preparations at the high do sage (1 10 m g/kg/day) were teratogenic and cm bryotoxic. A t the low dose level (50 m g/k g/d ay) the 2,4,5-T solution w as m ore h arm ful than the m ixture o f 2,4-D an d 2,4,5-T. T h e risks o f teratogenicity in hum an civilian u se an d the ro le o f dioxin s are d is cussed. Key-words: Terntogcncsis - em bryotoxlcity - herbicides - 2,4-D - 2,4,5-T. flic lively discussion of the possible hazard to man of the use of large inliiics of herbicides in agriculture and forestry has been focused upon chlorinated phenoxyacetic acids, even though these compounds have been sidered to be only moderately toxic in animals (Rowe & H y m a s 1954). : report by C o u r t n h y et al. (1970) on teratogenic and cmbryotoxic effects 2,4,5-trichIorophcnoxyacctic acid (2,4,5-T) and to some extent of 2,4ilorophenoxyacctic acid (2,4-D) in experimental ' animals was the basis the rapidly growing interest in the various adverse effects of these licidcs. The most significant malformations induced at high doses are t palate in mice and cystic kidneys in rals and mice. That the teratogenic ct is due not only to the 2,4,5-T but also to the high content (27 p.p.m.) lie dioxin contaminant 2,3,7,R-tclrnchlorodibcnzo-/r-dloxin (TCDD) was tinned in a lalcr study ( C o u r t n e y & Moontt J971). . \Si! studies on 2,4,5-T, 2,4-1) and dioxins have since been performed > 6768  'V fcMUKkUlOXlCITY OF HtROICIDES nu, . in different species and these essentially confirm that the two jhcnoxyacctic j acids at high doses are teratogenic and embryotoxic and show that TCDD i is teratogenic in the mouse (R oll 1971; Neubert & D illmAnn 1972). It is interesting to note that even with high doses (40 mg/kg 2,4,5-T and 80 mg/kg 2,4-D), it has not been possible to produce malformations in monkeys (Wilson 1972a). However, two out of four females aborted after 2,4-D j treatment (Wilson 1972b). T h e report that phenoxyacctic acids cause ! abortions in reindeers in Sweden was later nbt confirmed ih a study by ! . E hrne (personal communication). Several newspaper reports of human mal- ' formations in Sweden, Vietnam and Arizona which had been attributed to the mothers' alleged exposure to the phenoxyacetic acids have been evaluated on.more than one occation but it has not been possible to point at a causal relationship (L arsson 1971). 2,4,5-T and 2,4-D as different esters, salts or acids have b'efi commonly used as weed killers for more than 20 years. In Vietnam they have been j used as defoliants and crop-destroyers in quantities 10-15 times that re- j commended for civilian use. The regulations for use in th U. S. A. and ; Sweden have recently been restricted. For example, the use of phenoxyacctic acids in forestry was temporarily banned in March, 1971, by the Swedish Poisons and Pesticides Board in Sweden. The case was to b opened for re-evaluation at-the beginning of 1972 when additional data would be avail- , able. Most of the reports so far have dealt with studies ori 2,4,5-T with | various dioxin contents. However, since the phenoxyacctic adds used as herbicides for civilian purposes usually contain both 2,4,5-T and 2,4-D logether with emulsifiers and solvents, and since mechanisms of interaction, e. g. synergism, cannot be ruled out untried, it has been considered to be of practical interest to test the teratogenic effects of commercial samples. In the following study the effects on mice of two commercial phcnoxyacid formulations in Sweden, Hormoslyr 64 (2,4-D and 2,4,5-T, 2:1) and Hormostyr 500-T (2,4,5-T) have been tested in a double blind test. 0005154 Material and Methods Three-m onth-old random -bred N M R I m ice (obtain ed from G l. Boniholtfennrd, D K -36 ! R y , D e n m a rk ) w e r e 'u s e d . T h e n n im als w e r e k e p i in in a c r o l n c c a g e s in it ro o m w ith J con stan t-tem p eratu re (2 3 *) and au to m atically reg u lated light fro m 6 a. m. to 6 p. ni. J an d fed sta n d ard la b chow (E w o s, Sild crliilje,. Sw eden ) an d w ater ait lihinnn. I h c ' fem ales were m ated overnight and checked fo r vaginal plugs the next m orning, denoted , a s day zero o f pregnancy. T h e fem ales w ere weighed on this day anil also fiom 1 days 6 to 18. * T h e lest solutio n s w ere prepared nfnl co d ed for the doubl blind test at the S c d id i P o ison s an d P esticid es H oard. T h e su b stan ces (kindly supported front G ullvikx l-'ahiiks C* Jt C* I .w c w l^> f W V l * 4 4 4 >A- .... O 4^4 1 i w w ' i 9 \ 8 j d ** W* 't OOW748557 1 s w*1 d d 6 d ci `\M ' I f i * +1 +1 -H +1 +1 ^0 I 37 a* Mho>OdiMdhOVmiONJ B % of u se s w ith ft p a la te v At ' , T ablet is P 3 5 * * 5M*3 ia 6 No. of fo e tu s e s wi cleft palai 60 190 111 219 191 No. of living foetuses ! s ? MJ o f r e s o r b e d% im plantation sites 11 b! dh nHvHmien' <tmT| Hh 2 No. of im piantati site s 1* e n h p \o oo 3 ?. p a s z g Jl 0 hs fi N ^OS No. of litte n D a ily treatn S u b sta n c e M8 H om ionmo o o iie *r 3s ,, %$3 a `rt o* oe. < mU D ul .1 / / . . tlv iU K io i X lC IV OF HERBICIDES , A B , M atm S, Sw eden) used for preparing the solutions w ere the com m ercially available H o rtn o sly r JOO-T w hich co n tain s the b u to xy ety lester o f 2,4,5-lrictiloroph eno xyacetie a d d (500 g/l), an d H o rm osly r 64 w hich con tain s 2,4-dichlorophenoxyacetlc acid (330 g/l) an d 2,4,5-lrichtorophenoxyncellc acid (1 7 0 |/1). T h e content o f dioxih s, m ainly T C D D , w as less than 1 p.p.m . an d the m ost com m on dioxin w as 2,3,7,8-tetfadilorodibenzo-p* dioxin (verified a t the Sw edish P o iso n s an d P esticides B oard ). Dim fethylsulphoxide (D M SO ) w as used a s a vehicle o f the test solutions, w hich also contained 19 m g/m l of the solvent (a non-specified m ixture o f petroleum distillates). T h e ah in ials w ere given su b c u ta n e o u s in je c tio n s 0.1 m l/2 0 g b o d y w eig h t a t 10 a . m . fro m d a y 6 th ro u g h d a y 14 o f gestation. T h e d aily d o se o f phenoxyacetic acid fo r the different groups w as as follow s: . A : 110 m g/kg o f 2,4,5-T B : 50 m g/kg o f 2,4,5-T C : 110 m g/kg o f 2,4-D /2,4,5-T (2:1) D : 50 m g/kg o f 2,4-D /2,4,5-T (2:1) E : 0 m g/k g co n tro ls given 0.1 m l/20 g o f the veh icle). T h e n u m b e r o f a n im a ls tre ate d in the fiv e e x p e r im e n ta l g r o u p s a r e g iv e n in ta b le 1. -N one o f the treated dam s died o r show ed any signs o f adverse effects Xcept fo r local necroses at the injection sites which ap peared after a few days. O n 'd ay 18 o f gestation , the an im als w ere sacrificed b y cervical dislo catio n . T h e living foetuses w ere rem oved, w eighed an d exam ined fo r gross m alform ations. The num ber an d size o f the dead an d resorbed foetuses w ere noted. O ne third o f the living fo etu ses fro m each litter w as fixed in eth an ol, cleared in potassium hydroxide and stn ln cd w ith A liz arin red S fo r e x am in a tio n o f sk eletal m a lfo rm a tio n ! (Dawson 19261928). T h e rem ainder o f the living fo etu ses w as fixed in B ou ln 's solu tion fo r subsequent section in g an d exam in ation fo r internal m alfo rm atio n s (W it-soN 1965). Results Foetal mortality. Foetal mortality was significantly increased at both dose levels of 2,4,5-T and at the high dose of the 2,4-D/2,4,5-T mixture, see table 1. Tho most severe effect was obtained in the two grbtips given the highest phcnoxyncid dose, see table 2. Thus, every litter in group A and C showed resorptions and 33 % and 25 % of the litters respectively were totally resorbed. In group D only 2 of 19 litters showed more than 50 % resorption. Group D showed more litters with resorptions than the control group E , even if the resorption rate xvns almost similar in these groups. isolated cleft palate. An increased frequency of deft palate was also ob served in the same groups and to the same degree as described for the Incidence of foetal mortality, sec table 1. Litters with cleft palate in all surviving foetuses were obtained only in the high dose groups: in A, 6 out of 12 litters and in C, 6 out of 15 litters. Retarded foetal growth. The foetal weight was considerably reduced in animals treated with the high doses of the phenoxyacetic acids while no definite effect was registered after treatment with the low dbscs (tabic 1). OGOCisa O. U / w J ^ t L,. iv.AHG ik/ii 0OW748551: ribution o f resorption frequen cy.In litters from N M R 1 m ice after treatm ent w ith 2.4,5-T an d 2,4-D /2,4,5-T (2 :1 ) frolh d a y through d ay 14 o f gestation. 'o u p D aily treatm ent Sub- D ose s.c. stance m g/kg N O . OK litters A 2,4,5-T 110 B 2,4,5-T 50 C 2.4-D + 2,4,5-T 110 D 2.4-D 4- 2,4,5-T 50 B V ehicle 18 19 20 20 19 L itters with a tle a jt one foetus resorbed no. % 18 100 16 84 20 100 15 75 11 58 L itters with >50% resorbed foetuses no. % 13 72 24 12 60 0 0 L itters with all resorbed foetuses % no. 6 33 0 5 25 0 0 : slightly low-weight foetuses most often showed retarded skeletal develop 'd and reduced amounts of brown fat in the cervical region. Very few of foetuses in the control group showed these signs of retarded development. 'M etal malformations. After the highest doses of phenoxyacetic acids the centage of foetuses with malformations- of ribs and vertebrae was double t of the other three groups, including the controls (table 3). In addition, degree of malformations was much more severe in these two groups, ccially in group A. Table 3. Ictal an d internal m alfo rm atio n s in N M R I m ice a fte r treatm en t w ith 2 ,4,5-T an d - 2,4-13/2,4,5-T (2 :1 ) fro m d a y th rou gh d a y 14 o f gestation . roup R ib and D aily treatm ent vertebral Sub- D o se s.c. m alform ation s stance m g/kg no.*; % H aem orrhages su b c u ta n eo u s renal no. % no. % A 2,4,5-T it o D 2,4,5-T 50 6/24 25.0 7/70 10.0 10/60 16.7 9/190 4.7 1/36 2.8 0/120 C 2,4-D-b 2,4,5-T 110 11/44 25.0 21/111 18.9 11/67 16.4 D 2,4-13+ 2.4,S-T 50 7/R2 8.5 6/219 2.7 6/137 4.4 K Vehicle 8/r.R 11.8 10/191 5.2 3/123 2.4 to. wilt inalfnrniaflnns/nitmbcr examined. 6770 D ilated renal pelvis no. % 3/36 8.3 4/120 3.3 5/67 7.5 4/137 2.9 7/123 5.7 )' i I ^ >' Tabl4. ' ' .V I - E xam p les o f spon tan eously occurring m alfo rm atio n s In .N M R I m ice fter treatm ent ! w ith 2,4,5-T an d 2,4-D /2,4,5-T (2 :1 ) from d ay 6 through d ay 14 o f gestation. j G roup D ally treatm ent N o. or Sub- D o se s.c . living tan ce- m g/kg foetuses exenc e p h aly N o . o f fo etu ses with hydro- open m icro- c e p h aly eye g n a tia eelro-dactyly A 2,4,5-T 110 B 2,4,5-T 50 c - 2,4-D + r 2,4,5-T liO D 2,4-D + 2,4,5-T 50 E V ehicle (0 190 HI 219 191 2 I 1 . ,.3 3 .1 . 2 ' 4' ' 1 3 21 2 11 1 1 - 1 || - i Haemorrhages. Subcutaneous bleeding with a large variation iti location was found to be three times as common in the two high dos groups as in : the other three groups (table 3). The frequency of renal hctnorrhagc was I: increased only in the group given 2,4-D/2,4,5-T in the high dose (table 3). N o gastro-intestinal haemorrhages were detected. Renal malformations. Cystic kidneys were not found. Foettiscs with dif ferent degrees of dilated renal pelves were found, of which nly the more pronounced ones'were recorded (table 3). Only a slightly increased fre quency was observed in the high dose groups. The foetal kidneys in the two high dose groups usually appeared smaller and opaque in the sections as compared with the controls which were larger and had translucent mcdullac. - Spontaneous malformations. In the investigated material few malfor mations appeared in all the groups as shown in table 4. Discussion The present investigation confirms earlier studies, which have shown that high doses of phcnoxyacclic acids arc teratogenic, cause foetal death and retarded foetal growth in mite ( C o u r t n g y ct al. 1970; Uot.L 1971; N e u iiu r t & D m.lmann 1972). The substances tested which arc used aS weed killers in Sweden, do not, however, indicate any greater teratogenic risk than 2,4,5-T or 2,4-D compounds Used in other experimental studies. < The various types of malformations found in this study Were identical with those reported by other investigators. They^^t^conijncm^defect was O O W M "^ ol.ilcd* cleft pah Cystic kidneys which was one of the most character* j ^ o ' ^ tic malformations observed by C o u r t n e y e t a l . (1970) after 2,4,5-T tr e a U ^ ^ X ? cut were not found. Nor did we observe any marked dilatation of the renoT. 1 :lves. Less frequent, but probably also caused by the treatment, were :clctal malformations and subcutaneous haemorrhages. A frequent ob- rvntion was that the skin of the living,foetuses, especially the small ones, as very sensitive to handling.with easily occurring subcutaneous bleeding, lie phenomenon was mainly observed after treatment with the phenoxy- :etic acid mixture at the higher dose but it also occurred in the other groups, andling NMRI foetuses of the same age from untreated litters (unpublished nervation) did not cause this type of subcutaneous bleeding. It cannot be led out that the dimethylsulphoxide or the trace quantities (less than p.p.m.) of dioxin present caused some of the subcutaneous bleeding ( C a u - U O vH l l e e l a l. 1967). Gastrointestinal bleeding was not detected, though It s been reported to occur after 2,4,5-T treatment or dioxin treatment o l l i n s & W i l l i a m s 1971; C o u r t n e y e t a t . 1971; S p a r s c h u e t a l . 1971). ily the mixture of 2,4-D/2,4,5-T at the 110 mg/kg dose increased the :quency of renal haemorrhages. Earlier studies* indicated that 2,4,5-T was more teratogenic than 2,4-D Ir a k 1969). It is interesting to note that the foetal damaging effect of the mbination of 2,4-D/2,4,5-T did not exceed the effect of 2,4,5-T at ai ccoo mm - . rable dose of phcnoxyacctic acid. Concerning the teratogenic effects o f) $ 0 ; two substances in combination at 110 mg/kg it seems, however, as if the r l-D contributes more than would be expected from purely additive effect,^,) ice only few studies on 2,4-D derivatives are available tho results indicate*) a it fiylher studies should be carried out to investigate a possible synergistic I cct of the two phenoxyncetic acids. L in d q u i s t & U l l m -r o (1971) suggested an interference with embryonic irition as a possible mechanism for the teratogenic action of the herbicides ,5-T and 2,4-D. In spite of a rather, even distribution on whole-body oradiographs of the two substances there was a much more rapid dis- icarancc from all tissues for 2,4-D and a stronger accumulation in the k sac placenta for 2,4,5-T ( L i n d q u i s t & U l l b e r o 1971). The selective akc of 2,4,5-T in the yolk sac placenta and a lack of placental transfer in ly gestation was interpreted in favour of a similar teratogenic mode of ion for 2,4,5-T al that postulated for trypan blue ( L l o y d & D e c k 1969). wever, an inhibition of the embryonic nutrition seems to be a less likely1) isc for, c. g. cleft palate since even single injections of 2,4,5-T on day 13 ( "J, *m u iu .r t & D il l m a n n 1972) can cause cleft palate and at that stage the Y in nutritional role of the yolk sne placenta is over. --' n all animal experiments the dose resulting in teratogenic effects has been rzuclR high ns compared with the dose which a pregnant woman could Oi ttbKUiClU LS 415 normally be exposed to. Thus, the results of the present and other studies do hot substantiate any special risk to the human embrvo from the regular ^ise of phenoxyherbicidcs. -, ' Acknowledgements - This study was supported by grants from the Swedish Medical Research Council (14X and 14P-993-07) and from the Swedish Poisons and Pesticides Board. R EFER EN C ES ' C au jolte, F . M . E ., D . H . C au jolle, S. B. C ro s A M .-M . J. C alvet: L im its o f toxic and teratosenle tolerance o f dim ethyl su lfoxide. Ann. N .Y . A cad. Set. 1967, 141, 110126. C o llin s,T . F . X A C .H . W illiam s: T erato g en ic stu d ies with 2,4,5-T ad d 2,4-D in the. h am ster. Bull. Environ. Contam . Toxicol. 1971, 6, 559-567. C ourtney, K . D ., D .W .G a y lo r , M .D . H ogan , H . L . F a lk , R . R . B a te s. A J. M itchell: . T eratogen ic evalu ation o f 2,4,5-T. Science 1970, 168, 864-866. C ourtn ey, K . D . A J . A . M oore: T erato lo g y stud ies w ith 2,4,5-tricH lorophcnoxyacctic a d d and 2,3,7,8-tctrachlorodibenzo-p-dioxin. Toxicol, appl. Pharm acol. 1971, 20, 396-403. D aw son , A . B .: A n ote on the stainin g o f the skeleton o f cleared specim ens with A lizarin R ed S . Slain Techno!. 1926-28, 1, 123. L a r sso n , K . S .: Y tlra n d e O ver fe n o x lsy ro r. In : F en oxlsyror. G ransknlnj} ev ak tu ell In form ation. R apport fr ih en expertgrupp till Giftnilmnden. A B A llm Sn n a ftlrlaget, Stockholm , 1971. L in d qu ist, N . G . A S .U tlb c r g : D istribu tion o f the h erbicides 2,4,5-T dnd 2,4-D in p reg nant m ice. A ccum ulation in the y o lk sac epithelium . Experlentia 1971, 27, 14391441. L loy d, J. B . A F . Beck: Lysosom es and congenital m alform ation s, blothem . J . 1969, . 115, 32-34. M rak ,E . M .: R eport o f the Secretary's C om m ission on Pesticides an d T h eir R elation ship to Environm ental H ealth, 1969, p. 666. D epartm ent o f H ealth, Education, and W elfare, G overnm ent Printing O ffice, W ashington, D . C . N e u b e r t, D . A 1. D illm a n n : E m b r y o to x lc e ffe c ts in m ic e tr e a te d w ith 2 ,4 ,5 -trich io ro - phenoxyacetlc acid an d 2,3,7,8-lelrachlorodibcnzo-p-dioxin. N aanfn-Schm tedebcrg's Arch. Pharm acol. 1972, 272, 243-264. R o ll, R .: U n lc r su c h u n g c n U ber d ie te r a to g e n e W irk u n g v o n 2 ,4 ,5 -T btel M K u sen . Fit. Cosm et. Toxicol. 1 9 7 1,9, 671-676. R ow e, V . K . A T . A . H ym ns: Su m m ary o f toxico logical Inform ation on 2,4-D and 2,4,5-T type h erb icides an d an evalu ation o f the h azard s to livestock asso ciated with their . u se. A m er. J . vet. Res. 1954, 15, 622-629. S p a rsc h u , G . L ., F . L . D u n n , R . W . L iso w e A V . K . R ow e: Stu d y o f tKe effects o f high lev els o f 2,4,5-lrich lo roph cn oxy acetic a c id on foetal developm en t in the rat. Fd. Cosm et. Toxicol. 1971, 9, 527-530. W ilson , J . O .: E m b ry o lo gicn l co n sid eration s in teratology . In: Teratology. Principles ' -an d techniques. E d .: J . G . W ilson an d J . W arkany. T h e U niversity b f C h icago Press, C h icago and L ondon. 1965. p. 251-277. ' dj n 4,1111; U maI McK-iJL.mz 00 I 4itK O* U a U u , ii. Ilk .- W ilson, I. C .: !U r ^rt on (rentm ent o f pregnant R h esu s m onkeys with 2,4-D (2,4-dlchlorophenoxyacctic acid ) an d C M P A (4-chloro-2-m cthylphenoxyacetlc a d d ) to the Sw edish Posisons and Pcstjpldcs B oard , 1972a. W ilson ,J. G .: A bnorm alities o f intrauterine developm ent on non-hum an prim ates. In: The use o f non-human prim ates In research on hum an reproduction. W H O S y m * - posium . E d.: E . D iczfalusy and C . C . Standlcy. W H O R esearch and Training Centre on H um an Reproduction, Stockholm . 1972b, 261-292. / CD ^ <t to D O W 748560 Front the Institute o r Pharm acology and the D epartm ent o l Surgery L , U niversity o f A arh u s, A arh us, D enm ark Protein Binding o f Drugs in Plasma from Patients with Acute Renal Failtirfe By . ` Frederik Andreasen ' (R eceived Ju ly 19, 1972; A ccepted O ctober 30, 1972) A b stra c t: I t w a s fo u n d b y In vitro e x p e r im e n ts th a t th e p ro te in b in d in g o f a c e ty l* salicylic acid, salicylic a d d , phenylbutazone, diphenylhydanloin, sulphadiazine an d thiopental w as decreased in the p la sm a o f 10 su rg ical pdtiehts w ith acu te renal failure. T h e plasm a w as taken 4 to S days after the onset o f the renal failure. A t a drug concentration o f 4 0 0 pg/m l the d ecreases ran ged fro m 21.8 to 0.3 % a s com pared w ith a norm al con trol grod p. A t d ru g con cen tration s in o r n ear the norm al therapeutic level, the protein binding w as even m ore ' decreased fo r the salicylic acids ond sulphadiazine but le ts d ecreased fo r diphenylhydanloin and thiopental. I h e decreased binding cbuld on ly partly bo explained by the low er concentration o f album in in the patients: 3.0 g/100 m l 1 0 .4 8 (S.D .) com pared to 4.1 g/100 m l 0.53 (S .D .) ( P < 0 .0 1 ) . . ' j | j - Key-words: K idney failure - acute anuria - protein binding - drUg therapy. Acute renal failure (tubuio-interstitial nephritis (B run 1954)) is a com plication which may occur in patients suffering from severe injuries. Beside the renal changes the patients frequently show impaired hepatic funclio' (cf. B eecher el al. 1947; S herlock 1963; T horn 1969). Tte conceritratio of sehim proteins can be expected to be decreased (TiioitM 1969). Valut! for the scrum albumin concentration in patients with acute renal failure ai not givfcn in the literature. L anou el al. (1971) found in general ngrccmci with other investigators that the average protein breakdown in 10 paticn with acute renal failure was 105 g/24 hrs during the first eight days. K unin (1967) and B ennett ct al. (1970) have given practical guides drug usage in patients with impaired renal function and H edger (197 gives a similar guide for patients suffering front acute renal failure. Appa cutty drug therapy in these patients is usually based on knowledge of d biological half-life of the drug in normal subjects and oil the crcatinit clearance of the patients, (cf. B erne & BARnoim 1971). . The purpose of the in vitro experiments reported here Was to clucida the following questions: 0004158 17 A d i I'liim iicotoplni, nt. V , flic , t ZLL 8 ^l THE DOWCHEMICAL COMPANY RESTRICTED F O R U SE O F DOW E M P L O Y E E S O N LY DEPAg-E6rganics Department LAfi CODE L E T I f H i a m , RE POR 1 HUHKTP G H -P 7 8 3 January 5, 1973 E. E. Kenaga TITLE OF REPORT con ENVIRONMENTAL IMPACT OF HERBICIDES r* 38 PROBLEM n u m BER c h e c k e d at D Herbicide Study Group. HMAC. EPA SIS O * VjJ 'o IN FO R M A T IV E SUMMARY WITH CO NCLUSIO NS In April, 1972 at the request of the administrator of the 00 SN mo Environmental Protection Agency, the Hazardous Materials Advisory AS Committee was asked to appoint an expert committee called a OlUAO. OuoUc "Herbicide Study" group to determine and write a report on xo whether herbicides were being evaluated properly and impartially DOW718196 in regards to cost-benefits to man and hazards to the environment Areas of insufficient knowledge were to be pointed out. E. E. Kenaga was subsequently appointed to the "Environmental Impact" subcommittee. Kenaga's input, but not necessarily what will be issued in the final committee report, is covered here. Sub jects discussed are physical properties of widely used herbicides herbicide residues in the environment, toxicity of herbicides to wildlife and a summary of the evaluation of the uses and hazards of herbicides in various habitats. (COMPLETE CRI AG. ORG. INF. C EN T ER -2 AG. ORG. REGISTRATION SECTION AG. ORG. DEPT. R & D MANAGER AG. ORG. D E PT . MANAGER U.S. AREA R & D MANAGER (U.S. AREA RPT. ONLY) AG. - ORG. PLANNING rj K. C. Barrons E. R. Laning oo E. H. Blair G. E. Lynn L. Southwick T. R. Torkelson ooso L. L. Coulter J. H. Davidson D. D. McCollister .A. J. Watson L. R. McCutchen C. A. I. Goring - Walnut Creek_ J. P. Doyle R. D. Moss F. Dowell - Walnut"Creek R. L. Gantz W. R. Mullison J. E. Johnson - 2030 Bldg. M. E. Getzendaner J. K. Priddy M. A. Wolf - 1803 Bldg. T. W. Holmsen A. E. Schober V. K. Rowe - 1803 Bldg. T. A. Hymas H. W. Sheldon P. J. Gehring - 1803 Bldg. R. V. Johnston R. J. Shaver - Lake Jackson C MINTED 9-1-77 D IST R IB U T IO N C O N T IN U ED ON R E V E R S E SID E IF R E Q U IR E D . (Over') 677. DISTRIBUTION CONTINUED N. Bremner - Dow Canada ABCT, Sarnia E. S. Saunders - -Latin America ABCT, tloral Gables C. K. Kuyper/J. P. Giazzi - E/A Ag ABCT, Horgen J. Peskett - E/A Ag ABCT, King's Lynn R. H. Ferguson - Pacific ABCT, Hong Kong 0G0SG53 / 02. "776 rato ries, S ta te C ollege, P n .). C olum n tem p eratu res ranged from 150 to 220 (iso th erm al con d ition s) a n d the carrier g a s flow w as 40 tn l/m in (p rep u rified h eliu m ). P h o lo rcd u ctio n K a te s. M eth an ol and n -bu tyl alcohol so lu tio n s were 1 0 ` 4 M . W ater solu tio n s w ere 10-8 M . S a m p le s , 10 m l e a c h , w ere p la c e d in b o ro silic a te tu b e s Fit ted w ith T eflon sto p p ers (volu m e rem ain ed co n stan t through out the reaction period). Irradiation w as carried o u t for periods o f 1-32 hr a t 300 nm a t 40. A " m erry-goro u n d" ap p aratu s w as used to ensure equ al exposure o f all sam p les to uv irradiation . T h light source u sed w as a B ay o n et reactor eq u ip p ed w ith R U L 3000 la m p s (m a x i m um ou tp u t a t 300 nm ) (T he South ern N .E . U ltraviolet C o.). A fter irrad iatio n , 2-pl aliq u o ts w ere in jected in th e g as chrom atograph and s-triazin e d isap pearan ce w as m ea su red (p eak areas w ere m easured by w eight). From the am o u n t reacted (A 0 - A ) an d the elap sed tim e (t) the ra te con stan ts (h) were calcu lated (T ab le II). R E SU L T S AND D ISCUSSIO N A s show n in T a b le II an d F ig u re s 1-4, th e ra te o f p h o to redu ction is depen den t on a t least three facto rs. T h e n a tu re o f the h alogen su b stitu en t (F igu res 1 an d 2) h as a d efin ite influence on the rate. T h e value o f k calcu lated d e c re a se s rap id ly in th e order I-B r-C l-F. T h e se resu lts are in agreem en t w ith th e know n d isso ciatio n en ergies o f the correspon din g carbon -h alogen bonds. T h e large difference betw een th e rates for iodo- an d b rom o -su b stitu ted m a rin e s an d those for chloro- an d flu oro -'su b stitu ted on es m ay also be th e result o f the increased in teraction betw een the n on b on d in g electrons on iodine an d brom ine w ith th e xsy ste m in the rin g. B o th th ese h alogen s are know n to u n d e rg o v alen cy sh ell e x p a n sio n (F o rb e s, 1959) e sp e c ia lly in the excited state. T h is heavy atom effect facilitates in ter sy stem crossing. T h e rate of reaction depen ds on the n atu re o f the sol v e n t em ploy ed a s w ell. A d ecrease in k is ob served w hen n -bu ty l alcohol is used in stead of m eth anol or w ater. T h e ra te o f ph otoreaction in the latter solven ts is ap p ro x i m ately the sam e (T ab le II). T h is effect is p rob ab ly the re CROSBY. WONG su lt o f a d ifferen ce in p o la rity o f the so lv e n ts in volved a n d of their viscosity. P o lar solven ts are known to decrease the en ergy required for r - r * tran sitio n s (F o rb es, 1960). O O First absorption band Trituine Sim azine A trazin e Propazine X (m ethanol) 268 248 237 X (l-butanol) 262 244 23S s-T ria z in e s w ith eth y l su b stitu e n ts in th e 4 a n d 6 N p o sitio n s show a greater k valu e th an th ose w ith isopropyl gro u p s (T a b le II, F ig u re s 3 an d 4 ). T h e m ain d ifferen ce in these alkyl groups is their size. A s show n above, the in c re a se in ch ain len gth re su lts in a d e creased w av elen gth o f absorption . Steric effects are known to disturb the geom e try o f an excited sta te (R u zo et a i , 1973). T h is fact m ay exp lain our ob servation s; how ever, further in vestigation is n ecessary in order to a sse ss its im p ortan ce in th e case o f s-triazin e photochem istry. LITERA TU RE CITED f ccnn 5 00 cn v*i o h-- >o o C om es, R . D ., T im m on s, F . L ,, Weeds 13,81 (1965). D illing, W. L ., Chem. Rev. 66 ,3 7 3 (1966). _ Forbes, W. F ., Con. J . Chem. 37, 1977 (1959). Forbes, W. F ., Can. J . Chem. 38,1104 (1960). Jordan, L . S ., Farmer, W. J ., Day, B . E ., " Research Progress Re port of the Western Control Conference" 78 (1963). Jordan , L . S ., Farm er, W. J ., Goodin, J . R., Day, B . E ., "R esidue Reviews, Vol. 32, T h e T riazin e H erbicides," Springer-V erlag, New York, N . Y., 1970, p 267. Jo rd an , L . S ., M ann, J .D ., D ay, B .E ., Weeds 13,43 (1965). P ape, B . E ., Z abik, M . J . , J . Agr. Food Chem. 18,202 (1970). P ape, B . E .. Z abik, M . J ..J . Agr. Food Chem. 20,316 (1972). Ruzo, L . O ., Zabik, M . J ., Schuetz, R. D ., subm itted for pu b lica tion. Received for review M ay 29, 1973. A ccepted A ugust 6, 1973. T h is research w as supported in p art by funds provided by the Food and Drug Adm inistration D H EW under contract FDA 71-285 and the M ichigan Agricultural Experim ent Station, Article No. 6424. T h is research w as presented in p a n at the 160th N ational M eet ing o f the A m erican C h em ical S o c ie ty , C h icago, 111., S e p t 1970. Photodecomposition ofp-Chlorophenoxyacetic Acid D on ald G . C ro sb y * and A nthony S . W ong A queous solu tio n s o f 4-C P A (p-chlorophcnoxyacetic acid ) decom posed readily under su n ligh t or lab oratory u ltraviolet light (300-450 n m ) to p ro vide prin cipally p-chlorophenol, phenol, hydroquinone, p-chlorophcnyl form ate, ph en oxyacetic acid , p-h ydroxyph en oxyacctic acid , an d hum ic acid s. T h ese products represent oxidative rem ov al o f the side ch ain , rep lacem en t of the chlorine by hydroxyl or by hydrogen, an d polym erization o f u n sta b le in term ed iates. F o rm atio n o f p-chloro b en zo n itrile by irrad iatio n o f 4-C P A in th e presence o f cyanide ions su b stan tiated th at the correspon din g replacem ent o f th e ring chlorine by hydroxyl w as a phot n ucleoph ilic reaction. T h e effects o f u ltraviolet (uv) light on chlorinated ph enoxyacetic acid s have been reported by several in vesti g ato rs. K elly an d P in h ev (1964) irrad iate d p-chlorop h en o x y acetic acid (4-C P A ) in eth an o l w ith a m edium - D ep artm en t o f F.nvironm ental T oxicology, U n iversity of C aliforn ia, D avis. C aliforn ia 95616. p re ssu r e m e rc u ry a r c la m p a n d reco v ered p h e n o l a s -well a s o- an d p-h ydroxyph en ylacetic acid s from th e m igration of the side chain. C rosby an d T u la ss (1966) reported th at irra d ia tio n o f 2 ,4 -d ich lo ro p h e n o x y acctic a c id (2 ,4 -D ) in a q u e o u s so lu tio n w ith a lo w -p ressu re la m p re su lte d in 2,4-dichlorophenol, 4-ch lorocalech ol, 2-hydroxy-4-chlorop h en o xy acetic ac id , 1,2,4-b en zen elrio l. an d h u m ic aqizl/\ from p ro cesses in volvin g m n in lv the rem o val o f th e * ioe'^ " ~_ n p-CHLOROPHENOXYACETlC ACID Figure 1'. Photodecomposition rate of 4-CPA (O ), and simulta neous formation of p-chlorophenol ( ), at an initial pH of 8.0. ch ain an d th e rep lacem en t o f chlorines by h yd roxy ls; sim ilar resu lts also w ere o b tain ed from irrad iatio n o f chlorop h en ylacetic a c id s (C ro sb y an d L eitis, 1969). Few p h o to ly sis ex p e rim e n ts h ave b een co n d u cted w ith ch lo rin ated p h en o xy acetic acid s usin g th e low -energy uv ligh t ch aracteristic of n atu ral su n ligh t. 4-C P A is a w idely u sed grow th regulator an d the sim p lest o f th e ch lo rin ated p h en oxy acetic ac id s, m ak in g it an ideal m odel. T h e p u r p ose of th is in vestigation w as to exam in e the effect o f n at ural and sim u lated sunligh t (300-450 nm ) on d ilu te aq u e ous 4-C PA solutions an d establish a general m echan ism for the environm ental photolysis of phenoxy acid s. EX PER IM EN T A L SECTIO N M a te r ia ls. p-B rom oph en ol, p-ch loroanisole, p-chlorobenzonitrile, p-ch loroph en oxyisobutyric acid , p-chloroph en ol, p-cyan oph en ol, hydroquinone, p-h ydroxyph en oxyacetic acid , and phenol were com m ercial p ro d u cts used w ith out furth er pu rification . 4-C PA w as recrystallized three tim es from benzene, m p 157-158*. H u m ic acid w as prepared previously by C rosby and T u ta ss (1966). p-C hlorophenyl form ate w as prepared accord in g to V an E s an d S te v e n s (1965); m a ss sp ectru m m /e 156 (M + ), 127 (b ase , M + - C H O ); infrared (ir) sp ectru m 1200, 1757 (O C H O ) c m -1. p-C yanophenoxyacetic acid w as obtain ed by th e reactio n o f ch lo ro acetic ac id w ith p -cy an o p h e n o l in d ilu te b ase, m p 177-178* |H ay es an d B ran ch (1943) re ported 178.2-178.5*). Ir r a d ia tio n s. S ta n d a rd solutions o f 4-C P A (400 m g/1.), a d ju ste d to pH 8 w ith aq u e o u s so d iu m h y d ro xid e, w ere ir r a d ia te d for 6 5 -1 0 0 hr w ith uv ligh t (3 0 0 -4 5 0 n m ) in the p re se n c e o f e ith e r a ir o r n itro gen in` a 2-1. b o ro silic a te g la s s reactor (C rosb y an d T a n g , 1969) or w ith n atu ra l su n ligh t fo r 7 -1 0 d a y s d u rin g A p ril an d M a y in D a v is. C a lif. T h e brow nish-yellow irrad iated solu tio n s (p H 3 -4 ) were n e u tralize d w ith d ilu te aq u e o u s b ase an d e x tra c te d three tim e s w ith eth er; th e com b in ed o rgan ic p h ase s were w ash ed w ith w ater, d ried over sod iu m su lfa te , an d e v a p o rated to a sm all volum e on a rotary evap orator (n eu tral fraction ). T h e aq u eou s ph ase w as acidified to pH 2. e x trac te d w ith eth er, an d the e x tract d ried an d e v ap o ra te d a s above (acid ic fraction ). T h e aqueous portion rem ainin g a fte r th e secon d e x trac tio n w as evii|>orated alm o st to d ry n ess and taken up into 7:3 (v /v ) m ethylene ch lo rid e-ace tone (in soluble traction ). 0o W 556858 d etecto r as describ ed previously (M oilnnen an d C rosby, 1972). M ost se p aratio n s em ployed 2 ft x in. stain less ste e l colu m n s p ack ed w ith eith er 1% D E G A or 6% S E 3 0 on 60-80 m esh C h rom osorb G , helium Row rale 25 m l/ m in , an d an oven tem p eratu re program m ed from 75* to 2 0 0 * a t 5 */m in . A c id s w ere m eth y lated w ith eth ereal d ia zo m eth an e before ch rom atograph y. O th er aq u eou s so lu tio n s were irrad iated a s follow s: (A ) p-chlorophenol (100 m g/1.), ad ju sted to p H 8, irrad iated for 16 h r; (B ) 4 -C P A (400 m g/1.) in 1% so d iu m b isu lfite, ad ju ste d to pH 8, irrad iated for 68 hr; (C ) p-chlorophe n oxyisobu tyric acid (200 m g/1.), ad ju sted to pH 8, irra d ia te d for 64 hr; (D ) 4 -C P A (10 g/1.) in 5% p o ta ssiu m c y a n id e co n tain in g 0.5% p o ta ssiu m h yd roxid e, irrad iate d for 76 h r; (E ) 4-C P A (400 m g/1.) in 0.5% p o tassiu m brom ide, a d ju ste d to pH 9, irrad iated for 65 hr. R a te M e a su re m e n t. A queous solutions con tain in g 400 m g/1. o f 4-C PA an d ad ju ste d to pH 8 or 2 were irradiated for 200 hr. T h e tem p eratu re w as m ain tain ed a t a constant 3 5 * w ith a h eatin g tap e , an d th e voltage su p p ly to the uv ligh t w as stab ilized w ith a regu lator. N o atte m p t w as m a d e to control p H . A liq u ots (50 n d f w ere w ithdraw n at in te rv als, acid ified to p H 2, an d ex tracte d w ith eth er, p- C h lo ro p h en o l w as d e term in ed b y glc, .while u n re acte d 4-C P A w as sim ilarly determ in ed after m eth ylation . A n a ly tic a l v a lu e s w ere e stim a te d from a sta n d a r d curve for each com pound. R ecovery from un irradiated stan d ard s w as quan titative. Id e n tificatio n . Iden tification of all com poun ds except 2 ,4 '-d ih y d ro x y b ip h en y l w as b a se d on c o m p a riso n s o f p h y s ic al p ro p erties an d sp e c tr a o f fractio n s iso lated by glc w ith th o se o f au th en tic sta n d a rd s. 2,4'-D ih y d roxy b ip h en yl e lu te d a t 200*: m p 162-163* [O m ura an d M a tsu u ra (1969) reported 162-163*); m a ss spectrum m /e 186 (b ase and M + ); ir spectru m 3600, 3400 (O H ), 1600, 1525 ( ), 835 ( p - 0 ) , 7 7 0 (o - 0 ) c m " 1. R E SU L T S AND D ISCUSSIO N W hen dilute aqu eou s solutions of 4-C P A (sodium salt) w ere irrad iated in air w ith eith er uv light in the lab o rato ry o r w ith n atu ra l su n lig h t, th e n eu tral e x tract yielded p rim arily p-ch lorophenol (III) alo n g w ith p-ch loroph en yl form ate (II) an d phenol (V ). T h e acid ic ex tract contained o n ly p -h y d ro x v p h e n o x y a c e tic a c id (V II) a n d u n re a c te d 4- C P A . S u b seq u en t irrad iation o f p-ch lorophenol yielded ph en ol, hydroquinone, h u m ic acid , an d a sm a ll am ou n t of 2 ,4 '-d ih y d ro x y b ip h en y l (E x p e rim e n t A ). T h e p oly m eric h um ic acid w as insoluble. A s w ith 2,4-D (C ro sb y an d T u ta ss, 1966), in h ib ition of the fin al au toxidation ste p by sodium b isu lfite perm itted iso latio n o f p-ch lorophen ol, p-ch loroan isole, h ydroquinone (V I), an d ph enoxyacetic acid (IV ) (E xperim en t B ), al though control exp erim en ts show ed th a t 4-C P A also react ed w ith sod iu m b isu lfite in the d ark to form p-chlorophe- nol an d the p-ch loroanisole. T h e ra te s o f p h o to d e co m p o sitio n o f 4 -C P A in b a sic so lution (pH 8) an d the form ation of p-chlorophenol are il lu str a te d in F ig u re 1. T h e h ig h e st o b se rv e d y ie ld s o f p- chlorophenol an d p-ch lorophenyl form ate at an y given 00 tim e w ere 12 a n d 3% , resp ectiv ely , w hile th e oth er prod- I> * u cts w ere present in m uch sm alle r q u a n titie s. T h e photo- d e c o m p o sitio n ra te o f 4 -C P A in ac id ic so lu tio n (p H 2) w as a b o u t h a lf th a t in th e b a sic so lu tio n . U n d er lab o rato ry con dition s, the tim e for total d isap p earan ce of 4-C PA w as 250-3 0 0 hr, w hile an eq u al am ou n t o f p-ch loroph en ol d e c o m p o se d in h a lf th a t tim e . T h e se ob servation s su ggest th at the ph otodecom position re a c tio n s o f 4-C P A in w ater con sist o f th ree tyi>es: o x id a tiv e sid e chain reactio n s, ionic ring reactio n s, an d con d en 20,QUi267satio n s. S id e C h uin R e a c tio n s. T h e rate-determ in in CKOSHY, WONG o c h 2o 2' Cl o q + H2 0 < - *H + O H * o q + 2 H* --------------- - * 0 2 H H + 0 2 H h 2 0 2 0 b e D O W 556859 0X CM 1 o c0coo OH Cl Cl Figure 2. Mechanisms of 4-CPA photooxidation. Cl m e q + h 2 0 2 -------------- -- . O H + O H * d h 2 0 2 -------------- - Figure 3. Radical generation Irom solvated electrons. OCHO w hose ra te varied d irectly w ith pH . T w o o x id a tiv e m e c h a n ism s are plausible (Figure 2): (a) the norm al au toxid ation o f an eth er (W alling, 1957) follow ed by rearran g e m en t o f the resultin g peroxy radical and hydrolysis; and (b ) oxygenation of the radical generated by loss o f a h y d rated electron an d carbon dioxide from 4-C P A anion (Jo sch e k an d G rossw einer, 1966), ag ain follow ed by rear"ran gem en t. The second m echanism is preferred: the pre d icted in term ed iate II w as iso lated , an d rep lacem en t o f th e m eth y len e h yd rogen s o f th e p h en o xy acetic acid w ith m eth yl groups (p-chlorophenoxyisobutyric acid ) still p er m itted th e efficient form ation of p-chlorophenol upon ir rad iatio n (E xperim en t C ). However, the form ation o f trac e s o f p-chlorobenzonitrile during irrad iation o f 4-C P A in the presen ce of cyan ide ions (E xperim en t D ) su g g e sts th a t the p-ch loroph en ol ob served in n o rm al 4 -C P A p h o to l y s is co u ld be fo rm ed , in p a rt, by n u cleo p h ilic d is p la c e m e n t o f th e sid e ch ain by h yd roxide ion. T h e in tram olecu lar rearrangem ent, proposed to involve a ra d ic a l p a ir en clo sed in a solven t cag e, w hich p ro d u ced th e o- a n d p -h y d ro xy p h en y lacetic a c id s d u rin g irra d ia tio n o f 4 -C P A in eth an ol at 254 nm (K e lly an d P in h e y , 1964; K e lly et a i , 1969) w as not ob served in o u r e x p e rim e n ts (300-450 nm in aq u eo u s so lu tio n s), p e rh ap s d u e to the presen ce o f oxygen. An ionic process also h as been su g gested for the rearrangem ent (C oppinger an d B ell, 1966) b u t w as not in eviden ce. R in g R e ac tio n s. T h e m ost ch aracteristic ring reaction w as th e pH -d epen dent replacem ent o f the chlorine by-h y droxyl or by hydrogen. T h ese reactions w ere not affected b y or dependent on oxygen, although sim ilar processes h ave been considered by Jo sch ek and M iller (1966) to in volve free ra d icals. W hile the in ten sity o f sh o rt w ave len g th u v ra d ia tio n in su n lig h t is low , th ere w ou ld be s u f ficien t q u an tu m energy n ear the atm osph eric cu to ff (100 k eal/m o l at 286 nm ) to break the carbon-ch lorine bond (ab o u t 80 k eal/m o l) b u t ap p aren tly n ot en ough for h om oly tic d isso ciatio n o f w ater (116 k e a l/m o l). Furth er, th e rep laced chlorine api>carcd a s C l ' rath er than CI2, an d our s|>cci[k search failed to reveal b ip h en y ls. I lydroxy latio n ration alized on the b asis of som e en ergetic oxygen atin g species such a s those generated by the hydrated electro n (F ig u re 3) is not con sisten t w ith th e lac k o f an oxygen dependence. T h e source o f hydrogen to produce th e d cch lo rin ated p ro d u cts IV an d V II h as n ot been id e n tifie d . T h e reaction is licllcr explain ed a s a p b oton u d eop h ilic s u b s titu tio n (H a v in g a a n d K ro n e n lie rg , 196.8) in w h ich >iydroxide ion d isp lac e d ch loride from th e p h o to exictcd rin g, a p rocess alread y known to occur u n der high-energy irrad iatio n (N ijhofT an d lia v in g a , 1965). T h is m ech a n ism is sup|M iried by ou r oliservniion that 4-C P A reacted re a d i ly w ith c y a n id e io n s ii|Min ir r a d ia tio n (K x |H *riin en t l>) to form p-eyanophenot an d />-cynnophenoxyacctir acid a n a l ogou s to the sim ilar reaction of p-chlorophenol (O n iura 0Ct -- 0c. " / I\ 0 - - 0 - 0OCH.COOH O C H -C O O H OH OH OH tc 0CH.C00Hr ochcT P olym er $-0 he Figure 4. Proposed route of phenoxy acid photodecomposition based on 4-CPA photoproducts. an d M atsu u ra, 1969); a sm all am ou n t o f p-chlorobenzoni trile resulted from p b oton u deoph ilic d isp lacem en t o f the o xy acetate sid e ch ain (eq 1). In th e d ark , 4-C P A form ed trac e s o f p -cy an oan isole an d p-ch loroben zon itrile; it re a c t ed w ith p o tassiu m b ro m id e in th e ligh t (E x p e rim e n t E ) to give p-b rom oph en ol b u t w as u n reactiv e in th e d ark . C o n d e n satio n s. W hen the irradiation of aq u eou s p- chloroph en ol w ith a low -pressure m ercu ry arc la m p w as con d u cted a t su fficien tly high con cen tration s (> 1 % ), hy- droxylated biphenyls were produced (O m u ra an d M a tsu u ra , 1969). H o w ever, o n ly tr a c e s o f 2,*4'-d ih y d ro x y b ip h e n y l w ere d e tected a t th e low co n cen tratio n s o f p-ch loroph en ol in o u r e x p e rim e n ts (E x p e rim e n t D ), an d n on e w as form ed from 4-C P A . T h e ex p ected 4-ch loro-2,4'-dih yd roxy b iph cn - yl w as not observed at all. Form ation of the polym eric hum ic acid s represented the e v en tu al fate o f 4-C P A a s it did w ith o th er co m p o u n d s th at can be converted to quinoid produ cts (C rosby an d f)7 7 9 L c itis, 1969; C rosb y an d T u ta ss, 1966; M oilnncn an d C rosb y , 1972). U n der field con dition s, th is poly m erizatio n p ro b ab ly w ould involve both ionic an d o x id ativ e co m b in a tion w ith oth er su b sta n c e s in c o n ta c t w ith th e q u in o id in term ed iates to form a variety o f polym er typ es rep re se n tin g , in p a rt, th e c arb o n a to m s o f th e o rig in al 4 -C P A rin g . _ T h e p r o d u c t s i d e n t if ie d a n d t h e m e c h a n i s m s p r o p o s 0 0 "i O C 0 for th eir form ation su ggest the ph otolysis path w ay for * ' O u CIIOSUY, WONC 4 -C P A show n in F ig u re 4. H ow ever, th e clo se an a lo g y b e tw een these produ cts and those obtained from the ph otol y sis of 2,4-D (C rosb y an d T u ta ss, I960) an d 2.4,5-T (2.4,5-trich loroph enn xyacetic acid ) (C rosby an d W ong, 1973) in d ic a te s th a t th e p a th w ay w ill be g e n e ra l for the other com m ercial phenoxy h erbicides. AC K NOWLEDG M KNT T h e technical assistan ce o f G . F . M allet and C . J . Sode rq u ist in a sp e c ts o f th is w ork is gratefu lly ack n o w led g ed . LITERATURE CITED HCCCCCrrrraooooovpssssipbbbbnyyyivgn,.,.ag,DDDDE......GGGGG, ...K,,..,. rMTWTLoeaun.oni.tlenagBinsgs,e.b.slC.AEel.rH...g-E.SS.,./O.M...,RJA./....,.gJEAJAr....gg,AFrPrPg..ohruFoF.yrdosoFe.ooACoddCophdhCCepemhthCm.ee.hCm.me17h.m.70e,12.1,m73101,4..31417160.095645(9,11(216193(9(6(1176199969)(67.)169.396))6)...8). Hnyes, N. V.. Branch' G. E. K.. J. Amer. Chem. Soc. 65, 1555 (1943). Joschck. H.-I.. Grossweincr, L. 1.. J. Amer. Chem. Soc. 88, 3201 (1966). Joschck, H.-l., Miller, S. Kelly, D. P,, I'inhey. J .T Kelly, D. I\, Pinhcy, J . .I.T..T,,Je.RtAriamghbeeyrd..roCRn.helDm.c..ttGS. o.3,c4.A278u8n(,1t3.926J64.8).C(1h9e6m6.). 22, 977(1969). Moilanen, K. W,, Crosby, D. G., J. Agr. Food Chem. 20, 950 VONaim(jnh1u9nrE7faf2s.,),.KDA..,.F,M.,SaHttesavuveuinnrsag,.aT.WE.,,,,,CTRheeetemrla..hCTeodramrocm.nuLCneh.tit1m.34.414F9(9a1y9(s61.99H6).a5)s. 85. 1247 (1965). Walling, C., " Free Radicals in Solution," Wiley, New York, N. Y. 1957, Chapter 9. Received for review May 9, 1973. Accepted September 12, 1973. Presented at the Division of Pesticide Chemistry, 160lh National Meeting of the American Chemical Society. Chicago. III., Sept 1970. Supported in part by NIH grant ES-00054 and USDA Re- _ gional Research Project W-45. w O $ I Photodecomposition of 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T) in Water- D on ald G . C ro sb y * an d A nthony S . W ong cn cn 05 00 C5 P h o to d eco m p o sitio n o f the h erb icid e 2,4,5trich lo ro p h en o xy acetic acid (2 ,4 ,5 -T ) in aq u eo u s solu tio n prin cip ally involved cleav age o f th e eth er bond an d rep lacem en t o f the ring chlorines by hydroxyl an d by hydrogen. T h e m ajo r products w ere 2,4,5-trich loroph en ol an d 2-hydroxy-4,5dich loroph en oxyacetic acid ; 4,6-dichlororesorcinol, 4-chlororesorcinol, 2,5-dichlorophenol, an d a d ark polym eric product also w ere isolated . T h e to x ic 2,3,7,8-tetrach lorodiben zo-p-dioxin w as n ot d etected am on g th e ph otodecom position p ro d u c ts. 2 ,4 ,5 -T ph otolyzed very slow ly com p ared to its 4-chloro an d 2,4-d ich loro an alo g s, b u t th e 11fold in crease in ph otolysis rate cau sed by se n siti zatio n w ith aceton e or rib oflavin su g g ests th a t su n lig h t c an b e an im p o rta n t fac to r in the e n v i ro n m en tal d egrad atio n o f 2,4,5-T . F o r alm o st th ree d e c ad e s, 2,4,5-trich loroph en oxyacetic a c id (2 ,4 ,5 -T , 1) a n d it s d e r iv a tiv e s h a v e re c e iv e d w id e u se a s h erbicides, esp ecially for control o f b ru sh , ju n gle, an d aq u atic w eeds. F o r ex am p le, th e 1968 U . S . production of th ese co m p o u n d s exceed ed 60 m illion p o u n d s (U . S . T a riff C o m m issio n , 1970), a n d re sid u e s h ave b een d e te c te d in crops, soil, the atm osph ere (B am esberger an d A d am s, 1966), rain w ater (C oh en an d P in kerton, 1966), an d su rface w aters (T h o m an an d N icholson , 1963). T h e fate an d p er sisten ce o f 2 ,4 ,5 -T in th e en vironm ent h ave been the su b ject o f con siderable in vestigation (L oos, 1969; P S A C , 1971); th is rep ort d e a ls w ith th e in flu en ce o f su n ligh t, n atu ral an d sim u late d , on aq u eou s solu tio n s o f 2,4,5-T and its salts. EXPERIMENTAL SECTION M a t e r ia ls . 2 ,4 ,5 -T , 2.4 ,5 -trich lorop h en o l (II), 4,6d ich lororesorcin ol (III), 2,5-d ich loroph cn ol (IV ), an d 4chlororcsorcinol (V ) w ere purified com m ercial prod u cts. 2.4.5- T w as rc c ry stalliz e d th ree tim e s from b en zen e, m p 153.5- 5 4 .5 *. T h e trichlorophcnol w as freed o f persisten t im p u ritie s b y d isso lv in g it in a q u e o u s so d iu m h y d ro x id e solu tio n , ex trac tin g n eu tral im p u rities w ith sev eral por tions o f benzene, acid ification , rccrystollization three tim es from iienzcnc, an d subliin ution un der redu ced p res sure, m p 6 7 .5 \ D ep artm en t of E nviron m ental T oxicology, U n iversity of C a lifo rn ia a t D avit.. D a v is. C a lifo rn ia UGlli. Ir r a d ia tio n . Solu tio n s o f 2 ,4 ,5 -T (100 m g/1.) in d istilled w ater w ere a d ju ste d to p H 8 w ith 0.1 N so d iu m h yd roxid e an d irrad iate d o u td oors w ith su m m er su n lig h t in D av is, C alif., or in doors w ith u ltrav io let (u v) ligh t in the w ave len gth region o f 300-450 n m . T h e irrad iatio n s w ere con d u c te d in 3-1. b o ro silic a te g la s s fla sk s, th o se in d o o rs m o u n te d in a 14 in . ( i.d .) x 5 ft c y lin d rica l c h a m b e r (F ig ure 1) con stru cted o f T ra n sit p ip e cu t in h alv e s lon gitu d in ally , h in ged, an d lin ed w ith brigh t alu m in u m sh eet. T h e ch am b er w as fitted w ith six 4-ft F 4 0 B L flu orescen t uv lam p s (C rosb y an d T a n g , 1969), three clam p ed to the fix ed h alf an d three to th e hinged door. L igh t in ten sity m e a su re d w ith a Y S I rad io m eter (Y ello w -Sp rin g s In stru m en t C o ., Y ellow S p rin gs, O hio) w as 390 pW /cm * a t th e center o f th e ch am b er an d 785 pW /cm 3 halfw ay from th e center to the lam p su rface. T h e ch am b er tem p eratu re rem ained close to 30*, an d filtered a ir w as p assed through the so lu tion s to m ain tain oxygen satu ratio n and efficien t a g ita tion . T h e rem ovable hood an d vent for toxic com po u n ds w ere n ot u se d in th e p resen t e x p e rim e n ts. T h e in door irrad iatio n w as allow ed to proceed for 200 hr, an d 50-m l aliq u o ts w ere w ithdraw n a t in terv als an d an alyzed by gas-liq u id chrom atography (glc). E qu ivalen t ,, ^ 0 D Q Ue x p e r im e n t s w e re c o n d u c t e d w it h : (A ) 2 , 4 ,5 - T in 0 .5 % ( aq u eou s sodium b isu lfite; (B ) aq u eou s 4-chlorophcnoxya- c e tic ac id (4 -C l'A ) a t pH 8 ; an d (C ) aq u e o u s 2,4-dichloro- p h en o x y acetic acid (2,4-D ) a t pH 8 , a s w ell a s in th e d ark . T h e ex|)o su re tim e for th e ou td oor p rep arativ e scale e x p e rim e n ts a m o u n te d to a b o u t 9 hr e a c h d a y fo r 6tftiA >'A 7 < ) a a (540 hr). ^ v w < iC 0 9 tozA 6781 POVV718198 ENVIRONMENTAL IMPACT OF HERBICIDES Herbicide Study Group, HMAC, EPA Eugene E . Kena ga Ag-Organics Department The Dow Chemical Company Midland, Michigan 48640 January 5, 1973 SUMMARY In April, 1972 at the request of the administrator of the Environmental Protection Agency, the Hazardous Materials Advisory Committee was asked to appoint an expert committee called a "Herbicide Study" group to determine and write a report on whether herbicides were being evaluated properly and impartially in regards to cost-benefits to man and hazards to the environment. Areas of insufficient knowledge were to be pointed out. E. E. Kenaga was subsequently appointed to the "Environmental Impact" subcommittee. Kenaga's input, but not necessarily what will be issued in the final committee report, is covered here. Sub jects discussed are physical properties of widely used herbicides, herbicide residues in the environment, toxicity of herbicides to wildlife and a summary of the evaluation of the uses and hazards of herbicides in various habitats. 0G04GS4 6782 INTRODUCTION In April, 1972 I was appointed as a private citizen to the Environmental Subcommittee of the "Herbicide Study" group, sponsored by the Hazardous Materials Advisory Committee (HMAC) of the Environmental Protection Agency (EPA). The task of the "group" initially was to study the chemistry, toxicology, crop and other uses, and environmental impact of the most commonly used herbicides. Upon deciding which were the most heavily used pesticides (see Table 1) in the U.S. my initial assign ment was a section on residues in the environment. Assignments were given to other people and their reports were reviewed. I was not satisified with the wildlife toxicology data section, or the summary. Also needed (I thought) was a table of the physical properties, all of which I furnished. Thus, the information given here, while not a complete treatise on the subject, does represent my input to the Environmental Subcom mittee. The "Herbicide Study" report is still being written and revised and there is no guarantee that my information will be recognizable. I don't believe there is any "classified" information in my report. However, I believe that it is possibly of use to the herbicide research and development people as well as others so I am making it available. STRUCTURE AND PHYSICAL PROPERTIES OF SOME HERBICIDES USED IN LARGE QUANTITIES IN THE U.S. The compounds in Table 1 are those selected to have potential environmental impact because of their widespread use in the United States. Important physical properties given are those C0G4CS3 OOV/718200 3- - related to vapor pressure (volatility) and water-organic solvent partitioning Tatios (solubility data) of these herbicides: acrolein, alachlor, butylate, and propachlor are most volatile. Nitralin and trifluralin are the compounds which partition most strongly to organic solvents in preference to water (a factor related to bioaccumulation). See Table 1. RESIDUES OF HERBICIDES IN THE ENVIRONMENT The important use of residue data is the matching of residues in the environment with toxicity to plant and animal organisms. Persistence of residues and their decline should be related with toxicity which takes these factors into account. The level of residues of herbicides in the environment is related to dosage applied, number of applications, method of application, type of herbicide formulation, habitat, wind, sunlight, oxy gen, rain, soil type (especially to organic matter, clay minerals, pH, temperature), microorganisms (aerobic or anaerobic) and time after application, etc. Relative values of these residues can be predicted to a certain degree from a correlation of the physical, chemical and biological properties of the different kinds of herbicides with the above environmental variables and with herbicide uses and application methods (Freed, 1966; Van Middelen, 1966; Kenaga, 1968) . Determination of residues of herbicides must include the possibility of molecular transformation such as the hydrolysis, oxidation and reduction products of the chemical, if any, and metabolites, dgradtes, etc. Also, consideration must be made of the ingredients of the formulation and impurities 0784 0004088 TH70T/ M On 4- - present whether due to manufacture, packaging, or storage of the material. One need only to trace the development of pesticides such as DDT (and its more stable metabolite DDE), parathion (and its potent cholinesterase inhibitor paraoxon), heptachloro (and its more toxic oxidation product heptachlorepoxide), and 2,4,5-T (and its toxic tetrachlorodibenzop-dioxin trace impurity) to realize the importance of the above determinations. Reactions associated with microbial metabolism of herbicides include N-dealkylation of urea; ester or amide hydrolysis of carbamates; side chain degradation of the 5-triazine; dechlorination of 2,4-D and dalapon; etc. (Kearney, 1966). Not all herbicides involve metabolism directly to smaller molecules. For example, propanil converted microbially to 3,4-dichloroaniline, is thought to form a larger molecule by diazotization by the nitrate ion in the soil (Rosen, 1972). Few pesticides, including herbicides,have been subjected to detailed analysis for product impurities in the quantitative order of 1 ppm. Bioaccumulation of Residues The use of the terms bioaccumulation and bioconcentration often conjures up the image of the type of residue accumulation which occurs with DDT in animal organisms, and especially "785in their fat tissues. The bioconcentration factor (bio- 0GG4GS7 5- - yr\yoTfAAr> magnification) is the ratio of the measured residue held by animals or plants {or specific "tissues thereof) compared ( to the residues of the pesticide in the ambient air, water, or soil environment of the organism and/or the various species of food organisms consumed, as specified (Kenaga, 1972). The highest bioconcentration factors with insecticides usually occur in water. DDT and its degradate DDE may have a bio concentration factor of a million. Herbicides such as picloram (Hardy, 1966), silvex (Getzendaner, 1960), 2,4-D (Cope, 1970; Smith and Isom, 1970), and dichlorobenil (Cope et al., 1969) do not appear to bioconcentrate in fish or aquatic organisms more than ten-fold and often less than one-fold the concentrations found in water where fish were present. Diuron may have a bioconcentration factor of several hundred (McCraren et al., 1969). Although oysters and clams may accumulate a few ppm of 2,4-D in herbicidally treated areas such as Chesapeake Baj^ they cleanse themselves of residues before harvest time (Frank, 1971). Butler (1965) exposed oysters continously for 7 days to 0.1 ppm of 2,4-D, butoxy ethanol ester. The oysters accumulated 18 ppm and cleansed themselves in 7 days with fresh seawater. However, generally not much residue data is available for invertebrates. Fish may retain residues of some herbicides for weeks in treated ponds (McCraren et al., Cope et al., 1969). There appears to be little herbicide residue data pertaining to wild mam mals and birds, however domestic animal and bird tissues appear to be free of significant residues for human use. o786 CG04GS3 6- - The important fact, as far as chronic toxicity is con cerned is not how large the .bioconcentration factor is, but now much actual residue (in terms of ppm) bioaccumulates. While the terms bioaccumulation and bioconcentration are often applied to animals, it may also apply to plants, although it is rarely used in such context. Generally speaking, the properties of a chemical used to penetrate and kill insects are quite different than those needed to penetrate and kill plants. Among other things, insecticides are often quite fat soluble (in order to penetrate insects) and low in water solubility, thus stable compounds may be bioaccumulated in animal fats. Most herbicides tend to have greater water solubility and/or do not partition as greatly in favor of fat tissues as does DDT and other organochlorine insecticides. Further degradation of most herbi cides lead to even more water soluble products, for example, atrazine to hydroxy atrazine, and prometryne to hydroxypropazine (Frans et al., 1972). An insecticide like DDT is not systemic in plants and therefore does not accumulate inside the plant. However, many herbicides penetrate plants, and are systemic, and thus redistribute and accumulate residues especially in the growing portions of the plants, including leaves and roots. Since residues on treated plant foliage are often determined for the whole leaf, it is not then possible to determine the differences between surface residues and internal plant residues. 00040S3 *n?QT/MOCI 7- Assuming an even coverage of herbicide on the surface of various plants, the residues vary greatly ia ppm since the surface area to volume ratios of the different parts of plants and different species of plants vary greatly. In a literature study of many pesticides, maximum residues of pesticides were calculated on a one pound per acre basis by dividing the resi dues determined by analysis, by the dosage in pounds per acre applied (Hoerger and Kenaga, 1972). Such maximum residues, immediately after application, ranged from 6.6 ppm for fruits to 240 ppm for foliage such as grass, depending on the sur face area to volume ratio (size and shape of vegetation sprayed). The highest amounts of residues occurred with systemic herbi cides and insecticides on foliage. Most residues were far less than the maximum residues cited above. All residues de clined within days after application although some persisted for months at levels of analytical sensitivity. Duggan et al. (1971) reported on pesticide levels in foods and feeds in the United States from 1963 to 1969. The only herbicides found were 2,4-D, MCP, and dacthal. None were found commonly, or in amounts more than ppb quantitatively. Residues in Soil A major problem in working with herbicide residues in soil is the accurate measurement of actual amounts of the com pound and/or its degradation products in varying time periods after application. Very few studies are made which DOW718205 8- - include a number of herbicides tested comparatively in the field. A southern -state regional cooperative study (Frans et al., 1972) showed that no phytotoxic residues of atrazine, chlorpropham, DCP^diphenamid, diuron, linuron, norea, prometryne or trifluralin persist in soil after one year when the herbi cides are applied at recommended application rates, however, no analytical residue data accompanied this phytotoxicity data. Herbicide residues are known to persist longer in the colder areas of the United States. In Oregon, studies of chemical brush control with 2,4-D, amitrole; 2,4,5-T and picloram showed residues of picloram to persist for more than a year while the rest were more rapidly degraded (Norris, 1971). Analytical work shows that even though phytotoxicity may not occur on the target crop in soil treated year after year, residues of the herbicide (and dgradtes) may still be bound to soil and may be subject to release under certain circumstances. The nature and mechanism of binding and releasing of pesticide residues held by lignin, humus, cellulose, and other organic matter is uncertain and residues may sometimes be a problem in the rotation of more herbicidally sensitive crops. Organic material (principally humus) in soil is the major soil constituent which reduces leaching because of strong adsorption. Two major components of humus may act entirely 6789 OCC4G30 flO^STiMOa -9- different. For example, sodium humate apparently sorbs and solubilizes the nonpolar compound DDT, while humic acid strongly sorbs the polar compound 2,4,5-T from solution (Vershaw et al., 1969). Organic material is capable of sorbing most organic compounds from water, thus in practice, leaching which causes phytotoxicity away from the treated area is a rare problem even when water soluble herbicides are applied to organic soil. There is no compound which does not leach to some degree. Anderson et al. (1968) discussed the various factors related to leachability, relating it to soil pH, mineral, and organic content. Two closely related compounds, trifluralin and nitralin, vary greatly in leach ability. Even mineral soils are capable of strong sorption such as with phenolic and phenoxyacetic acid herbicides. Sorption of such compounds is near a maximum when the cal culated ionization is about 15 to 30% and when the pH approaches the pKa of the herbicide in water (Miller, 1972). Herbicide residues in bottom muds of cold lakes or those of low oxygen content, may persist for months (Smith and Isom, 1971). The relative order of persistence of the subject herbicides has been reported in the Herbicide Handbook of the Weed Society of America (1970) and by Sheets and Harris (1965). The most persistent herbicides of this group are atrazine, diuron, linuron, nitralin, propazine and trifluralin, ail of which may be phytotoxic for over 2 months (see Table 2). 0G04G31 6790 - 10- I Residues in Water The most common .herbicides used for -control of aquatic weeds are xylene, copper sulfate, 2,4-D, acrolein, endothall and diuron (Anon., 1971). Very few materials are registered for this use because of the risks that the manufacturer takes in such use. The main sources of herbicidal residues in water are from direct application, aerial drift from application, run off from land, and from control of ditchbank vegetation (Frank, 1971). Johnson et al. (1967) discussed the pesticide residue monitoring studies carried on by the various U. S. agencies and concluded that a number of insecticides were important residue indicators, but none were herbicides. Eleven streams in western United States were monitored for nine insecticides, 2,4-D, 2,4,5-T, and silvex. No herbicide residues were found (Brown and Nishioka, 1967). Dosages of most herbicides applied for aquatic weed control are initially in the order 0.1 to 4 ppm in water, except for aromatic solvents (xylene) which are as high as 600 to 700 ppm. The latter residues, however, are highly volatile and diminish rapidly in water. Residues of many herbicides are detectable (>0.1 ppm) for days or weeks after application in treated pond water, but are rarely greater than 0.5 ppm 6791 0004092 - 11- within a few days (Table 4, Frank, 1971). Fenac, 3-aminotriazole, and dichlobenil appear to b more persistant (Grzenda et al., 1966; Van Valin, 1966). Herbicide con centrations in excess of 0.1 ppm are seldom encountered in streams close to treatment areas, even immediately after spraying (Norris, 1971). pn^RT/MOa Interpretation of analytical data concerning pesticide residues in water can sometimes be misleading expecially if the water contains organic or other particulate matter. In such cases, the herbicide may be strongly adsorbed to solids suspended in water by the same mechanism by which they are sorbed on particles in soil, and thus may exaggerate the true amount of herbicide solubilized in water, if partic ulates are not first filtered out. In such cases there may be more herbicide on the suspended particulate than sol ubilized in the water. The relative mobility of pesticides in soil leaching experiments show phenoxy and picloram herbicides to be most mobile, fol lowed by a group of miscellaneous herbicides, then by phenyl urea, triazine and other related herbicides, and then by CIPC and toluidine herbicides. Insecticides, especially chlorinated hydrocarbons were least mobile (Norris and Moore, 1970). It is important to determine the half-life of herbicide residues in water. This will vary considerably depending n o 6792 DOW718209 - 12- on temperature, ultraviolet light, pH of the water, aeration, size .and nature of the particulate matter in the water, the amount of vegetation in water, and surface-depth relation ships of the body of water treated, etc. The half-life of chemical residues in water is not necessarily linear with time, depending on the effects of various concentrations of the herbicide on degradative organisms and other factors. A number of chemicals which are persistent in soil are less persistent in water due to the action of ultraviolet light. A herbicide such as picloram which is resistant to degrada tion in soil is more easily decomposed by sunlight in water with half-lives ranging from 2 to 41 days depending on water depth, amount of sunlight, and water quality (Hedlund, 1970). More studies on various herbicides concerning de composition by sunlight are needed to help calculate a half-life or a "disappearance rate" from water. Residues in Air Herbicide residues in air may be from three sources: (1) those caused at the time of application of the pesti cide, such as occurs with spray and dust drift. Only 20-80 percent of the pesticides dispersed in air may reach the target (Norris and Moore, 1970); (2) those related to dis persal of pesticides on particles due to wind erosion after application; (3) those related to gaseous dispersion because of volatility from treated areas (soil, water, plants, etc.) GGO^OS** 6793 - DOW718210 - 13- or other areas contaminated due to the above three mechanisms-. It is generally supposed that items 1 and 3 are most important with herbicides. Very little work has been done concerning the measurement of residues of herbicides in air except near the point of application. Airborne concentrations which are harmful to non-target crops are most frequently due to drift at the time of application of the herbicide to the target. Usually residues on plants contaminated from drift are far less than those on the target species of plants, however, sensitive plants such as cotton and grapes are particularly vulnerable to 2,4-0 and may occasionally be damaged by residues as much as 15 miles from the point of application under rare weather circumstances, but are most frequently important within the nearest 500 feet (Van Middelem, 1966). Herbicide residues due to drift are most closely correlated with the type of equipment and herbicide formulation used, and wind velocity. Most serious drift problems can be solved by modification of equipment and formulations such as granules, inverted emulsions, and spray thickeners, and avoiding small particulates and windy days for application (Butler et al., 1969; Akesson et al., 1971; Akesson et al, 1972). Occasionally during periods of great winds, the accompanying dust storms may deposit herbicides presumably carried on contaminated or treated soil particles over great distances such as from Texas to Ohio (Cohen and Pinkerton, 1966). 0004035 - f 6794 - 14- Even during normal weather particulate matter suspended in air may sorb volatile herbicides as they do DDT, lindane et al., and then be redeposited on bodies of water or on the ground by rain storms some distance from the original source of the particulate or herbicide application (Abbott et al., 1965). DOW718211 Particles of soil, spray droplets, and rain droplets, which are able to be carried by normal wind velocities of 5 mph for over 100 feet vary from <5 to 100 microns in diameter (Reimer et al., 1966). Assuming particles to have nearly equal deposition of the herbicide on treated soil, a particle with a diameter of 10 microns will have a surface area 100 times greater per volume than a particle with a diameter of 100 microns and consequently about 100 times the residue in ppm, assuming no volatility. Particles over 5 microns are not normally expected to be carried very far while particles of 0.5 to 1 micron may be transported on transoceanic trade winds (Risebrough et al., 1968). In order to be carried on small particles in air, and subject to light, wind and rain, a pesticide must be very chemically stable, and low in volatility. Analyses of particles from air have been shown to contain 2,4-D, 2,4,5-T and atrazine in amounts of 10 ppb or less. Residues of herbicides are not known to build up to high levels by air dispersal at great distances from the point of application. They may be of less consequence than DDT 0G04G3S 6795 rf o t > A A O f l - 15- for example, since the herbicides do not appear to build up in the animal food -chain -of life. To a lesser extent than concentrations caused by drift, con centrations caused by volatility may be responsible for damage of sensitive non-target plants surrounding the target plants. The presence of wide spread air concentrations in Washington has been studied with various salts and esters of 2,4-D (Adams et al., 1964). The role of volatility of herbicides is very little understood and is indicated mostly where herbicidal damage occurs on valuable crops. Volatility of 2,4-D varies greatly depending on its form. For example the vapor pressure of the methyl ester is greater than 100 fold that of 2,4-D (Hamaker and Kerlinger, 1969). Volatility of a given herbicide from various treated surfaces varies greatly depending on the weather, formulation, and sorptive capacity of the surface from which the herbicide volatilizes. Generally speaking soil and other surfaces which adsorb herbi cides the strongest, allow the least volatility. Many soils lose a large percentage of a herbicide within a few hours or days by volatility. Little is known of the fate of herbicides or other pesticides in air except that they are rapidly diluted. The dilution factor is the main reason for the difficulty in detecting herbicides in air since the lower analytical sensitivity limits for most compounds are soon reached. 0004037 679G fiTZRUMOa - 16- It is known that sunlight alters the structure of many pesticides especially in the presence of moisture and parti culate matter as occurs normally in air, however, the techniques for simulating these effects are not well estab lished. Compounds like 2,4-D have been shown to be reduced to polymeric humic acids similar to soil humic acids by either artificial light or sunlight (Crosby and Tutass, 1966). In general, the metabolites and dgradtes of herbicides have been shown to be less of a problem than the original com pound. Knowledge of all routes and rates of degradation for all herbicides is incomplete as it is with other pesticides, however, the major facts concerning the eventual degradation of herbicides is quite well known except for soil binding and air degradation. Not much is known about minor impurities formed during the production of herbicides. THE TOXICITY OF HERBICIDES TO MAMMALS, BIRDS, FISH AND INVERTEBRATES The amount of data available for some herbicides is not very much, while on the other hand the data reported here in Table 3 is by no means complete. An attempt was made to find acute and especially chronic toxicity data on all four of the major zoological groups mentioned in the above title. 0C04G3S 6797 DOW718214 - 17- Toxicity data on mammals indicates that only acrolein, copper sulfate, and possibly endothall,; are acutely toxic. Apparently none of the herbicides are chronically toxic at residue levels likely to persist in the environment. Toxicity data on birds indicates that only endothall (no data on acrolein) may be acutely toxic. There is no indication of chronic toxicity to birds at residue levels likely to persist in the environment. Acute toxicity data on fish indicates that several compounds have LC50rs of 1 ppm or less in water. Among these are acrolein (aerates rapidly from water), alachlor, copper sulfate, certain esters of 2,4-D, certain esters and salts of endothall, and trifluralin. Acrolein, copper sulfate and endothall are applied directly to water. None or very little acute or chronic toxicity data was found on insects, shrimp, and oysters for some herbicides such as acrolein, alachlor, butylate, copper sul fate, linuron, nitralin, propachlor, propazine, or xylene. Copper sulfate and xylene were acutely toxic to Daphnia at 0.1 ppm. Trifluralin was acutely toxic to certain species of Daphnia and Gammerus below 1 ppm. SUMMARY STATEMENT FOR THE ENVIRONMENT IMPACT SECTION REPORT, HERBICIDE STUDY, HMAC, EPA When the members of the environmental impact subcommittee turned in their reports (including mine), the chairman (Dr. David Pimentel) assembled a summary. Pimentel's summary pin pointed many adverse effects known to have occurred with herbicides without putting these effects into perspective in regard to the use of the herbicides in crop and non-crop area (ecological niches) and the likelihood of environmental contamination. Since I did not like the chairman's summary, I wrote my own as follows: 0C04033 6798 DOW718215 - 18- "The earth's environment is nearly all managed in part by jnan, either directly through crop production, construction, mining, etc. or indirectly through pollution, elimination by carelessness such as by fires, or overuse of certain parts of the environment and by many other ways. The use of herbicides is only one of man's many technological tools which influence the environment and manage ecosystems. Basic to the use of herbicides is the desire of man to raise essential and sufficient food crops with a minimum of effort. cost,and space. Where food is raised only food plant species are desired and the aboriginal plant and animal ecosystem has often long since been usurped. It is not reasonable to consider food croplands as part of a natural ecosystem or to expect it to maintain the diversity of life fou,nd in uncultivated * areas. The main requirement which might be made of a herbicide on such croplands is that they be used for efficient production of quality food which would not result in non-target damage such as might occur from toxic residues encountered by a diversity of organisms on or off the treated area via contact with air, water, soil or food. Thus, if the use of herbicides result in competitive food production of crops without side effects of economic or non target significance, the criteria for ecological evaluation are not the same as for the use of herbicides on aquatic sites, right-of-ways, parks, and forested areas, which include natural ecosystems. Even in the latter areas herbicides are rarely 0C041G0 6799 o t ? q t ; kkrsn -19- used to eliminate all vegetation, but are mainly needed to control economic pest plants. Herbicides do not always create ecosystems containing less plant species. When used to create right-of-way strips within forest climax areas;herbicides create more overall species diversity and varied habitats within such areas because climax species may be locally eliminated and replaced by different plant species. Man's uses for herbicides are designed to change or manage various environments and may be divided into general habitats as follows: a. Selective plant control in row crops which are grown annually as large acreages of single species such as corn, cotton, soybeans, sugarbeets, sorghum, tobacco, peanuts, sugar cane, sweet corn and other vegetables and are subject to cultivation for weed control. Herbicides used here are selective to control grasses and/or broadleaf plants without adversely effecting the desired commercial crop. (There is no need to be concerned for herbicides which are not persistent nor leave the target area in significants amounts). Such areas are at best only temporary wildlife habitats and not the crops intended as primary food for wildlife. b . Selective control in grains planted annually which cover the ground like wheat, barley, oats, and rice, etc. Con trol of weeds by cultivation in such crops would be im possible. These areas serve at best as temporary wild life habitats and the crops are not intended as primary food for such wildlife. c . Selective plant control to maintain a habitat dominated by grass or forage plants which annually or perennially cover the ground such as rangeland and pasture plants, 6800 0004101 DOVV718217 -20- lawns, hay, alfalfa, legumes, airports, roadsides and utility rights-of-way (non-cropland). These uses while designed to eliminate trees and tall shrubs for domestic animals or for human safety purposes(furnish .a great deal of food and habitat for wildlife. d. Selective plant control in woody vegetation areas such as forest plantings, fruit and nut trees,and shrubs and ornamental plants for elimination of certain competitive plants. Wild or mature forest lands are rarely treated with herbicides. Taller plants such as trees and shrubs may serve as nesting places or homes for birds and animals. Ground areas under trees and shrubs may also be useful for wildlife habitats depending on whether the ground cover is eliminated or kept in grass, etc. e . Non-selective pest plant control in sites where no vegetation is wanted such as parking lots, railroad beds, transformer areas, industrial areas, etc. The areas are not suitable for wildlife habitats. f . Selective plant control in land and marsh areas managed for growing wildlife food or improving wildlife habitat for game animals such as deer, grouse, geese, ducks, etc. These herbicides are used to enlarge the food carrying capacity for individual game species which need man's aid for such directed ecological changes. g . Pest plant control of weeds and algae in aquatic sites .such as rivers, ponds, lakes, irrigation and drainage ditches, etc. Weeds such as these cause blockage of channels by boats, ships, etc., add to eutrophication of water, alter water flow rates. Such weeds change the ecosystems for wildlife. These areas are wildlife habitats unless polluted beyond suitability by man. 6801 CCG^O* -y ft I -21- Using the above description of habitats and the 1968-census of crop acreages (Ref. 1972, USDA, Crop Research, Extent and Cost of Weed Control with Herbicides and an Evaluation of Im portant Weeds, 1968. Table 4. ARS, USDA. In press.) the areas treated with herbicides i'n the USA have been grouped as shown in Table 4. Habitats "a", "b" and "e" are not considered to be reservoirs for natural wildlife and have little environmental po- t tential except possibly for temporary food and cover. The herbicide treatments on these areas account for about 85.2% of the total U.S. acreage treated. In addition to being the largest crop habitat, the areas may receive several to many annual treat ments. The major volume of the herbicides is expended in this habitat. Very few major herbicides (atrazine and 2,4-D) are used in natural environments. Most of the herbicides used on such habitats do not leach readily and are degraded in soil. Major environmental effects on non-target areas are unlikely and have not been found to be significant to date with such herbicides. Habitats "c" and "dM are selectively managed by herbicides to allow grassy and forage vegetation to grow in preference to woody vegetation, particularly trees and tall shrubs. Wild life is often encouraged or present in such areas. Herbicide treatments may be made much less frequently than in the pre vious category and represent about 14.6% of the area in U.S. treated with herbicides. Habitats "f" and "g" represent terrestrial and aquatic areas which are commonly used by wildlife and aquatic organisms. However, these areas represent only a very small part (about 0.2%) of the U.S. area treated with herbicides. All of the areas treated with herbicides in U.S. in 1968 represented about 5.62% of the total land area. 0004103 6802 -22- Herbicides have certain benefits environmentally in that they are selective and in that plant species can be manipulated for the -selection t>f beneficial food of game or other animals, in addition to man. Such selective plant control may result in better ground cover, and consequently less erosion and silting in rivers, and more stabilized plant and wildlife communities. The use of herbicides results in more efficient use of farm land, which allows the use of more marginal farm land for other purposes, including wildlife areas. The majority of herbicide usages do not appear to directly involve natural environmental habitats. Very few incidences of serious damage to wildlife and natural environments have occurred beyond that intended by man, which would not be equally damaging by use of non-chemical methods. Herbicide residues do not seem to be building up in the air, water, and soil environment. Fortunately, the chemical and physical character istics of herbicides allow most of them to degrade within about 3 months. A few herbicides bioconcentrate temporarily in animals but the level seldom is more than 10-fold. In spite of the beneficial uses of herbicides, their effects need to be kept under constant surveillance and in balance to prevent trends which could lead to serious environmental disruptions, disastrous to man as well as his environment. 0GG4i04 680a OZZPJIMOCI -23- RESIDUE REFERENCES CITED Abbott, JD,C,, R.B. Harrison, J.O. Tatton, J. Thomson. 1965 Organochlorine pesticides in the atmospheric environ ment. Nature 208(5017): 1317-1318. Adams, D.F., C.M. Jackson, and W.L. Bamesberger. 1964 Quantitative studies of 2,4-D esters in air. Weeds 12: 280-283. Akesson, N.B., S.E. Wilce, and W.E. Yates. 1971 Atomization control to confine sprays to treated fields. Paper 71-662. 1971 Annual Meeting American Society of Agricultural Engineers. Chicago, Illinois. Dec. 7-10, 1971. Akesson, N.B., W.E. Yates, and P. Christensen. 1972 Aerial dispersion of pesticide chemicals of known emissions, particle size and weather conditions. 163rd National Meeting, American Chemical Society. Boston, Mass. Unpublished manuscript. Anderson, W.P., A.B. Richards, and J.W. Whitworth. 1969, Leaching of trifluaralin, benefin, and nitralin in soil columns. Weed Science. _X6(2) : 165-169. \ Anon. 1971 Aquatic herbicides. Priviledged source. Brown, E., and Y.A. Nishioka. 1967 Pesticides in selected western streams - A contribution to the national program. Pest. Mon. J. _1: 38-41. Butler, B.J., N.B. Akesson, and W.E. Yates. 1969 Use of spray adjuvants to reduce drift. Trans. Amer. Soc. Engineers. _12(2): 186. Butler, P.A. 1965 Effects of herbicides on estuarine fauna. Proc. So. Weed Conf. 18: 576-580. Cohen, J*M., and C. Pinkerton. 1966 Widespread translocation of pesticides by air transport and rain-out. Adv. in Chemistry Series 60, American Chemical Society. Washington, D.C. 163-176. Cope, O.B., J.P. McCraren, L. Eller. 1969 Effects of dichlobenil on two fish pond environments. Weed Science. 17(2): 158-165. 0004105 6804 Tf'T'RTtMOn -2 4 - Cope, O.B., E.M. Wood, and G.H. Wallen. 1971 Some chronic effects of 2,4-D on bluegills. Tran. Amer. Fish'. Soc. 19(1): 1-12. Crosby, D.G., and H.O. Tutass. 1966 Photodecomposition of 2,4-dichlorophenoxyacetic acid. Ag. Food Chem. 14(6): 596-9. Frank, P.A. 1971 Herbicidal residues in the aquatic environ ment. 161st National Meeting of American Chemical Society. Los Angeles, Calif. March, 1971. Preprint. Frans, R.E., D.E. Davis, and J.B. Weber. 1972 Behavior of specific herbicides in plants and soils. Southern Cooperative Series Bulletin No. 167. Ark. Agr. Exp. Sta. Univ. of Arkansas, Fayetteville, Ark. 22201. 26 pp. Freed, V.H. 1966 Chemistry of herbicides. ASA Publication No. 8. Soil Science Society of America. Madison, Wis. pp. 25-43. Getzendaner, M.E. 1960 A residue study of silvex in fish living in Kuron containing water. Report GH-490. The Dow Chemical Co., Midland, Mich. Unpublished report. Grzenda, A.R., H.P. Nicholson, and W.S. Cox. 1966 Persistence of four herbicides in pond water. J. Amer. Water Works Assoc. j>8(3): 326-332. Hamaker, J.W. and H.O. Kerlinger. 1969 Vapor pressure of pesticides. Advances in Chemistry No. 86. American Chemical Society. Washington, D.C. pp 39-54. Hardy, J.L. 1966 Effect of TORDON herbicides on aquatic chain organisms. Down to Earth. Fall issue: 11-13. Hedlund, R.T. 1970 The rate of photodegradation of picloram in aqueous systems. Report GS-1089. The Dow Chemical Co., Walnut Creek, Calif. Unpublished report. Hoerger, F. and E.E. Kenaga. 1972 Pesticide residues on plants: Correlation of representative data as a basis for estimation of their magnitude in the environment. Environmental Quality and Safety. 9-28. Johnson, R.E., T.C. Carver, and E.H. Dustman. 1967 Indicator species near top of food chain chosen for assessment of pesticide base levels in fish and wildlife - clams, oysters and sediment in estuarine environment. Pest. Mon. J. 1: 7-13. 0004105 6805 zzmMoo -2 5 - Kearney, P.C. 1966 Metabolism of herbicides in soils. Advances in Chemistry, Series 60. American Chemical Society, Washington, D. C . pp .250-263, Kenaga, E.E. 1968 Guidelines for evaluating the properties of pesticides for safe use in the wildlife environment. Down to Earth. 23(4): 11-14, 16-18. Kenaga, E.E. 1972 Guidelines for environmental study of pesticides: Determination of bioconcentration potential. Residue Reviews. 4 4 (In press). McCraren, J.P., O.B. Cope, L. Eller. 1969 Some chronic effects of diuron on bluegills. Weed Science. r7(4): 497-504. Miller, R.W. 1972 Adsorption of phenols and phenoxyacetic acids by organo-clays in water. American Chemical Society, NW Section Meeting, Corvallis, Ore. June, 1972. Abstract. Norris, L.A. 1971 Chemical brush control: Assessing the hazard. J. Forestry. 19(10): 715-720. Norris, L.A. and D. Moore. 1970 The entry and fate of forest chemicals in streams. Symposium: Forest Land Uses and Stream Environment. Forestry Extension. Oregon State University. Corvallis, Ore. 97331. pp 138-158. Reimer, C.A., B.C. Byrd, and J.H. Davidson. 1966 An improved helicopter system for the aerial application of sprays containing TORDON 101 mixture particulated with NORBAK. Down to Earth. 22(1): 3-6. Risebrough, R.W., R.J. Huggett, J.J. Griffen, E.D. Goldberg. 1968 Pesticides: Transatlantic movements in the northeast trades. Science. 159: 1223-4. Rosen, J.D. 1972 Conversion of pesticides under environmental conditions. Environ. Quality and Safety. 85-96. Sheets, T. J. and C. I. Harris. 1965. Herbicide.Residues in Soils and Their Phytotoxicities to Crops Grown in Rotation. Residue Reviews, 11:123-4. F. A. Gunther (Ed), SpringerVerlag, New York. Smith, G.E. and B.G. Isom. 1967 Investigation of effects of large scale applications of 2,4-D on aquatic fauna and water quality. Pest. Mon. J. 1(3): 16-21. CG04107 6806 -26- Van Middelem, C.H. 1966 Fate and persistence of organic pesti cides in the environment. Advances in Chemistry Series 60. American Chemical Society. 'Washington, D.C. pp ^228-^249. Van Valin, C.C. 1966 Persistence of 2,6-dichlorobenzonitrile in aquatic environments. Advances in Chemistry Series 60. American Chemical Society. Washington, D.C. pp 271-279. Wershaw, R.L., P.J. Burcar, and M.C. Goldberg. 1969 Interaction of pesticides with- natural organic material. Env. Sci. Tech. 3(3): 271-3. WSSA Monograph 3. 1970. 2nd Edition. Urbana Illinois. Average residual phytotoxic life given for stated dosages, which are the recommended crop use dosages. 0GQ4108 6807 -2 7 - HERBICIDE TOXICOLOGICAL REFERENCES Anon, 1970. Herbicide Handbook of the Weed Society of America. Second Edition. The W. F. Humphrey Press, Inc. Geneva, N. Y. I Applegate, V. C., J. H. Howell, A. E. Hall, Jr., and M. A. Smith. 1957. Toxicity of 4,346 Chemicals to Larval Lampreys and Fishes. Spec. Sci. Rep. - Fisheries No. 207. Fish and Wildlife Service. USDI. Washington, D. C. Batelle's Columbus Laboratory. 1971. Water Quality Criteria Book. Vol. 3. Effects of Chemicals on Aquatic Life. Water Control Research Service 18050 GW V05/71. Environmental Protection Agency. Washington, D. C. ( V E.P.A. 1972. Data from Files of the Pesticide Regulation Division, Environmental Protection Agency. Washington, D. C. Hansen, W. M., M. L. Quaife, R. T. Habermann, and 0. G. Fitzhugh. 1971. Chronic Toxicity of 2,4-Dichlorophenoxyacetic Acid in Rats and Dogs. Tox. Appl. Pharm. 20:122-129. Heath, R. G., J. W. Spann, E. F. Hill, and J. F. Kreitzer. 1972. Comparative Dietary Toxlcities of Pesticides to Birds. Special Scientific Report, Wildlife No. 152. Patuxent Wildlife Research Center, Bureau of Sport Fisheries and Wildlife. Laurel, Md. Lawrence, J. M. 1962. Aquatic Herbicide Data. Agricultural Handbook No. 231. USDA, Washington, D. C. Lawrence, J. H. 1963. Aquatic Herbicide Data. Supplement 1, Agr. Exper. Sta. Auburn University, Auburn, Alabama. 0G0403 6808 -2 8 - Jtfartin, -H. 1968. Pesticide Manual. First Edition. British Crop Protection Council.' London, England. McKee, J. E., and H. W. Wolf. 1971. Water Quality Criteria. Second Edition. Publication 3-A. The Resources Agency of California, State Water Resources Control Board. Palmer, J. S. and R. D. Radeleff. 1969. The Toxicity of Some Organic Herbicides to Cattle,.Sheep, and Chickens. Production Research Report No. 106. ARS-USDA. U. S. Superintendent of Documents, Washington, D. C. Palmer, J. S. 1972. Toxicity of 45 Organic Herbicides to Cattle, Sheep, and Chickens. Production Research Repbrt No. 137. ARS-USDA. U. S. Superintendent of Documents, Washington, D. C. Pimentel, D. 1972. Ecological Effects of Pesticides on NonTarget Species. Executive Office of the President, Office of Science and Technology. Washington, D. C. Sanders, H. O. 1970. Toxicities of Some Herbicides to Six Species of Freshwater Crustaceans. J. Water Pollution Control Fed. 42(8):1544. 0G0410 6809 -2 9 - TABLE 1 Connoti Nane of Herbicide Acrolein STRU CTU RE AND P H Y SIC A L P R O P E R T IE S O F SOME H E R B IC ID E S Structure "Vapor Pressure mm Hg (C) Solubility of Herbicide in Xylene (x) Water Acetone Benzene <b) CH2 - CH-CIIO 325 (30) 25% mise. mise, (x) Alachlor Atrazlne Butylate f2H5 / T i H ^ 0 "200113 \ = / ''C-CHjCl 0.02 (100) C2H5 0 Cl (CHg^HCHN NHC2H5 3xl0"7 (20) 1.4x10" (30) 0 CHa N 1J CHj -CHj -S-CN(CH-CH-CH3)2 13x10" (25) 148 ppm sol. sol. (b) 33 ppm 1.8g/100g 360 ppm (Methanol) (ji-Pentane) 45 ppm mise. mise.(x) Cbloramben ? 700 ppm 23g/100g 0.02g/100g (: C opper S u lfa te C uS04 2 ,4 -D Cl c i- Q - och. c ' ^ ? so l. 7 7 6 x l0 "7 (2 5 ) 6 0 0 -7 0 0 ppm 4 5 g /1 0 0 c c 1 .0 7 / g l0 0 c c ( D iu ro n E n d o th a ll Cl ci \= / 0 .3 1 X 1 0 "5 (5 0 ) n ( c h 3 :*2 42- ppm 5 .3 g /1 0 0 g 0 . 12 g / 10 0 g (t c^ tCpr ^O H 7 1.0% 7 g /1 0 0 g 0 . 0 1 g / 1 0 0 g_ (L- L ln u ro n Cl -- >=/ Cl NHC- N^ 0"3 ^ C ll3 7 7 5 ppm 5 0 g /1 0 0 g 1 3 g /1 0 0 g (b ) 00041116810 ` TABLE 1 (Cont.) Common Name of Herbicide Nitralin STRUCTURE AND PHYSICAL PROPERTIES OF SOME HERBICIDES Structure Vapor Pressure mm Hg (*C) Solubility of Herbicide in Xylene (x; Vater Acetone Benzene (t N2 S02 " A / "N <C3H7>2 -6 <1.5x 10 (25) 0.6 ppm 37g/100g <12.5g/100g NO. Propachlor CHiCH3>2 C~0 CHjCl 0.03 (110) 700 ppm 30.9% Propesine (CH^) gHCIIN N 2.9x10,"-8 (20) NHCH(CH3)2 8.6 ppm dlff. 50% (b) diff. (x,b Trifluralin r C f "CF. ( C g H ^ NO_ 1.99xl0"4 (29.5) <1 ppm 1.19 1.73 (x) (% Molarity) (% Molarity Xylene CH- isomer 0 CHj m 2 6.62 (25) 8.30 (25) 8.76 (25) 196 ppm 198 ppm inf. inf. inf. inf.(b) inf.(b) inf.(b) 000411" 6811 -3 1 - TABLE 2. PERSISTENCE OF HERBICIDES IN SOIL IN FIELD TESTS Herbicide lb/A Residual Phytotoxicity Applied lo Soil (Months) Re Terence Alachlor Atrazine ......... . Butylate Chloramben 2,4-D Diuron ............................... - - Endothall Linuron Nitralin Propachlor Propazine Trifluralin 1-4 2-4 2-3 3-8 2-4 3-4 2-4 3-4 3.6-4 1-2 2 0.6-6.4 12 4 0.5-3 0.5-1.5 3-6 1-4 3 1.8 4 0.5-1 1.5-2.5 4-7 4-7 12 <12->12 0.12-0.75* 1.5-2 0.25-1 5-7 4-8 15 One Growing Season 1 <4 4 "Moderately Persistent" 1-1.5 <12 2 14 5 4-6 HH S&H S&H S&H HH HH ? HH HH S&H -- S&H r S&H HH ^ S&H ' "S&H ' HH HH HH HH S&H S&H S&H HH HH - Herbicide Handbook of the Weed Society of America. 1970. S&H - Sheets, T. J. and C. I. Harris. 1965. o o o iiia 6812 -3 2 - table 3 . TOXICITY OF JIEHUICIDES TO MAMMALS, BIRDS, FISH, AND INVEKTEmUTKS Comoon N a m (Trade Nano) of Herbicide Nano of Organism Typo of Test Expoauro Dosage .le Effect Rofernnoo Acrolein (Aqualln, Hydrotha1) Rat Rabbit Rat Bluegill Chinook Salaon Coho Salaon Rainbear Trout Largenouth Bass Mosquito Fish Fathead Minnow Bluegill Brown Trout Continuous Flow Acute Oral Acute Oral Water Dietary, 90 Day Acute, 96 hr Acute, 24 hr Acute, 0.25 hr Aeuto, 24 hr Acute, 96 hr. Acute, 48 hr Acute, 48 hr Acute*, 24 hr Acute*, 24 hr 46 ag/kg T ag/kg 200 ppa 0.1 ppa 0.08 ppa 2.0 ppa 0.14 ppa 0.16 ppa 0.06 ppa 0.01S ppa 0.079 ppn 0.046 ppa " so " so No Effect Herbicide Uandbook " so " so 100% Mort. " so " so " so " so " so " so Lawrence, : Plaontel Lawrence, 1963 Batelle Alachlor (Lasso) Rat Pheasant Rainbow Trout Bluegill Acute Oral 90 Day Dietary 5 Day Dietary ? Acute, 96 hr Acute, 96 hr 774 ag/kg* 200 ppa >4300 ppn 1.0 ppn* 5.8 ppa* " so No Effect " so " so " so Corrected for 4 lb/gal emulslftable composition, probably 43% a.l. Herbicide liandbook Rat Mice Rat Cattle Sheep Bobwhlte Mallard Japanese Quail Ring-necked Pheasant Chicken Spot Harlequin Fish Rainbow Trout Bluegill E. Oyster Brown Shrlap Water Flea (Ikiphnln nngna) Acute Oral Acute Oral 2 yr Dietary Acute Oral 2 cdod! / 10 CDOD Acute Oral 3 CDOD 8 CDOD S Day Dietary 5 Day Dietary S Day Dietary S Day Dietary 10 CDOD 10 CDOD Acute, 48 hr Acute, 24 hr Acute, 96 hr Acute, 48 hr Acute, 96 hr Flowing Sea Water Acuto, 48 hr Acute, 48 hr 3080 ag/kg 1750 ag/kg 100 ppa " so " so No Effoct Herbicide Handbook 50 a g A g Slgnlf Effect-- Palner ct a 25 a g A g Slgnlf Effect 10 a g A g No Effect 100 a g A g Slgnlf Effect SO ag/kg Slgnlf Effect 2S a g A g Slgnlf Effect >5000 ppa >3000 ppa >5000 ppa >5000 ppa " so " so " so " so Heath 50 ag/kg 25 ag/kg 1 ppa 0.S5 ppa 4.5 ppa 26 ppa 1 ppa Slgnlf Effect No Effect No Mortality Palner et al Plaentel " so " 50 " so No Effect EPA Pimentel 1 ppa 3.6 ppa 30% Mortality or Loss of Equlllbrlun " 50 Plaentel 6813 000411*5; 33- TADLE 3 . TOXICITY OK IIKfMICIDES TO MAMMALS, DIRUS, KISH, AND 1NVKRTKOIIATKS (CONT.; Common Nam* (Trado Name) of Herbicide Nano of Organism Typo of Tout Expofturo Doango a.l. Effect Rcforonce Butylato (Sutan) Rat Guinea Pig Rat Do; Bobehlto Blueglll Rainbo Trout Ganna rus Acute Oral Acute Oral 4659-5431 agAg 1659 a g A g 90 Day Dietary 32mgAg/day 90 Day Dlotary ' 40agAg/day 7 Day Dietary 40,000 ppa Acute, 96 hr 6.9 ppa Acute, 96 hr 4.2 ppa Acute, 96 hr 10.0 ppm " 50 LD50 No Effect No Effect " so " so " 50 " so Herbicide Handbook Sanders Chloramben (Aalben) Rat Cattle Sheep Bobvhlte Chicken Ra Inbo* Trout Blueglll ' Acute Oral 2 yr Dietary 10 CDOD 10 CDOD 2 CDOD 10 CDOD Acute Oral 7 CDOD 10 CDOD 10 CDOD Aeute, 96 hr Acute, 96 hr 5620 a g A g 50 10,000 ppa No Effect Herbicide Handbook 250 mg/kg Slgnlf Effect Palmer 175 mg/kg No Effect SO mg/kg Slgnlf Effect 25 mg/kg No Effect >700 mg/kg LDS0 EPA 500 mg/kg Slgnlf Effect Palmer 375 mg/kg Slgnlf Effect 250 a g A g No Effect 4.2 ppa 1000 ppa " 50 " so EPA . . ------- Copper Sulfate* Sheep Mallard Ring-necked Pheasant Blueglll Striped Bass Blueglll Bass Goldfish Fathead Minnow Daphnla Daphnla Dlotary Acute Oral Acute Oral Acute, 24 hr Acute, 48 hr Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute, 16 hr , Acute, 2 hr 25 ag/day Jaundice >2000 a g A g 1** " 50 >2000 agAe'** LDS0 1.5 ppm 0.15 ppa 3.0 ppa 3.0 ppa 1.0 ppa i.O ppa 0.1 ppa 100 ppa " 50 " 50 Tolerated Tolerated Tolerated Tolerated " **50 Killed Toxicity varies greatly, with pH and turbidity. e* Bordeaux aixture. Dead or Immobilized. Pimentel Lawrence, 1962 2,4-D (Acid)* r t f c\r ^ -"i Rat Mouse Rabbit Dos Guinea Pig Mulo Deer Rat Dog sut Cattlo Acute Oral Acute Oral Acute Oral Acute Oral Acute Oral Acuto Oral 2 yr Dietary 2 yr Dietary 3 Generation Reproduction Acuto Oral 25 CDOD 86 CDOD 112 CDOD 666 a g A g 375 a g A g 800 a g A g 100 m g A g 1000 a g A g 400-800 a g A g 1250 ppa 500 ppa 500 ppm " so " 50 " 50 " SO " so LDS0 .. No Effect No Effect No Effect Pimentel -- Hansen et al 250mgS//kg 200ag-//kg lOOmg-' A g SOag^Ag Slgnlf Eifect Slgnlf Effect Slgnlf Effect No Effect Pa lner rU Oq ii. 34- table 3 . toxicity o r jikiidicides to m ilmals, in n s r is ii, akd' invuitkijhatf.s (cont. Connon Nane (Trado Nano) o f Horblclde Nano of Orgnnlsn Typo of Test Kxpoauro Dosage a.l. Effoct Rafnrunen 2 ,4 -D Cont. Sheep Mallard Ring-necked Pheasant Japanese Quail Pigeon Mallard Ring-necked Pheasant Japanese Quail Chicken Chorus Frog Tadpole Blueglll Rainbow Trout Daphnia nagna Gannarus iasciatus 7 CD0D 10 CDOD 8 CDOD Acute Oral Acute Oral Acute Oral Acute Oral S Day Dietary 5 Day Dietary 5 Day Dietary 10 CDOD 10 CDOD 10 CDOD Acute, 24 hr Acute, 24 hr Acute, 24 hr Acute, 48 -hr Acute, 48 hr SOOng-- 'Vkg ' Slgnlf Effect 250mg /A g Slgnlf Effoct lOOmgS'Vkg No Effect >1000ng/kg 472 ng/kg " 50 " so Pn lner Pimentel 668 ng/kg 668 ng/kg >5000*ppn >5000*ppn " 50 " so " so " 50 >5000*ppn SOOngS'Vkg 250ng-- ''Vkg lOOng^/kg 100 ppn " 50 Slgnlf Effect Slgnlf Effect No Effect 50 Palner Plnentel 5 ppn 5 ppn >100 ppn 3.2 ppn O 10 No Mortality No Mortality " 50 Applegate Sanders Very little variation in toxicity occurs between various salts and esters of 2,4-D in nannals and birds, however, certain esters are auch more toxic than the acid to fish (around 1 ppn). c. b ^ c f- a3 Dluron (Karncx) Rat Acute Oral Rat (Low Chronic Toxicity) Cattle 10 CDOD 10 CDOD Sheep Acute Oral 2 CDOD 10 CDOD Bobwhlte 5 Day Dietary Japanese Quail 5 Day Dietary Ring-necked Pheasant 5 Day Dietary Mallard 5 Day Dietary Chicken 10 CDOD 10 CDOD Bullfrog Tadpole Acute Rainbow Trout Acute, 48 hr Coho Saloon Acute, 48 hr Largenouth Baas Acute, 48 hr Rlueglll Acute, 24 hr White Crapple Acute, 24 hr Striped Bass Acuto, 96 hr Dnphnla puicx Acute, 48 hr Gannarus E. Oyster Acute, 48 hr Acute, 9G hr Flowing Salt Water Brown Shrlnp Acute, 48 hr riowlRC Salt Water Different tenperatures. 3400 ng/kg Om Herbicide Handbook 100 ng/kg 50 ng/kg 250 ng/kg 100 ng/kg 50 ng/kg 1730 ppn >5000 ppn >5000 ppn Slgnlf Effect No Effect Signif Effect Slgnlf Effect No Effect " 50 " so " 50 . Palmer et al Heath >5000 ppn 10 ng/kg 5 ng/kg 10 ppn 4.3 ppn 42 ppn 16 ppn 9.7-27 ppn* 8.75 ppn 3.1 ppn 1.4 ppn 0.38 ppn 1.8 ppn " 50 Slgnlf Effect Pa lner et al No Effect All Killed Lawrence, 1963 LC 50 LC50 LC 50 LC'.50 501 Mortality Plnentel LC50 LC!so" LCSO Shell Growth Batcllc Decreased 1.0 ppn No Effect 00041G 681 r rr 35- TABLE 3 . TOXICITY OK HERBICIDES TO MANUALS, BIRDS, FISH, AND INVERTEDHATES (CONT. CoBinon Nano (Trade Name) of llerblcldo Name of Organism Type of Test Esposuro Dosco a.le Effoct Reference Endothall (Endothal) Rat Dog Cattlo Sheep Chicken Blueglll Carp Largeamuth Bass Fathead Minnow Daphnla magna Cammarus lacustrls Acuto Oral 2 yr Dietary 2 yr Dietary 2 CDOD 10 CDOD 2 CDOD 2 CDOD 10 CDOD 10 CDOD 10 CDOD Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute; 96 hr Acute, 24 hr ? Acute, 24 hr 38-51 mg/kg LD50 >300 ppm* No Effect >300 ppm* No Effect 25 m g ^ / k g Slgnif Effect 10 w g ^ / k g No Effect SO mg'/kg Signif Effect 25 m g ^ / k g Slgnif Effect 10 m g ^ / k g Slgnif Effect 5 m g ^ / k g No Effect 10 mg-- ^/kg Slgnif Effect 125 ppm** " 50 175 ppm** " so 120-200 ppm** LCjq 320-610 ppm*<" " so 46 ppm " ***50 2 ppm " so. Herbicide Ha ndbook Palmer p V C m m * Pimentel y c C i Lawrence, 196 Pimentel Sanders Dlsodlum Salt. ** Cocoamlne, dlmethylamine and copper salts any be toxic to sono fish at less than 1 ppm. *** Imaobi 1lza tlut. Llnuron (Loros) Rat Doc Cattle Sheep Chicken Carp Acute Oral Acute Oral Acute Oral 6 CDOD 10 CDOD 10 CDOD 4 CDOD 1 CDOD 10 CDOD 10 CDOD Acute, 48 hr 1500 mg/kg 4000 mg/kg 500 mg/kg 100 mg/kg 50 mg/kg 25 mg/kg 100 mg/kg SO e g A g 25 mg/kg 10 m gAg >10 ppm LDS0 " so " so Slgnif Effect Slgnif Effect No Effect Slgnif Effect Slgnif Effect No Effect Slgnif Effect " 50 Martin Palmer et al EPA Mltralln (Planavin) Rat Mouse Rat Cattle Sheep Chicken Bluoglll ? Trout Goldfish Silver Salmon Acute Oral Acute Oral 2 yr Dlotary 2 CDOD 10 CDOD 4 CDOD 10 CDOD Acute Oral 10 CDOD Acute, 48 hr Acute, 48 hr Acute, 48 hr Acute, 48 hr >2000 mg/kg >2000 mg/kg 2000 ppm " so " so No Effect? 250 mg/kg Slgnif Effect 175 mg/kc No Effect 500 mg/kg Slgnif Effect 250 mg/kg No Effect >1000 mg/kg " so 500 m gAg No Effect 20 ppm No Mortality 20 ppm No Mortality 20 ppm Mo Mortality 20 ppm No Mortality Herbicide Handbook Palmer Martin Palmer Martin 0004117 6816 36* TABLE 3 . TOXICITY OF IIKMJICIDES TO MAMMALS, BIRDS, FISII, AND INVKRTEnRATES (CONT Common Name (Trado Kane) of Herbicide Name of Organism Type-of Test Exposuro So sik a .1. Effect Reforcnco Propachlor (Baerod) Rat Cattle Sheep Ring-necked Pheasant Chicken Blueglll Fathead Minnow Acute Oral 90 Day Dietary 3 CDOD 10 CDOD 4 CDOD 9 CDOD 10 CDOD Acuta Oral 3 CDOD 10 CDOD 10 CDOD Acute, 96 hr Acute, 96 hr 710 ag/kg " so 133mg/kg/day No Effect 25 ag/kg Slgnlf Effect 10 Bg/kg No Effect 50 Bg/kg Slgnlf Effect 10 Bg/kg Slgnlf Effect 5 ag/kg No Effect 735 ag/kg " so 1000 a g A g 10 a g A g 5 agAg 1.3 ppa 0.49 ppa Slgnlf Effect Slgnlf Effect No Effect " so " 50 Horblcldc Handbook' Palacr Herbicide Handbook Palmer Herbicide Handbook Propazine (Hilogard) Hat House Rat Cattle Sheep Bobvhite Mallard Chicken Trlfluralln (Tref lan) Rainbow Trout Goldfish Blueglll Rat Mouse Dor Rabbit Rat Dog Cattle Sheep Ring-necked Pheasant Chicken Mallards Chicken Acute Oral Acute Oral 130 CDOD 2 CDOD 3 CDOD 10 CDOD Acute Oral 10 CDOD S CDOD 10 CDOD (Low Toxicity) (Low Toxicity) 6 CDOD 10 CDOD 25 CDOD Acute, 96 hr Acute, 48 hr Acute, 48 hr Acute, 48 hr >5000 ag/kg >5000 ag/kg " 50 " so Horblcldc Handbook 250 a g A g No Effect 250 a g A g 25 ag/kg 10 ag/kg 500 ag/kg 100 ag/kg 25 a g A c 10 ag/kg Slgnlf Effect Slgni'f Effect No Efiect Slgnlf Effect Slgnlf Effect Slgnlf Effect No Effect Palmer et al nerblclde Handbook 250 a g A g 100 eg/kg 25 ag/kg >100 ppa 7.8 ppa >32 ppa >100 ppa Slgnlf Effect Slgnlf Effect No Effect " so " so " so " so Palaer et al EPA Plaentel EPA Acute Oral >10,000 a g A g LD,n Acute Oral Acute Oral Acute Oral 5000 ag/kg >2000 ag/kg >2000 ag/kg " so " so 2 yr Dietary >2000ppa/day No Effect 2 yr Dally >1000 ppa Dietary Capsule No Effect 2 CDOD 175 a g A g Slgnlf Effect 10 CDOD 100 a g A g No Effect 2 CDOD 175 ag/kg Slgnlf Effect 10 CDOD 100 ag/kg No Effect Acute Oral >2000 ag/kg . U>50 Plaentel Herbicide Handbook Palaer et a 1 Plaentel Acute Oral Acute Oral 10 CDOD 10 CDOD 10 CDOD >2000 a g A R " so >2000 ag/kg " so 500 a g A g Slgnlf Effect 250 a g A g Slgnlf Effect 100 Bg/k bG&a Palaer et 1 \ 6817 37- DOVV7I8234 t a b l e 3. TOXICITY or HERBICIDES TO MAMMALS, BIRDS, FISH, AND INVERTEDRATES (CONT.) Common Xaae (Trade Name) of Herbicide Haae of Organisms Type of Tost Exposure Dosage a.i. Effect Reference Trlfluralln Coot. Rainbow Trout Fathead Hlnaow Blueglll Channel Catfish Goldfish Daphnla amcna Daphnla pulex Gammarus lacustrls Ganma rus fasciatus Crayfish (Orconectes nals) Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute, 96 hr Acute, 48 hr Acute, 48 hr Acute, 48 hr Acute, 48 hr Acute, 48 hr 0.01-0.086ppm 0.093 0.019-0.089ppa 0.2S4 ppm 0.282 ppm 0.86 ppm 0.24 ppm 8.6 ppm " so " 50 " 50 " so " so " 50 " 50 " so 1.0 ppm " so 50.0 ppm " so EPA Batelle EPA Sanders Pimentel Sanders .Xylene Rat Various Species of Fish Blueglll Rainbow Trout Sea Lamprey Orange-spotted Sunflsh Daphnla maena Acute Oral Acute Acute, 24 hr Acute, 24 hr Acute, 24 hr Acute, 1 hr Acute, 48 hr e Ooually a mixture of o, a, and ;i xylene. m-Xylooo - usually most toxic-of' isomers. 4300 mg/kg 10-90 ppm 5 ppm 5 ppm 5 ppa 47 ppm " so* Rill Kill Fish* Sick No Mortality No Mortality Kill McKee and Rolf . Lawrence, 1962 0.01-0.1 ppm Kill a/ CDOD - Couccntlrt dally oral doses each at the given ag/kg. b/ Signlf Effect " Significant effect (weight loss, reduced weight gala or Illness). f/ Alkanol aalne salts, d/ Potasslua salt. 0004113 681)? UUW78235 } -3 8 - TABLE 4. RELATIVE ACREAGE OF HERBICIDAL USE CLASSIFIED BY HABITAT Habitat or No. of 1000 Crop Acres Treated Classification 1968 % of Herbicidally Treated Acres for % of Total Each Crop or Group U.S. Treated of Crops with Herbicides a . Row Crops 72,693 1. Corn 2. Soybeans 39,302 ---17,167 b. Grain Cover Crops 36,157 [l. Wheat 20,915 c . Grass and Forage 15,475 Dominated Areas 1. Rangeland 2. Pastures 4,373 4,525 . d. Woody Plant Crops 3,188 e. Land Bare of Vegetation ? f . Wildlife Manage ment Areas ? ? Aquatic 216 56.91 30.77 13.44 28.30 16.37 12.12 3.42 3.54 2.50 <0.1 <0.1 0.17 3.20 1.73] 0.7G 1.65 0.92] 0.68 0.19 0.20 0.14 T0.01 <0.01 0.01 U.S . Total 127,729 100.00 5.62 OGGSiSO \o3 0820 18.316 Chloracne from the accidental production of tetrachlorodibenzodioxin GEORGE MAY' G e n e ra l P ra c tic e . B o lso v e r, D erb y sh ire a n d C o a lite a n d C h e m ic a l P ro d u c ts L im ited \* O % CO CO o o i. M a y , G . (1 9 7 3 ). British Jo u rn al o f Industrial M edicine, 3 0 , 276-283. C h loracn e from the accid en tal production o f tctrachlorndibcnzodloxin. F o llo w in g the accid en tal p rod u ctio n o f 2,3,7,8-tctrach lorod ib cn zod ioxin (d ioxin ) a s the result o f an exotherm ic reaction at a ch em ical p lan t in D erb ysh ire, 79 case s o f ch lo racn e w ere recorded, m any o f them severe. C o n trary to the u su a l experience they h ave respon d ed very favou rab ly to treatm ent an d there w ere n o c a se s o f c o n ta c t ch lo racn e a m o n g relativ es o r d o m e stic an im a ls in the in itial o u t* b reak . H ow ever, tw o cases o f con tact ch loracn e w ere recorded three years later. S im ilar in ciden ts are know n to h ave occu rred in b oth E u rop e an d the U n ited S tates o f A m erica, alm o st in v ariab ly acco m p an ied by w id esp read severe illn ess an d w ith fatalities. A p art fro m o n e d eath du e to an e x p lo sio n w hich follow ed the exoth erm ic reaction the m ore serio u s seq u elae, w hich m ay ran ge from d ep ressio n an d lo ss o f w eight to liver, kidn ey, an d c a rd ia c failu re a s w ell a s m align an t d isease, h ave n ot occu rred. A q u ic k an d reliab le m eth od o f b io lo gical a ssa y fo r the presence o f d ioxin in p ro d u ced trich lo ro p h en o l w as d ev elo p ed b ase d o n o ra l d o sa g e to ra b b its w ith assessm en t o f liver fu n ction a t fixed tim e intervals th ereafter. T h is test h as alread y been superseded by in stan * tan eou s gas-liqu id ch rom atography. A n en tirely new p lan t w ith su ita b le m o d ificatio n s an d m u ltip le safe ty featu res h a s n ow been in satisfac to ry o p eratio n fo r three y ears. It > j" 1 ^ no 2.4, wa 12C * cor PIT f liqi ,\ ess- the sta P ro d u ctio n o f 2,4,S-trich loroph en ol (2 ,4 ,5 -T C P ) tetrach lorob en zen e. A fter the o rth o dichloroben zen e b egan in A u g u st 196S a t B olsov er, D erbysh ire at ceased to be evolved, the reaction m ixture w as run th e F in e C h em icals U n it o f C o alite an d C h em ical in to w ater in the sep arato r. T h e con ten ts o f the P rod u cts Lim ited. se p a r a to r w ere acid ified w ith co n cen trated su lph u ric 1,2,4,5 ,-T efrach lorob en zen e an d eth ylene glycol ac id at a tem peratu re o f ab o u t 70 to 8 0 ' C . A fter the w ere ch arg ed to the re a c to r togeth er w ith o rth o se p aratio n o f the o rgan ic layer co n tain in g 2 ,4 ,5 -T C P dichloroben zen e an d lO O T w cau stic so d a solu tion fro m the aq u eou s ph ase, agitation w as continued (F ig . 1). fo r a fu rth er 15 m in u tes an d the c o n te n ts o f the T h e vessel w as h eated b y oil an d reaction took vessel w ere allow ed to stan d fo r tw o h ou rs. The p lace a t a b o u t 1 8 0 C w ith a n arb itrary u p p er lim it aq u e o u s p h ase w as then sy p h o n ed o ff an d tran sferred o f 2 0 0 Q T h e p ro gress o f the reaction w as m on itored to a sto rag e vessel, an d a secon d w aterw ash w as b y sam p lin g, an d o n com pletion the vessel con ten ts ad d ed to the organ ic layer. A third w ash w as also w ere c o o le d to I 4 0 C S te a m w as th en in jected into req u ired , an d finally a w ash o f tow n w ater w as run the re actio n v essel in o rd e r to recover the o rth o to th e effluent treatm ent d rain . Sin ce ab o u t 1966-67 d ich lo ro b en zen e w h ich w a s u sed p u rely a s a so lv en t c a u stic s o d a flake h as been ch arg ed d irectly to the to prevent b lock in g o f the con den ser by so lid reactor an d n o w ater h as been ad ded at this stage. T h e aq u e o u s p h ase con sistin g o f the w ash in gs, Request* for reprints to D r. O . M ay, la Wclbeck R oad, Bolsover, nr. Chesterfield. w hich con tain ed so d iu m ch lo rid e, eth ylen e glycol, an d d igo l w as distilled under vacu u m to rem ove 276 \ f* , l, T Th d is res w e: pre agr ap 2 .4 of sec d is cul till: chl ace wa ch! k S. :r 4l'r.'.p y ` * *a \ .r ,*. ', .* / .r * a C00.Tr/-7i 6821 D O W 994301 * *' I Chloranw from ihr acni/riital prmlucthm oj trtrarhhiriii/ihrii;mhoxiii 277 I.J.4.5*Tefro<Mofobnfen N o O H tolid of oqueout -- Ethylene Clycol O rth o dicKiorobciGene - I iteom - R EA CTIO N recovered -- I Ortho dichlorobcnien P rod u ction h ad been m aintained for three y ears o n the a b o v e b asis. If the arb itrary tem p eratu re dead lin e w as reached the process w as co o led by low erin g the tem perature o f the h ealing oil. T h is regulation w as m anu ally controlled. U se s o f finished products voter Aqueouf ACid<fiCOtn Sepgarnodfion |sglptHiric ocid O'q*onic T h e m o st im p o rtan t ap p licatio n o f 2 ,4 ,5 -T is fo r killin g w o o d y p lan ts, several species o f w hich have been sh ow n to be m ore susceptible to 2,4,5-T than to 2-m cthyl, 4-chlorophcnoxyacctic acid (M C P A ) r ZE - o r * 2 ,4 - d ic h to r o p h c n o x y a c c tic a c id ( 2 ,4 - D ) . B o th Evaporation water --p vo te r* Woihinq * - , e t i a m i n e a n d e s t e r d e r iv a tiv e s a r c a v a i l a b l e c o m I to drain m ercially, the latter being the m ost im portan t. T h e e ste rs a r c a p p lie d c ith er in em u lsio n s fo r overall Filtration cryitolhne NoCI NojSq, wcoKiad CItcoI lljcol dittil lotion -- rendu 1 a drem d.p. . K ^ * 2A5*-TCP Agricultural qrade Secondary . freBt, ^ dittillotion 2AS-TCP pharmaceutical qrade fo lia g e sp r a y s o r in so lu tio n in a m ineral oil su ch a s vaporizin g o il o r diesel oil for local ap plication to frill gird les, o r to stu m p s, o r by the 'b a sa l b ark ' technique. C om m ercial products often con tain a m ixtu re o f 2 ,4 -D an d 2,4,5-T esters for reaso n s o f eco n o m y an d the greater range o f effectiveness on different plan t sp ecies gain ed by including the tw o ch em icals. L ik e the o th er au xin type herbicides, 2,4 ,5 -T is o f n o . 1. S ch em atic rep resen tatio n o f the reaction in lo w m a m m a lia n to x ic ity . It is g e n e ra lly le ss e ffe c tiv e 2,4,5-trichlorophcnol production. o n h e r b a c e o u s sp e c ie s th a n M C P A a n d 2 ,4 -D b u t it is m u ch m o re p e rsisten t in the so il. H exach loroph an e has for m any years been used w ater. W h en th e b a s e tem p e ratu re o f th e still reach ed a s a b a c te r io sta tic a g e n t in o p e ratin g th eatres an d it 12 0 *C d istilla tio n w as d isco n tin u e d a n d the still is m o st lik ely th at th is u se w ill co n tin u e. It is a lso con ten ts w ere tran sferred to a cen trifu ge w here the u sed in to p ical ap p licatio n s in d erm ab rasio n , an d in precipitated sa lts w ere rem o ved by filtration . T h e a larg e ran ge o f toilet prep aratio n s ran gin g from so a p liquor rem ain in g a fte r filtration , w hich co n sists to v agin al sp ray s an d b ab y pow ders. R ecen tly essentially o f eth ylen e gly col, w as fraction ated an d so m e b ab y p o w d ers h ave contained h exach lorophane the gly col th u s reco v ered w as recycled to the first in a co n ce n tra tio n o f 6 % in stead o f the recom m en d ed stage o f the p rocess. m a x i m u m o f 0 -6 % , a n d its u s e in t o ile t p r e p a r a t i o n s is u n d er review . D istillation T h e o rg an ic m ate rial w hich w as se p arate d o ff w as I distilled at a vacu u m o f 2 0 m m o f m ercury. T h e residues fro m the still w ere run to w aste, the fron ts were recycled to th e o rgan ic lay er fro m the w ash in g process, an d the m ain fraction produ ced w as agricultural grad e 2 ,4 ,5 -T C P . In the C o alite process a proportion o f the pure o r ph arm aceu tical grad e o f 2.4;5-T C P w as p ro d u ced by a fu rth er fraction atio n o f the agricu ltu ral g rad e. T h e residu es from this second d istillation w ere fed b ack in to the prim ary d istillatio n a n d th e fro n ts w ere a d d e d to- the a g ricultural g rad e m aterial fo r sale. T h e m ain d is tillation w as the p h arm ace u tical g rad e m aterial. I T h e ag ricu ltu ral grad e w as reacted w ith m on ochloroacetic acid to give 2,4,5-trich loroph en oxyacetic acid (2 ,4 ,5 -T ) a n d th e p h arm aceu tical grad e * w as co n d en sed w ith form ald eh y d e to fo rm h exa- c h lo ro p h a n e . In c id e n t A t m idnigh t o n 23 A pril 1968, the reaction tem pera tu re reached I7 5 *C an d thereafter it ro se con tin u o u sly fo r 5 0 m in u tes. W hen it h ad reach ed a p o in t som ew here in excess o f 2 5 0 C an explosion o f con sid erab le violence occurred an d the supervisin g ch em ist w as killed by fallin g m asonry. Su b seq u en t an alysis indicates that the explosion w as o f a d ou b le n atu re. W hen the tem perature had reached 225C the process began to generate its ow n heat an d the in itial ru ptu rin g o f the vessel w as du e to the ad van ce o f this exoth erm ic reaction . Ethylene glycol an d orth odich lorobenzen e vap ou rs, w ere released in qu an tity into the atm osph ere. In the presence o r atm o sp h eric oxygen these constituted an explosive m ixtu re w hich ap p e a rs to h ave been d eton ated by a n overhead electric lam p . Extensive local d am age was d o n e t o b o t h p l a n t a n d b u ild in g a n d a c o n - r pjqmayqnwqjjniany i>m* wmasum COO5772 J .I i, ; I 1 ` 278 C . S l a v sid c ra b lc part o f the w all in the p ro x im ity o f the exp losion w as dem olished. By far the greater p a il o f all four floors, how ever, had apparen tly been com pletely unaffected by b last o r residue. T h e entire Fin e C h em icals U nit w as clo sed dow n pending clinical an d lab oratory in vestigation on 14 em p lo y ees w h o w ere in the b u ild in g at the lim e o f the incident o r w h o h ad h ad to en ter in its afterm ath. For a time work continued normally. Pmcev. operators were watched particularly carefully hut they all remained well. However, certain men, all of whom had been in the Fine Chemicals Unit, developed a mild but peculiar skin condition. This consisted of a slight malar erythema and the eruption of liny vesicles around the borders of the pinnae (Fig. 2). Occasionally there was evidence of mild Clinical and laboratory (indines Tw o men com plained o f tiredness, and three o f slight tightness o f the chest, but clinical exam ination revealed nothing o f note. The investigations included liver function tests, full blood coun ts, and urinalysis. T w o m en, the w orks engineer and a senior laboratory technician, both o f w hom had been closely associated w ith process developm ent an d with con tin uin g pilot plant research, did n ot enter the building until som e hours after the explosion. These two men were the m ost likely to show an y w ork-induced condition which pre existed the explosion but neither o f them had any abnorm ality. O f the rem aining 12 m en, only one showed no abnor m ality in an y test. T h ree m en show ed on e ab n orm ality , four showed two, and another four show ed three ab n o rm alities. A m o n g these w ere 13 ab n o rm a l liver function tests: Z n S O ( turbidity in six m en, thymol turbidity in two m en, an d serum tran sam in ase in five m en were abnorm ally high (T able). O ne m an had album inuria and one had. an abnorm al serum protein level. W hite cell cou n ts in five m en were regarded-by the haem atologist as being som ew hat too high or to o low an d there were three m en with glycosuria. T h ese findings persu aded u s to treat the incident with extrem e caution and to w ait and see whether the changes w ere progressive. Fortu n ately , all the m en reported fit very shortly after, an d 10 d ays later practically all the tests w ere within n orm al lim its. O ne m an with \*/% glycosuria w as know n to have had this before the explbsion. In view o f these satisfactory findings the building w as re-opened. A ll dam aged and obviously dirty areas, were sealed o ff to be decontam inated properly an d at leisure, while the rem ainder o f the building w as subjected to a thorough sp rin g clean . P roduction w as then resum ed in the greater part o f the p lan t tiu . 2. Typical established chloracne o f pinna show ing cysts and com edones. conjunctivitis. These men were not continuously occupied in the sam e b uilding a s o p erato rs w ho alw ays w ore gloves, but were m ostly fitters, plum bers, and electricians w ho only entered the unit on occasion to d o specified jo b s w ith b are h an ds. O b v io u sly , therefore, it w as so m e thing which had been deposited o r em bedded in the m atrix o f the building itself which w as cau sin g the reaction an d which w as being absorbed by contact. The d ia g n o sis qu ick ly estab lish ed itse lf a s ch lo racn e a n d it becam e w idespread. L ater it w as established that the toxic m aterial w as 2^,7,8-tetrachlorodibenzodioxin (dioxin) (F ig . 3), a reaction product o f the sodium salt o f trichlorophenol (M ilnes, 1971). F ro m 8 M ay to 8 D ecem ber 1968,79 cases o f chlor acn e w ere recorded. N o t all individuals presented with m alar erythem a, etc., but those w ho did generally becam e severe an d extensive cases. Som e men appeared TABLE L iver F unction T ests in 14 Men in B uilding at T ime of E xplosion or who entered it SOON AFTERWARDS -- T h y m ol t u r b id ity ............................................ (norm al upper mil 2-3 units) Z in c turbidity ............................................ (norm al upper limit 10 units) SO FT .............................. (norm al upper limit 35 units) ,/ Patient i 2 J 4 5 6 7 9 10 11 12 13 14 1-2 0-7 0-5 0-7 0-5 1-2 0-S 0-6 1-8 1-1 1-2 2-5 3-1 3 0 5 7 8 17 I 13 3 12-8 9-9 12-7 7 ? 13 5 3 12-5 3-3 9 4 18 20 32 16 IS 36 4 2 22 4 0 6 28 4 0 6 36 > FIO. sm a tribi. area a s t area 4Is t r i a r In s w h i1< edor m iss, hue. diag Th im the r thigh In fi' V.V..U . . G G 0 5 '? 7 .0 3 2 3 D O W 994303 ? Ch!orunie front the accidenta! production o f tctrach/oroi/ihcnzodioxin 219 r?"'rs i in r skin ta a n d o f the f m ild 4 O ONo Cl L *J dwim 2-4-VTnfMoeooh<wote 2.17.8 -T efrocM orodibctuodtonn n o . 3. Form ation o f on e m olecule o f 2,3,7,8-tctrachlorodibcnzodioxin from tw o m olecules o f sodium 2.4.5-lrichloropltcnol. (T his is the International U nion o f Pure an d A pplied Chem istry nom en clatu re: in som e earlier literature the com pound h as been referred to as 2 J.6 .7 -tc tra c h lo ro d ib e n z o d io x in .) 1 ow ing to react w ithin d a y s w hile oth ers d id not present with established sym ptom s until betw een tw o and three m onths after the last known exposure. Y ounger m en, particularly those with fair com plexions, w ere first alTcctcd, while the reaction w as m ost persistent in sallow -skin n ed m en in the 23-40 age g r o u p .. particularly if their personal hygiene w as neglected. The established clinical condition w as present on the face in all cases. Inclusion type cy sts, ran gin g fro m I to 7 m m in diam eter, were diagn ostic (F ig . 4). T h e m alar fig. 5. C h loracn e affectin g the antecubital fossae. it pied w ore kian s X3r iO\ n the g the .. T h e ind it it th e lioxin .alt o f :h lo rw ith rally eared 14 705 9-4 36 t 4 n o . 4. F acial chloracne o f the m ultiple com edone and sm all cystic typo show ing well the m o st com m on dis tribution. areas w ere w orst affected, the cy sts dim in ish in g in num ber t v the condition extended on to the cheeks, m axillary a r e a s, a n d sid e s o f -the n eck : T h e e a r lo b e s , p o st-a u r ic u la r 4: triangles, an d suboccipital hair-line w ere also involved, in som e m en the condition w as predom inantly cystic, while in oth ers there w as a liberal sm atterin g o f com e d o n e s, ev e n to th e -e x te n t-th a t n o t - p o r e h a d been m issed an d the w hole side o f the face bore a d u sk y grey hue. The facial condition varied from very m ild an d ju st diagnosablc to severe and obvious. The other parts o f the body m ost often involved were the extensor asp ects o f the a rm s, the lateral asp ects o f thighs an d calv es, the b ack , an d the stern u m in that order. In five case s the antecubita! fo ssae w ere involved (F ig . 5), a condition which h as not previously been described, and in two cases the m edial asp ects o f the thighs were affected. Postular folliculitis, presum ably from secondary infection, w as present in a m oderate number o f cases. Treatm ent In the early stag es it w as foun d that the use o f ointm ents, cream s, p astes, an d steroids tended to aggravate the con dition . O ral oxytetracydin e, 250 m g, tw o to fo u r tim es daily, w as used fo r pu stu lar follicles, depen din g o n the severity an d extent o f the in fection. O therw ise, in th e m ore severe cases a I % solu tio n o f cetrim ide w as applied a fter th e. d aily b a th ; in m oderately severe cases zin c su lp h id e lo tio n , B P C , w as u sed w hile m ild case s W ere given n o lo cal m ed icatio n w hatever. O x y letracycline a n d cetrim id e w ere often used togeth er. Severe to m o d e rate c a se s w ere a lso treated w ith ultraviolet ligh t a t th e w ork s. T reatm en t w as reinforced by the m o st rig o ro u s cam p aign o f w orks an d persona! clean lin ess. A system fo r lau ndering overalls w as introduced and the m en w ere advised to use the show ers regularly, and to keep the garm en ts they w ore u n der their overalls clean . T h ey w ere also ask ed to ch an ge in to d e a n cloth in g before leaving the w orks. H air oils w ere discouraged an d thorough rin sin g o f th e skin after w ash in g, p ar ticularly sh avin g, w as recom m ended. F in ger n ails w ere kept sh ort an d d e a n an d steam ing o f the face .005774 682<! * H I F G 6 - ! - M O < 3 ----- 1 280 (.M a y a t h o m e, fo llow ed by gcn ilc sq u eezin g o f co m e d o n e s w ere iw ;irc, u sed both hut a t a low er op eratin g a n d c y sts w ith su b se q u e n t a p p lic a tio n o f cau -tlc- tem p e ratu re. It w as b eliev ed th at th e C o a lite m eth o d co lo g n c o r astringent lotion, w as ad vised. T h e m en w ere also recom m ended to get as m uch sunshine as could not produce an explosion an d . furtherm ore, in the event o f an u n fo reseen tem p eratu re rise, that p o ssib le . W ithin the next fo u r to six m on th s the great there w ould he few er an d less d an gerou s toxic b y p ro d u cts. A s far a s is kn o w n , the C o a lite m eth od m a jo r ity o f c a s e s m a d e a n a lm o st c o m p le te re c o v e r y , w a s u n iq u e in th at it u se d g ly c o l (b o ilin g p o in t 1 9 7 'C ) in reverse o rd e r, w ith the y ou n ger fairer in d iv id u als w hile oth er m eth od s u tilized m eth anol (b o ilin g lead in g . T en m en w ho h ad show n d isap p o in tin g poin t 64' C ). O w ing to the low er boilin g point o f the p ro g ress u n d er the general regim e w ere given intensive ste a m b ath in g follow ed by u ltraviolet latter, high p ressu res w ere requ ired b efo re a .suitable reaction tem p eratu re c o u ld b e reach ed in the liqu id ra d iatio n , after w hich even their ch lo racn c b egan to resolve. ph ase. T h e C oalite reaction took place at little m ore than atm ospheric pressure. A ttem pts to produce e x p lo sio n s in g ro ssly o v erh eated p ilo t p la n ts h ad i i T reatm en t o f the building T h e F in e C hem icals- U n it w as ag ain clo sed a n d n o faile d a n d it w as n ot until a fte r th e in ciden t th at the m o re to x ic b y p ro d u cts w ere iso lated in su ch pilot p la n ts. N eith er h as it sin ce been p o ssib le to re p ro c o n e w as allo w ed in to it w ith out sp e c ial p ro tectiv e clo th in g, inclu ding full face m ask , glo v es, an d b o o ls. duce an explosion. T h e only av ailab le reference to dan gerou s by r 'i A n y o n e c o m i n g o u t o f th e b u i ld i n g h a d t o d o s o p r o d u c t s f r o m th e in te r a c tio n o f s i m i la r c h e m ic a ls th rou gh a special decon tam in ation ro om w here he h ad assistan ce to change. The presence o f dioxin o n the w alls, ro o f, an d oth er p arts o f the plant w as sh o w n by paintin g a solution prepared from ru b b in gs tak en from different p arts o f the b u ildin g at explosive tem perature and force (Q uarterly Safety Su m m ary o f the A ssociation o f British C h em ical M an u factu rers, I9 6 0 ) briefly described tw o in ciden ts. In the first, a ch arg e d au to clav e w as h eat ing u p w hen the pressure an d tem peratu re increased L ( r < I o n the in n er sid e o f rab b its' e a rs, a s first dem on * is t r a t e d b y A d a m s , I r i s h , S p e n c e r , a n d R o w e (1 9 4 1 ). rap id ly an d an ex p lo sio n e n su e d , resu ltin g in fatal in ju ries. In the se c o n d in cid en t, w hich o ccu rred in r> T h e results w ere so consistent that they w ere checked b y testin g the plain solvent and even ru bb in gs from new asb esto s to dem on strate that these, on their o w n , cou ld n o t p rod u ce chloracn e. A s the ra b b it's e a r test to o k eight w eeks a m ethod o f o ral d o sage a n d co n tro l w as developed w ith estim atio n o f the a sim ilar p rocess, the tem perature ro se unexpectedly to 4 0 0 C w ith e scap e o f v a p o u rs. A fte r 10 d a y s all 20 p e rso n s e x p o se d fell ill an d h ad m o re o r less severe liver d am age. It w as th ought that sp o n taneous decom position h ad taken place and that pen tach lorooxydiph en yloxide w as form ed. T h e | ^ ii ci < seru m alan in e am in o tran sferase at fixed tim e residu es w ere very toxic, very stick y , an d difficult to I in terv als. N o t o n ly w a s this m eth o d fa ste r b u t it clean u p . w as m ore reproducible an d reliable. W hen a plan t in G erm an y w as to ld the reaso n fo r \ t It w as o b v io u s that the entire building w ould have to b e thorough ly clean ed and resurfaced. H eavy an d exp en sive equ ipm en t w hich h ad been affected by b la s r o r w as heavily con tam in ated w as bu ried 120 feet (42 m etres) deep at the b ase o f an open-cast c o a l m in e. D io x in is w ater in so lu b le an d w ill n ot b e leach ed ou t. A fter the m o st extensive an d v igo ro u s decon tam in atio n , sw ab s from the a n a s previously con tam in ated w e n tested on rabbits an d all w ere fo u n d to be n egative. R ab b its w ere also ex p o sed to th e atm osph ere o f th e building itself fo r on e w eek w ith n egative resu lts. P rodu ction b egan on ce m ore a n d since then n o new case o f ch loracn e h as devel o p e d in th a t b u ild in g , n o r h as c h lo racn e a p p e a re d am o n g the decon tam in ation sq u ad . D iscussion the cessatio n o f their su p p lies o f an o th er chem ical, n o rm ally prod u ced in the F in e C h em icals U n it, they provided detailed in form ation ab o u t a sim ilar experience som e years earlier in w hich they had identified dioxin a s the to xic m aterial. D io xin is a n atu ral, very m in or con tam in an t in the routine m anu facture o f irichloroph en ol. It d o es not ap p ear to an y great extent unless the reaction tem perature exceeds 2 0 0 C an d it is n orm ally rem oved in the pu rificatio n p ro ce ss. T h e re a fte r it b eco m es an in creasin g con stituent o f th e reaction p ro d u cts. In the G e rm an incident there h ad been so m e m on th s o f ch ron ic overh eatin g d u rin g w hich a ll the w orkers an d m an y o f their w ives, ch ildren , a n d dom estic an im als h ad developed ch loracn e. It w as only after p rod u ctio n w as finally term in ated b y a n explosion th at the grav ity o f the situ atio n w as fully realized a n d in ten sive research w a s c a rrie d o u t to iso late the to x in . *. i ^ ' Y > 1 r c c t ct 5 1 1 t \ r r c c 2 C M an u factu rin g p roced u re at C o a lite d id n ot utilize eith er p ressu re o r ag itatio n in th e au to clav e sta g e. E x p lo sio n s elsew h ere, o f w hich the research sta ff E xact figures o f in d ivid u als involved or o f m orbidity o r m ortality rates w ere n ot given , but there w ere a n um ber o f cases o f severe liver disord er, som e l T r c 0G0S77S 5 1 V .' Chiaritene Jiom the ucriilenial pitHluelion aj letrut hlorotlilH-n:oilio\in 2X1 D O W 994305 ip ' H 're. hat xic ru>d C) lin g th e ible llou irde lu c e had the lilo t pro- byc a ls crly itish tw o :c a t.ised a tal J in e d ly s all le ss or th The It t o i for cal, th e y n ilar had n is itin e pear ttu re i the an s. In m ths 'k e rs e stic after io n li/e d : the m orth ere .om e o f them fatal. Fifteen y ears later o n e in dividual w as expected to tlic so o n from this an d oth ers w ere still under treatm ent. O ne death from pulm onary carcinom a h ad been rejected a s bein g attrib u tab le V w hile on e o f intestinal sa rc o m a h ad been accep ted . T h e w orks m edical olliecr considered that dioxin passed in to the sy stem by iu lialatio n a s w ell a s orally ,, an d tran sd c rm ally . H e w'as in d e e d su rp rise d th at o u r ehloracnc case s h ad developed n o oth er sym ptom s. T h e G erm an literatu re on the su b ject o f d ioxin an d its toxicity m ak es n o sp ecific reference to this t particular p lan t. T h ere can be little d o u b t, how ever, that this incident alo n g w ith tw o o th ers fo rm s the b asis o f a scries o f articles (K im m ig and Sch ulz, 1957: B auer, Schulz, an d Spiegclbcrg. 1961; and Sch ulz. 1968) w hich d escrib e in great d etail n ot on ly chloracnc but all the toxic reaction s attribu ted to d ioxin , w hich include liver d a m a g e , em p h y sem a, . m yocardial degeneration, raised blood pressure, renal d am ag e, reflex irregu larities, d ep ressio n , disturbances o f m em ory an d con cen tration , etc. O cttal (H o fm an n , 1957) rep orted liver n ecrosis in rab b its kept in c ag e s ad jacen t to th o se e x p o se d to dioxin . F u rth erm o re, u n exposed an im als w hich w ere put into cages previously occupied by exposed i \ anim als a lso suffered liver n ecrosis so that a virus infection w as a t first su sp ected , i C row (1970) defined ch loracn c a s the `produ ction in th e h u m an su b je c t o f a ty p e o f a c n e w ith c h a ra c te r istic clinical featu res, so far produ ced on ly by aro m atic chlorinated h yd rocarb on s o f varyin g structures*. It is quite distinct from all the oth er acn es o f external o r ig in . T h e unch lorinated d ioxin m olecule d o es not cau se acne an d is a lso non-toxic. It h as been argu ed that the toxicity is related to the degree o f ch lorin ation . T his is d em on strab ly untrue b ecau se the 2,3,7,8tetrachlorinated dioxin h as often been show n to be not onl> th e m o st to xic but a lso th e m o st pow erful cause o f acn e. T h e interrelationsh ip o r positionin g o f ch lorin e a to m s in d io x in m o le c u lar sp a c e is, therefore, o f p aram o u n t im portan ce. A s in the "V d io x in s , s o m e m e m b e r s o f th e c h lo r n a p h t h a le n e s a n d the d ip h en yloxid es cau se ch lo racn e. T h e re is a stro n g school o f th o u g h t w hich believes th at it is n o t on ly the p o sitio n o f the ch lo rin e a to m s in m o le cu lar sp a ce but their sy m m etry w hich is resp o n sib le fo r their biological activity . W hether it be o n e o r b o th , w e have a w hole series o f co m p o u n d s w h o se unch lori* * nated m olecules are inert but w hich , w hen ch lori nated, an d depen din g b n the n u m b er an d position o f their ch lorin e ato m s, cau se an identical clinical ac o n d itio n . T h e y h a v e , t h e r e f o r e , c o m m o n f a c t o r and I su ggest that th is sh o u ld be called `the chloracne factor*. T h e literature sh ow s that ch loracn e is extrem ely resistant to a ll fo rm s o f treatm en t. W e sa w c a se s in i G erm an y w hich w ere still activ e a fte r 15 y ears' treatm ent and freedom from con tact. A t the present tim e, fo u r y ears after the incident at U olsovcr, c h lo racn c is ju st diagn o.sablc in seven c a se s. A t no tim e did we have to icsort to the in cision o f cysts, neither h as there been any legacy o f severe scarring. W hether this is du e to the com p aratively short period o f exp osu re to dioxin , to the com prehensive perso n al anti en viron m en tal hygiene m easu res or to a m ixture o f both , is dillicult to say. T h ough eh loracn c w as the presenting sy m ptom , the possibility o f underlying seriou s d isease w as not ign ored. N o o th er ailm ent presented w hich cou ld reaso n ab ly h ave been held attrib u tab le to d ioxin exp osu re an d co n tin u o u s m on itorin g o f liver function tests an d u rin alysis w as satisfactory . T h e G erm an com pan y had indicated that the brom sulph th alcin test o f liver function an d scrum gam n ia-globuiin levels w ere a lso im portan t. T h ese w ere ch eck ed in so m e o f the m o re sev ere c a se s w ithout revealin g an y ab n o rm ality . T h eir recom m en dation that w e sh ould carry o u t a few liver b io p sies w as d iscard ed , fo r the tim e bein g a t an y rate, ow ing to the con tin ued fitness o f the m en, the norm ality o f the oth er investigations, an d o u r desire not to create undue alarm . T im e ap p ears to h ave proved this decision to be correct. D u rin g the m on th s that follow ed n o co n tact, su ch a s w ife, child o r dom estic pel, w as reported to have c h lo ra c n e . R ab b its w ere used to dem onstrate the presence o f dioxin a s H o fm an n an d N eum ann (1952), dealing w ith a sim ila r incident, h ad in dicated th at ra ts introduced to the au toclav e ro om h ad show n no reaction an d gu in ea-p igs on ly non-specific reaction s. aC a t s , h o w e v e r , s u f fe r e d d if f u s e l o s s o f h a i r a f t e r lengthy laten t p e rio d , w hile rab b its d ied o f acu te liver n ecrosis after a latent period o f on e to three w eeks. O b viou sly , rab b its are extrem ely sensitive to ad i o x i p a s liv e r i n t o x i c a n t W hen the G erm an com pan y h ad resum ed its ow n p ro d u ctio n in the e arly 1950s they h ad n ot th ereafter ar e le a s e d s in g le b a t c h o f t r ic h lo r o p h c n o l w it h o u t as u b m i t t in g s a m p l e t o a n e ig h t- w e e k b io lo g ic a l a ssa y ag ain st the ra b b it's e ar. I f ch lo racn e w as then p rod u ced the d ioxin content w as to o high. T richioroph en ol is very im portan t com m ercially a n d it w a s th erefore n ecessary to c arry o u t to xicity tests o n C o a lite 's ow n com m ercial p ro d u ct before go in g into produ ction once m ore. A great deal o f in form ation w as availab le o n an im al exposu re (A nderson, A m d t, G oldbcy, an d Jo n es, 1949; Farqu h arson , G age, an d N orth over, 1958; M cC o llister, L o ck w o o d , an d R o w e, 1961) w hich indicated that trichloroph cnol w as not toxic unless given in m assiv e d o sa g e . In deed, M cC o lliste r et at. (1961) h ad show n that the rab b it, though not a s resistan t a s the ra t, d id h ave a high toleran ce to at r ic h io r o p h e n o l. I t w a s s h o w n t h a t d o s e o f th e 0005776 282 O'. M ay C oalite com m ercial product o f abou t I g/k g had to be given b efo re it w as leth al. In the incident describ ed here, the ch lorin e ato m s in d io x in w ere situ a te d at p o in ts 2,3 .7 , an d 8, w hich ap p ears to be the m ost toxic o f the d ioxin gro u p . T h ere ore, how ever, eigh t poin ts o n to w hich a chlorine atom can be h ooked an d therefore there c a n b e o v er 60 dilTcrcnt ch lo rin ated d io x in s, d ep en d * ing on the n um ber an d positio n o f the chlorine ato m s. V ery little is know n ab ou t the other chlori n ate d d io x in s in w hich the b io lo g ic al activ ity is a lso d ictated by the n u m b er an d positio n o f the chlorine atom s. A lth ou gh C o alite chem ists failed to prod u ce an exp losion experim en tally u sin g the o ld p rocess, on e d id take place eventually in the production p lan t, despite the absence o f pressure an d agitation . W hether the sh eer size o f the com m ercial plant cou ld excite an exotherm ic reaction in to becom ing explosive o r w hether there w as an y oth er con stituent is not p o ssib le to say . A n exotherm h as been ap p aren t experim en tally a t 4 0 0 'C , an d d io xin h as been recovered an d purified from this production (M iln e s, 1971). A n en tirely new p lan t to p rod u ce 2,4,5-trich loroph en ol in w hich a ll p ro cesses, in clu ding tem perature con trol an d oth er new techniques, are au tom ated h as been in o p e ratio n fo r three y e ars w ith ou t the occurrence o f ch loracn c o r production o f dioxin . A ll batches are now subm itted to gas liquid ch rom a to grap h ic ( G L Q an aly sis b y a technique specially dev elo p ed to test fo r d io xin an d w hich is sen sitiv e to 0-2 p .p .m . T h is level is con sid ered to b e w ell w ithin the lim its o f acceptab ility . In the process fo r trichlorophenol produ ction currently in u se orth odichlorobenzin e h as been replaced' by petroleum eth er. T h e form er process involved a total con den ser an d extern al sep arato r (g lass condenser). T h e petrol ether process incor p o rate s a reflex co n d en ser fab ricated fro m steel an d reflex d ivid er w hich is m uch sim p ler an d safer. A d d itio n ally , each re a c to r h a s five fail-safe fu n ctio n s b u ilt in to the in stru m entation independent o f the program m e con trol. T h ese functions are designed to sh u t dow n a re a c to r au to m atically in the even t o f 'a fail-safe con d ition being reached an d to shut dow n th at reacto r in su ch a w ay that an exoth erm ic reaction is not possible. T h e program m ing com p u ters m em orize the sligh test d eviation to w ard s a fail-safe situ ation an d are therefore continuously u p d ated . T h e tem perature o f the reaction is held at 1 8 0 *C until tests sh o w th a t there is n o d io x in in the reaction m ixture. A fine p o w d er su ch a s purified d io xin co u ld ob v io u sly b e ab so rb ed b y inhalation an d o ral ingestion. In o u r ow n case, the con tam in an t w as trap p ed in a h eavy greasy d e p o sit s o w e are inclined to ru le o u t in h alation com pletely. E arly liver fu n ctio n tests ap p eared to in dicate (he p resen ce o f a toxin w hich m ight have been inhaled b u (..ih csc d ep artu res from norm al w ere transient an d none o f the in dividuals concerned developed chloracnc. In the event the w orkers w ho did develop ch loracn c w ere those w ho had to get dow n to h andling pipes, jo in ts, c a b le s, an d the like w ith (h eir h are h an d s, an d this su ggests that these m en ab sorbed the dioxin th ro u gh the sk in . In o u r o p in io n , there m u st then be so n ic excretion o f ab sorbed dioxin o r its prod u cts by th e facial pores. It is su ggested th at in these {ight-exposed areas, such a s pinnae an d m alar region , n* chem ical reaction takes place, prob ab ly itself light-induced, cau sin g oedem a an d ob stru ction o f the follicles w ith subsequen t fo rm atio n o f reten tion c y sts an d co m ed o n es. It is not until the fo llicles re-open an d function norm ally that ch loracn c begins to d e a r , an d it is fully accepted that the m e th o d s by w hich this becom es possible w ere not, in o u r case a t least, th ose necessarily ap plicab le to o rd in ary acn e. A t the C oalite R efinery ap p ro xim ately 300 m en are em ployed and no new ease o f chloracne has ap p eared am o n g them since D ecem ber 1968. In M arch 1971, con stru ction began o f an entirely new building fo r an unrelated product using, alm o st th rou gh ou t, factory fresh m aterials. T h is building w as erected by outside con tractors using their ow n em ploy ees. A fter only three w eeks a t this w ork tw o o f these em ployees developed a skin condition w hich they reported to their ow n d o cto rs an d thereafter to separate derm atologists. T h e so n o f o n e ( F i g . 6) a n d th e w ife o f th e o t h e r a l s o d e v e lo p e d th is co n d itio n w hich w as u n d ou bted ly ch lo racn e (Jen sen an d W alker, 1972). T h e on ly piece o f eq u ip m en t w ith w hich they h ad an y au th o rity to c o m e in co n tact, w hich co u ld po ssib ly h av e been co n tam in ated w ith d io x in , w as a large m etal vessel w hich h ad been su b ject to the m ost rig o ro u s clean in g. C o alite 's ow n em ployees h ad n ot on ly w elded n ew c o lla rs to it a fte r b u rn in g new ven ts b u t h ad actu ally been inside it. L ate r tests (G L C an d dou ble b io lo gical assa y ) o n th is vessel failed to reveal the presence o f d ioxin . T h e only con tact these individuals h ad w as w hen they connected u p new equ ipm en t to the new collars. Furth erm ore, they only w orked to g eth er fo r a d a y o r tw o an d yet they d ev elo p ed the m o st p ersisten t ch lo racn e o f a ll; it is b elieved they a re still un dergoin g intensive treatm en t, a n d they w ere the on ly people to p ass the con dition on at h om e. W e h ave been un able to satisfy ourselves w h ere o r h ow th is con tam in atio n occu rred . C o alite's ow n w orkers m ust have been at very m uch greater r isk o v e r m an y m o n th s b efo re. E ven i f it c o u ld be p o stu lated th at toleran ce can develop to dioxin, w h ic h is n o t s o , e x a m p le s sh o u ld h a v e b e e n se e n in n ew co m ers to the C o m p an y stalT. It is certain ly the c a se th at th e m en affected d id n ot av ail them selves o f the h igh -grade p erso n al h ygiene facilities a t the cc i CO CO .i.og f 05 I r nc cor pte * b ull I 9 \ we thi: de' anc It v in t \ hac *> F d ie Ms pro con R et pro n> n ic e tarn Ser F lo ; eigh trac D io lim i eag! CG05777 6827 yiik D O W 994307 IX aT C h io m a te f r o m the a c e h lm t a l proihirtion o j ictr<ulili`rotUlH-it:otli<i.\iii 2X3 i f e I wish to thank the Directors of Coalite and Chemical Products l.imilcd for permission to publish this article, the Coalite Research Laboratory, where pure dioxide was isolated, the late Dr. Herbert llondy, chemical adviser, and Dr. Ken Payne, research director, for com ments on the technical aspects of the paper, and The Huntingdon Research Institute where extensive animal i investigations were carried out. largely by Ronald E. n Davies. Dr. Doris Fletcher, formerly consultant derma c tologist nt Chesterfield Royal Hospital, advised on the s treatment of the patients. c r y n n References -s Adams, E. M-, Irish, D. D.. Spencer, Il C.. and Rowe, V. K. va y -c (t1o9h41av).eTchaeurseesdpoancnseefoofrrmabdbeitrmskaintittios.cIonmdupsoturinadl sMreedpiocritneed, Industrial Hygiene Section, 2,1-4.. Anderson. < . W,, Arndt. C. It., Goldhcy, E. J.. and Jones. y o .s TtMr.iecCdh.ilco(a1rol9Ap49hs)sc.onCcoiaalt.ttiloJenof,ueIreIndSain,l g1o2tr1fi-a1tl2hs3ew. itAhmdeerriicvaaativeVseotefr2i,n4a,5ry- Bauer, H.. Schutz. K. H., and Spicgelberg. U. (1961). y s n o no. 6. Case o f contact chloracne showing fairly typical CChroeGMHpBwrhreeoa,yrevsru,neKpkfeoRiler.tilahtca-ilVDehnlphegeD.hyrgVe(hRL1irieeem9n.nrp7(gdaeo10i,utfr9o)ntt.17luego82rnCcg,),gn.hi5ec1.Wl.na61oAl8rOaSrba-s5ccoech5hnicti5ioneidv.eb.getfetryTor,r,nrH5ap6entG.,ars19selatk3.wce5stelil0roiutb-nn5eosgp1voa.vetfroh.nSoCltoC.hghJeieomlohuirncnoa'sd-l ^^^o.bsrR'CLQ-i'.ao^fto. n d >f comedone distribution but no cysts. There is some malar pigmentation, a condition frequently described elsewhere but one which did not develop among the original cases. Farquharson, M. PThhaermbiaocloogloicgayl, E.. Gage, action of cJh. lCor,opahncdnNolos.rtBhroitvieshr,JJo.u(r1n9a5l8o).f 13, 20-24. --H--o--fkbtmooe,xlraoaignsgn'cnsidhe,Ae, nNrH2c3e.h2uCi,Tvmh2.fla2on8r(rn1k-29o,e35xhW07pl.e)e..nriw(mN1aee9sun5stee2err)lse.lieoEEfPilrncafnetah.horMlNuoneagtugiheenonyudnne-mdScziPthuhmrahiroetmicdehaer--- w orks fo r eith er their person s o r their cloth in g an d this m ay e x p la in th e h om e tran sferen ce. T h e w ife developed sy m p to m s n in ejn o n th s after h er h u sb an d , and eight m on th s alte r his last em ploym en t here. esNexauppehhrtiztm,h2ea,lnitn1ee6l.l9e-Zn1e7n3Pt.rralfbulnagtt fderrArHbeauittswmierdkiuzinnguncdhlAorrbieerittesrJensceanse, sN. P. rEo-c.eaednidngWs aolfkethr,eAR.oyEa.l(S1o97c2ie).tyCohfloMraecdnicei:net,h6re5e, It w as not p o ssib le to d em o n strate d io xin b y G L C 687-688.- in th e c o n te n ts o f h is fa c ia l c y sts, so m e th in g w h ich had not been tried on the origin al cases. 4 R ecently, en viron m en talists h ave been w atch in g Kimmig, J.. and Schulz. K. H. (1957). Berufliche Akne (zsyocgleisncahnentetnherC. hDloeriamkantoel)ogdiucrac,hIISch, l5o4r0ie-5rt4e6.aromatische McCollisier, D. D,, Lockwood. D. T ,, and Rowe. V. K. dioxin co n tam in atio n o f 2 ,4,5-T v ery carefu lly . M any o f the early b atch es o f th is h erbicide h ad been produced at excessive tem peratures an d , o n release, contained u p to 4 0 o r even 6 0 p .p .m . o f d io xin . (1961). phenol. TTooxxicicoolologgyicandinAfpoprmlieadtiPonharmonaco2lo,4g,y5,-3ir,ic6h3l-o7r0o.- Milnes, M. H. (1971). Formation of 2.3.7,8-tetrachloro* d2,i4b,e5n-ztroidchiolxoirnophbeynatthe.erNmaatlured.e2c3o2m,p3o9s5i-t3io9n6. of sodium R e g u la tio n s , q u a l i t y c o n t r o l, a n d c e s s a t i o n o f Quarterly Safety Summary o f the Association o f British at 2S production b y co m p a n ies w ithout ad eq u ate tech * nical k n o w led g e h av e n o w elim in ated d io x in c o n * Chemical Manufacturers (I960). Fire and explosion-- Trichlorophenol (a) Case history No. 620. (6) Supplement to Case No. 620. Voi. 31. p. 22-23. 's la m in a tio n . T h e U n it e d S t a t e s A g r ic u lt u r a l R e s e a r c h Schulz, K. H. (1968). Zur Klinic und tiologie der Chlor Arbeitsmedizin Sodalmedizin Arbeitshygiene,ZT TC Service (C h e rry , 1972) h a s an aly sed sa m p le s fro m Florida experim en tal p lo ts that h ad received fo r akne. 25*29 2, n , eight y e ars m a ssiv e 2 ,4 ,5 -T sp r a y s w ith d io x in in traces u p to 4 0 p .p .m . w ith out fin d in g re sid u e s. * TC D io x in r e s i d u e s h a v e n o t b e e n f o u n d a t t h e l o w e r e s limit o f d e t e c t io n (0 -0 5 p .p .m .) in t is s u e s o f th e b a ld Received for publication May 2, 1972. .te eagle co llected in 15 w id ely se p arate d sta te s. Accepted for publication December II, 1972. GG0H7S Ij w W i i -- m m wiiiWi rrf" wrr HUJNIU'J Io*t DOW352579 M u t a t io n R e s ta r c h , 2 1 (1973) 335-340 >0 ElsevierScientificPublishingCompany. Amsterdam - Printed inThe Netherlands 335 LY M PH O C Y TE CH RO M O SO M E A N A L Y SIS O F A G R IC U L T U R A L W O R K E R S D U R IN G E X T E N S IV E O C C U P A T IO N A L E X P O S U R E TO P E ST IC ID E S* JULIE YODER, MICHAEL WATSON and W. W. BENSON ESenrvviircoens,mBeonitsael, PIdraohteoctSio3n720Ag(eUn.cSy.,A.I)daho Statt Department of Environmental and Community (Received March ist. 1973) (RevisionreceivedJune 4th. 1973) SUMMARY L y m p h o cy te cu ltu res from 42 p esticid e ap p licato rs an d 16 con trols w ere sc ru tin iz ed fo r c h ro m o so m al a b e rra tio n s d u rin g th e m id w in ter e b b in p e stic id e sp ray in g o p eratio n s, an d ag ain d u rin g the p e ak su m m er period o f in ten se sp ray in g activ ity . C ultures p repared from the ex p o sed in d ivid u als du ring th e h eavy sp ray in g p erio d show ed a m ark e d in crease in th e freq u en cy of ch ro m atid lesio n s. T h is tren d w as esp ecially n oticeab le am o n g w ork ers e x p o se d p rim arily to h erb icid es. A few ch rom atid exch an ge figu res w ere also n oted am o n g th e exp o sed gro u p . A lso d iscu ssed is the n eed for b etter m eth od s of estim a tin g th e p o ten tial m u tagen ic th reat, if an y , o f su ch o b served clastogen icity . INTRODUCTION T h e Id ah o C om m u n ity S tu d y on P esticid es is one o f th irteen sim ilar research facilities th ro u gh o u t the U n ite d S ta te s in a re a s w here p esticid es are e x te n siv e ly u sed . Sp o n so red b y the E n v iro n m en tal P rotection A g en cy , the p rim ary ta sk of th is un ified in v e stig a tiv e effort is to e v a lu a te th e effects o f p esticid e ex p o su re on h u m an h ealth . T h e Id a h o p ro g ra m is n ow in its six th y e a r o f in te n se clin ical, b io c h e m ic al a n d e p id e m io logical scru tin y of a core gro u p of carefu lly selected v o lu n teers h av in g extrem e o ccu p atio n al ex p o su re to v a rio u s p esticid es. A n ad d itio n al p a ra m e te r cu rren tly bein g e x am in e d in d e ta il is th e p o ssib ility o f u sin g c y to g cn ic a n a ly sis of ly m p h o c y te ch ro m o so m e s a s an in d e x o f p e stic id e m u ta g e n ic ity in m an . C y to g e n etic a n a ly sis o f c h ro m o so m al a b e rr a tio n s in so m a tic ce ll c u ltu re s is o n e of th e m a jo r m eth od s of testin g cu rren tly recom m en ded fo r e v alu atin g chem ical m u t a g e n e s i s in m a m m a l i a n s y s t e m s 4" -" . M o s t i n v e s t i g a t o r s a g r e e t h a t t h e d o m i n a n t leth al test an d the h ost-m ed iated a ssa y are also im p ortan t test p roced u res, a n d th at *Thisresearchwas supported underContract No. 68-02-0332by the Divisiono l PesticideCom munity Studies. Officeof Pesticides Programs, Environmental Protection Agency, through the Idaho StateDepartmentofEnvironmentaland Community Services. 0002130 " 336 ' J. Yoder et a l. a ll th re e sh o u ld b e u tiliz e d in c o n firm in g m u ta g e n ic ity in m a m m a ls. H o w e v e r, th e la tte r tw o tech n iqu es are n o t su ite d fo r u se w ith h u m an su b je c ts, an d so m a tic cell cu ltu res th u s rem ain th e on ly p rac tic a l m eth o d of assay in g m orph ological eviden ce o f a p p a re n t d a m a g e to th e h u m an g e n e tic m a te ria l, per sc. W ith re sp e ct to su ch c u ltu r e s , in vivo te c h n iq u e s a r e p r e fe r re d o v e r in vitro t e s ts b e c a u se th e y p ro v id e th e a d d e d a d v a n t a g e o f t a k i n g m e t a b o l i s m i n t o a c c o u n t * - *. A l t h o u g h s u c h a n a p p r o a c h b y itself can n ot provid e u n assailab le proof o f concurrent g am etic in volvem en t, it a p p e a rs likely th a t au to so m a l ab e rratio n s m a y in d icate parallel go n o so m al even ts of a s i m i l a r n a t u r e 14. E v e n a s a p u r e l y s o m a t i c i n d i c a t o r , a n a l y s i s o f l y m p h o c y t e c h r o m o so m es w ould seem h igh ly p ertin en t w hen on e con siders th at m an y sp o n tan eo u sly occu rrin g d iseases, certain n eo p lasm s in clu d ed , are now believed to be cau sed b y en viron m en tal pollu tan ts*. T h e trem en d o u s a r ra y o f a g ric u ltu ra l x e n o b io tics now in com m on u se co n sti tu te s a larg e p o rtio n o f th e v a rio u s ch em ical a g e n ts to w hich m an is en v iro n m en tally e x p o se d . T h e in d iscrim in ate u se o f su ch su b sta n c e s w ould lo gically p resen t the p o s sib ility o f a gen etic legacy of in creased m u tatio n frequen cy an d su b seq u en t h um an h azard . In creased ab erration s of ly m ph ocyte chrom osom es h av e been recen tly re p o r te d * in a g r o u p o f M ic h ig an fr u it p r o d u c e r s in v o lv e d in h e a v y p e stic id e sp r a y in g . A ccord in gly , w e h av e ex am in ed ch rom osom es o f periph eral ly m p h ocy tes (leukocytes) cu ltu red from persons selected from our occu pation ally exposed stu d y grou p. T h e p re v ale n ce of ch ro m a tid lesio n s a m o n g th ese v o lu n te e rs h as then been co m p a red w ith th at o f a con trol grou p, b oth prio r to an d d u rin g th e h eavy sp ray season . MATERIALS AND METHODS I t w as in itially h oped th a t sufficient n u m b ers of persons h av in g exclu sive occu p atio n al exp o su re to a single ch em ical series cou ld be ob tain ed . H ow ever, b ecau se of the g re at ag ricu ltu ral d iv e rsity o f th is region , su ch a sam p lin g tech n ique p ro v ed to be im p o ssib le . W ith in C an y o n C o u n ty a lo n e , in w h ich m o st o f th e e x p o se d p a r tic ip a n ts resid e, m ore th an one m illion p o u n d s of p e sticid e s w ere ap p lied to cro p s d u rin g 1972 (ref. 10 ). T h is fig u re, irresp ec tiv e o f o th e r a g ric u ltu ra l ch em icals su cli a s fertilizers, am o u n ts to ap p ro x . 4 p o u n d s of p e sticid e s fo r each acre of farm lan d . P erso n s highly e x p o se d to p esticid es in th is a re a u su a lly a p p ly a larg e v arie ty of ch em icals th rou gh ou t th e grow in g seaso n , an d m u ltip le e x p o su re is th e ru le rath er than th e excep tio n . F o r th ese re a so n s, d ose or d u ratio n of e x p o su re is an o th er v ariab le th a t can n ot b e rigid ly defin ed b y su ch an ap p ro ach . B e c a u se of th eir local p rev alen ce in p esticid e-related o ccu p atio n s, on ly m ale C au ca sian s w ere sam p led . P erso n s w ith recen t h isto ries o f viral in fectio n s, chem o th e ra p y o r X - r a y tre a tm e n t w ere re je c te d , a s w ere th o se w ith h isto ries o f m a lig n an cies. A to ta l o f 16 co n tro l an d 42 e x p o se d p erso n s, m atch ed a s closely a s p o ssib le a s to ag e s an d p h y sical c h aracte ristics, w ere ch osen . H igh exp o su re w as defin ed a s sp ray in g w ith p e stic id e s for a t le a st 4 h p e r d a y on th ree o r m ore d a y s jk t w eek th ro u g h o u t the p eak sp ray season of Ju n e 15 th th rou gh A u gu st 30th. T h e control grou p w as com p osed of b u sin essm an , stu d e n ts, h ealth p ro fessio n als an d teach ers w ho h ad no h istory o f in v o lv e m e n t w ith p e stic id e s. A g e s o f th e co n tro l g r o u p ra n g e d fro m 19 to 64 (m ean 39.S ). P e r so n s in th e e x p o se d g r o u p ra n g e d in a g e fro m 18 to 70 (m e an 38.6) a n d h a d from I to 25 y e a rs (m ean 8 .J) o f prio r o ccu p atio n al exp o su re to pesticid es. A s show n o o * PESTICIDES AND CK C O TABLE I on ro POERSDTEIRCIDOEPSFRMEOQSUTECNOCMY? onon froup A o h tsrcticv lc-n f'o srd Com m on nam e Dcmcton Ethyl parathion Trichlorofon Nalcd Malathion Dicofol < Diazinon < Emlosulfan 0 DNOC F Methyl parathion C Toxaphene C Dimcthoate O DDT Oi Chlordimeform CK Oxvdcmctomnethyl Of TEPP OF Carbary! C 1Nomenclature after Berg bOP. organicphosphorous series. in T a b l e I , p e r s o n s i n tl~ acco rd an ce w ith th e ir p a g e , 42.3 ; m ean y e a r s o f . fs p r a y r ig o p e r a t o r s a n d chem icals m ost frequ en t, pounds dem eton, eth y l p : applied less frequ en tly b; 36.5 ; m e a n y e a r s o f p rio r of local w eed con trol a g h e r b ic id e s ; e s p e c ia lly 2 ,4 - B lo o d sa m p le s (10 during the off-season , Ja i pation allv exposed for a ob tain ed from all ]>crsons the peak season of p cstic d aily exposu re am on g tlu exten siv ely prior to botl fully noted. Lym ph ocyte cu itu i kit?. (D ifco L a b o r a to rie s, p r e v i o u s l y r e p o r t e d b y .1 proceduie w as to in crcas facilitate the tech n ique. 1 m ito tic d iv isio n in o rd e r 0002131 J. YODER et al. lam m als. H ow ever, the S je c ts. a n d so m a tic cell m orph ological evidence . W i t h r e s p e c t t o s u c h -. cause t h e y p r o v id e th e =ough such an approach .am e tic in v o lv em en t, it llel go n o so m al ev en ts of ; o f lym ph ocyte chrom oa t m an y sp on tan eou sly :lieved to be cau sed b y .- in c o m m o n u s e c o n s t i m an is en viron m en tally gically p resen t th e pos- - and subsequent hum an - h av e been recen tly rc:eav y pesticid e spray in g, y m p h ocytes (leukocytes) posed stu d y group. The th en b een co m p ared w ith sp ray season. :is h av in g exclu sive occuied. H ow ever, b ecau se of ;g tech n ique proved to be the exposed p articip an ts lied to cro p s d u rin g 1973 m icals su ch as fertilizers, farm lan d . P erson s highly y of chem icals th rou gh ou t r th a n th e e x c e p tio n . I; o r ble th at can n ot be rigid ly d occu pation s, on ly m ale i v iral in fections, chcm oith h istories of m align an is c lo se ly a s p o ssib le a s to re w as den n ed a s sp ray in g s p er w eek th rough out the : control group w as com * ichers w ho had no h istory m g ed from in to 64 (m ean to 70 (m e an 38.6) a n d h ad re to p e stic id e s. A s sh ow n PESTICIDES AND CHROMOSOMAL ABERRATIONS 337 TABLE I PESTICIDES MOST COMMONLY USED BY EXPOSED SUBJECTS IN THIS STUDY* (LISTED IN DECREASING ORDER OP FREQUENCY) C r o u p .1 in secticid e-exp o sed Croup B H erbicide-exp osed Com m on name Dcmcton Ethyl parathion Trichlorofon Xated Malathion Dicoiol Oiazinon Endosulfan DNOC Methvl parathion Toxaphcne Dimuthoate DDT Chlordimcform Oxvdemctonmethyl TEPP Carbary! Tvpe OP OP OP OP OP OC OP . OC PH OP OC OP OC OC OP OP C Com m on nam t .4-I) Aniitrole Atrazine Zineb (fungicide) DCPA Dicamba Chiorpropham Alachlor Metiram (fungicide) Trifluralin Maneb (fungicide) Pidoram Diquat S.4.3-T *NomenclatureafterBerg1and Frcar*. *OP, organicphosphorousseries;OC, organicchlorineseries;PH. phenolicscries;C.carbamate series. in T a b le I , p e rso n s in th e e x p o se d g r o u p w ere fu rth e r d iv id e d in to tw o su b g r o u p s in acco rd an ce w ith th eir pred o m in an t ty p e of o ccu p atio n al ex p o su re. G ro u p A (m ean a g e , 42.3 ; m e a n y e a r s o f p rio r e x p o su r e , 1 2 .0) c o n siste d o f 16 c r o p d u ste r s, fo r m u la to r s, sp ra y rig o p erato rs an d farm ers w ho w ere chiefly ex p o sed to in secticid es. T h e specific ch em icals m ost frequ en tly d isp ersed b y G rou p A w ere th e organ ic ph osp h oro u s com p o u n d s dem eton , eth y l parath io n , trich lorofon an d n aled. T h ese an d oth er in secticid es ap p lie d less freq u en tly b y the g ro u p a re su m m arized in T a b le I. G ro u p B (m ean ag e, 36.5 ; m e a n y e a r s o f p rio r a g r ic u ltu r a l e x p o su r e , 6.3 ) w a s c o m p r ise d o f 26 e m p lo y e e s of local w eed con trol agen cies. T h ese perso n s w ere all p red o m in an tly e x p o se d to h e r b ic i d e s ; e s p e c ia lly 2 ,4 - D , a n iit r o le a n d a t r a z i n c ( T a b le I ) . B lo o d sa m p le s (10 m l h ep arin ized ) w ere first o b ta in e d fro m a ll v o lu n te e rs .d u rin g th e off-season, Ja n u ary -F e b ru ary , 19 71, w hen none of th em h ad been occu p a tio n a lly e x p o se d for a period o f a t le a st 30 d a y s. A seco n d b lo o d sa m p le w as th en o b tain ed from all perso n s d u rin g the p erio d o f Ju ly th rou gh A u g u st, w hich is w ell in to th e p eak season o f pesticid e ap p licatio n an d rep resen ts a com m on in te rv al of in ten se d a ily ex p o su re am o n g the ag ricu ltu ral w ork ers selected . B ach person w as in terv iew ed ex ten siv ely prior to b oth sam p lin g s an d all p esticid e-related in fo rm ation w as care fully n oted . L y m p h o cy te cultu res w ere p rep ared b y m ean s o f D ifco T C ch rom osom e cu ltm e k its (D ifco L ab o rato rie s, D etro it, M ich igan ). T h e tech n iqu e is b ase d on th e proced u re p r e v i o u s l y r e p o r t e d b y M oorhead et a l . 13. O u r o n l y m o d i f i c a t i o n o f t h e i r s t a n d a r d procedu re w as to in crease in cu bation tcm p e ratu ic fro m 37* to 38, w hich seem ed to fa c ilita te th e tech n iq u e . T h e sa m p le s w ere c u ltu re d fo r 48 h to o b ta in c e lls in th e first m ito tic d iv isio n in order to m in im ize th e v a ria b ility in ab e rratio n y ield th a t is som e- 6832 03)02132 D O W 352581 333 J. YODER Ct a l . t i m e s a s s o c i a t e d w it h l o n g e r c u l t u r e t i m e s 1. F l a m e - d r i e d s l i d e s w e r e s t a i n e d w ith G iem sa an d rearran ged an d coded b y a person other than the scorer to av o id bias. 25 m e ta p h a se sp re a d s fro m each person w ere then ex am in e d m icro sco p ically for ch rom osom al ab erratio n s. O nly k a rv o ty p ab le cells h av in g w id esp read ch rom atid s w ith m in im al o v e rla p p in g w ere se lecte d fo r sco rin g . P h o to g rap h s w ere m a d e o f all ab n o rm al m etap h ase figu res an d k ary o ty p in g w as perform ed w h en ever there ap p eared to be eviden ce o f o b v io u s ch rom osom al rearran gem en t. T h e criteria of Geb- hardt'w ere follow ed in th e d e term in atio n o f c h ro m a tid a c h ro m a tic lesio n s (gap s), b re ak s a n d 'c h ro m atid e x ch an g e figu res. RESULTS AND DISCUSSION O th er th an ran d o m loss of chrom osom es, h eteroploidy w as n ot d em on strated in a n y o f th e 1450 m ito tic b u r sts e x am in e d . W ith re sp e c t to ch ro m o so m al lesio n s, th e m a jo r ity o f a c h ro m a tic a re a s (gap s) an d b re a k s w ere confined to sin g le ch ro m atid s. A lth o u gh iso ch ro m atid lesio n s w ere o ccasio n ally en cou n tered in b o th con trol an d ex p o sed in d iv id u als, th is ap p aren t tren d tow ard ch rom atid ab erration s rath er than ch rom osom e ab e rratio n s w ould a p p e a r to m erit fu rth er stu d y . T h e in cid en ce o f c h r o m a tid le sio n s on a p e r p erso n b a sis is p re se n te d in T a b le II. T h e m ean n u m b er o f g a p s an d b re ak s p e r p erso n p er 25 cells e x am in e d d id n ot ap p re ciab ly ch an ge fo r th e con trol gro u p a t eith er sam p lin g period. A m o n g th e e x p o se d g ro u p , h ow ever, th e m e an n u m b e r o f g a p s p e r p erso n n e arly d o u b le d in cul- TABLE II SEASONAL VARIATION IN TH E OCCURRENCE OR CHROMATIS LESIONS G roup Sum btr 2J( p e r p e r s o n p e r cells scored) C hrom a tid gaps C h rom a tid breaks O ff-season M id -sea son O ff-season M id-season Controls Totalexposed 26 41 Group A 16 (Insccticide-exposcd) 16 Group B 36 (Herbicide-exposcd) Mein S.E. 0.63 0.23 Range >-3 Mean S.E. 0.62 0 .11 Range 0-2 Mean S.E. I.OO 0.23 Range 0 -3 Mean S.E. 0.38 0.10 Range -X 0.69 0.24 0-3 1.14 0 .17 0-3 0.75 0 .17 0-3 1.38 0.22 0-3 0.31 0.12 O--I 0.17 4-0.08 -3 0.31 0.18 0-3 0.07 0.03 0-1 0.44 0.22 0-3 *71 0.24 0-6 1.36 0.39 0-3 1.81 0.33 0-6 tu res from th e m idsu m m er sam p les, an d the m ean n um ber of b reak s observed per p erso n in c re ase d b y a fa c to r o f 10 . T h e m a x im u m n u m b e r o f g a p s fo u n d in a n y sin gle e x p o se d in d iv id u a l (T a b le I I ) in c re a se d fro m 2 in th e sla c k se a so n c u ltu re s to 5 in th e m id seaso n cu ltu res. M axim u m b reak s for an y single exp osed volu n teer rose from 2 to 6 d u rin g th e sa m e p erio d . (D ifferen t p e rso n s w ere in v o lv ed in each o f th e fo u r c ase s cited .) T h o se ex p o sed p rim arily to in secticides (G rou p A) exh ib ited a 25% redu ction in th e m e an n u m b e r o f g a p s p e r p e rso n in c u ltu re s fro m th e h igh e x p o su re p erio d . H o w e v e r, th e m e a n n u m b e r o f b r e a k s in c re ase d 5 -fold in th e se sa m e sa m p le s. M em b ers o f th e g r o u p fre q u e n tly in v o lv e d w ith h erb icid e sp ra y in g (G ro u p B ) sh o w ed an in- PESTICIDES AND CHUO> crease of n early 4 tim e; season . M ean b reak s p period (T able II). A lso b reak s, respectively, d ex p o sed grouj). O n the n o te d in G ro u p B w orl b ility of in du ced com ] hanccd chrom osom al r CO D uring th e Ju h CJl c e l l s e x a m i n e d ) , a s m ro in clu ded one G rou p A < OT 00 an d a possib le G ro u p * to g e n e r a l p o p u l a t i o n a t in o u r ex p o sed gro u p a n y of these con figu rt w ere sam p led in th e cc v e rsio n in th e A 2 ch r p e a r in a n y o f th e oth From these res d a m a g e in ly m p h o cy t g re ater during the h er ever, the p ossib ility 0 lativ c. B ecau se of the the direct ph en otypic retro sp ectiv e techniqi. existin g h ealth sta tist com piled w ith th e ger in ciden ce of su ch par p la s m s , etc., a m o n g a b e cau se of insufficient estim ate m u tagen icity view tech n iques. H ow research er a n d su b ject REFERENCES 1 R entier. M. A., and human peripheral leu 7 B erg, G. L.(Ed.). F a 3 Epstein, S.S..Contr 4 E pstein, S.S..and M M.I.T..Cambridge. M 5 Evans. H. J., Popul. Brice and V . Law (E 1 0 7 0 . pp. 10 9 - 1 0 *. 6 F rear. IV E. H.. P e s . 7 GF.miARDT. K..The t: K o h r d o h x (Eds.),C i PP- JO7-3S*. 8 G ebhardt, E.. Mut; 6833 0062133 J. YODER C t a l. les w ere stain ed w ith - scorer to avoid bias, d m icroscopically for id espread ch rom atid s ;phs w ere m ade of all l w henever there ap. The criteria of G e b -o m atic lesions (g a p s). w as not dem onstrated > ch rom osom al lesion s, d to single ch rom atid s. 1 in b o th co n tro l a n d berration s rath er th an a is p resen ted in T a b le cells exam in ed d id n ot period. A m on g th e exn early d ou b led in cul- Chromatid breaks Off-season Mid-season 031 0.12 O-I 0.170.08 0-2 031 0.18 0-2 0.07 O.Oi O-I 0.44 0.22 0-3 t-7*0.24 0-6 1.560.29 0-3 1.St0.33 0-6 of breaks observed per tap s fo u n d in a n y sin g le ason cu ltu res to 5 in th e volunteer rose from 2 to n each of the fou r case s iiibitcd a 25% red u ctio n .e h igh e x p o su re p e rio d . sam e sam ples. M em bers 'ro u p B ) show ed an in PESTICIDES AND CHROMOSOMAL ABERRATIONS 339 crease o f n early 4 tim es the m ean n u m b er of g a p s per person du rin g the p eak sp ray in g se aso n . M ean b re a k s p er perso n for G ro u p B in c re ase d b y a fa c to r o f 25 d u rin g th is perio d (T ab le II). A lso n otew orth y is th a t th e tw o su b je cts h av in g the m o st g a p s an d b re ak s, resp ectiv ely , d u rin g th e high ex p o su re p erio d w ere m em b ers of th e h erbicidee x p o s e d g r o u p . O n th e o t h e r h a n d , t h e m a r k e d l y lo w fr e q u e n c y o f b r e a k s (0.07 0 .0 5 ) n o ted in G ro u p B w ork ers d u rin g th e slack seaso n in v ites sp ecu latio n a s to th e p o ssi b ility o f in d u ced c o m p e n sato ry p ro tectio n o ccu rrin g a t th is tim e in th e fo rm of en h an ced ch rom osom al repair. D u rin g th e J u ly - A u g u s t sc o rin g o f th e 42 h ig h ly e x p o se d in d iv id u a ls (1050 cells exam in ed ), a sm all n u m b er of ch ro m atid exch an ge figu res w ere n oted . T h ese in clu ded one G rou p A q u ad rirad ial. tw o G ro u p C trirad ials, tw o G ro u p F q u a d rirad ials an d a p o ssib le G ro u p G trira d ia l. S u ch in terch an g es h ave been fo u n d to o ccu r in th e g e n e ra l p o p u la tio n a t a fre q u e n c y o f a b o u t o n e p e r 3000 cells*. T h e fre q u e n c y n o te d in ou r e x p o se d g ro u p ap p ro ach es on e p er 200 cells. N on e of th e co n tro ls e x h ib ite d a n y o f th e s e c o n fig u r a tio n s , b u t , u n fo r t u n a t e ly , fe w e r in d iv id u a ls (400 c e lls e x a m in e d ) w ere sa m p le d in th e co n tro l gro u p . O ne e x p o se d in d iv id u al also h a d a p ericen tric in versio n in th e A 2 ch rom osom e. T h is w as con firm ed b y k a ry o ty p in g b u t d id n o t a p p e a r in an y of th e o th er 23 cells sco red for th a t person. F ro m these resu lts, it is ap p aren t th a t the exten t of v isible ch rom osom al d am ag e in ly m p h o cy te cu ltu res p rep ared from th e e x p o se d g ro u p is co n sid e rab ly greater du rin g the h eav y sp ray season . W ith ou t ad d ition al correlativ e stu d ies, how ever, th e p o ssib ility of con curren t m u tag en ic in volvem en t m u st rem ain on ly sp ecu lative. B ecau se of th e trem en dou s lim itin g facto rs im posed by long gen eration tim es, th e d ire ct p h en o ty p ic su rv eillan ce o f h u m an p o p u latio n s is re stric te d to o n ly a few retro sp ectiv e tech n iqu es. O ne su ch m eth od w ou ld logically in clu de the an a ly sis of ex istin g h ealth sta tistic s. U n fortu n ately , co n tem p orary v ita l record s are n ot u su ally co m p iled w ith th e g e n e ticist in m in d . W e h a v e recen tly a tte m p te d to e x a m in e th e in ciden ce o f su ch p a ra m e te rs a s stillb irth s, teratism s, re tard atio n etio lo g ies, n eo p la s m s , etc., a m o n g a g r ic u ltu r a l w o rk e rs in t h is s t a t e , a n d h a v e h a d little s u c c e s s b ecau se o f in su fficien t d a ta . O n a m ore p erso n al level, an o th er a id in se e k in g to e stim a te m u ta g en ic ity in su sp e c te d p o p u la tio n s is th e large scale u se of d e ta ile d in te r view tech n iqu es. H ow ever, a m a jo r o b stacle here is th e e x ten t o f cred ib ility betw een research er an d su b ject. R EFER EN C ES 1 1 3 4 hBBEEuiebmnmrstdtageen,eziGrxxp.,,,eMrSSLi....pSSh(A..eE,..rd,a.aC)nalo,ndnltFdeMrauo.Jkrl.moScC.oyf.CLtcheBehegrsemaymeistixctocavealresmlx,.H,ipnFMoalaTnlucdhuttbetaooatrMniosotkunsi,t.naMRfgeSleieaussnel.t,uniccctSriie,tn(.yg1W2o9ic2f6l0h9Pl)reo(osu13mt9gSio7ch30isb-d)y3oe,Qms91e:O.6Ch-aifobotnei,craer.>pa0tt7si3oa.nndyiEelvdasluiantitohne, M.I.T.. Cambridge. Mass., 1971,p.47. j Evans, H. Price and PJ..,LPaowpul(Eadtsi.o)n,HcyutmogaennePtiocpsualantdionenCvyitroognemneentitcas.lWfialcltoirasm,sianndP.WiAl.kiJnasc,oBbasl.tWim.orHe.. 6 7 GFR19rOe7er0hah.rrabp.rpoD.dr.tn1,E9(.E9E.-d,H2s.0.T.2)h.,PeeCsthtriecemiaditcemaIelnnMdteuoxft,a4hgteuhnmecasdin.s.ciCnhorlMloemagoemsSmocmaielessncaiennPdvubiMtlrioas:nh,erreSsspu.rltiSsnt,gaetiren.CIo;Nl.elYewgoegY.fo.Prlak..,.\n11o9967G0... PP- 367-331. 8 Gebhakdt. E.. Mutagcnithtsuntersuchungcn an menschlichen Zcllkulturcn, Limschau, 71 (1972) 123-124. 0002134 U O W 352583 t 340 J. YODER e t a l . 9 10 eIHdxoaphooosimeCdnogimnadrminvueinrdi.utyaRlSs...tuIadnnytedornnA. P.CfoWjniig.c.uBPilfljao,noAtmnPenrrou.taeLlcytCimoonnpthProaaccrytist,Pe7roc(hg1rr9eo7sm0s)oRs7oe7mpao.erta, nIadlayhsios of pesticide Department o( Aericulturc. a120 Warm Springs Avenue, Boise, Idaho 8 370 2 . December iSth. 1 9 7 1 . pp. 2- 3 . t( Ma ,M. l u .v g . H. 1. Harris (VE.d. sM)o, nMituotraignegnoicf cEhffeemctiscoalf mEnuvtiargoennmseinntaoluCr oenntvaimroinnmanetns,t, in H. U. Sutton Academic Press, and New York. 1 9 7 2 , pp. 2 7- 39. u Moorhead. P. S ,, P. C. Nowell. W. I. Mellman. D. M. B attips and D. A. Hukgerpord, Exptl. Ceil.Chromosome preparations of leukocytes cultured from human peripheral blood, Res., to (1060) 6 13 -6 1 6 . >3 EM S Sewsletter,Nichols. W. W.. p. Moorhead and C. testing. 3 (19 71) ao-23. Tonhol..R ohrborn, G.. Possibilities of routine 2 8 (19 7 1) 120- 118 . B rxw ex, Chromosome methodologies in mutation in vivo mutagenicity testing in mammals, Arch. Mutation Research, 21 (1. lilscvicr Scientific Put SHORT C O M M I Lack of mutagenic Oi O C yclam ate, a cc rab bits, dogs an d m an p l a c e 1* . C H A a n d N - O l CO t o b e a c o n s i d e r a b l e in cn is an in d icatio n th a t tl O t r a t i o n 1* . cn C y c l a m a t e h a s t 00 cytogenetic a ssa y sy st m u tation s to S-azagua C H A on chrom osom e t in vivo a r e c o n tr a d ic to r C h in ese h am ste r bon e it N -O H -C H A fa ile d to iru larly n e g ativ e in a h ost im p la n tatio n lo ss in ra t m a t i o n s w a s d e t e c t e d 1*, b u t did n ot in d u ce dom In view o f the con regard in g th e u se o f eye effects of the cy clam ate c o n s t it u t io n R(ijs,yBly c o n c e n tra tio n s ra n g in g fr from the first la rv a l in str s u b s t a n c e {0 .0 1 - 0 .2 0 % ) . tw o or th ree fem ales of den oted (b) w here m a ss m th e m ales w ere m ate d for cell sta g e s, w h ereas in the fo r a period o f 5 d a y s. ~ tran slo catio n s w ere scorec C h em ical d am ag e t ; gen eration s an d becom es ch em ical m u tag en s is, the of M X X G h ave even been; te sts for scx-lin k cd recesi d etect m osaic lcth als. F o r In T ab les I an d II t' I t c a n b e se e n t h a t th e f: elu d in g p o stm cio tic (A , B j la rv a l feedin g. A lth o u gh t Abbreviation: CHA, cvcloh incthyl-iY'-nitro-iV-nitrosogui| 6835 0002135 lO^A 683 fiOO(V^o a ^ 7 1974 JUL 77-5 the health hazards OF defoliants 4*^2 o n c e r ^ p vr'S.'rvc.v OK*- C .O V-\ \ T t CI-'^V rj-T ST , ..___ of the Committee Investigatfiionnnss and Recommendations V H* THE Jiaipfx J f a E f r o y COMPANY. <NC. 2103 P IO G flA N O E P O BO X 7592. AUSTIN. TE X A S 7S705 C O R R E C T E D AND C O M P U r . T T r ^ r r * .fc-V. R- r i I [, it. i ,y \ \ 68o < /TO rnnnAAO^ To: Ministery of Social and Health Affairs The working group on health hazards and labor safety of defoliants has learned that the safety rules concerning defoliants have been neglected. It is also known that the defoliants are toxic for man and animal if taken in large quantities. Therefore, it is possible that people exposed to the defoliants - in particular those spraying them have been in danger of their health. The suspected health hazards may be occupational poisonings or the defoliants might have chronic effects such as severe types of tumor, chronic lung illnesses or other chronic diseases. The Institute has planned the enclosed medical investigation to begin with. Based on the data from this investigation one can conclude if further investigation is necessary. Funding for these further possible investigations will be requested separately. The present goals of the investigation are: 1. To determine the health of the sprayers of defoliant in Oulu area (30 persons) in order to get statistics about various symptoms and diseases. This part will .be called the preliminary investigation. 2. To determine the cause of death arnon^ workers using defoliants in the largest possible span of time. The investigation will concern at least one, thousand workers. The results will be compared with a control group 43f the same size.. The goal is to find out if the distribution of the cause of death among workers. 0000270 6838. 8TC G O O # 00 Page 2 who are exposed to defoliants, differ from those who are not. This part will be :ailed the cause of death investigation. 3. To classify the symptoms among those workers which have contacted the Institute because of the use of defoliants (50 persons). The purpose of this part of the investigation is to identify the symptoms caused by the defoliants and to determine the irregularities in various organs. This part will be called the health investigation. Based on the results from the above investigation one can conclude if a more extensive investigation is necessary. Since the planning of activities of the Institute for the year 1972 does not include the cost of this investigation, it is necessary to hire additional staff to replace those that will do this investigation. There are also other direct and indirect expenses as indicated in the enclosed estimate of cost (Appendix 1). Referring to the above reasons the Institute of Labor Health asks for the special grant of 79,690 FMk for the investiga tion from the Ministery of Social and Health Affairs. M. J. Karvonen, professor Chairman of the Institute of Labor Health S. Hernberg, M.O. Chairman of Department V. Riihimaki, M.D. Assistant physician 0000271 OV0OO9 319 Appendix 1 Estimate of cost on the health investigation of people exposed to defoliants. I. Planning 5,000 II. Preliminary investigation 30 VR workers from Oulun area according to plan 1, 523 FMk/person Possible additional costs Computer costs, summary andreport to the workers Incidental (heating, electricity, cleaning, phone, typing, mailing,...) 15,690 1,500 4,000 5,000 III. Cause of death investigation for about 1,000 persons exposed to defoliants and a control group of equal size. Collection of data, computer cost and summary 5,000 IV. Health investigation The medical check-up of the 50 persons reported to . the Institute for symptoms of defoliant poisonings. **' 55*0 FMk/personv 27,500 - Computer cost, summary and report to the participants 8,000 Incidental (heating, electricity. cleaning, phone. typing, mailing, etc.) Total FMk 8,000 76,690 6840 0000272 07P m n rM Plan 1 C THE PLAN OF THE PRELIMINARY INVESTIGATION IN THE INSTITUTE OF LABOR HEALTH Medical check-up (1 hour) Neurological test Neurophysiological special test ENMG EEG Lab test: LA PVK s _ GOT ' GPT M. Alkalic phosphatase Creatinine of the serum Urine albumin Urine glucose 2 hour sugar test SPJ T3 test Cholesterol * The test for the lung functioning: spirometry Heart investigation: EXG X-ray investigation: thorax X-ray(heart thorax) neck X-ray Total 30 67 97 48 4 9 10 10 10 9 3 3 37 16 33 10 31 18 40 38 FMk 523 6841 0000273 THE HEALTH HAZARDS OF DEFOLIANTS Investigations and Recommendations of the Committee S. \ Helsinki 1973 6842 0000274 Z?.v, finnrVW i In March 1972, there were unusually frequent complaints of sickness among employees of the State Railway System who have been working to clear out bushes using herbicides. That is why the General Director of the Health Department of the State, Professor Leo Noro, asked a group of experts to investigate the health hazards and labor safety connected with the use of herbicides of that kind. Dr. Juhana Idanpaan-Heikkila, M.D., from the Health Department was appointed as the chairman of the working group. Other members were Ms. Kirsti Arjanne and Mr. Heikki Loppi, inspectors of the State Health Department, Dr. Holger Tornroth, M.D., from the State P.ailway Department, and Dr. Sven Hernberg, M.D., from the Institute of Labor Health. The secretaries were Mr. Vesa Riihimaki from the 0 Institute of Labor Health and Mr. Pekka Hillman from the State Railway Department. Dr. TQrnroth was later replaced by Dr. Matti Romo, also from the State Railway Department. On the first of May the group was enlarged to include two more members: Mr. Oiva Lyytinen to represent the State Forestry Department and Mr. Kaarlo Laaksonen to represent the State Highway Construction Department. As an specialist involved in poisons, Dr. Jorma RautapSa attended the meetings of the group. The task of the working group was changed in May 1972 by excluding the matters concerning labor safety, which were committed to another working group set up by the Health Department. The working group has further heard as specialists Dr. Pekka * Nuortevaa, Professor Marja Sorsa, and Mr. Ukko Rummukainen, all from the Institute of Forestry, Professor Pekka Koivistonen from the Food Research and Technology Department of the Helsinki University and Professor Jaakko Mukkula from the Institute of Plant Ecology. The chemical analyses were done in the State Institute of 0000275 pyo m n r M ^ I Agricultural Chemistry and in the Institute of Labor Health. The working group has met with the other working group on labor safety# as well as# with a working group of the Institute of Plant Ecology. From Sweden the working group has been in contact with Dr. Ake Swensson, from the Labor Health Department# Karolinska Insti tute# Dr. Lennart Sundell# Labor Health Department, from the Regional Hospital in rebro# and Dr. Sten Rosenqvist from the Swedish State Railway System. The working group has met 20 times. Upon completion of the job# the working group submitted this report to the State Health Department. Helsinki# February 23, 1973 Kirsti Arjanne K. J . Laaksonen Oiva Lyytinen Juhana Idanp&an-Heikkila Sven Hernberg Heikki Loppi Matti Romo Vesa Riihimaki Pekka Hillman sy 6844 0000276 nann> Page 1 1. DEFINING THE TASK AND PRELIMINARY RESEARCH 1.1. Preliminary investigations and analysis First, the working group tried to relate the suspected cases of illness and their possible connection to the defoliants, to find out statistics about persons working with herbicides, either spray ing or handling them in the factory, and to verify how completely the existing health regulations were followed. The statistics were gathered immediately among the State Railway System, the State Highway Construction Department, the Forestry Department and some private Power Companies. The Labor Health Institute issued a questionnaire to investigate the known health hazards that occurred to workmen during and after spraying the herbicides. The working group procured the health reports and docu ments related to the suspected cases of illness or death. Based on these documents, six cases of illness were reviewed in detail by the'working group. Five of these cases were from Haukipudas, and one was from Jyvaskyla. The working group became familiar with the working methods, spraying equipment and safety procedures in Oulu and Haukipudas on April 7, 1972. At the same time, informa tion from the workers was collected on the use of defoliants and their possible health hazards for workers. The known cases of sickness were reviewed and the patients interviewed in detail. The Committee on Toxic Materials issued a memorandum in March 1972 for the working group (referenced under Myrkkyasiain Neuvottelukunta). The working group also obtained the memoranda from a corre sponding committee in Sweden. To complete the information from Sweden, Dr. Idanpaan-Heikkila and Dr. Hernberg visited Stockholm and drew up a plan of cooperation concerning task assignments for * finding out about health hazards of the defoliants concerned. 1.2. Conclusions Based on preliminary research, it was concluded that it was highly improbable' that defoliants were the cause of serious illness 0000277 S'&FbOOO Page 2 (e.g. tumor or emphysema), although the case histories reviewed could not give reliable data about the causality. It was concluded in the preliminary report that some workers had shown symptoms of poisoning obviously due to defective safety methods and substandard equipment. Further, it was noted that toxic impurities (e.g. dioxin) has been used earlier in Finland in herbicides from 1950 to 1960. The total number of workers in contact with herbicides was estimated ; to be a couple of thousand. 2. PROPOSALS FOR THE PROJECT Based on the preliminary research, the working group divided its project into three phases in order to find out about the cur rent situation and facts concerning defoliants and about their alleged health hazards (see Appendix 1 for the project proposal) . The working group submitted the project proposal and cost evalua tion to Mr. Osmo Kaipainen, the Minister of Health and Social Affairs, on April 22, 1972, and he considered the research urgent. Mr. Matti Louekoski and Mr. Reino Lindroos, representing the Government, supported this opinion on April 28, 1972. The Govern ment granted 80,000 FMk for the preliminary investigation. The Labor Health Institute initiated the urgent parts of the project according to the recommendations of the working group. . On May 25, the working group proposed to halt temporarily spraying from a backpack and to use other methods until further information about the health hazards had been gathered. This method of spraying was considered the most dangerous. 3. BACKGROUND INFORMATION ABOUT THE LABOR GROUPS UNDER INVESTIGATION AND THE REPORT OF THEIR EMPLOYER'S DEFOLIATI*ON METHODS 3.1. Defoliation in Finland The herbicides to clear out tree shoots and bushes appeared commercially in Finland from the beginning of the 1950's and, since 1960, the main method to clear out bushes has been by the use of defoliants. The main users of herbicides have been the Govern ment Departments for Forestry, Agriculture, Highway Maintenance and 6846 0000278 92T C fU \nA * Page 3 Construction, the State Railway System as well as private power companies. Private owners of farms or forests have used in significant amounts of herbicides compared to the above users. In the beginning of 1973, about 50 herbicides were granted permission to be sold. The active ingredients were - 2,4-D - 2,4,5-T - MCPA - pichlorame amine salt and ester amine salt and ester salt and ester The use of MCPA has been very insignificantforclearing out tree shoots since it has no effect on themostfrequent types oftrees, aspen and mountain ash. In 1973 there were three herbicides con taining pichloram and 2,4-D that were approved for clearing out aspen and mountain ash shoots. According to statistics on its use, the amounts of pichloram is rather small. So, over the past 20 years, the main ingredients for these kinds of herbicides have been 2,4-D and 2,4,5-T. According to the statistics gathered by the Institute of Plant Ecology, the sale of these two compounds for the past three years has been as follows: Compound 2 ,4 - D 2 ,4 ,5-T 1969 5 3 .* 6 9 .8 (of Sthaele as citniv emeintrgicredtoiennst') 1970 5 5 .9 5 2 .1 1971 4 2 .2 48*2 3.2. Defoliation methods used by the State Railway System The use of herbicides was started in 1953, when 16,400 litres of commercial products were purchased. During the peak years of 1962-63 the quantity of herbicides was 27,000 litres annually. In 1971, only 5,200 litres were used. In 1972, no defoliants were sprayed or purchas>ed. Spraying has annually employed about 90 persons, railroad 6847 0000279 ,' V O O A O O ' Page 4 workers and others (e.g. students). The herbicides are commercial products, whose active ingredients have been the phenoxy compounds 2,4-D and 2,4,5-T in a ratio of 2:1. . One commercial product con tained DCA, TCA, and MCPA. The concentration of active ingredients has been 500 g/i with a spraying concentration of 1-2%. The herbicides have been sprayed primarily on the leaves (85%) using hand-operated backpack type sprayers. Some experience has been collected concerning spraying from a train coach. For visibility, it is necessary to clear out bushes in the railway. Tree shoots that are too long also make communication difficult. In winter these bushes collect snow drifts and block the railway tracks. The use of herbicides is necessary since no good mechanical methods for clearing out the bushes have been developed so far. The mechanical methods to clear out railway shoulders are not developed due to the inaccessibility of the terrain. Use of manual labor is impossible since it is expensive and not available. The backpack type sprays will be replaced by train coaches. The backpack type sprays will be used only when other methods fail. Near lakes and rivers each tree is treated separately. The same is true for overly long tree shoots. The use of train coaches will reduce the number of people used for spraying. The allergic reactions will be confined to a smaller number of people. ..The use of safety regulations will be strictly enforced. 3.3. Defoliation methods used bv the State Highway Construction Department For traffic safety clearing out bushes from the highway shoulders is necessary, mainly to improve visibility and to pre vent snow drifts from blocking the roads. The herbicides are used because they destroy the roots as well as other parts of the tree shoots. The mechanical methods only increase the amount of shoot growing. Experimentally, the spraying was started in 1951 and was adopted as the primary method in 1953. The peak year was 1969 when about 10,000 ha were 0000280 Page 5 DO sprayed. In 1972 the total area sprayed was 4,500 ha, which is about 30% of the total area cleared out. Annually this project employs about 150 persons in the country. The active ingredients are phenoxy compounds in which the ratio of 2,4-D to 2,4,5-T is 2:1, which corresponds to about 500 g/ of pure acid since there is difficulty in evaporating the esters. The commercial product is diluted in water to give a spraying .emulsion of 2.5%. The spraying is done from the highways using car or truck sprayers. Very infrequently the backpack type sprayer is used. It is estimated that according to the prices in 1972 it would cost an additional 2,000,000 FMk annually to give up entirely the use of herbicides. Until another solution is found, spraying of herbicides will continue' since it is fast, effective, and economic. 3.4. Defoliation methods used by the State Forestry Department The defoliation was experimented with first in 1955 over an area of a few acres. Larger areas were used in 1958. Chemical defoliation was first used in soil renewal. Only a few acres each year were sprayed. The activity really started in 1960 when the airplane was also adopted. In 1969 the use of the airplane was tempdrarily halted as a method of spraying. Consequently, during ten years the area defoliated totalled about 60,000 ha. Up until 1971, the area sprayed from the ground totalled 28,000 ha. In addition, other methods have been used in an area of 6,000 ha. In 1972 defoliation by herbicides was completely stopped. For the current year about 2,000 ha will be treated using a tree by tree method, 3,000 ha will be sprayed. Annually, the total area defoliated is about 50,000 ha. A tree by tree method would employ about 50 men for two months. The time for spraying is one month and employs 200 men using motor sprayers. In the future the tree by tree method will be employed as much as it has been until today. Where it can be used, it is the 0000281 6849 raye o cheapest way to defoliate. Spraying from an airplane or from the ground is by far cheaper than any mechanical method. Depending on circumstances/ mechanical methods may be 2-3 times more expensive than the chemical methods. Chemical-methods will be used on an area that is just 10-20% of the total area to be cleared out, be cause the question of selectivity is not well known according to .present knowledge concerning the spraying method. Safety in spraying will be considered in the future. That would favor spraying from an airplane. ( c, 3.5. Defoliation methods used by Imatran Voima Co. The use of herbicides in clearing out tree shoots under open power lines was started in 1958. From 1965-1969 3/000-4,500 l of herbicides were used annually. In 1971 only 1,800 i was used. In 1972 no chemicals were used. Annually, the area treated has been about 1 ,000-2,000 ha. The spraying has been subcontracted. The company employs about 10 persons to supervise and plan the activity. The herbicides are commercially available, with the main active ingredients being the phenoxy compounds 2,4-D and 2,4,5-T in a ratio of 2 :1 . The spraying has been done almost exclusively using backpack type sprayers. Some tractors and airplanes have also been used. For the time being it appears that power lines cannot be protected by any other methods. Mechanical methods would add 50% to the cost in the long run.. From terrain vehicles the spraying cannot be done extensively enough. This is due to the location of the power lines in the terrain. Spraying from the air, however, is possible and this method is employed to some extent. * 4. THE PROPERTIES AND EFFECTS OF THE HERBICIDES USED TO DEFOLIATE (PHENOXY COMPOUNDS) 4.1. General The phytotoxic chemicals used in defoliation are chlorinated y phenoxy acids, mainly 2,4-D and 2,4,5-T. To a lesser extent it 0000282 also contains an important weed killer MCPA. In addition to phenoxy acids some defoliants also contain pichloram. Since this chemical is in frequent use in Finland and since there is current knowledge about the toxicity of pichloram (e.g.'it is known that the acute toxicity of pichloram is small (Aamisepp et al. 1970, Johnson 1971)], this compound will not be considered in this discussion. The literature on 2,4-D or 2,4,5-T does not always indicate what kind of chemical compound is being discussed (the compound may be a water soluble amine or a Na-salt or one of the several esters that are commercially available). Furthermore, the addi tives or impurities are seldom mentioned. It is important to note that the composition of the phenoxy acid as well as its additives and contaminants can drastically change its effect on metabolism and health (e.g. acutic toxicity, chronic toxicity or teratogenicity) ;as will be indicated later on. In particular, the contaminant of '2,4,5-T, called 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD), has been an important factor and made it difficult to evaluate the results; it will be treated in a separate chapter. The survey of the literature on the properties and effects of these compounds (2,4-D and 2,4,5-T) does not necessarily discuss pure compounds only. It appears.that the purity of the chemical compounds has become important only after 1969-1970. 4.2. Metabolism of 2,4-D and 2,4,5-T in animals and men Experimerts with animals (rats, pigs, cows, hens) has shown that the water- solutions or free acids of 2,4-D and 2,4,5-T will be absorbed effectively and almost completely in the digestive system. The absorption of esters, however, has been slower and less complete and the absorption has obviously been preceded by hydrolyzation into a free acid (Erne 1966a). The concentration in plasma reached a peak in 3-10 hours, when high concentrations were observed in the kidney, liver, and lungs. Pigs also showed high concentrations in their metabolic glands. The excretion of 2,4-D and 2,4,5-T was mainly via the kidney in the urine (pigs showed only minor/amounts in their feces or gall liquid) and the 0000283 6851 Page 8 process was rapid (e.g. the half times of 2,4-D were, depending on the dose, 5-10 hours in rats, 10-30 hours in pigs and cows). The half time of 2,4,5-T was somewhat longer. The corresponding process for esters was slower than that for water soluble salts. Seventytwo hours after intake there were only insignificant amounts of these compounds in the animal tissues. Experiments in pigs indi cated that 2,4-D can go through the placenta and reach the embryo. On the other hand, the amounts in the brain were very low, indicating the slow penetration through the blood-brain barrier (Erne 1966a). Erne (1966b).noted that the metabolites of 2,4-D in the urine of the pig were mainly free acids but were also to some extent protein conjugates. The experiments in 2,4-D metabolism in sheep, using isotopes (Clark et al., 1964), and rats (Khanna and Fang 1966) in dicated that the compound showed up unchanged in the urine; the experiment using rats further indicated that larger quantities slowed down the absorption and secretion times. Contradicting the previously mentioned observations, Clark and Palmer (1971) report that 86% of the 2,4,5-T ester given to sheep was still an ester in the urine whereas 1.4% had changed to a free acid. Even though it is evident that the major part of 2,4-D and 2,4,5-T that enters an animal will pass through unchanged, small quantities of metabolites can be found. Wilson (1971) observed that pregnant :rhesus monkeys under 2,4,5-T feeding showed an occurrence of trichlorophenol equal to about 1% of the amount of 2,4,5-T in the urine. Several 2,4-D and 2,4,5-T experiments on the metabolism of animals indicate no storage of these compounds in fatty tissues. There is no theoretical likelihood for this storing property due to the polaric nature of the compounds. If the daily dose that gets into the organism is large enough, there is some slow accumulation, since the half time of 2,4-D and 2,4,5-T varies in different mammals from 10-30 hours according to Erne (1966a) if the daily dose is high (over 50 mg/kg). The chronic feeding experiments have indicated that the animals will die of an acute type poisoning due to the accumulation of the phenoxy compounds. 0000284 terhooo Page 9 According to Erne (1966a) the esters will be transformed into their corresponding acids in the digestive system and will be ab sorbed in that form. There are no papers about the water soluble phenoxy compounds or esters to indicate how they will be absorbed through the lungs or the skin and what happens subsequently. It is supposed that the absorption through the lungs is almost total and that the absorption through the skin is less significant. Ester compounds can be expected to be absorbed through the skin more easily than the water soluble and polar acid or salt. Little data is available on the metabolism of 2,4-0 and 2,4,5-T in men and mostly comes from blood, urine, and tissue tests in suicide and attempted suicide cases. Observations [e.g. Nielsen et al. (1965)] indicate no essential difference between human and animal metabolism in this case. Conclusion There is data about the metabolism in various mammals. Ex periments have been conducted almost entirely concerning absorption through the digestive system, excluding absorption through the lungs or skin. The metabolic change of the compounds into more toxic products, even in lesser quantities, cannot be excluded at the present. The metabolism of the compounds may indicate signifi cant differences between different animal species as well as between animals of the same species with different genetic properties. The flow of the phenoxy compounds in various organs and their access to various important organs (e.g. genital glands) has not been adequately investigated. The metabolism of phenoxy compounds in man is nearly unknown and the estimates and beliefs are based on animal investigations. * 4'.*3. The toxic mechanism of 2,4-D and 2,4,5-T in animals The effect of 2,4-D and 2,4,5-T on cellular metabolism is hardly known at a*ll. Stanosz (1969) noticed reduction in the consumption of oxygen in the heart, liver, and kidney when rats were given 10 mg/kg/day of 2,4-D over a period of 120 days. Kuhn .. ' 0000285 . 1 685.'* Page 10 and Stein (1965) observed acute poisoning causing amounts of 2,4-D to induce in the muscles of rats an increase in glycolycis. They also observed the occurrence of traces of oxydative phosphory lation. Furthermore, Philleo and Fang (1967) discovered that the results were dependent on the amounts and that, even with small doses of 2,4-D, there were occurrences of disturbances in the acetate metabolism. Based on current theoretical knowledge of the chemical structure of the phenoxy compounds, they seem to have no influence on the hereditary material changes of the cell (ability to chemically react with DNA) (Giftnamnden, 1971). Desi et al. (1962) observed EEG-irregularities by using very large amounts (200 mg/kg), when they studied the neurotoxicity of 2,4-D in various animals. No microscopic changes in the neurons could be shown in animals that died by poisoning. The same authors thought that 2,4-D caused changes in the thyroid glands originating from the brain or the thalamus, but later on Florsheim et al. (1963) showed that the actual cause was the inability of thyroxine to bind in the plasma of the carrier protein. Conclusion The mechanism of the toxic effects of the phenoxy compounds (enzyme inhibition) in animal cells and tissue is virtually unknown according to the current literature. 4.4. Toxicity of phenoxy compounds 4.4.1. Acute toxic effects The acute toxic effects on animals of the most common phenoxy compounds are known, when the chemical has entered their digestive system (see Table I). Acute toxicity is expressed in units of LD^q which gives that amount of poison in milligrams per kilogram of the weight of the animal that kills 50% of the animal population under investigation. Some defoliants and chemicals go through the skin to the organism and, if the compound is very toxic, even small amounts on the skin can be dangerous for the health of a person 000028 yterbooo /aor* Page 11 who is handling them. In the case in which the chemical comes in contact with skir\ acute toxicity is expressed as dermal LD,-q . According to the current available literature, the dermal toxicity of phenoxy compounds is scantily investigated. According to Aamisepp et al. (1970), the dermal LD^q of 2,4-D for rats is fairly low, about 1,500 mg/kg. Various evaluations for LDj.q give different results even for animals of the same species. Some mammals, in particular dogs and pigs, seem to be particularly sensitive to the toxicity of the phenoxy compounds. For the dog the oral 2,4-D LD^q value is about 100 mg/kg (Drill and Hirazka, 1953). v TABLE I. The acute toxicity of defoliants and TCDD contaminants contained, when given once orally to rats and gerbils. LD^q per kg of body weight ______________________________ rat_______________________ gerbil 2,4-D 2,4,5-T MCPA pichloram 375 - 700 500 600 - 800 8 200 i 000 380 3 000 2,3,7,8-tetrachlorodibenzo- p-dioxine 0.022 - 0 .0^5 (TCDD) 0.0006 The symptoms of acute poisoning are very similar for different mammals. A few hours after the introduction of the phenoxy com pounds in the digestive system, a reduction in appetite, vomiting and diarrhea appear, as well as, motor disturbances, stiffness of the muscles and ataxia. There has been observed, in dogs (Drill and Hirazka 1953) after death some non-specific congestive and toxic degenerative changes in their digestive system, liver, and lungs. The cause of death among apes (Hill and Carlisle 1947) was supposed to be cardiac arrythmia. Brandt (1971) has done a survey of the literature on poisoning cases in human beings and gives one case 0000237 6855 boooiAO Page 12 of his own observations. All poisonings (total 9) have been caused by swallowing some phenoxy compounds and were primarily in connec tion with suicide attempts. In six cases the compound was 2,4-D and in three cases MCPA. In Finland only one suicide has been committed with a 2,4-D preparation (ALHA, private communication). The symptoms of acute poisoning among men resemble those obtained in animal experiments. Non-specific toxicity and changes has been observed in the liver and kidney. Nielsen et al. (1965) indicated some pathological changes in the central nervous system due to poisoning, but these may have been due to a lack of oxygen. Brandt's (1971) own description of a case, when a person recovered after having taken at least 300 mg/kg.of 2,4-D, showed the only permanent damage to be mono-neuropathy in one leg. Based on the above case histories the estimated dangerous dose of 2,4-D for man is 50-100 mg/kg. 4.4.2. Chronic toxic effects When animals were fed with 2,4-D and 2,4,5-T over several weeks and even months, the symptoms of poisoning were observed among dogs for doses of 20 mg/kg/day (Drill and Hirazka 1953) and among pigs for doses of 50 mg/kg/day (Bjorklund and Erne 1966). Other mammals have tolerated repeated intake of much larger quan tities. In the above investigations the chronic symptoms resemble the acute symptoms. It appears that the accumulation of poison in tissues causes "acute poisoning" and there is essentially no dif ferent mechanism for chronic toxicity. Some animals have shown toxic degenerative changes in the liver and kidney as observed in some feeding experiments. 4.4.3. Embryotoxic and teratogenic effects The teratogenic and mutagenic effects of 2,~4-D and 2,4,5-T have been extensively discussed in the publications by Giftnamnd t (1971) and Myrkkyasiain Neuvottelukunta (1972). It seems evident that 2,4-D and 2,4,5-T prevent the development of the embryo and cause deformed embryos. Some 2,4,5-T preparations previously con tained 2,3,7,8-tetrachloridilbenzo-p dioxine (TCDD) which also has a 0000288 >eri>ooorhoa Page 13 strongly teratogenic influence. The results of the investigations on damage to the embryo are difficult to interpret and are am biguous. The influence of the phenoxy compounds on various mammal embryos differs. Mice are the most strongly influenced out of six mammals (mice/ rats, gerbils, rabbits, sheep, and rhesus monkeys); toxic effects have been observed in 1971 with a dose of 100 mg/kg (NIEHS investiations, Giftnamnd 1971) . Rats showed embryotoxic effects with 2,4-D and 2,4,5-T doses over 100-150 mg/kg/day (Khera and McKinley 1972). The observed effects included mild cases of defects in the bone structure and did not cause death. Wilson's (1971) investigations for the Swedish Giftnamnd on the embryonic toxicity of 2,4,5-T showed no defective development among the rhesus monkeys even with large quantities, 40 mg/kg/day. Several chemicals are known to cause defective embryonic de velopments when given in large enough quantities [e.g. regular salt (NaCl), cafein, salicylates, and leucine amino acid are some of these (Giftnamnd 1971)]. It should be pointed out, too, that the most affected mammal in 2,4,5-T experiments (mice) can tolerate a dose (100 mg/kg) that would be lethal for a man even if taken only once. It is possible that the frequency of birth defects has been increased in Vietnam due to the chemical warfare. Such effects are not due to phenoxy compounds alone, since other chemicals with teratogenic side effects have been used in large quantities and since "the phenoxy compounds that were used have contained contaminants such as dioxine. Swedish investigations in 1970 on two cases of alleged embryonic defects showed, according to the experts in teratology, no obvious connection with the minor allergic reactions and the defects (Giftnamnd 1971) . 4.4.4. Mutagenic and carcinogenic effects * 2,4-D and 2,4,5-T are mutagenic in plants. It is not certain yet whether this phenomenon is secondary in its influence in a way similar to that of the growth hormone or whether it is of primary mutagenic influence. Due to their chemical structure, the phenoxy 00.00239 6857  nr> Page 14 compounds cannot be expected to react with the hereditary material of the cell (DNA), whereas some other currently used chemical could (Myrkkyasian Neuvottelukunta 1972) . Dioxines could also react with DNA. The mutagenic influence of the chemical can be caused by its interaction with the synthesis of the hereditary material in the splitting and doubling of the cell; in this case, however (unlike the previous case), the influence will show up only after the con centration has risen to a high enough degree. Even though the hereditary material of the animal and plant cells does not differ significantly, the metabolism of chemicals in plants and animals is so differnet that mutagenic effects in plants do not imply similar effects in animals. Davring and Sunner (1971) observed, however, genetic defects in drosophila that were fed with 250 ppm of 2,4,5-T and Davring (1971) observed traces of similar phenomenon in mice fed with 250 ppm of 2,4,5-T. The significance of these ob servations is not clear at the moment. The occurrence of cancer cells also involves the mutation of the hereditary material. Consequently, if a chemical is mutagenic, it should be able to cause cancer cells-- and further cancer, pro vided the cells will not be destroyed. 2,4-D and 2,4,5-T have never been observed in animal experiments-to cause severe cancer. The evidence, however, cannot be considered conclusive enough to exclude the carcinogenic possibilities. Summary The acute toxic effects of 2,4-D and 2,4,5-T on various mammals and men is known. Chronically toxic effects have been investigated with animal experiments and based on this evidence it appears that the chronic and acute toxic effects are similar. It should be pointed out that most of the experiments have been of short duration (a few days or weeks) and only a few have been long term (months or years). The damage to the embryo by 2,4-D and 2,4,5-T seems to be so negligible that, under usual circumstances, man is not likely to be exposed to a potentially dangerous dose. Dioxines are very 000029.0 Page 15 toxic and teratogenic compounds. They could increase the effects of the phenoxy compounds. The mutagenic effects of 2,4-D and 2,4,5-T are mild, according to current knowledge. Carcinogenic effects cannot be shown, but there is not enough evidence to exclude the possibility with cer tainty. The mutagenic or carcinogenic effects of dioxines have not been investigated according to the literature available. So far there is no evidence that phenoxy compounds have caused embryonic defects, genetic damage or cancer in humans. 5. SURVEY OF THE LITERATURE CONCERNING THE HEALTH HAZARDS TO HUMANS THAT HAVE BEEN EXPOSED TO PHENOXY COMPOUNDS 5.1. Symptoms following exposure Russian researchers have frequently reported that persons exposed to phenoxy compounds both in spraying (e.g. Fetisov 1966) and in industrial preparation (e.g. Basirov 1969) have ex perienced unusual fatigue, headaches, giddiness, loss of apetite and stomach aches as well as irritation in the respiratory organs and the skin. 5.2. Illness and poisonings The irritation in respiratory organs (asthma) by defoliants containing phenoxy compounds has been studied by Forbes et al. (1966). In this case, however, the asthma was not considered to be caused by the defoliant. In the late 1950's and early 1960's six cases of illness caused by 2,4-D were published, some of which developed into severely disabling polyneuropathy (Goldstein et al. 1959, Todd 1962, Foissac-Gegoun et al. 1962, Berkley and Magee 1963). In most of these cases the duration of exposure to 2,4-D was reported as being rather short, just a few days, and in one case it consisted of just a very brief splashing of the legs with the preparation; in this case, however, there was an intensive rash. In three cases the authors point out/that an earlier contact had caused various symptoms in the patients, although it was only after the second contact that 0000291 or-" heeb ooor^ort Page 16 the neurologic illnesses showed up after a latent period of a few days. It is strange that after 1963 there have been no reports of similar symptoms in the literature. The cases indicate an illness based on allergic reactions or other idiosyncracies. It is also possible that 2,4-D was not the only cause of the illness, but that some impurity in the preparation might have been a factor. Lorenzen and Lyngs^e (1957) have published a case history about two workers that became ill after having sprayed MCPA; the problem was in sugar metabolism (in one case a temporary problem and in the other case permanent diabetes). Animal experiments did not indicate diabetogenic effects. Other cases are not reported in the literature. Monarca and Oivito (1961) have described one case of acute poisoning without permanent damage due to the spraying of 2,4-D. Statistics on the health factors in the industrial processing of phenoxy compounds exist in Germany (Kimmig and Schulz 1957, Bauer et'al. 1961, Kleu and Gotz 1971), in the Soviet Union (Basirov 1969) and in the United States (Bleiberg et al. 1964, Poland et al. 1971, Johnson 1971). Kimming and Schulz reported as early as 1957 that among the 31 workers of a factory producing 2,4,5-T there occurred chloric acne and it turned out that three of the workers had damaged livers. Ten persons of the group were investigated later on (Bauer et al. 1961) and again about 15 years after the first observations (Kleu and Goltz 1971). During these years it was observed that all ten showed symptoms of a psychopathologic state that corresponds to a known psychoorganic syndrome caused by damage in the brain. Kimming and Schulz observed in animal experiments that pure 2,4,5-T or its initial compound trichlorophenol did not cause chloric acne. The synthesis of trichlorophenol did not result in a pure product, * but the authors did observe such impurities as tri- and tetrachlordibenzofuran and tetrachlordibenzodioxine. These compounds, in particular the last one, caused chloric acne and'damage to the liver in rabbits even in small quantities. Schulz even experi mented with his owh skin and observed the above mentioned rash caused by tetrachlordibenzodioxine (TCDD). 0000292 ' 6860 Page 17 c Unfortunately these investigations by Rimming and Schulz were not noticed. It was only in the late 60's that it was re discovered in the U. S. that commercial 2,4,5-T contained the dangerous T C D D and that further investigations on the properties and effects of the dioxines were initiated. It is very likely that the illnesses reported among the workers producing 2,4,5-T in the U. S. (Bleiberg et al. 1964, Poland et al. 1971) are due to the impurities o the synthesis (e.g. dioxines) and/or the initial or intermediary compounds (tetrachlorobenzene, di- and trichlorophenol). In addition to the chloric acne, the workers were diagnosed as suffering from hyper pigmentation of the skin, hirsutism and tardive cutaneous prophyria, a disturbance in porphyria metabolims caused by the injured liver. On the other hand, Johnson (1971) could not observe anything ab normal in his investigation concerning 220 workers of a factory producing 2,4-D (where the estimated daily dose was 30-40 mg/day) and in another investigation on 64 workers dealing with the syn thesis of 2,4,5-T (where the estimated daily dose was as low as 2-8 mg/day). Even though the investigation is not well described, it is reported to be based on extensive lab tests. Ten 2,4-D cases and 52 2,4,5-T cases were given karyotype test of lympho cytes with regular results. Basirov (1969) reports that the workers of a factory pro ducing 2,4-D compounds commonly had neurasthenic syndromes and symptoms of imbalances in the autonomic nervous system. Several epidemiological investigations have been reported among farm labor spraying defoliants (Long et al. 1969) but the cases described have always been due to several different kinds of defoliants and the results are not important. c c C Summary Use of phenoxy compounds causes several temporary symptoms such as fatigue, headaches, giddiness and irritation of the skin and of the respiratory system. The irritation can cause asthma if a patient has a disposition to it. In the early 60's there were 0000293 descriptions concerning injuries to the peripheral nervous system 4 due to acute and mild or repeated exposure to the .skin- No reports exist later on almost similar cases. It seems evident that the defoliants contained chemical impurities; for example, chlorinated phenols have caused similar injuries (Richert 1962). The best evidence comes from the investigations concerning people who are employed in the industrial production of phenoxy compounds, since the exposures are usually strong and take place over years and, in the ideal case, the effects of other chemicals are insignificant or easy to evaluate. In the processing of 2,4,5-T the workers have been exposed to very damaging impurities, dioxines, and initial chemicals (tetrachlorobenzene), as well as to inter mediary compounds (chlorinated phenols)- It is very likely that the observed illnesses are due to some chemicals other than 2,4,5-T. The health hazards of 2,4-D and 2,4,5-T should be investigated epidemiologically in the future under conditions where the above confusing factors are removed. The possible mutagenic and carcino genic effects should be investigated. The epidemiological in vestigations about the health hazards to workers spraying defoliants are inconclusive concerning the knowledge of the effects of the phenoxy compounds. 6 . ' DEFOLIANTS USED IN FINLAND 6.1. Chemical structure Currently, approval of a defoliant requires the filing of the complete chemical composition of the defoliant with the Institute of Plant Ecology. No exact knowledge about the chemical composition of the defoliants is available from the 50*s and the 60* s. The active ingredients of the defoliants, the phenoxy compounds form about one-half of the spraying liquid by weight; the rest con sists of various additives and solvents. The spraying liquid usually contains additives that lower the surface tension (surfac tants) to spread the liquid evenly on the applied surface. The amine salts of 2,4-D and 2,4,5-T dissolve in water. The defoliant used contains abou 0.5-1.3% of the active ingredient. ^* 0 0000291 Page 19 The additives of the phenoxyacetic acid esters are, in addi tion to the surfactants, various emulsifiers that make the dissolu tion in water possible. The solvent is usually gasoline. The esters are dissolved in water (concentrations as above for the salts), but the prepara tion for trees is dissolved in diesel oil or gasoline, and the con centration of the active ingredient is 2% (tree trunk spray) or 15% (tree stump spray). y.w nnnrM C 6.2. Effective compounds Currently the 2,4-D and 2,4,5-T compounds in use are either amine salts or macromolecular esters,- e.g. isooctyle, butoxethyl and butoxyethanol esters. Previously small molecular esters were used, e.g. butyl ester, but these are easy, to gasify, dangerous "for the health of the user and difficult to use on warm days. The vapor pressures of isooctyl and butoxiethanol esters are so low under normal conditions {e.g. the vapor pressure of isooxtyl-2,4,5-T is 0.54 x 1(T5 mmHg at 25C (Jensen and Schall 1966), and isooctyl-2,4-D 0.2 x 10~ 5 mmHg, and butoxyethanol-2,4-D is 0.45 x 10 ^ mmHg (Flint et al. 1968)] that they render no problems from gasifica tion . The .amine salt, isooctyl ester, but butylester preparations of 2,4-D were not observed to cause systematic poisoning during daily application on the skin over a three-week period (Kay et al. .1965). The solutions were of two kind, 0.63% and 3.13%. The water solution caused mild or moderate irritation, rash, and even tissue necrosis. 6.3. Effect on health of various compound, additives, and solvents There are no investigations available to clarify sufficiently the metabolism (absorption, transportation, secretion and dissolu tion) and the differences in effects of the salts and esters of phenoxyacetic aci*d. The polaric salts can be assumed to penetrate the skin and various membranes less easily than the esters. The penetration property of esters may be increased by various additives 0000295 that reduce the surface tension [e.g. anionic and cationic deter gents which damage the skin very much (Schleuplein and Blank 1971)]. The effect of the oil based ester solutions is particularly damaging both locally for the skin and for the mucous membrane as well as internally for the organs. Conclusion In addition to the phenoxy compounds, the defoliants contain various additives (e.g. ones to reduce the surface tension, emulsifiers for esters, and solvents such as oil). The differences in their effects and their metabolism are not well known. The esters .contain detergents that can significantly irritate the skin and the mucous membranes, and the irritation is even worse with oil based solutions. 7. IMPURITIES OF DEFOLIANTS 7.1. 2.3.7.8-Tetrachlorodibenzo-p-dioxine (TCDD) content in ' defoliants TCDD can be developed in the synthesis of 2,4,5-T from chloro benzene using alcalic hydrolysis if the reaction temperature exceeds 160C (Schulz 1968) (see Figure 1). chloro benzene 2,4,5-T Na-salt 2,3,7,8-cetrachlorodibenzo-p-dioxine // Figure 1. The by-product of 2,4,5-T synthesis (2,3,7,8-tetrachlorodibenzo-p-dioxine). 0000296 Page 21 This way of synthesis has been widely used previously and in the U.S. it was abandoned in the late 60's. Woolson et al. (1972) have recently investigated the dioxine content of 17 defoliants containing chlorinated phenols and the results are in Table II. It is significant that TCDD was observed primarily in 2,4,5-T; in 22 samples the content was less than 0.5 ppm but in 20 samples it was greater than that. The high values all came from the same manufacturer during 1966-1970 and towards the end of this period there was a decrease in comparison to the previous values (20-30 ppm). This company later gave up the production of 2,4,5-T. At the time of the investigation the products never exceeded 0.5 ppm in the content of TCDD. One sample of 2,4,5-trichlorophenoxypropioneacid contained TCDD over 0.5 ppm. No sample of 2,4-D contained TCDD, but one of them a small amount of hexa-CDD. Hexa-, hepta-, and octachlorodiben2o-p-dioxine were observed in large quantities in some chlorinated phenols (e.g. pentachlorophenols). Table II. The content of dioxin in samples of defoliants (Woolson e t al. 1572) Pt-uicut tetra <10 <100 <10 ppm o f chlorodibenio^^ioxin bexn hepta <100 <1000 <10 < 1 0 0 <1000 <10 S o . o f Total sam ples ao. of octa contam u in p iM <100 <1000 inated tn te d 1 - .5 - T S itic i 2 .-O ID H .-O P ) D iv in ih a CM orpphenol *ri- tom - perns- O lliers 7 t ND ND NO ND NO ND 13 0 e 3 ND 1 ND 4 1 0 1 1 0 # 0 0 ND* ND ND ND ... e ND ND ND ND 0 0l 1 01 7 00 0 13 0 02 2 0 .1 4 60 0 13 Any inmplc m) contain on* or mere different dioxins. * NO < 0 J ppm ofiny on* chlorodioxiti. 00 20 46 10 23 <2 17 1 28 08 46 33 10 11 7 24 J . AGR. POOD CHEM.. VOL. 20. NO. 2. 1972 3S3 In Finland beginning on October 31, 1971, the Chamber of Agriculture and Forestry has prohibited (703/71) the sale and use of those defoliants which contain TCDD over 0.5 mg/kg computed from the 0000297 OVt/009 3^5* Page 22 content of 2,4,5-T acid. Currently, it is not likely that the defoliants containing 2,4,5-T have TCDD more than 0.5 ppm. The TCDD content of older defoliants has been determined in the State Institute for Agricultural Chemistry, contracted by the working group, and those results follow later on. The defoliants may contain small quantities of other contaminants. Huston (1972) observed 3 neutral contaminants in 2,4-D, one of which contained 30 ppm of them. The toxology of these compounds is not known. 7.2 The effects of TCDD TCDD is one of the most poisonous compounds known today. It's LD5q value for the gerbil is 0.0006 (Table I). In other words, 0.6 micrograms per kg is lethal. TCDD is strongly teratogenic. Sparschan et al. (1971) observed 0.5 yg/kg daily to be teratogenic. It should be pointed out, too, that embryotoxic effects of 2,4-D and 2,4,5-T among rats occurred when the daily dose exceeded 100150 mg/kg (Khera and McKinley 1972). Above, the chloric acne and liver damages caused by TCDD were discussed. The illnesses among workers producing 2,4,5-T could be caused primarily by TCDD, but other (unknown) contaminants and chemicals that are used may be partially involved. The possible mutagenic and carcinogenic properties of TCDD are not known. Several Cl atoms (6,7,8) containing dibenzo-p-dioxines may be contaminants in the defoliants, but they also appear in many other widely used chemicals such as pentachlorophenol. There is no ' exact knowledge of the properties of these compounds, but, evidently, they are toxic. Several dibenzoparadioxines have caused in hens the 'so-called "Chick-Edema" disease (Kimbourgh 1972). TCDD is a very stable, insoluble, and lipophilic compound. It might accumulate in living organisms. Investigations of this are under way and the first observations (Kearney 1970) on radio active TCDD indicate that the compound will be absorbed in the ground and that the plants will collect it only in small quantities. 00002698866 8. THE INVESTIGATIONS OF THE INSTITUTE OF LABOR HEALTH 8.1 Questionnaires 8.1.1 The purpose of the investigation After the health hazards of the defoliants gained publicity, the personnel in health care and occupational hygiene received several reports from individuals about alleged illnesses. Via the media, people were advised to file the reports with the Institute of Labor Health. 8.1.2 Method i The Institute sent questionnaires to these individuals and to some employers (see Appendix 8) in which such questions as the context and duration of the exposure, the symptoms of the alleged illness, and its duration were asked, as well as information about the possible time and place of hospitalization or of care by private doctor. This investigation was supposed to reach as many users of defoliants as have suffered from the use of defoliants. The data depended on the initiative of the individuals. The main points to be clarified by this method were: what are the most common symptoms in connection with the defoli ants? - are there cases of acute poisoning? - are there cases that are similar to those reported in the litera ture or that could be connected with poisoning by defoliants? 8.1.3 Material The Institute received 213 replies. The distribution of the persons according to age, sex, method of contact and time of * exposure are described in Figures 1 and 2. 33 persons reported no symptoms during and after defoliation. / 0000299 Figure 1. The s t a t i s t i c s on persons who returned the q u estio n n aire, according to age and sex NO. OF PERSONS t C c C w 19 29 39 49 59 69 SEX jo. or PERSO NS Flguru 2. The s t a t i s t i c s on persons who returned the qu estio n n aire, according to. the uethod and duration o f the exposure (in work 0 0 0 . 0 3 0 0 w k >- Page 25 The majority of replies comes from old people and the distribution of age differs significantly from that concerning the cause of death in the population (see p. 44, Table IX). In some cases the time elapsed since the last contact with herbicides was several years. The total population clearly contained longer exposure times (about half of them, more than 40 days or 8 weeks) in comparison to the population in the cause of death investigation (see Figure 7, p. 43). About three-fourths of those replying were exposed to defoliants when spraying them with the backpack type spray. C 8.1.4 Reported symptoms and illnesses The most common symptoms reportedly found in persons that were exposed to the defoliants. Table III. The symptoms of the 180 persons participating in the inquiry* At the moment of exposure At the moment of investigation no. of persons(% from total) no. of persons(% from total) Headache Giddiness Fatique Stomach. Ache Irritation of respiratory organs Difficulties in breathing Irritation of the skin Rash Irritation of eyes 99 22 18 13 20 14 20 13 16 (55.0) (12.2) (10.0) ( 7.2) - (11.2) ( 7.8) (11.2) ( 7.2) ( 8.9) 59 (32.8) 20 (11.2) 7 ( 3.9) 18 (10.0) One person might ha one or more! sympton * _ J-JS" 00003I r b-b?bOoo c*\<~ Some of the infrequent symptoms and illnesses reported were (the number of cases are in parentheses): allergic reactions such as asthma, allergic cold or rash (5), insensitivity of legs (6), weakness in legs (4), decreased sense of smell or taste (2), impotence (1) and increased perspiration (2). No acute poisoning cases showed up in the inquiry. One person# however, reported a headache, a stomache ache and an intensive rash so. as to cause him to go to a hospital. No mention of disabling peripheral nervous illnesses was contained in the material returned. One person reportedly became diabetic some months after the defoliation, and another contracted a liver disease (nephrosis) during the work. Severe cases of cancer were also reported (acute leukemia and cancer of the bladder). 8.1.5 Summary and conclusions Two hundred thirteen people responded to the inquiry of which 180 reported symptoms and illnesses connected with the defoliants and 33 reported no symptoms. The questionnaire was distributed mainly among persons who voluntarily reported to the officials in health care or occupational hygiene or at the Institute, but some were sent by the employers. The population was rather old, primarily male (only 10 women), and were exposed while spraying the defoliant with a backpack type spray. The duration of the - exposure was rather long (compared to the death cases) and about half have had an exposure of more than 8 weeks. In evaluating the significance of the symptoms# one must note that the inquiry was addressed to persons who had some symptoms. That is why no external reference could be found. It should be pointed out that about one-fourth of the people [40] reported the headache only during spraying. The headache might be caused by the defoliant but it could also be due to the strenuous physical work, the temperature, the unpleasantness of the spraying (the liquid stinks), or the constriction around the neck.caused by the backpack. The exhaust gases of the engine could also cause a headache. The irritation of the respiratory system, the eyes and 0000302 6870 Page 27 C n r o rnfi the skin was less frequent. These symptoms did not last long. The allergic reactions should also be considered, since in three cases the trouble resembled asthma. The symptoms reported in the inquiry matched well with the previous reports in the literature (e.g. Fetisov 1966) concerning the phenoxy compounds. On the other hand, there was no report of acute poisoning. There was no ill ness that resembled previous reports (e.g. Goldstein et al, 1959) on neurological illnesses. 8.2 Clinic research 8.2.1 Introduction The clinical medical investigation on people exposed to the defoliants was conducted in two parts. The first one (8.2.2 Preliminary investigation) was intended to describe the health of people intensively exposed (spraying method, during long periods of work) to the defoliants regardless of the reported symptoms. The second one (8.2.3 Health investigation) was intended to find out about the background and other results pertaining to the illnesses, according to the reported symptoms. The mutual goals of both investigations were a) to find out traces of poisoning by looking at different organisms, b) to look for functional changes in the respiratory system, c) to investigate the possible diseases of the skin and the mucous membranes, and d) to identify a possible group of symptoms characteristic of the poisoning. It should be pointed out that the investigation started only after one year had elapsed since these persons had been exposed to the defoliants (the summer of 1971). During the months after the exposure, the organism is expected, to a large extent, to recover from the alleged illness or malfunction. Some organs recover slowly and incompletely; this is the case with the nervous system. The clinical investigations have been emphasized to study the nervous system. The justification for this is that, in addition to a slow recovery, the nervous system is generally injured in poisonings (e.g. "solvents, heavy metals). There have been ITT' " Tt" ---.-a . -.4#* 0000303 6871 bOOOj?G descriptions about the injury to the nerves caused by 2,4-D (e.g. Goldstein et al. 1959) and the neurophysiology related to it [e.g. by Fullerton (P. M. Fullerton: Toxic chemicals and peripheral neuropathy, Proc. Ray. Soc. Med. 62^ (1969), 201-204)]. A clinical investigation immediately during or after the spraying of defoli ants would have been well justified, but that could not be included in the planning, since it was not known if the use of defoliants would be prohibited in 1972. Also, the initiation of the investiga tion was considered important. Table IV The symptoms of the 30 persons participating in the preliminary investigation* Symptom No. of cases No. of cases No. of cases having at the time still having the symptom before of exposure the symptom exposure Headache Giddiness Fatigue Stomach ache Irritation of respiratory organs Irritation of the le i n Rash Irritation of the eye 10 4 4 1 6 4 6 6 73 30 21 1 0 *One person might have one or more symptoms TT7T ' 0000304 6872 rsebooo Page 29 8.2.2 Preliminary investigation Material The population was chosen as 30 workers among the State Railway System, of which 25 were currently working and 5 were retired due to various illnesses. The persons were chosen from among those with the longest exposures to the defoliants accord ing to the employee records. Out of the original group, 4 refused. According to their health records they had no symptoms. The statistics according to age and sex, as well as the method and duration of exposure are represented in Figures 3 and 4. Their most common symptoms ar listed in Table 4. Out of the group of 30, 13 were over 50. The exposure to the defoliants was over 8 weeks in 12 cases. The most common subjective symptoms that were reported were headaches and various irritations. The method of investigation The attending doctor made a detailed sickness and working history (see attached form 8b) and made the physical investigation (see attached form 8a) ,- excluding neurology. The lab tests, X-ray's, and lungs were investigated according to Appendix 1. The clinic neurologist recorded subjective symptoms, the occurrence of any organic psycho-symptoms# exceptional findings from the autonomic, central and peripheral nervous systems, and the condition of the muscles. The neuro-physiological exam included the usual EEG, and the peripheral nervous system was investigated using a method called ENMG, developed in the Institute of Labor Health. For a comparison, a sample of neurologically unspecified indus trial workers were investigated (50 with EEG, 100 with ENMG). Results The most important results are in Table 5. The deviations from 0000305 6873 Page 30 v the normal were few and were mild. Only one person out of 30* was observed to respond positively to the sugar test indicating symptoms of latent diabetes. This was a new discovery for that one person. The performance of the lungs indicated two substandard cases. No asthma was recorded. According to the project, other [.than the lab investigations were given, e.g. the blood composi tion, the cholesterol content of the serum, EKG, and X-ray of the chest and throat. These were used to describe the general health condition (the usual health checking) and in diagnostic analysis. With the exception of the blood composition test, abnormalities were observed in the fat content of the serum and in the blood pressure among the old persons, as well as changes in the shape of the heart due to malfunctioning, all of which had no obvious con nection with the use of defoliants. The neurologic investigation indicated two cases of an organic psycho-syndrome, 8 cases of mild irregularities in the autonomic nervous system (increased perspiration, variations in the blood circulation in the limbs and cardiac arythmia) , five cases of motoric disturbances, and 8 cases of malfunctioning of the peripheral nervous system. Except in one case the findings for the central nervous system could be ascribed to obvious causes (large intakes of alcohol or medicines, blood circulation problems). The symptoms of the autonomic nervous system were probably due to the situation (e.g., excitement, fatigue, etc.) In the case of the peripheral nervous system, the anomalies were confined to one or two nerve fibers which indicates a local injury, a com pression of the nerve fiber or base. In the neurophysiological investigations the EEG was irre gular in 2 cases and the ENMG in 8 cases showed slowed conductivity in the nerves. The irregularities in the EEG were mild, except for . *Hernberg et a l. (1971) found in a random group of indus trial workers a frequency of 4.5% that deviated from the normal. 0000306 Page 31 Figure 3. The distribution according to age and sex in the preliminary investigation NUMBER OF PEOPLE NUMBER OF PEOPLE 30t __________ 25- 15" ; ii19 20- 3020 39 40- 50- 60- Age in 49 59 years 5-- C? Sox Figure A. The distribution according to the method and duration of exposure DURATION OF EXPOSURE (in weeks) 6875, 0000307 Page 32 ( one case, and could be normal. The irregularities in ENMG could be explained in 6 cases due to local injury, ischialgia, or use of vibrating power tools. TABLE V. In the preliminary investigation observed malfunction ing in the lungs and Lab tests Organ Liver Kidney What was looked at Aspartateaminotra n sfe ra se (COT) Alanineaminotra n sfe ra se (GPT) A lcalic phos phatase (S-AFOS) Normal range 12-35 Wr units/m l 4-30 Wr unlts/ml 0 .8 -2 .9 B-L units Creatinine (S-Krea) Urine glucose Urine albumin 0 .7 -1 .3 mg/100 ml - mm I r r e g u la r it ie s found Number of Quantity 0 1 38 1 3.2 2 1.4; 1.4 0 1 + (weak) Sugar Metabolism Thyroid Sugar t e s t 1 hr. 210 mg/100 ml Iodine bound in protein (S-PSJ) Trliodo thyronine binding te s t (S-T3 te st) 4 .0 -8 .0 yg/100 ml 80-1202 1 0 0 223 Respiratory System Spirometry VC, FEV1>0 802 FEV1>071;8OZ 2 Ve 108; 75Z 0000308 ' '6876 Page 33 Conclusions The investigation convinced us that the health of the sample of 30 persons was normal, corresponding to a similar random group of same age. The neurologically exceptional cases were old persons, although mild irregularities in EEG were also observed among the young. In the case of the peripheral nervous system, the neurological results corresponded well to those of the ENMG. In particular, the neurophysiological tests are very sensitive, and similar irregular ities can be obtained from a "normal population". In this case, the control group consisted of a sample of neurologically un specified industrial workers. The frequency of exceptional EEG's among the 30 workers was about twice the frequency of the control sample. The difference is not significant (p > 0.05). ENMG did not show any difference between the two groups. Five persons were referred for further investigation and care after this investigation. CCr <? 8.2.3 The Health investigation Material The participants were chosen from the 180 who had reported -symptoms. The criteria were a) duration of the exposure, b) oc currence of "typical" symptoms and c) occurrence of other interesting symptoms (reported in the literature). Since age is crucial in evaluating the results, no one over 50 was chosen. The statistics on age, sex, and the method of exposure, as well as its duration are shown in Figures 5 and 6. It should be pointed out that out of 18 persons 11 have been exposed to the defoliants 17 weeks or more and 14 persons have been using the most hazardous method, the backpack type sprayer. Methods / The plan described in the preliminary investigations was 0000303 a {( /.CP Page 34 Figure 5. The distribution of age and sex in the health investigation NO. OF PEOPLE 1 y NO. OF PEOPLE 8 6- 4 .. 2-. 10- " 2 0 - 3 0 - 4 0 - 5 0 - 6 0 - Age in 19 29 39 49 59 years 9 ' cf Sex Figure 6. The distribution of age and sex in the health investigation NO. OF PEOPLE - NO. OF PEOPLE T 3Siv... ^*5* ooq3i^e78 *v. *'" _ ^ f OW09 353 Page 35 followed. In lab tests, the ones concerning the malfunctioning of the liver and thyroid were removed, since the working group considered them insignificant. Instead, as a new program, all persons were submitted to a toxi-psychological test, given by Mr. Kari Lindstrom, MSc. That includes the following usual sequence of tests used in the Institute of Labor Health: 1. General Intelligence performance. This is tested using the Wechsler intelligence test for adults (WAIS): similarity test, number sequences, sign test, completion of pictures and the cube test. 2. Memory and learning test. . This was tested using Wechler's memory tests (WMS), limited to the three areas of memory: -mechanical-verbal memory -logical-verbal memory -visual memory 3. Sensorimotor response has been measured using three different tests of speed, which were combined to give one variable. The methods are: -the Bourdon Wiersma test -the Santa Ana test -the sign test (WAIS) 4. Psychomotor disturbances have been estimated by the MIRA-test which is a test of motor performance without using vision as a control. 5. The characteristic reactions were estimated using the Rorschach projection test and another one specially designed to describe the personality of workers exposed to industrial poisonings. This measures primarily affectivity, amount of psychic energy and sense of reality. The results are transformed to a standard scale to make it possible to compare with a standard sample of male 0000311 . - 6879' j 0 Vf)009 364 Pago 41 weeks and 8 weeks and one year and more than one year. The deaths were divided into similar groups according to the age of death. From the above statistics the expected number of deaths were obtained using the mortality statistics of the whole country. The expected total mortality (mortality due to natural causes, to tumor, and to cancer of the respiratory systems) was evaluated for the total mortality, it has been possible to evaluate the expectancy among the working population. This has been done using the statistics on the probability of the cause of death in one year collected by the Department of Health and Social Affairs using their health insurance statistics. 8.3.2. Results The mortality for various causes (Table III) was almost consistently lower than the expected values, and even in those cases where the values were high they were not statistically signi ficant. There was no significant difference in mortality between the two groups from 1955-1965 and from 1966-1971. The duration of exposure (2-7 weeks vs. 8 or more weeks of defoliation) did not increase mortality (Table X). 8.3.3 Further Considerations There were some difficulties in collecting the statistics due to the lack of a file containing job assignments. It seems that, in particular, the defoliation in the Department of Forestry is poorly documented, because their labor force is more transient than that among other employers. It seems that, due to the physical requirements of the work, only workers in good health were selected for the job. The workers were fairly young at the beginning of the defoliation, one-third being 24 or younger (see Table VII). The majority (61%) were exposed to the defoliants in 1966 or after. The defoliation reached its peak years in the late 60's. Since for the majority the exposure to defoliants took place during the 4 0000317 6880 jS\^V)O0)O Page 42 past 5-6 years, one would not be able to detect illnesses that take theoretically a long time to show up. It is known that cancer can take 10 years to develop. It is justifiable that the investigation will be repeated in the future (e.g., after some 3-5 years). On the other hand, it should be pointed out that for three-fourths of the workers (See Figure 7), the time of exposure was brief (2-7 weeks) and the influence would, therefore, be small. When the mortality rate of the group of workers is compared, to the mortality rate in Finland, it should be kept in mind that the total population contains such cases as birth defects and the chronically ill who are not (completely) able to work. Hence, it is *-o be expected that the mortality rate of the working population is lower than the mortality rate of the total population. The insurance statistics used as an alternative reference are more representative of the mortality rate of the labor population, but since this system has been in existence for a short time (since 1962) it might contain "an over-representation of healthy persons". The results of this investigation show that, even though the mortality rate due to other illnesses is low, the mortality rate for tumor and lung cancer'is almost as expected from the total population. It cannot be assumed that the general risk among this group (due to smoking) to contract cancer and lung cancer is less than that among the total population. The caus2 of death investigation shows that among those exposed to defoliants the mortality rate due to natural causes or various groups of illnesses does not exceed the total mortality rate in Finland or that among labor. In other words, there is no evidence that the defoliants increase mortality. An investigation in the Swedish State Railway System on epi demiologic mortality (Axelson, et a t . , 1972) did not indicate any excess mortality due to natural causes or cancer or* lung cancer among people exposed to the phenoxy compounds. ,/ .. 0000318 Page 43 Figure 7. The distribution _i the time of exposure in defoliation ao %oo CD CO a 05 TABLE VII: Distribution of the cause of death Employer Defoliation started 1955--1965 1 9 6 6 -1 9 7 1 * State Railway System 251 5 2 .4 190 1 5 . 9 Dept, of High way Construction 211 27.3 291 240 Dept, of Forestry ?4l 3 1 . 1 693 57.9. IMATRAN VOIMA CO., 71 9.2 23 1.9 Total 77^ 100 1 1 9 7 100 Total 441 502 934 94 56 22.4 25.5 47.4 4.8 1971 100 TABLE VIII: The age distribution in the cause of death Age when start ing the defolia tion 15 - 24 25 - 34 35 - 44 45 - 54 55-60 Defoliation started 1955-1965 1966-1971 ** 189 24.4 408 34.1 201 26.0 238 1 9 . 9 ,491 24.7 279 23.3 146 18.9 199 16.6 47 6.1 73 6.1 Total 774 100 1197 100 Total . 597 439 470 345 120 1971 * 300 22.3 23*8 I7 . 5 r0 0 oj O C O A-,1 100 00.00319 TABLE IX. The cause of death distribution in various illnesses and their expectancy in the cause of death investigation Starting 19L.5-1965 Years Starting 1966-1971 Years Expec Expec Deaths tancy tancy I II Expec Deaths tancy I II Cancer in respiratory systcn 6 5.0 e n e X S# X 3 3.7 ee Total Expec Expec- X Deaths tancy tancy I II 9 6.6 . n. All severe cases of tumor 10 12.9 e e 3 1.5 e e n.s. 13 1 6 .*i ee Irregularities in the blood circulation All natural causes Total of causes 11 2*1.2 ee % 2 6 5 *1 . 8 e# 30 6 6 .1 37.1 6 * 8.3 10 .1 6 . 6 15 21.9 e # 17 e e 36 11 5 n.a. *3 33.5 71.* 88.2 ee e *8.7 Page 44 x The expectancy la computed in different age groups using the mortality rate of: I Total population in Finland II Those belonging to the health insurance system. xx n.a. non-significant difference 02E0D00 oe co 00 9C 6000MOC CO TABLE X. The mortality* distribution according o the duration of the defoliation and the expectancy Starting 1955-1965 years Starting 1966-1971 years Total Expec- Expec- Expec- Expec- Expec- Expec- Deaths tancy tancy xx Deaths tancy tancy xx Deaths tancy tancy xx 2-7 weeks of defolia I II I II I 11 tion Cancer in respiratory system 2 XXX 1.7 n *s 3 1.0 n s 5 2.7 n s All severe cases of tumor * *.6 .. 3 2.* n s 7 7.0 Irregularities in the blood circulation All natural causes 5 11 8.6 19.8 .. .. 3 5.1* 7 10.9 8 1*.3 18 30.7 Total of causes 13 2*1.7 13.6 12 l*i.6 7.7 25 39.* 21.3 % 8 weeks of defoliation * Cancer in respiratory system 1 1.* - 0.* * 1 1.8 All severe casos of tumor 2 3.6 - 0.9 2 *.* Irregularities in the blood circulation 2 6.8 All natural causes 5 15.* 1 1.9 1 3.9 3 9.0 6 19.2 o Total of causes 6 18.9 JO.* 1 5.0 2.7 7 23.9 13.1 x Only includes cases where the duration of the defoliation is known xx The expectancy is computed in different age groups using the mortality rate of: I Total population in Finland II Those belonging to the health 6884 insurance system, xxx n.s. non-significant difference J 9FOOOWOO page 45 0000321 Page 46 9. INVESTIGATION IN SWEDEN 9.1 Swedish mortality investigation i r i n 9.1.1 Material ^ In 1972 the Clinic of Occupational Medicine, OREBRO, inves tigated the mortality among people using herbicides in the Swedish State Railway System (Axelson, et al. 1972). The workers had sprayed various herbicides such as compounds containing a mixture of amitroie and diuron, amitroie, a mixture of amitroie and monuron, and a mixture of atrasin and phenoxy. The total population was 324 workers, of which 194 has been exposed to the phenoxy compounds and 143 to amitrole or its mixtures. 9.1.2 Results and conclusions The results are in Table VIII. The data is displayed in such a way that the effect of time can be seen after 3 and 5 years for those times when the duration of exposure exceeds 46 days or more. In case of amitrole and mixtures, 4 cases of cancer were observed with an expectancy of 1.07, and out of these, two were lung cancer, with an expectancy of 0.19. It is significant that no excess in mortality rate was observed among those exposed to the phenoxy compounds. 10. URINE TEST RESULTS CONCERNING ALLERGIC REACTIONS AMONG DEFOLIANT SPRAYING GROUPS (A case history from the Summer of 1972) 10.1. Method and Results The working group thought it would be important to investi gate, even if only superficially, the allergic reactions during spraying of the defoliants. Urine tests were performed on four persons working in various ways with spraying.' From the urine test, the quantities of 2,4-D and 2,4,5-T were analyzed. The liquid which w^s sprayed is called VESAKONTUHO 500, containing . g885 0000322 TABLE til. OBSERVED AND EXPECTED TOTAL AMOUNT ? DEATHS, THE NUMBER OF DEATHS IN TUMOR (WHO II) AND LUNG WITH RESPECT TO THE LATENCY TIME AMONG THOSE WHO HAVE BEEN EXPOSED (46 or MORE). CANCER CASES (WHO A50) 1957-72 IN SWEDISH STATE RAILWAY SYSTEM (SJ) IN 1947-71. THE EXPECTANCY BAND ON MORTALITY REPRESENTING THE WHOLE COUNTRY (SWEDEN) DURING THE PERIOD. PREPARATION VUUL ur DEATH 0 Year OBS. PERS EXP. PER YEAR OBS, TOTAL TOTAL MOR TALITY TUMOR 2,802 19/33 *1/59 0/78 17 5 2 OBSER. PERS/YR. \ 3 EXP. Year OBS, 1,341 14/41 3/47 0/60 15 5 .2 PHENOXY ACID TOTAL MOR AND COMBINA- TALITY 1,621 TIOMS TUMOR AMITROLE TOTAL MOR AND COMBIN- TALITY ATIONS TUMOR * *v * 1.223 11/58. 2/76 0/47 7,94 1/87 0,32 9 1 0' 9 4% 2x ) 1,046 807 8,55 2/06 0/36 7 1 0 5/90 1/41 0/24 . 8 24 , ><x) 5 Year OBS. PERS/YR EXP, OSS, 1,289 712 561 10/96 2/66 0/47 6,36 1/55 0/27 4/46 1/07 0/19 13 5 2 6 1 0 6 4xx) _ 9LX X X ) OTHER PREPARATIONS AND COMBINATIONS TOTAL MORTALITY TUMOR 295 1 ,6 3 I 0,33 S'000*5 0,CG 1 209 1,39 9x) 156 1 ,0 9 4XX) 0,32 3xxxxx) 0,26 3^ 0,03 l x) 0.09 l x) 6886 0. <0,05 <0,03 m ) p-0.C2 xxxx^p<0,01 xxxxx)p<c0,005 - a A a rage 47 zeoooo Page 48 2,4-D and 2,4,5-T in a ratio of 2:1. The samples were collected the next morning and evening after work and the second morning after the spraying. The jobs, working conditions and duration of the work was as follows: 1. Driver of a tractor spray, one door of the tractor open, no prevention. Three consecutive days. -2. Driver of a tractor spray, closed doors, a mask used for respiration. Five consecutive working / days. 3. A man on board a truck. No mask used. Three consecutive working days. 4. Driver of a spraying truck, no mask used, windows open. Three consecutive working days. TABLE XII. The content of 2,4-D and 2, 4,5-T in urine among the defoliant sprays in State Department of Highway Construction . Determinations individually and using spectrophotometry. (Behm. E. and Schroder, I. Weed Res. 9 (1969) 43-7). ` C 1 . F ir s t day a f t e r spraying morning evening 2. Second day afte r spraying morning 1 . 19* 57 22 2. 3 3 3 3. 8 17 13 4. 3 11 4 3. Estimate o f 2,4-D and 2,4,5-T (in mg) in Che organism a fte r the spraying xx c .a . 98 c.a. 8 c . a . 35 c .a . 18 x concent o f 2,4-D and 2, 4,5-T in urine mg/i xx che samples are over a period of 40 hours a f t e r Che sp ray in g. T otal amount of urine i s assumed to be 2 , that i s , 0.5 i during Che f i r s t n igh t, 1 .0 A during the next day and 0.5 A during the second nigh c. The h a lf l i f e o f 2.4-D and 2.4.5-T i s assumed Co be 1 day (24 hours). 0000324 34S, G o o i & o c L Paga 49 \0.2 Discussion of the results The amourts were measured only once and the accuracy of the results cannot be verified. The sampling, mailing, and storing may be sources of errors that cannot be considered here. It was intended to obtain only a rough estimate of the concentration of the phenoxy compounds in urine after spray ing. The estimate of the amounts of 2,4-D and 2,4,5-T immedia tely after the spraying was only meant to give some indication. The largest amount was found in worker 1 (96 mg), which is the amount accumulated in the organism over 3 days if the daily dose is 50 mg. on the average. This is about 1/70 of an immedia tely dangerous amount (a lethal dose in man is about 50-100 mg/kg). Disregarding the adaptation that takes place in connec tion with chronic exposure, the amount accumulated over 3 working days would be about 1/35 of the lethal amount. Sprayer number 3 collected a dose in his body about 1/100 of a dangerous dose. The rest of the amounts were much smaller. The results correspond to the expectations about exposure in various tasks of defoliation, and the small amounts in sprayer number 2 should be pointed out, which demonstrates that closing the windows and using a mask seems to help. Even though one sprayer, according to the data, was sig nificantly exposed to the defoliants during work, there is no method for interpreting the amount of exposure in relation to any alleged health hazards. In general, the influence of chronic exposure is poorly known. 11. THE CONTENT OF DIOXIN IN THE DEFOLIANTS USED IN FINLAND DURING 1960-1971* 11.1 Investigation contracted by the working group The working group contracted an evaluation of the content of *The working roup would like to thank the following companies for financial aid during this investigation: Berner Co., Farmos Co., Kemira Co., Nykema Co., and Eero Helkavaara Co. 6888 0090325 or>0>N\On Page 50 dioxin in seven different samples of the commercial defoliants. The results were given on February 16, 1973 (see Appendix 2 and table below). The method of evaluation is described in Elvidge, 0. A. Analyst, October, 1971, 9_6, pp. 721-727. The content of dioxin (TCDD or 2,3,7,8-tetrachlorodibenzo-p-dioxin) is in a table below: D efo lian t (Brand Name) Vesakontuho 500 Tributon DT Bruspon Spray 201 E/3 Origin and Years o f use VR 1967 - 70 VR 1971 VR 1968 VR 1960 - 64 KVK/KjJge H oraoslyr 500 T Regulan KSL sample from year 1961 KSL " " 1965 KSL " " 1962 VR S ta te Railway System KSL The I n stitu te o f Plant Ecology Active Ingredient 2, 4-D 2,4,5-T 1 VI If tl It DCA Vf MCPA Dioxin content ppm (mg/kg) 0.31 0.20 0.10 0.31 2,4,5-T n 0.14 0.21 0.51 11.2 Conclusions The data in the above table shows that the most common defoliants from 1960-1971 did not exceed the upper limit of 0.5 ppm of dioxin (TCDD) set up by the Ministry of Agriculture and Forestry (decision 703/71}. The only sample exceeding this limit is not statistically significant since the evaluation was inaccurate. / 6889 0000326 Page 51 12. CONCLUSION 12.1 General 12.1.1 The phenoxy compounds, which are usually the active ingredients in the defoliants, are the most commonly used in herbicides. That is why the hazards in connec tion with the use of the phenoxy compounds should be investigated. 12.1.2 The phenoxy compounds have been called "action hormones*, because their influence resembles that of a natural growth hormone of plants called 3-indoleacetic acid. Previously, it was believed that since the defoliants .are "action hormones" they are not poisonous. This is wrong. One should always consider all herbicides to be toxic. The phenoxy compounds in the defoliants are fairly poisonous based on evidence of acute poisonings. 12.2 Earlier reports on phenoxy compounds', a survey of the literature 12.2.1 Connected with the spraying and production of the phenoxy compounds, there had been descriptions of various acute symptoms such as fatigue, headaches, stomach troubles, and irritation of the skin and the respiratory system. 12.2.2 In spite of the common use of the phenoxy compounds there are few published observations about dangerous poisonings or illnesses among the sprayers. 12.2.3 In the industrial synthesis of one of the important ingredients of the defoliants, 2,4,5-T, the workers have been reported as suffering from skin disease, liver illness, and, probably, a nervous illness. The probable cause of these illnesses is not pure 2,4,5-T, but contam inants used during the industrial process (e.g. TCOD, as well as the initial and intermediary chemicals) . 6890 0000327 2 SL 5 6000MCX Page 52 12.2.4 The liquids containing 2,4,5-T might have a contaminant called TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin) . The TCDD is very toxic and causes deformation in the embryos. 12.2.5 2, 4-D and 2,4,5-T have been observed (in animal experi ments) , to damage the embryo slightly and interact with the hereditary material of the cell. No carcinogenic effects could be detected in animal experiments. No evidence exists that the phenoxy compounds have caused cancer, hereditary diseases, or deformation of the embryo. 12.3 Research of the working group 12.3.1 Sometimes the workers spraying defoliants have shown symptoms and, in general, acute effects such as head aches, vomiting, stomach trouble, giddiness, and irritation of the respiratory organs, the eyes, and the skin. 12.3.2 In some cases these irritations caused by the defoliants could lead to asthma or allergic rash in some particularly sensitive persons. Many years after the beginning of the illness, it is visually impossible to verify the cause in these persons. 12.3.3 In the investigation the effect of defoliants on the nervous system was tested using very sensitive methods. This was considered important, since previously it has been suspected that the phenoxy compounds cause damage to the nerves. On the other hand, the persons under investigation have shown nervous symptoms (e.g. headaches). One can expect to detect, at the moment of investigation, only those injuries for which recovery is slow or incom plete; the time since the last exposure to the defoliants was at least a year. The team from the Institute of Labor Health, with experience in the neurophysiology of various poisons (e.g., solvents and heavy metals) could 6891 0000328 1009 376 Page 53 uo\y- not detect a single case of poisoning among the persons under investigation. In delicate tests to describe the functioning of the nervous system, it was discovered that there was twice the normal frequency of disturbances in the electrical functioning of the brain as compared to a group of workers that had not been exposed to chemicals affecting the nerves. In statistical analysis, the difference was not significant (0.5 < p < 0.10), but, however, it was indicative. The observed irregularities were generally mild and they do not necessarily indicate illness. In the severe cases, further investigations and follow-up studies were considered necessary. 12.3.4 The investigation of the cause of death showed that the mortality rate from all natural causes or various specific groups of illnesses among persons exposed to the defoliants did not exceed the figures of the total population in Finland or those of the working age population. This discovery applies also for the mortality rate from cancer. Consequently, the defoliants do not seem to increase the mortality rate. It should be pointed out that the number of people in the investigation, about 2,000, is rather small. It is possible, too, that chronic illness will show up in the future, and it is recommended that the investigation be repeated after 3-5 years. 12.4 Results on the content of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in defoliants used in Finland In the State Institute of Agricultural Chemistry, seven samples from 1960-1971 were analyzed to contain TCOO less than 0.5 ppm in six cases, this amount has been recommended the upper limit by the Ministry of Agriculture 6392 0000329 .j1? . ' ` m.---1 tr-- - ---- US. GOO Page 54 UV- and Forestry (decision 703/71). One sample contained 0.51 ppm. Random samples of old defoliants did not reveal any dangerous quantities of the contaminant TCDD. 12.5 The TVL Case history of allergic reactions among defoliant . spraying groups The urine tests given to workers of TVL exposed to phenoxy compounds show that significant quantities of the poison can get into their bodies if adequate measures for protection are not used. No health hazards were found during these investigations. 13. RECOMMENDATIONS BY THE WORKING GROUP 13.1 Protection for the sprayers of defoliants from exposure should be improved. The practical recommendations for this have been given to the committee on safety regulations set up by the Ministry of Social and Health Affairs. 13.2 13.3 The working group has seen the recommendations by this committee on safety regulations and considers that they will protect the sprayer and any by-stander from health hazards, if followed carefully in using and dealing with defoliants. The effects of the proposed safety measures should be checked among the sprayers in the future (e.g., by measuring the individual exposure in various spraying methods). The working group could not investigate the environmental impact of the use of defoliants including points other than human health. The impact of the defoliants on nature should be investigated separately. 13.4 The working group considers that workers, other than transient workers in defoliations, should be given a health check as ^required by the work safety law, S 44 (the decision 0000330 Page 55 009 378 637/71 by the State Council, subsection 21 in the list of cases). The preliminary check on the workers should screen out persons who are exceptionally sensitive to the hazards of the above chemicals, (e.g., pregnant women or persons with illnesses or allergies or injuries of the respiratory passages, skin, nerves, liver or kidney). Consequently, the preliminary check should precede the beginning of any work of this kind. At regular intervals, a check should be done immediately after the completion of the spraying season. This check should include, in particular, any symptoms of the respira tory passage, eye and skin, the neurological status and lab tests in order to evaluate the functioning of the liver and kidney (evaluation of the transaminase, albumin and glucose in the urine). 13.5 The working group considers it important to have reliable tests on the content of the poisonous contaminants, such as dioxins, to be done prior to granting sales permission. The chemical composition of these products should be checked repeatedly in the future. 13.6 The working group expects that world wide research on the phenoxy compounds will be followed. Special emphasis should be placed on the epidemiological investigations of the health hazards among people such as cancer, injuries to y embryos, and hereditary diseases. The epidemiological mortality investigation by the working group is proposed to be followed by another one (e.g., after three years). At the same time, the possible chronic health hazards should be considered. 13.7 The follow-up check for chronic injuries requires that the employers ke^p detailed records on defoliation which indicate the workers, the methods, the duration of the work 6894 0000331 W0009 379 Page 56 and the defoliants used. 13.8 If new discoveries on the health hazards of the defoliants show up in the future, the health hazards connected with these chemicals should be re-evaluated. C 68 0000332 vvuuoy 380 Appendix 3 I LITERATURE Aamisepp, A., et al.i Survey on the use and the effects of modern chemical herbicides in civil and military use. FOA 1/ Report A 1519-31, Sept. 1970. (Swedish) Defense Research Insititue, Dept. 1. Johnson, J. E.: The public health implications of widespread use of the phenoxy herbicides and picloram. Bioscience 21 (1971): 17, 899-905. Erne, K. 1966a. Distribution and elimination of chlorinated phenoxyacetic acid in animals. Acta vet. scand. 7: 240-256. Erne, K . : Studies on the animal metabolism of phenoxyacetic herbicides. Acta vet. scand. 1966b, 7, 264-271. Clark, D. E. & al: The fate of 2 ,4-dichlorophenoxyacetic acid in sheep. J. Agr. Food Chem. 12:43-45. 1964. Khanna, S. & al: Metabolism of C14-labelled 2,4-dichlorophenoxyacetic acid in rats. J. Agr. Food Chem., 1: 500-503. Clark, D. E. & al: Residual aspects of 2,4,5-T and an ester in sheep and cattle with observations on concomitant toxicological effects. J. Agr. Food Chem. 19 (1971): 4, 761-764. Wilson, J. S. 1971. Report on the treatment of pregnant Rhesus monkeys with 2,4,5,-T acid. Ruotsin Giftnarandille tehty raportti tutkimuksista, 5 s . Nielson, K. & al: Fatal poisoning in man by 2,4-dichlorophenoxyacetic acid (2,4-D): determination of '-he agents in forensic materials. Acta pharmacol. toxical., 22: 224-234, 1965. Stanosz, S. Effect of sodium 2,4-dichlorophenoxyacetate on tissue respiration in parenchymatous organs of rats. (in Polish). Med. Piacy 20 (6): 600-605: chem. Abstr. 73:13387p (1970) 1969. Kuhn, E. u.a.: Modellmyotonie nach 2,4-Dichlorphenoxyacetat (2,4-D) V.v ber der Ratte Klin. Wschr. 4_3 (1965) , 673-677. Philleo, W. W. 6 al: 1967. Effect of 2,4-dichlorophenoxyacetic acid on the in vivo metabolism of acetate in adult rats. J. Agr. Food Chem. 15:256-260. y Desi, I. & al: Nervous system effects of a chemical herbicide Arch. Environm. Hlth. 4(1962): 1, 95-102. 0000333 TOO II Giftnamnden: Phenoxy acids. Discussion on current information. Report of a group of experts. Stockholm 1971. (Swedish) Florsheim, W. H. & al: Some effects of 2,4-dichlorophenoxyacetic acid on thyroid function in the rat: Effects on peripheral thyroxine Endocrinology 72(1963), 327-333. Drill, V. A. & al: Toxicity of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid, a report on their acute and chronic toxicity in dogs. Arch. Ind. Hyg. Occupt. Med. 7 (61-67) 1953. Hill, E. V. & al: Toxicity of 2,4-dichlorophenoxyacetic acid for experimental animals. J. industr. Hyg. Toxicol., 29: 85-95 1947. Mogens R. Brandt: Herbatox poisoning. A brief review and report of a new case. Ugeskr. Laeg. 1971: 133:500-503. Bjorklund, N-E. & al: Toxicological studies of phenoxyacetic herbicides in animals. Acta vet. scand., 1_-. 364-490 1966. Myrkkyasiain Neuvottelukunta (a committee on poisonous affairs) The health hazards of the phenoxy compounds 2.4-D and 2,4,5-T used in defoliation. Helsinki, 1972. (Finnish) Khera, K. S. & al: Pre- and Postnatal Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4-Dichlorophenoxyacetic Acid and their Derivatives in Rats. Toxicology and applied pharmacology 22, 14-28 (1972):1. Davring, L. & al: Cytogenetic effects of 2,4,S-Trichloropheno^ xyacetic acid on oogenesis and early embryogenesis in Drosophila roelanogaster. Hereditas 6 8 : 115-122. 1971. Dvring, L. Cytogenic effects of 2,4,5-trichlorophenoxyacetic acid on Drosophila m. and Mus m. A preliminary comparison study for the Swedish GIFTNAMND (Poison committee) Nov. 1971 7p. . Bashirov, A. A. Health condition of workers producing herbidices of amine salt and butyl ether of 2,4,-D acid. Vraeebnoe delo 1969: 10, 92-95. Todd, R. L. A case of 2,4-D intoxication Iowa st. med. soc. 52 (1962) 663-664. Foissac-Gegoun, P. & Al: Polynevrite aprs usage d'un desherbant: 1*acide 2,4-D Lille medical .7 (1962) :10, 1049-1051. Flint, G. W. 4 af: Vapor pressures of low-volatile esters of 2,4-D Weed Sci. 16:541-543. 1963. 0000334 ms? 0OW0003 382 Ill Jensen, D. J. & al: Determination of vapor pressure of soma phenoxyacetic herbicides gas-liquid chromatography. J. Agr. Food Chem. 14:123-126. 1966. Fetisov, M. I. Problems of occupational hygiene in work with herbicides of 2,4-D group (in Russian) . Gig. sanit. 9_: 28-31 1966. Kimming, J. and Schulz, K. H.: Dermatologica 115 (1957) 540-546. Lorenzen, I. and Lygsjie, J. En undersdgelse over virkningen af et kloreret fenoxyacetat pa kulhydratstofskiftet pa kaniner. Nord. hyg. T., 38!: 153-157. 1957. Bleiberg, J. & al: Industrially acquired porphyria. Arch. Dermatol., 89: 793-797. 1964. Long, K. R. & al: the epidemiology of pesticides in a rural area. Amer. Im.. Hyg. Assoc. Jour. 30:298-304. 1969. Forbes, J. D. & al: Weed spraying and coincident asthma. J. Occup. Med. 8(1966): 12, 643-650. Goldstein, N. P. 4 al: Peripheral Neuropathy After Exposure to an Ester of Dichloroohenoxyacetic Acid, J.A.M.A. 171:1306-1309, * 1959. Monarca, G. and DiVito, G.: Sull'intossicazione acuta da diserbante (acido 2,4-dichlorofenossiacetico) Folia medica 44 (1961): 6 , 480-485. Berkley, M. C. and Magee, K. R.: Neuropathy following exposure to a- dimethylamine salt of 2,4-D arch. Int. Med. 1963:351-352. Bauer, H. & al: Occupational intoxications in manufacturing chlorophenol compounds. Arch. Gewerbepath. 18(1961) 538-555. Poland, A. P. & al: A health survey of workers in a 2,4-D Plant and 2,4,5-T plant. Arch Environ Health 22:316-327, 1971. Richert, J . : Uber neurologische Komplikationen einer mit Chlorakne einhergehenden ChlorkohlenwasserstoffVergiftung. Nervenarzt 33 (1962), 180. Kleu, G. and Gltz, R.: Spat- und Dauerschden nach chronisch gewerblicher Einwirkung von Chlorphenolverbindungen Med. Xlin. 66(1971): 2, 53-58. 0000335 . iSSS IV Kimbrough, R. D. Toxicity of chlorinated hydrocarbons and related compounds. A review including chlorinated dibenzodioxins and chlorinated dibenzofurans. Arch. Environ Health 25(1972): 2, 125-131. Huston, B. L. Identification of three neutral contaminants in production grade 2,4-D. J. Agr. Food Chem., 20 (1972):3, 724-727. Woolson, E. A. et a l . : Survey of polychlorodibenzo-p-dioxin content in selected pesticides. J. Agr. Food Chem., 20 (1972):2, 351-354. Schulz, K. H. Zur klinik und tiologie der Chlorakne. Arbeitsmedizin-Socialmedizin-Arbeitshygiene 3(1968):2, 25. Sparschu, G. L. & al: Study of teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Food Cosmet Toxic. 9:405-412, 1971. Kay, J. H. et a l .* Subacute dermal toxicity of 2,4-D. Arch Environ Health 11 (1965):5, 648-651. Schleuplein, R. J. and Blank, I. H . : Permeability of the skin Physiological Reviews 51 (1971):4, 702-747. Kearney, P. C . : Chlorinated dioxin research Presented before a joint meeting on pesticides. United Kingdom, Canada, United States. Sponsored by the Council on Environmental Quality and President's Cabinet Committee on the Environment Working Group on Pesticides, Washington, D. C., November 5, 1970. Hernberg, S. & _1: Blood lipids, glucose tolerance and plasma creatinine in workers exposed to carbon disulphide Work-Environment-Health 8(1971), 11-16. Axelson, 0., e t a l . i Herbicides and mortality - an epidemologic investigation of the workers of SJ(Swedish Railway System) OREBRO 1972. Fullerton, P. M . : Toxic Chemicals and peripheral neuropathy. Proc. Roy. Soc. med. 62(1969), 201-204. 0000336 6899 t'8C 600CMO Appendix 2 v. C E R T IF IC A T E NO.: A 121-127/73 Ordered by: the working group on health hazards and working safety with defoliants Date: January 5, 1973 Sample: 9 samples of defoliants Task: 2 , 2 , 7 , 8-tetrachlorodibenzo-p-dioxin analysis Results: Brand A 121/73 Vesakontuho 500 A 122/73 Tributon DT A 123/73 Bruspon A 124/73 Spray 201 E/3 A 125/73 KVK-K0~e A 126/73 Hormoskyr 500 T A 127/73 Regulan Maximum amount of dioxin (from the amount of 2,4,5-T) ppm (mg/kg) 0.31 0.20 0.10 0.31 0.14 0.21 .0.51 Method of analysis: Elvidge, A. A., Analyst, October 1971, 96, pp. 721-727 Due to shortages of time two defoliants "Vesakonhauite F 500" and "Bruspon" (from 1957) could not be received by February 16, 1973. The result on "Regulan" A 127/73 is based on one measurement only. Helsinki, February 16, 1973 // . Veijo Mattinen 0000337 '--'VVtK)09 385 Appendix CURRENT KNOWLEDGE ABOUT THE EFFECT OF DEFOLIANTS ON HUMAN HEALTH In Finland the main defoliants are 2,4,5-T and 2,4-D as esters (which dissolve in oil) or salts (which dissolve in water). These phenoxy compounds are considered second class poisons based on LD50 values in animal investigations. During the past two years the question has been raised whether these compounds - in particular 2,4,5-T - cause damage to the human embryo. In Vietnam, these phenoxy compounds have been extensively used to defoliate, and it has allegedly caused de formed embryos. The investigation results are not conclusive so far. However, it is known that 2,4,5-T compound might have con tained large quantities (up to 30 ppm) dioxin, which is a known poison and causes deformation of embryos. Recent investigations with mice indicated that pure 2,4,5-T could cause changes in the chromosomes. But the animal experiments did not indicate any carcinogenic effects for 2,4-D or 2,4,5-T [Myrkkyasian neuvottelukunta, March, 1972]. It has been alleged that the workers of defoliation in SJ (State Railway System) have become ill or died in Sweden in winter 1972. An investigation was started there. The medical investigation was done by Regional Hospital in rebro (Department of Occupational Medicine, Dr. Lannart Sundell) and a committee of the Parliament was set up (including some experts). In Sweden the main ingredi ents in defoliants are other than phenoxy compounds, namely, amitrol and diuron. t In Finland the working group on defoliation has kept touch with the medical investigation group in Sweden. 0000338 6301 OW0009 386 Page 2 In literature there are reports of some twenty poisonings of human beings by phenoxy compounds of which some have been exposures during work, some have been suicides. These cases have been described in literature as neurological cases with usually the similar kind of symptoms (disabling neuro pathy) . In some cases there have been disturbances in the per formance of the liver and the kidney. The closer details about the poisonings for people are described in Appendix 3 "The effect of defoliants on people". It seems pos sible that the illnesses in these cases were due to some allergic reactions, since 2,4-D had usually very little effect. The working group considers that there is enough medical evidence and allegations about the health hazards of the defoliants that the most exposed workers should be brought into medical investiga tion . Plan of investigation Preliminary investigation For the preliminary investigation, 30 workers of VR from Oulu, area will be chosen according to the length of exposure to defoliants. This group will contain both currently working, retired, and persons in illness pension. The medical check-up will be done in the Institute of Labor Health in Helsinki according to Appendix 2 and the investigation will include lab, X-ray, lung functioning, and neurophysiology as well as check-up by a specialist in occupational health and neurology. The program takes one day for each worker. ,/ 000C3"9 630,2 -V0009 387 Page 3 The occurring symptoms and findings of illnesses will be processed statistically (possibly multivariable analysis) in computer. The final statistical analysis will be determined only after it is known how frequent abnormal findings are. In the first phase, no control group is intended to be used, but if more than expected pathological findings show up, one has to compare this with a normal control group (which is not included in the cost estimate). The results will be reported individually to each worker. The primary purpose of the preliminary investigation is to find facts about poisonings caused by the defoliants and the results are hoped to help in planning and guiding, further investigations. The cause of death investigation Simultaneously with the preliminary investigation an extensive cause of death investigation is carried over among workers exposed to defoliants. Special emphasis will be exercised to get workers who sprayed defoliants in 1950, if reliable information of them is available. At this moment, it seems that the most reliable data about workers .exposed to the defoliants comes from VR (State Railway System). Preliminary estimates puts 1000 the number of people to be inves tigated, but the final figure may change since the data is not yet complete. For comparison, another group of 1000 persons should be chosen as a control group. This group, not exposed to the defoliants, should closely resemble the group exposed to defoliants (as far as age, sex, geographic location, social status, similarity of the work is concerned). ,/ u-- ' i` 0000340 6S03 Page 4 The cause of death is purely statistical file research. The pur pose is to determine the cause of death among workers who were exposed to the defoliants and died in 1953-1971 based on the death certificates and reports of autopsies. Special emphases will be exerted on finding out the possible over mortality in cancer, chronic lung diseases, cardiovascular dis eases, and other chronic illnesses. This kind of investigation is the only easy method to find out if the defoliants increase the rate of mortality as compared to the so-called usual illnesses. Health investigation The Institute of Labor Health will choose 50 workers among those who responded to the questionnaire as becoming ill due to the defoliants, and who have had the most common symptoms of the whole group. The purpose of the health investigation is to identify the pos sible joint symptoms of poisonings and the connected permanent or chronic changes in the functioning of the organism. It should be pointed out that only permanent and chronic changes can come out of the investigation, since the workers have not been exposed to the defoliants at least for a year. The final decision about the program for investigation is not yet done, but it should probably include neurological investigations, since that kind of injury is permanent. The estimate of cost is just preliminary for one patient. 0000341 C O O a* Page 5 The principal investigators: .The responsible investigator Sven Hernberg, assistant professor .Occupational medicine investigations Vesa Riihimaki, M.D. Juhani Hassi, M.D. Neurophysiology (EEG and EMG) Anna-Maria Seppalainen, M.D., specialist ^ Neurology Pauli Karli, M.D., specialist Internal diseases Simo Tarpila, assistant professor Statistical analyses Sisko Asp, MS. In addition to the above, some staff is required for other jobs (labs, files, typing, mailing, etc.) and computer time. Schedule The preliminary investigation can be started right away, if funds are available.* The investigation will take about 2 months ex cluding the statistical analysis. The cause of death investiga tion will be carried over simultaneously with the preliminary investigation. It will take more time depending on how quickly the material will be gathered. In the data collection the local civil servants of VR will do troublesome and tedious chores Page 5 rincipal investigators: sponsible investigator iernberg, assistant professor itional medicine investigations Uihimaki, M.D. L Hassi, M.D. Physiology (EEG and EMG) laria Seppalainen, M.D., specialist gy Karli, M.D., specialist al diseases arpila, assistant professor ti I analyses \sp, MS. Ltion to the above, some staff is required for other jobs files, typing, mailing, etc.) and computer time. .e liminary investigation can be started right away, if funds ilable.' The investigation will take about 2 months ex- the statistical analysis. The cause of death investiga li be carried over simultaneously with the preliminary gation. It will take more time depending on how quickly erial will be gathered. In the data collection the local ervants of VR will do troublesome and tedious chores ,/ OOOG34 O Oiay take O CJ GO C> \ 0000343 'O'C- ' DOV0OO9 391 INSTITUTE OF LABOR HEALTH Medical Department HEALTH HAZARDS OF THE DEFOLIANTS Name : ______________ Prof essio n /o ccu p atio n : Address: ____________ . Who has employed you to d e fo lia te ? Appendix 8a Social Security No.: Phone: _____________ Dace: When (hov many y ears, how many days a year)? Which brand names o f d e fo lia n ts have you used? Hov have you used d e fo lia n ts? Have you used a mask? _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Have you used a rain coat? _ _ _ _ _ _____________ Other p o ssib le ways being exposed to d e fo lia n ts: </ 0000344 ' 6807 ; DOW 1 1 3 5 8 c c N i ,,- " .-- Reprinted by the U S . D EPARTM ENT O F H EA LTH , EDUCATION. A N D W ELFARE PUBLIC HEALTH SERVICE from CRC CRITICAL REVIEWS IN TOXICOLOGY, January 1974 THE TOXICITY OF POLYCHLORINATED POLYCYCLIC COMPOUNDS AND RELATED CHEMICALS Author: Route D. Kimbrough* Center for Disease Control Atlanta, Ga. Referees: M. M. Barnes J. B. Greif Medical Research Council MRC Toxicology Unit Medical Research Council Laboratories Carshalton, Surrey England INTRODUCTION Only the small group of the polychlorinated polycyclic compounds given in Figures 1 and 2 will be reviewed in this article. Several years ago when (his laboratory was the Atlanta Toxicology Branch of the Food and Drug Administration, concern was voiced over the possible long-term effects of polychlorinated biphenyls, and we began to study the toxicity of Aroclor 1254 and Aroclor 1260. About this time a follow-up study was performed on a group of workers in New Jersey who were engaged in the production of 2,4,5-T.1 These workers had very severe chloracne and had previously been shown to have porphyria.3 During that same time, a petition was filed with the Food and Drug Administration'for a residue level of hexachlorophene on certain food crops. Hcxachlorophene, as well as 2,4,5-T, is made from trichlorophenol, although the chemical reactions are quite different. When we checked the available literature on the toxicity of hexachlorophene we felt that adequate toxicity data by present standards were not available, particularly not if the compound was meant to be ingested. Further more, Larson3 had pointed out that hexachloro phene was more toxic than commonly acknowl edged. Hexachlorophene and similar chemicals behave differently from such polycyclic polychlorinated compounds as the chlorinated biphenyls (PCBs). Hexachlorophene in particular is acutely much more toxic than the PCBs and is fairly rapidly eliminated from the body, while the PCBs have a very low acute toxicity, but are very poorly metabolized, particularly those with more than four chlorine atoms on the phenyl ring?. The long-term effects are therefore more critical for compounds such as PCBs. The chlorinated dibenzodioxins and furans, some of which are extremely toxic on an acute basis, may also show persistence and their long-term effects may be equally as important. These observations show that it would be erroneous to make predictions on long-term effects from results ohtained in acute toxicity studies. 'Formerly with Ihc Environmental Protection Agency, Chamblee Toxicology Laboratory, 4770 Buford Highway, Chamblee, Ga 30341. CGOoiSi January 1974 44S 6909 X e 98261 l>MOO FIGURE 1. The materials shown in this figure are mixtures of compounds in which several but usually not all o f the positions (-X) are chlorine and the rest are hydrogen. The properties of the materials depend on the degree of chlorination o f the mixture. All are insoluble in water but soluble in varying degrees in organic solvents and in fats. The trade name for the U.S. product (Monsanto) of the chlorinated biphenyls and terphenyls is Arodor. The trade name is followed by the number. For the biphenyls, the first 2 digits of this number are 12, and for the terphenyls, 54. in each instance the last 2 digits give the percentage of chlorine. OH OH OH OH OH OH Cl Cl Cl Cl Cl HEXAOLOROPHENE DICXLOROPHENE 2,2 1-Meth y 1ene-bls(3.4, 6 - t r 1 chlorophenol) 2 ,2 ' -Methylene-b1s(4-ehlorophenol) S1THI0N0L 2.2' -TMo-b1s(4,6-dtchlorophenol) v OH OH Cl Cl 2 ,4 ,4 ' trtchloro- 2' hydroxy-diphenyl ether (Trlclosan > !ntin DP300) Cl Cl FENT1C10R 2 ,2 ' -TMo-b1s(4-chlorophenol) Cl ci 3,3'.4,5'-T etrch loros11cy U n tlid e (TCC) 3,4,4'-Tr1chlorcarbin1l1de (TCP) FIGURE 2. The compounds in this figure are white crystalline solids with clearly defined melting points above 160*G They are practically insoluble in water and soluble in varying amounts in organic solvents and in fats. Those containing an OH group, all the above excepting 3,4,4'-trichlorocarbanilide, are soluble in aqueous alkali. 446 CRC Critical Reviews in Toxicology GGOGioG 6910 898fy'tM)Q v- ; / H p rsen t review is a n atte m p t t o correlate v ario u s to x ic e ffe c ts p ro d u ce d toy th e ch em icals listed in F igu res 1 an d 2 in o rd er to b etter u n d erstan d an d ap preciate their behavior. T h e su b jects covered in this article have lately en jo y ed great in terest and a rapid ly grow ing n um ber o f scien tific rep orts are available. Sin ce a n um ber o f the ch em icals discussed p rod u ce sim ilar o r related e ffe c ts, their to x ico lo gy is d iscu ssed . together rather than listing each com pou n d sep a rately , w hich w ould have led to a great deal o f re p e titio n . A d etailed d iscussion o f the variou s chem ical reaction s, an alytical m eth ods for the determ in a tion o f the co m p o u n d s, is n o t given. T h e review h as been w ritten w ith the to x ic e ffe c ts o f the variou s co m p o u n d s as the focal p o in t and the literatu re cited serves to illustrate these p o in ts. N o attem p t has been m ade to present a com plete review o f the literatu re in th is area. DEVELOPMENT, USAGE, AND OCCURRENCE A d e sc rip tio n o f the sy n th e sis o f ch lo rin ated b ip h e n y ls ( P C B s ) c a n b e f o u n d in L ie b ig 's A n n a le n as early as 1881.4 Su ccessfu l p rod u ctio n o f d iph en yl in com m ercial qu an tities w as n o t re p o rt ed un til 1 9 3 0 .5 A t that tim e the ch iorin ation o f tech n ical b ip h en y l w as a lso d e sc rib e d .4 B ip h en y l itse lf is fu n gistatic an d u sed as a preservative fo r citru s fru its.7 C h lorin ated biphen yls are very stab le, an d have been u sed fo r protective coatin gs, as plastic resins o r gu m s fo r varnishes, an d for w aterproofin g and flam eproofin g o f w ood. T h e excellen t dielectric properties o f these co m p o u n d s w ere n o ted as early as 1 9 3 0 . U n til the advent o f the P C B s, m ineral oil w as u sed as a co o lin g an d d ielectric liq u id im prgn an t fo r high voltage electric ap p aratu s su ch as tran sform ers and cap ac ito rs. Sin ce m ineral oil is flam m ab le an d flam m able gases co u ld also be prod u ced , restric tion s fo r the use an d in stallation s o f these trans form ers h ad to be se t, particu larly fo r in d o o r in stallatio n . W hen m ineral oils w ere replaced by the A ro clo rs, the fire an d ex p lo sio n h azard s w ere elim in ated. T his w as recognized by the N ation al B oard o f Fire U nderw riters and the N ational E lec tric C o d e w as m o d ifie d .8 U se o f A ro c lo rs a lso g re a tly redu ced the ph ysical size o f the c a p a c i t o r s .9 A recent su m m ary o f the varied u ses o f PC B s *v t h a s b e e n p ro v id e d toy rfiro ad h u rst.1 0 W ith .a p e a k p ro d u c tio n o f 4 2 ,5 2 7 sh o rt to n s o f P C B s in 1 9 7 0 , M onsanto is the sole produ cer o f A roclors (U .S . trad en am e fo r P C B s) in the U .S . Y e arly P C B prod u ctio n figures for the period 1960-1971 w ere reported by M on san to.1 1 The production increas ed steadily until 1 9 7 0 an d d ro p p ed to 2 0 ,2 3 6 sh o rt to n s in 1971 du e to a v o lu n tary restriction o f the use o f PC B s by the co m p an y. T rade nam es in oth er cou n tries in clu de K an n eclor and S a n th a te r m in Ja p a n , C loph en, F len d or, P h e n o d o r, an d Sovo l in E u ro p e. The polych lorin ated cap acitor liquids, com m on ly called askarels, are m ixtu res o f chlorinated biphenyls and chlorinated benzenes. A variety o f oth er in du strial ap p lic atio n s10 have been fou n d for PC B s. T h ey have served as flu ids fo r h ydrau lic, gas turbine, an d vacuum pu m ps, as heat transfer flu ids, as plasticizers, adhesives, textile coatin gs, su rfa ce c o atin g s in p a in ts an d v arn ish es, as se al an ts, and as part o f form u lation s to prepare pressure sensitive reco rd s an d co lo r copyin g p a p e rs. T h e e x te n t o f th e cu rren t u se o f P C B s in prin tin g processes is u n know n . O th er suggested uses over the y ears have in clu ded cataly st carrier fo r polym erization o f olefin s, conversion o f w ater perm eable soil to a n on perm eable state, and com bin ed in secticide an d bactericide form u lation s. M ixtures o f chlorinated biphenyls and chlorinated n aph th alenes have been em ploy ed to insulate electric w ires an d cab les, particu larly w hen they w ere u sed in the m in ing in d u stry an d on w arsh ip s.13 R esid u es o f polych lorin ated biph en yls have b een rep o rted in the en viron m en t b y a n u m b er o f au th o rs in d iffe re n t p a rts o f the w o rld . T h ese reports w ere usually from areas w here industrial pollu tion w as responsible for the environm ental c o n ta m in a tio n . H o ld en ,13 afte r fin d in g P C B s in m arine fish an d m ollu sk s from S c o ttish w aters, n o ticed that th ey w ere generally highest in sam p les from the F irth o f C ly d e an d he discovered that the PC B con tam in ation p rob ab ly originated from industrial sew age. T h e sew age sludge w as trans ported from tw o C lasgow purification w orks by b o a ts an d d u m p e d in d e e p w ater s ix m iles so u th o f G arroch H ead. T h e d isp osal o f sew age sludge on land could lead to the in troduction o f PCB residues in to the terrestrial eco sy stem by transfer through fo o d chains. S o far PC B residues have not b e en d e te c te d in fresh w a te r fish in S c o tla n d . Polychlorinated biphenyls have also been found 58 January 1974 447 631S i 135863DOW 1 in wildlife .samples collected along the Dutch coast and the Rhine River14 and in fish and seal in different parts of the Baltic Sea along the Swedish coast.15 Risebrough et al.16 compared the presence of PCBs with that of DDT and its metabolites in various birds. These authors found very low concentrations of PCBs in wildlife species in Baja, California, Mexico, and the Gulf of California, which are very remote areas with a high ratio of DDT to PCB of approximately 9 or 10. In sea birds from the Pacific, the ratio was between S and 10 and at that time, PCBs were not found in eggs of the Adelie penguin from Cape Crozier, Antarctica. Relatively high PCB concentrations were found in San Francisco Bay, Puget Sound, and San Diego Bay and the ratio of DDT to PCBs in wildlife samples from this area was between 1 and 2. This is again emphasized by the findings of Dustman et al.17 Twelve Alaskan bald eagle eggs had median PCB residues of 1.6S ppm while 11 eggs collected in Maine, Michigan, Minnesota, and Florida showed a median concentration of 9.7 ppm. Zitko1* found low concentrations of PCBs in different types of fish taken from several lakes and the St. John River System, New Brunswick, Canada, and the Nova Scotia banks. Fish from the Milwaukee River and Lake Michigan in the mid west part of the U.S., where the river enters into the lake, contained varying amounts of PCBs19 while fish taken from various small lakes in Wisconsin without industrial discharge failed to show PCBs. Large predators from Lake Michigan contained as much as 10 to 25 ppm PCBs on a fresh weight basis expressed as Aroclor 12S4 and salmon eggs contained as much as 12 to 17 ppm PCBs on a fresh weight basis. Further studies by Veith30 of the concentration of PCBs in different rivers emptying into Green Bay indicated a trend towards a decrease in PCB concentrations since the voluntary partial ban on PCB sales by Monsanto was imple mented in September of 1970. If PCB discharges are very high a fishkill may result such as the one in Escambia Bay, Florida in 1969 31 In that particular episode the PCBs had entered a plant's effluent through accidental leak age of heat exchange fluid. The PCB found in this case resembled Aroclor 12S4 most closely. The peak amount found in water of the Escambia River at the outfall amounted to 275 ppb and the peak concentration found in oysters from Escambia Bay was 3.0 ppm. Aroclor residues in sediment samples taken near the outfall- reached 486 ppm. When the leakage from the plant was -corrected, Aroclor levels gradually decreased in this area. The wildlife portion of the U.S. National Monitoring Program analyzed starlings, mallard and black ducks, bald eagles, and fish of several species for PCBs. According to Stickel,33 monitor ing for terphenyis is also done but not for any of the other chlorinated compounds in this report. Research surveys that include fisheating birds, insectivorous birds, ospreys, woodcocks, and cer tain ducks also monitor only for PCBs and for pesticides. Oysters {Crassostrea virginica) are a good indicator of PCB and other chlorinated hydrocarbon concentrations in water and a nation al monitoring program in these mollusks in IS coastal areas has been developed.33 So far, mollusks have only very occasionally contained low levels of PCBs. In summary, the PCB concentrations in water are low or nonexistent in remote lakes and fresh water streams and can be as high as SOMg/1 (5 ppb) or higher in polluted rivers such as the Hudson River.34 The PCB concentration is usually higher in sediment from these polluted waters because PCBs are only very slightly soluble in water.35 Since PCBs are stored in living matter, they are concentrated in the food chain and the usual " biomagnification" is encountered as we know it from other poorly metabolized and excreted sub stances. The concentration of PCBs in fish, par ticularly those in the upper end of the food chain from polluted rivers and lakes, is usually estimated in the ppm range and fisheating birds will have somewhat higher levels of PCBs than the fish from the same geographic location.36 Young antmak may contain less PCBs than their older counter parts of the same species in a particular location. PCBs are also infrequently encountered in dairy products, meat, fish, and poultry in ready-to-eat human food.37* * Occasionally, human food and domestic animal feed have been more, extensively contaminated with PCBs. In these instances, the ' contamination could usually be traced to local misuse of transformer fluid, leakage of PCBs from heat exchange fluid, or contamination of recycled paper with PCBs where the paper had been used for food packaging such as cereal boxes.39 The U.S. Food and Drug Administration has recently set temporary tolerances for PCBs in certain food products (see Table 1). 448 CRC Critical Reviews in Toxicology 0006137 6912 s^ oisseijJI/lgg TABLE 1 Temporary Tolerances Expressed is Parts per Million (ppm) Established by U.S. Food end Drug Administration (1) Milk (fat basis) (2) Dairy products (fat basis) (3) Poultry (fat basis) (4) Eggs (5) Complete and finished animal feeds (6 ) Animal feed components (7) Fish and shellfish (edible portion) (8) Infant and junior food (9) Paper food-packaging material 2.5 2-5 5.0 0.5 0.2 2.0 5.0 0.2 10.0 Note: The tolerance for paper food-packaging material shall not apply to such material separated from the packaged food by a functional barrier impermeable to PCB migration. The Food and Drug Administration will provide upon request the analytical methods it will use for enforcing the temporary tolerances. All of these aspects of PCBs in the environment have been the subject of a number of reports, reviews,30 and conferences.31-35 Terphenyls and chlorinated terphenyls are used as reactor coolants. The terphenyls consist of mixtures of ortho-, meta-, and paraterphenyls. Chlorinated terphenyls are produced by Monsanto and termed Arodor, followed by numbers. The first 2 digits of these numbers are 54 and the last 2 indicate the percent of chlorine such as Arodor 5460. Arodor series 2S and 44 consist of mixtures of polychlorinated biphenyls and polychlorinated terphenyls.36 According to Zitko et al.,37 poly chlorinated terphenyls have been found in environ mental samples. Polychlorinated terphenyls do not elute from GLC columns under conditions used for PCBs,3* which may explain why they have only been detected occasionally, but the way they are used probably also predudes their extensive spread in the environment. Chlorinated dibenzodioxins and chlorinated dibenzofumns may occur as contaminants of cyclic chlorinated hydrocarbons. In 1959 Tomita et al.39 reported on the synthesis of polyhalodibenzo-p-dioxins. According to these authors, chlorophenols, when heated to about 200C or more under alkaline conditions and pressure, will form chlorodibenzodioxins, as shown below. ON* a. + Clw a. Cl N* O' +2N*a Under similar conditions a chlorodibenzofuran may also be formed. .OH H. a X <V a NiO a. H ,0 + NC1 ay Chlorinated dibenzodioxins and chlorinated dibenzofurans are formed, or can be formed, as contam inants in the manufacture of a variety of products which use chlorophenols and chlorobenzenes as starting materials, such as 2,4,5-T, pentachlorophenol, nitrofen, hexa- and pentachlorobenzene, and tri- and tetrachlorophenoi. Polychlorinated biphenyls may also be contaminated with chlor inated dibcnzofurans.40,41 and chlorinated $benzofurans may result from polychlorinated biphenyls under UV irradiation.43 The toxicity of these products varies depending on the position and number of chlorines attached to the phenyl rings. Products from different manufacturers may show a great deal of difference in the type and amount of contamination and different lots of the same product may vary in composition, making toxicological evaluations of these products very difficult. Rigorous production control and cleanup procedures need to be implemented so that the composition of different lots manufactured by the same company becomes more uniform. Villanueva et al.43 and Jensen and Renberg44 reported the presence of chlorinated dibenzo-pdioxin in pentachlorophenol. Hydroxynonachlorodiphenyl ether was also found and referred to as " predioxin" under the assumption that it represented a precursor of dioxin. COOGioS 6*13 V * >; i According to Johnson et al.,45 the predom presence of dioxins in fish -taken from four inant dioxin in a commercial pentachloropheno! locations in Vietnam in Military Region III, which was octachlorodibenzodioxin while trace amounts includes Saigon and several provinces to the i'- 00 of hexachlorodibenzodioxin and no tetrachlorodibenzodioxin were found. Since the technical north.54 The concentrations ranged from 18 to 814 ppt. in CO pentachlorophenol was in some respects more Zitko55 was not able to demonstrate chlor toxic in animal studies, than the pure product, the inated dibenzodioxins and dibenzofurans in production process of pentachlorophenol was aquatic animals from various locations in Canada. changed to minimize the dioxin concentration and Chlorinated naphthalenes may so far have a new product has now been registered under the remained unidentified since they interfere with Federal Insecticide Fungicide and Rodentidde Act other chlorinated compounds in any multiresidue with a lower dioxin content. analysis and are detected only when mass spec Technical 2,4,5-T has also been found to be trometry is also performed.54 contaminated with a dioxin. In this instance, The various germicides given in Figure 2 have unfortunately, the very toxic 2,3,7,8 tetrachloro- been used as antiseptics, deodorants, and particu dibenzodioxin,44-4* was present. The contami larly, as far as hexachlorophene is concerned, also nation of the 2,4,5-T with 2,3,7,8-tetrachlorodi- as preservatives for various cosmetics, soaps, and benzodioxin ranged from.< 1 ppm to as much as lotions. A listing of different products that 32 ppm over the years. In newly manufactured contained hexachlorophene prior to the new regu 2.4.5- T in the U.S. the content of tetrachlorodi- lations was given by the American Pharmaceutical benzo-p-dioxin has been substantially reduced and Association.57 Registration of bithionol was is usually kept below 0.1 ppm. Whether other cancelled in the U.S.5* because it produced dioxins or furans have ever been detected in photosensitivity reactions in humans using soaps 2.4.5- T in the U.S. has not been reported in the that contained this product. None of the poly literature. chlorinated polycyclic compounds used as germi Chlorinated dioxins have also been found in cides have been shown to accumulate in the so-called " toxic fat," which produces chick edema environment. Hexachlorophene has additional uses disease when fed to chickens. After several years as a fungicide on ornamental plants, on conveyor of study by industry and the Food and Drug chains as a mildewstat, in laundry rinses as a Administration, Flick et al.49 were able to mildewstat, in shoes and air filters and assorted separate a purified crystalline product which industrial uses as a fungistat, and on leather, paper, produced chick edema disease. Two years later, and textiles.59 It is also registered as a pesticide Cantrell et al.s0 announced that 1,2,3,'7,8,9- for use on cucumbers, peppers, and tomatoes,40 chlorodibenzo-p-dioxin was one of the toxic and for cotton. compounds that produced chick edema. The origin Dichbrophene is used as a mildew preventive o f the chlorinated dibenzodioxin in " toxic fat" on cotton and is recommended as a fungicide and was not clear. It was suspected that it arose from bactericide for the protection of textiles and chlorophenols which were used to preserve hides materials from molds.41 for the manufacture of leather. Tallows and greases were obtained from these hides54 and a CHEMISTRY: METHODS FOR more recent outbreak of chick edema disease was CHEMICAL DETERMINATION traced to the contamination with chlorophenols of AND PHOTOLYSIS soapstock used as feed fats. Higginbotham et al.53 showed that chlorophenols and their salts, when Much of the chemistry of polychlorinated heated, undergo condensation reactions and form biphenyls has recently been reviewed.33-34 chlorinated derivatives of dibenzo-p-dioxin; Commercial chlorinated biphenyls are mixtures of furthermore, chlorinated dioxins and predioxins biphenyl groups with different numbers of were originally present in some of the chlorinated chlorine atoms (Figure 1). When hydrogen atoms phenols. Generally no efforts have been made to are replaced by chlorine on the biphenyl ring, a determine whether various chlorinated dioxins and large number o f substitution combinations can fitrans are present in the environment. However. arise since the number o f chlorine atoms on the Baughman and Mcselson53 recently reported the molecules can vary and the chlorines can also be 450 CXC Critical Reviews in Toxicology CG0G133 * 6 9 1 4  *. . DOW ^ '135872 -attached to the molecule in different positions, resulting in a variety of isomers. Even if the chlorination may be the same, different commer cial batches may vary in the relative concentration of isomers. The PCBs produced in the U.S. by Monsanto fall under the trade name Aroclor. The various Aroclor mixtures have a great number of constituents, and when the biphenyl is chlori nated, 210 different chemicals are theoretically possible. Aroclor 12S4, for instance, yielded 69 constituents while the fractionation of Aroclor 1242 and 1260 yielded 45 and 78 components, respectively.34 The chlorinated biphenyls (PCBs) are chemi cally very inert, resistant to corrosive chemicals, insoluble in water, and have a low vapor pressure. Aroclor 1221, for instance, has a boiling point of 278C. The boiling point increases with increasing chlorination. The chlorinated biphenyls are deter mined in biological and environmental samples by a combination of electron capture gas liquid chromatography and mass spectrometry. Nuclear magnetic resonance spectroscopy is used in addition to the above two procedures in some laboratories. PCBs were first identified in environ mental samples by Jensen in 1966*1 They were detected as interfering peaks on GLC analysis of environmental samples that were analyzed for chlorinated pesticide residues.43 The PCBs interfered with routine pesticide analyses for chlorinated hydrocarbons. Various authors have therefore published separation methods64 and Reynolds31 discussed this exten sively in a review. According to Safe and Hutzinger,63 the use of mass spectrometry for structural studies of PCBs is limi ed because the chlorine atoms may be ran domized between the phenyl groups when the PCBs are fragmented by electrons. The only , exception to this is 2,2'-dichlorobiphenyl. The quantitation of polychlorinated biphenyl in environmental samples has met with difficulty since the PCBs represent mixtures. Several workers have compared PCB components in field samples to Aroclor 1254 because the sample chroma tograms were most similar to these mixtures.46-6* Rote and Murphy63 quantitated individual peaks of different Aroclors by means of a response curve. The semilogarithmic relationship of detector response (total peak area/16 ng) to average chlorine content was obtained for each Aroclor by the method of least squares. From this relationship the theoretical response of the detector to each chlorinated biphenyl was calcu lated. When this approach was used, the levels of PCBs were lower than when the PCB levels were compared to' the Aroclor 1254 standard. Because of these difficulties in quantitation, the PCB levels reported from different laboratories may vary greatly. The various problems encountered in the analytical methodology of polychlorinated biphenyls and their quantitation are also described in a U.S. FDA report which can be obtained upon request from the U.S. Food and Drug Adminis tration in Rockville, Md. The title of the report is Analytical Methodology for Polychlorinated Biphenyls (Feb. 1973). Polychlorinated terphenyls (Figure 1) are not eluted from gas chromatography columns under conditions used for polychlorinated biphenyls.31 By altering their gas chromatograph column, Zitko et al.37 were able to detect polychlorinated terphenyls. Chlorinated naphthalenes (Figure 1) elute at the same time and cause interference with the determination of chlorinated biphenyls and chlori nated hydrocarbon pesticides in general, unless mass spectrometry is performed to establish the identity of the various chemicals involved.56 The methods presently available for the deter mination of trichiorocarbanilide and other germi cides (Figure 2) are not very well developed. Graber et al.69 outlined a thin layer chroma tography method for the determination of these chemicals in soap and very briefly mentioned other methods that have been employed in the past. Several methods for the determination of chlor inated dibenzodioxins and chlorinated dibenzofurans have been described. The goal of these methods is two-fold. First of all, quite a number of technical polychlorinated organic compounds may contain one or several of these compounds70-73 as contaminants and methods have been developed to determine them in various technical products. Other methods dealt primarily with toxic fats where many attempts were made to isolate and identify the compounds responsible for chick edema disease.73 Some of these methods were described in the papers that will be cited in connection with the description of chick edema disease in this article. When preparing the various chlorinated diben zodioxins and dibenzofurans for.toxicity studies in CG06150 January 1974 DOW 1135873 order to obtain pure products, methods also need to be available for their separation and identifi cation.74 One of the problems with tetrachlorodibenzodioxin as well as tetrachlorodibenzofuran is their severe toxicity in the microgram range which will probably not result in an accumulation within the ppm in biological tissues, for instance, for residue analysis. Baughman and Meselson43 have recently described a method for the determination of these materials, within the ppt range. Hexachbrophene is made from trichlorophenol. A process for its production was patented by Gump.7S Additional methods for the produc tion and purification for hexachlorophene have been published and are dted in Chemical Abstracts. Recently several methods74* 7* have been developed for the determination of small amounts of hexachlorophene in blood and biological tissues. Some methods were based on extraction of hexachlorophene with ether or ethanol or a mixture of ether and ethanol. An acetyl or methyl derivative was formed and the hexachlorophene was determined with electron capture gas chroma tography.79 In an earlier method described by Bachmann and Shetlar,*0 simple benzene extraction was used and no derivative of hexa chlorophene was made, which led to very variable results. The various advantages and disadvantages of these methods were discussed by Ulsamer.79 In earlier work my co-workers used ether for extraction and prepared a dimethyl ether deriv ative of hexachlorophene.*1 Recovery with this method was only about 75% and we have recently altered our method of extraction to obtain better recovery.*3 A method developed by Kabacoff et al.7* for the determination o f hexachlorophene is, according to the authors, also suitable for deter mination of dichlorophene and Gutenmann-and Lisk*3 have described a method for the determin ation of hexachlorophene in several agricultural products. The production of hexachlorophene from trichlorophenoi proceeds at low temperature and under acidic conditions which makes the produc tion of a chlorinated dibenzodioxin as an impurity highly unlikely. Since chemicals that are found in the environ ment are exposed to sunlight, the effect of UV light on the compounds under discussion has been given considerable attention. Hustert and Korte34 synthetized a few PCB isomers, namely, 2 ,4 ,6,2',4f,6'-hexachlorobiphenyl, 2,4,5,2',4',6'hexachlorobiphenyl, 2,4,2',4'-tetrachlorobiphenyl, 2t5,2',5,-ietrachlorobiphenyl, and irradiated the various, isomers with UV light. A mercury vapor lamp with quartz filters was used as a light source. The UV spectrum of the synthetized chlorinated biphenyls only shows significant absorption below 300 nm. When these compounds were irradiated in a solvent such as hexane, acetone, methanol, or a mixture of methanol and water, dechlorination and polymerization of the compounds was observed. When the PCB isomers were irradiated in perfluorinated dimethylcydohexane, an inert medium, isomerization, and chlorination dechlor ination were observed. These experiments indicate that, under environmental conditions, photolysis products may result with a lower chlorine content but small amounts of higher chlorinated biphenyls may also occur in low concentrations. Safe and Hutzinger*5 reported that the irradiation of 2,4,6,2' ,4',6'-hexachlorobiphenyl at 310 nm resulted in compounds of lower chlorine content, such as di-, tri-, tetra-. and pentachlorobiphenyls. In these investigations hexane and methanol were used as solvents. It is possible that new compounds are formed by loss of chlorine. Rearrangement and condensation may lead to chlorobiphenyls in environmental samples that are not present in commercial PCB mixtures. The 24-hr irradiation of 3,3',4,4'-tetrachlorobiphenyl, 2,2',6,6'tetrachlorobiphenyl, 2,2' ,5,5*-tetrachlorobiphenyL dissolved in hexane resulted in about 70% loss of the starting material, while with 2,2\4,4',5,5'hexachlorobiphenyl, only 3.8% remained and less than 1% of 2,2',3,3',4,4',5,5'-octadilorobiphenyl remained. The irradiation of Arodor 1254 in hexane resulted in a change of the composition of the Arodor mixture. The gas chromatogram obtained from the irradiated Arodor showed shorter retention times than the standard Arodor 1254. The irradiation of Aroclor 1254 in a dioxane water mixture also containing sodium bicarbonate did not lead to the formation of chlorodibenzofurans or hydroxychlorobiphenyls. The compounds that were observed corresponded to PCB molecules to which water was added and a more polar " carboxylic" acid fraction. Irradiation of Aroclor as a thin film with a frequent addition of water led to newly formed hydroxylated compounds and a more polar " carboxylic" fraction. The results obtained from irradiating 452 CRC Critical Review* in Toxicology <r-^ r' oxtu. 6816 D O W , I i 35874 PCBs therefore depended greatly on the solvents used and the pH of the solution in addition to the light source used and may result in dechlorination, formation of polymers, hydroxylation, and carboxylic products.86 Exposure of various chlorinated dibenzodioxins to summer sunlight or fluorescent UV light with an intensity of roughly 100 fiw/cm2 in the presence of organic solvents such as methanol led to rapid photolysis of 2,3,7,8-tetrachloro- and 2 ,7 di c h Io r o d i b e n zo d io x in , while 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin produced a series of chlorinated dioxins with uniformly decreasing chlorine content. Some evi dence was also found that reactions other than reductive dechlorination occurred.87 When 2.3.7.8- tetrachiorodibenzodioxin was spread on a glass plate in a methanol solution and the meth anol was allowed to dry, quantitative recovery of the dioxin was possible even after 14 days of UV irradiation. Similar results were obtained when 2.3.7.8- tetrachlorodibenzodioxin was dissolved in methanol, put on a glass plate, covered with soil, and irradiated.87 These results demonstrated that under favorable environmental conditions, when organic hydrogen donors are present, photolysis will take place. Bare surfaces of soil, concrete, or water are not effective inducers of photolysis. Chlorinated dibenzodioxins could at times prove to be quite persistent. Partial dechlorination may also occur with chlorinated diphenyls or the higher chlorinated dibenzodioxins and may result in less chlorinated compounds. The possible long lasting presence of the dioxins is also emphasized by Kearney et al.,88 who recovered 56 and 63% of originally applied tetrachlorodibenzodioxin after one year in Hagerstown and Lakeland, Md. soils. T etrachlorosalicylanilide and similar compounds split off chlorine atoms when exposed to sunlight. This is briefly mentioned in connec tion with photosensitivity reactions of the skin. Shaffer et al.89 irradiated hexachlorophene in absolute ethanol with UV light and obtained a number of dechlorinaled bisphenols resulting from loss of chlorine in the ortho and para positions relative to the hydroxy group. GENERAL TOXICITY AND EFFECT ON REPRODUCTION Diphcnv! itself, without any chlorine atoms, is a fungistatic agent which is used as a preservative for citrus fruits, and is usually impregnated into the wrapping paper. The protected citrus fruit contains a certain amount of biphenyl residue. In different countries the amount allowed as a residue on citrus fruit varies between 70 to 110 ppm of biphenyl. Booth et al.90 found a reversible nephrotoxic effect of biphenyl on the rat kidney when rats were fed 0.5% or 1% (10,000 ppm biphenyl in the diet). None of these effects was observed on diets containing 0.1% (1000 ppm biphenyl). Deichmann et al.9> showed that pro longed exposure tc air containing diphenyl in high concentrations of 5,40, and 300 mg/m3 produced liver and kidney injury as well as broncho pulmonary lesions in mice and rats. In general, diphenyl has always been considered to be relatively nontoxic. In 1969, in Finland, a man who had been intensively exposed to diphenyl for 11 years became ill and died.92 Diphenyl impreg nated wrapping paper had been manufactured in Finland for about 15 years in the plant where the man was employed. The equipment used in the impregnating process was cleaned 5 to 10 times a year with tri- or tetrachloroethylene. The cause of death of the worker in this particular report was not satisfactorily explained and neither were the complaints of 31 other workers engaged in the same factory. Since chlorinated ethylene com pounds were used for cleaning, it should be established whether organic solvents containing halogens could lead to the production of toxic substances when they are brought in contact with diphenyls. If this proves to be the case, then care should be taken that this situation is avoided. The PCBs are also not very toxic when given as a single or a few repeated doses to birds and mammals. However, the juvenile pink shrimp is very sensitive to PCBs in water, while trout and catfish can tolerate higher doses; insects are also not very sensitive to PCBs. Pin fish are not quite as sensitive to a single dose, but will die when exposed for 14 to 45 days to 5 ppb of Aroclor 1254.3 PCBs are of a low order of toxicity to rats and rabbits when given as a single dose. The acute oral toxicity decreases in rats with increasing chlorine content, while this is not as obvious when the dermal toxicity is tested in rabbits.33 This may be due to poorer absorption of the higher chlorinated compounds. In studies with adult Sherman strain rats, we found that the oral LD*o for Aroclor 1254 and 1260 was somewhere in the January 1974 453 J range of 4 to 10 g. These two compounds proved to be more toxic in weanling rots. The single oral LDjo for weanlings was 1295 mg/kg, and 1315 l O mg/kg for Arodor 1254 and 1260, respectively, 5^" and the lowest lethal doses were 1000 mg/kg for ! Arodor 1254, and 1200 mg/kg for Arocior 1260. The intravenous LDf0 for Arodor 1254 in adult ` | female rats was 358 mg/kg.*4 ^ A number of shortterm studies have been reported where the PCBs were given to animals over a period of one or two weeks. When the PCBs C 3 were given in this fashion they were somewhat more toxic than when they were given in a single dose. These studies are reviewed in great detail by Nelson et al.33 and will not be reported here since they do not give a great deal of additional information. Vos and Koeman** studied the toxicity of three PCB preparations in chickens, Phenodor DB6, a French product, Gophen A60, a German product, and Arocior 1260, a product from Monsanto in the U.S. The birds were fed 400 ppm of PCBs in their diets for 60 days; the Phenodor and Gophen were much more toxic than Arocior. The birds fed the two European samples died between 12 and 60 days after onset of exposure. The findings at autopsy consisted of hydro* pericardium, ascites, subcutaneous edema, and liver necrosis. Only 3 of 20 birds fed Arocior 1260 died during this time with hydropericardium. None o f the Arodor fed birds showed liver necrosis. With the help of mass spectrometric and micro* coulometric analyses, the authors*5 determined that the two European samples were contaminated v with tetra- and pentachlorodibenzofurans. As an additional contaminant, chlorinated naphthalenes were found. Dermal toxidty studies with the three com pounds, Clophen. Phenodor, and Arocior 1260, in rabbits*4 after two days led to reddening of the skin where the materials were applied. The skin reaction was more pronounced after one week, desquamation o f the external epidermal layers of the skin was observed, the hair grew at a reduced rate, the skin became thickened, and prominent transverse wrinkles developed. The test animals showed a gradual loss of weight, and one of the rabbits given Phenodor and three of those given Gophen died. All but one rabbit showed fluores cence o f the liver under UV light, and microscopic 454 CRC Critical Revieh i in Toxicology changes were also observed in the liven and the treated skin. Vos and Notenboom-Ram*7 compared the dermal toxicity of the isomer 2,4,5,2',4',5'hexachlorobiphenyl with that of a PCB mixture (Arodor 1260) in rabbits. Their dermal application resulted in early microscopic skin lesions in the Arodor group. The lesions in the 2,4,5,2'.4',5'hexachlorobipheny! group appeared later and were leu severe. Both experimental groups showed liver changes. The liver damage was more pronounced in the animals that had been exposed to hexachlorobiphenyl tha,i in those that had been exposed to the Arodor. In inhalation studies with Arocior 1242 and 1254, rats, mice, rabbits, guinea pigs, and. one cat were exposed to PCB vapors five days a week for several weeks. The concentration of Arocior 1242 vapor was 1.90 to 8.63 pg/1 and no ill effects were observed. The exposure of animals to the vapor of Arocior 1254 in the concentration of 5.4 or 1.5 jig/1 produced enlarged liven in rats. An inter current respiratory infection in some control as well u experimental rats made it difficult to interpret the results.** Since it is now known that the chronic toxidty of PCBs is a more important parameter in establishing their effects than short term exposures, further studies with lower levels over longer periods of time would give more meaningful information. Fortunately, most of the compounds under* discusin have a low vapor presure so that exposure by inhalation probably does not present a great problem. A number of reproduction studies in various spedes have been conducted with PCBs; usually, embryo toxicity was observed but there were no malformations. Heath et al.*' were unable to demonstrate an effect on reproduction with diet ary levels of 25 and 50 ppm of Arocior 1254 in mallards and bobwhites. An increase in eggshell cracking or reduction in shell thicknes was also not observed while DDE, which was Used as a positive control, induced significant shell thinning and cracking and a reduction in hatching successes in mallards but not in bobwhite. In field studies with brown pelican shells, it was found that thinning could Be best correlated to DDE residues rather than dieldrin or PCBs. This finding indicates, according to Blus et al.,100 the PCBs are not responsible for eggshell thinning in most birds following environmental exposure. es isPcakall101 fed ringdoves 10 ppm Arodor 1254 Cg 0 6 6 3 for 6 months. He injected others with 160 mg/kg intraperitoneally for 4 days, and evaluated the ashed eggshell weights of these birds. He found no difference in eggshell thickness between the experimental and the control groups. In a pheasant reproduction study with Aroclor 1254,'02,103 a significantly greater difference in the number of eggs that were pipped but not hatched was found in the group of hens that received 50 mg of PCBs weekly. Egg production and hatchability was lower in this group, the survival of chickens that did hatch at six weeks of age was reduced, and the ones that did survive were significantly lighter than the controls. Aroclor 1242 at 10 ppm or 100 ppm and Aroclor 1254 at 100 ppm in the diets of chickens did reduce egg production and hatchability and caused thin eggshells, while Aroclor 1242 at 1 ppm, Aroclor 1254 at 10 ppm, and Aroclor 1260 at 100 ppm did not affect reproduction in chickens.104 In a rat reproduction study, decreas ed survival of pups at a dietary level of 100 ppm of either Aroclor 1242 o r 1254 was observed. The results obtained from the reproduction studies in birds particularly seem to indicate that the lower chlorinated biphenyls affect reproduction more than the higher chlorinated biphenyls, and it is very important to determine whether the lower chlorinated biphenyls that have been suggested as replacements for presently employed chlorinated biphenyls have an effect on reproduction. In another rat reproduction study94 the lowest dose of Aroclor 1254 that affected reproduction was 20 ppm (1.5 mg/kg/day) which manifested itself in a decreased number of litters and less pups per litter. Neither 1 ppm nor 5 ppm of Aroclor 1254 had an effect on reproduction in the Sherman strain rat; however, an exposure of the dams to the Arodors at 5 ppm or higher increased the liver-to-body weight ratios in weanling rats of both sexes. At 1 ppm the increase in liver weight was observed only in Fla and Fib weanling male rats. The dietary levels of 100. 20, and 5 ppm of Aroclor 1260 did not affect reproduction; how ever, the liver-to-body weight ratio of 21-day-old pups was increased at ail exposure levels. Mink are highly susceptible to the toxic effects of PCBs. A daily dietary intake of 30 ppm resulted in death in about 6 months. Exposure to 5 ppm Aroclor 1254 in the daily diet severely affected reproduction.103 Unfortunately, it is often very difficult to compare the various dietary levels that affect different species, since the food consumption may vary decisively from one animal species to another and the daily food intake in g/kg body wt. is often not included in the scientific report. Some mice strains, for instance, will be exposed to as much of a given substance that is present in the diet at the concentration of 300 ppm as rats on a diet containing 600 ppm or more. With some species it is of course difficult or almost impossible to - GO * Cl CO measure the food consumption, but a greater effort in establishing these very basic facts would C3 be helpful for the interpretation of toxic effects in different species. Not very much is known at present about the toxicity of terphenyls or chlorinated terphenyls. These materials have not been studied to any great extent. The few studies that are available only describe very specific effects such as microsomal enzyme induction in the liver and give no overall evaluation of their toxicity. The most toxic of the various chlorinated dibenzodioxins and dibenzofurans is 2,3,7,8-tetra- chlorodibenzodioxin which, in the German litera ture, is sometimes referred to as 2,3,6,7-tetrachlor- odibenzodioxin. Single oral LD]0 values are female rats: 44.7 Mg/kg; male rats: 22.5 Mg/kg; male guinea pigs: 0.72 Mg/kg: rabbits: < 30 Mg/kg; and dogs: between 30 and 300 Mg/kg.106 It is possible that young animals are more susceptible to the toxic effects of this compound than older animals and LDso values should therefore also be determined in weanling animals. One of the characteristics of this compound was that it caused delayed death, sometimes as delayed as 40 days after a single dose. During the period of intoxica tion, the animals lost a great deal of weight.106 On the other hand, 2.0 g/kg of dichloro- dibenzo-p-dioxin and 4.0 g/kg body weight octa- chlorodibenzo-p-dioxin were not lethal to rats. Hexa-, hepta-. and trichlorodibenzo-p-dioxin were more toxic.104 Schulz107 in earlier experiments found that oral doses of 20 to 50 Mg/kg/body weight of 2,3,7,8-tetrachlorodibenzodioxin resulted in fatal liver necrosis in rabbits. More recently Milnes10* reported that 10 Mg/kg was lethal'to rabbits. The 2,3,7,8-tetrachlorodibenzodioxin has a variety of specific toxic effects. Whether these effects vary in different species is presently not well defined. With the interest in this and the other related compounds, more information on January 1974 455 DOW 1135877 their toxicity probably will toon become available. The problems inherent in studying these com pounds are the unavailability of some of them, the fact that they may not be completely pure, particularly the hex- and hepta- preparations, and of course with respect to the tetrachlorodibenzodioxin, its extreme toxicity, which makes it difficult to handle. In contrast to PCBs, teratogenic as well as fetotoxic effects have been reported for 2,3,7,8tetrachlorodibenzodioxin. Sparschu et al.10' gave 0. 0.03, 0.125, 0.5, 2.0, and 8.0 Mg/kg/day of 2,3,7,8-tetrachlorodibenzo-p-dioxin to pregnant rats on days 6 to 15 of pregnancy. With the 0.03 Mg/kg dosage level, an effect on the fetus was not observed, but doses from 0.125 Mg/kg on up resulted.in fetal mortality, early and late resorp tions, and fetal intestinal hemorrhage. Khera and Ruddick110 gave doses rangihg from 16 to 0.125 Mg/kg to Wistar rats on days 6 to 15 of pregnancy. These authors also observed pronounced fetotoxicity at all dosage levels except 0.125 Mg/kg- The affected fetuses showed cerebral and intestinal hemorrhage as well as subcutaneous edema at autopsy. The offspring of mothers given 0.5 and 1 Mg/kg of the tetrachlorodibenzo-p-dioxin gained less weight during their suckling period and their survival to weaning was reduced. This could have been caused by the intrauterine effect of the dioxin or by excretion of the dioxin in the milk of the dams given the material during pregnancy. Courtney and Moore'1' observed cleft palates in 3 strains of mice given 3 pg/kg TCDD subcutane ously from days 6 to 15 of pregnancy and an increased incidence of kidney anomalies. Hydronephrotic kidneys were also produced in mouse pups that were nursed by dams treated with TCDD during pregnancy or at time of parturition.'' 3 The 2 ,3 ,7 ,8-tetrachlorodibenzo-p-dioxin is extremely toxic in the chick embryo assay.33 In the rat, hexachlurodibenzodioxin caused fetotoxic effects at 1 and 10 pg/kg and teratogenic effects at doses, of 100 pg/kg.'06 Harris et al." 3 gave female rats daily doses, of 0.1, 1, or 10 pg/kg TCDD for 3 1 days. Rats given 10 pg/kg lost weight and most of them were moribund within 3 weeks. Weight gain was also less at the I pg/kg dietary level. The rats receiving 0.1 pg/kg -were not affected. In experiments with guinea pigs. 9 of 10 animals died after a single dose of 3 pg/kg TCDD. while guinea pigs receiving a single dose of 1 pg/kg gained less weight. All guinea pigs receiving I pg/kg/week died within 24 to 32 days and a weekly dose of 0.2 pg/kg depressed their weight. Of the various animal species tested, the guinea pig teemed to be most susceptible to the toxic effects of TCDD. Reduced thymus and spleen weights were observed in rats that received a single dose of 25 Pg/kg TCDD. The rats that died at the various dosage levels showed massive hemorrhages into the heart, liver, brain, adrenal glands, and gastrointesti nal tract. Necrosis and ulceration of the glandular part of the stomach were observed. Occasionally organized thrombi were noted and the liver reveal ed necrosis of parenchymal cells. Sublethal doses of TCDD produced microscopic changes in liver, kidneys, and thyroid. In the kidneys the collecting tubules were primarily affected. Doses o f 0.1 Hgfkg/day for 31 days, a single dose of 5 pg/kg, and multiple weekly doses of 1.0 and 0.2 pg/kg did not produce changes in rats discernible with the light microscope. Toxicity studies were conducted in our labora tory with dichlorophene} ' 4 The oral LD$o for adult female rats when the material was given in peanut oil by stomach tube was 1,660 mg/kg and in males it was 1,500 mg/kg. The only signs of toxicity observed in these rats were diarrhea and some depression. When 200 mg/kg was applied daily in propylene glycol to the skin of a clipped area of the back of male adult rats as a 12.5% solution, local irritation of the skin with some ulceration resulted but there was no systemic toxicity. The acute intravenous LDso in adult male rats was 16.8 mg/kg when it was given in a saline lecithin suspension. In a two generation rat reproduction study, a dietary level as high as 1,000 ppm (50 mg/kg/day) had no effect. Gross and microscopic examination of the various organs including the brains of the parents as well as the offspring did not reveal any morphological changes that could be related to dichlorophene exposure. These findings indicated that dichlorophene was not very toxic to rats. The great difference between the oral and the intravenous LDS0 suggested either poor absorption or rapid break down of the materia] in the gastrointestinal tract. Martins6' lists the acute L D j0 for guinea pigs as 1,250 mg/kg and for dogs,. 2,000 mg/kg. Rats fed for 90 days on a diet containing-2,000 ppm showed no evidence of toxicity. The acute toxicity of hexachlorophene is much greater and varies in different animal species and is 456 CRC Critical Reviewi in Toxicology CCG65.GS DOW U35878 also dependent on the route of administration and the solvent in which it is given. Gump115 lists the acute oral LD50 in mice as 168 mg/kg and cites LDSo values reported by other investigators rang ing from 80 mg/kg to 215 mg/kg. Dogs and sheep were more susceptible to the toxic effects of hexachlorophene. The interest in the effect of hexachlorophene on sheep in particular stemmed from the fact that it is effective against liver flukes116 and advocated for its anthelmintic properties. This even led to the treatment of children with orally administered hexachlorophene in China.117 Intravenously administered hexa chlorophene in rats resulted in an LDSo value of 9.1 mg/kg and in rabbits of 8.5 mg/kg. More recently we found that the oral LDjo (single dose) for adult male rats was 66 mg/kg and for adult female rats 56 mg/kg; for weanlings it was 120 mg/kg when the material was given in peanut oil by stomach tube to Sherman strain rats. The acute intravenous LDS0 was 7.5 mg/kg in adult male rats.118 Nakaue et al.119 reported similar oral LDS0 values in Wistar rats. The single oral LDjo for sheep and cattle lies between 30 and 60 mg/kg.130 Single dermal LDS0 values have not been reported in the literature. We found in rats118 that single applications of 600 mg/kg hexachloro phene in 95% ethyl alcohol solution caused death in 1 of 10 rats. Dermal application of 24 and 48 mg/kg/day for 30 days in propylene glycol or in a detergent as a 3% solution caused ulceration of the skin and affected the white matter of the central' nervous system in at least half of the rats. A dose of 12 mg/kg/day as a 1.5% solution caused only mild erythema and desquamation of the skin of rats. No effect on the central nervous system was observed. Whether a single dose given in either propylene glycol or a detergent would be more toxic than one given in ethyl alcohol was not established. The signs of acute hexachlorophene poisoning following a single oral dose varied with the age of the rats.118 Adult rats suffered from severe depression and diarrhea, but showed few signs of leg weakness. Weanlings, on the other hand, developed posterior leg weakness as well as de pression. The leg weakness became more.apparent in the adults when they were given repeated doses of hexachlorophene. Death was usually preceded by convulsions in rats dosed intravenously. In rats killed with hexachlorophene, rigor mortis -developed very rapidly and was very pronounced. Body temperature elevation following hexachloro phene poisoning has also been recorded in rats.119 Both changes, the pronounced rigor mortis and the temperature elevation, may be related to the fact that hexachlorophene uncouples oxidative phosphoiylation. Additional symptoms of hexachloro phene poisoning particularly in other species are discussed under neurotoxicity. Nakaue et al.119 fed 400 ppm hexachloro phene (28.9 mg/kg/day) to Wistar rats. They developed leg weakness within 5 days, diarrhea, emaciation, and they died in 7 to 10 days. No deaths occurred in the rats that were fed 200 ppm (about 13 mg/kg/day) for 16 weeks. In a two generation rat reproduction study which we conducted in our laboratory, we found a reduced survival in the offspring of the F, generation fed 100 ppm (11.8 to 5.5 mg/kg/day) in their diet for 54 and 166 days. This was statistically significant in the second breeding of the F t generation. In the second generation the litters were slightly smaller, but the survival to weaning was not significantly reduced.118 A dietary level of 20 ppm (2.3 to 1.1 mg/kg) did not influence reproduction. Thorpe131 reported that the oral administration of hexachlorophene to male rats as well as single doses of 25 and 50 mg/kg hexachlorophene to sheep produced de generation of spermatogenic cells; multinucleated cells were observed within the tubules of the testes. The doses given by Thorpe were higher than the ones we used in the reproduction study, and microscopic examination of the testes of the rats used in our reproduction study did not reveal any abnormalities. Toxic doses of hexachlorophene given to the dam during pregnancy may result in malformations of the offspring. Thus far, two studies have been reported in the literature; both studies were conducted in rats.133'133 It should be determined whether malformations can also be produced in other species and with lower doses before any conclusion can be drawn from these findings. A number of human poisoning cases have been reported in the literature and some of these were fatal. Most of the poisoning cases in humans have either been caused by accidental ingestion, because a hexachlorophene detergent - solution was mistaken for milk of magnesia, or by the applica tion of hexachlorophene to large areas of burned or otherwise damaged skin.115,134 Larson3 was January 1974 4S7 CGOGiSG ' 8S21 the first to recognize that burned patients observed in these patients were mainly confined to developed convulsions and other toxic symptoms the central nervous system and to the gastro after repeated exposure to hexachlorophene which intestinal tract. The group that received 3 doses of was applied to burned areas of the skin. The 20 mg/kg/day had the most severe reaction. One incidence of convulsions in burned patients patient in this group became comatose on the DOW '1135879 decreased when the use of hexachlorophene was fourth day with loss of light reflex and positive discontinued.125 pyramidal signs. Fundus examination of the eyes Eleven cases have been reported124,127 in revealed mild papillary edema. A low grade fever which a single dose of 3% hexachlorophene deter was also observed. Complete recovery occurred gent solution had been taken orally. Gastro within three days. One half to two thirds of the intestinal symptoms including nausea and vomiting patients had diarrhea within 1 to 2 hr after were observed in ten of these cases; the eleventh medication and in some cases a marked depressive case, a child who drank about 250 mg/kg hexa effect on the central nervous system was also chlorophene, became comatose -and died. One observed. An additional case of poisoning after additional death occurred in this group, however, oral ingestion was reported by Korlof and this was most likely due to other causes. A Winsten12' in Scandinavia and two fairly recent 17-day-old infant was accidentally given daily oral cases with fatal outcome occurred in the U.S.130 doses of 37 mg/kg hexachlorophene for 7 days. On Herter131 was probably the first to report a the third day the child started to have loose stools. human poisoning case caused by the dermal On the fourth day of hexachlorophene administra absorption of hexachlorophene. Because of a tion the child developed spasms of the extremities language barrier, the mother of a newborn infant with twitching of the face, sucking movements of applied 3% hexachlorophene as a lotion afteT the the lips, and lateral shaking movements of the infant's bath without rinsing the baby. After four head. It was also noted in the case report that the days, excoriations appeared on the child's face and child refused to take the medicine, there was buttocks, the infant developed convulsions, his drooling and frequent vomiting, and it is not quite face twitched and his extremities jerked. A roving dear whether the child received 37 mg/kg/day. nystagmus was present. This child recovered with The child survived. A week after the hexachloro in 16 days after exposure to hexachlorophene had phene suspension was discontinued, persistent been discontinued. spastic flexion of the fingers, hands, and arms was In addition to the two cases already mentioned still observed. After a month the hands were in which poisoning occurred because of oral slightly flexed at the wrists but were not spastic. ingestion, Mullick130 also reported four cases - Horizontal nystagmus was observed but cleared children who died following the treatment for later. The child was discharged when it was two bums in two instances and the treatment for months and three weeks old. At this point it had severe ichthiosis with a 3% hexachlorophene bath. shown great improvement and when checked at six Larson3 reported six burned patients who months the child was healthy looking.12* Pilapil developed central nervous system symptoms mentioned in his discussion a number of other following the dermal exposure to hexachloro -k unpublished cases, some of which were reported to phene. All six patients recovered. Korlof and the National Gearing House for Poison Control Winsten129 reported two cases of poisoning Centers. Among these was the case of an infant following the dermal application of hexachloro who by mistake got a spoonful of PhisoHex in phene. Both patients were fairly extensively each feeding bottle for approximately two weeks, burned, and developed central and peripheral when it suddenly died. nerve damage as well as some kidney impairment. Chung et al.117 treated a total of 105 patients Lockhart132 mentioned six deaths that were 5 to 15 years old with hexachlorophene for reported to the FDA caused by the topical chlonorchiasis sinensis infestation. Sixty of these application o f hexachlorophene on burned patients received 1 dose of 20 mg/kg, 32 received patients. In all of these cases the skin -was 2 doses of 20 mg/kg/day, 8 patients-received 3 damaged, probably enhancing absorption of hexa daily doses o f 20 mg/kg, and 5 patients received chlorophene, and although skin does represent a 20 mg/kg body weight on the first day and 10 barrier, it is not impermeable - a fact we tend to mg/kg on the second day. The toxicity reactions forget at times. Damaged or abnormal skin may be 458 CRC Critical Reviews in Toxicology CG065.67 RQOO C. /a# 088S8T l MOO m o re perm eable, and the skin o f prem atures, n ew b orn s, and ch ild ren in gen eral m ay have ab sorption ch aracteristics oth er than those o f ad u lt h u m an b ein g s, an o th e r fa c to r th at is o fte n neglected. R ecen tly 1 33,134 41 death s due to h e x a c h lo ro p h e n e p o iso n in g h av e b e en re p o rte d in F ran ce. T h e d eath s occu rred in in fan ts w h o w ere treated w ith a pow d er to w hich 6% h ex ach lo ro phene had been added b y m istake. T h is pow der w as m ainly ap plied in the d iap er area w here the skin b ecam e in flam ed , th is a s w ell a s th e d iap er w hich served as an occlu sive dressin g, p ro b ab ly en h an ced ab so rp tio n . T h ese cases w ill n o t be m ade p u b lic until litigation p ro b lem s have b een settled . L ittle published in fo rm atio n is av ailab le on the to x icity o f th e germ icid es in F igu re 2 asid e from h exach lorophen e and dichloroph en e. T h e acute LD;0o ral fo r triclo san (T C C ) in m ice an d rats is ab ou t 4 ,0 0 0 m g/k g.,3 s The acute subcutan eous to xicity for rats w as 1 4 ,7 0 0 m g/k g and the acu te d erm al to x ic ity w as a b o u t 9 .3 g in ra b b its. C on cen trations o f 1 to 5% solu tio n s cau sed eye irritation in rab b its. D aily o ral d o ses o f T C C up to 1,000 m g/k g/d ay fo r 4 w eeks did not prod u ce an e ffe c t in rats. W hen 2 .5 an d 5% su sp e n sio n s in gu m arabic w ere applied d erm ally to rats fo r 4 w eeks, the anim als did n ot gain as m u ch w eight. Un pu b lish ed m aterial cited in the m in u tes o f th e O T C (O ver the C oun ter D ru gs) Panel listed a " no e ffe c t " level a s 1 7 0 m g /k g /d a y in ra ts a n d fo r a 9 0 -d ay feed in g stu d y in ra b b its, 1 3 8 m g /k g /d ay . In b ab o o n s a d aily oral d o se o f 100 m g /k g /d ay did not produce a toxic reaction. T h e m in utes also m en tion ed som e w ork w here low er d o ses show ed an effect on rats and d ogs. Since so m e o f these results are con flictin g, fu rth er stu d ies are indicated and sh ou ld b e perform ed w ith p ro d u cts used as in g red ien ts in s o a p s : a ch e m ic a l a n a ly sis o f these p ro d u c ts is n ecessary to d eterm in e w h eth er im purities m ight be respon sible fo r varying results. C h loroan ilin e as a result o f h eatin g du rin g m anu factu re cou ld be present as such an im purity. O f the an ilin es, accord in g to H am blin ,1 3 6 on ly p- and m -chloroaniline m ay cau se m eth em oglobin em ia if ab so rb ed th rough th e sk in . T h e o- an d m -ch loro aniline m ay cau se liver and kidn ey d am age. T ri clo san itse lf is also ab so rb ed through the skin an d a b so rp tio n is g re ate r w hen ap p lie d to d am ag ed sk in . SPECIALTOXIC EFFECTS Sk in R eactio n s C h loracn e T h is occu pation al skin disease can be p rodu ced by a n um ber o f chem ical com po u n d s.137 P o ly ch lo rin ated b iph en y ls as w ell as the ch lo rin ated n ap h th alen es, a few isom ers o f the ch lorin ated dib en zod ioxin s, and chlorinated dibenzofuran s are all ab le to p ro d u c e ch lo racn e in a certain p r o p o rtio n o f the h um an p o p u latio n . O ily skin an d large p ores seem to pred isp ose to the disease w hile the o p p o site is true fo r sm o o th , tender skin an d it is p o ssib le th at y o u n g ch ildren m ight be lesssu scep tib le b ecau se o f the n ature o f their skin . C h lo racn e h as also o ccu rred in w ork ers en gaged in the p ro d u ctio n o f 2 ,4 ,5 -T 1 . D uring the in d u s trial p ro d u ctio n o f trich loroph en ol a su dden rise in tem p eratu re w ith in the reactor h as occasion ally resu lted in accid e n ts. In on e in stan ce th is e x o th e rm ic re a c tio n c au se d an e x p lo sio n . ! 04 W hen the rise in tem p eratu re o ccu rred , 2 ,3 ,7 ,8 -tetrach lorodiben zodioxin w as form ed and severe o u t b reak s o f chloracn e w ere observed am o n g the w ork ers.1 3 7 * T h e term chloracne w as first used b y H erxheim er in 1 8 9 9 ,1 3 * w h o th ou gh t it w as cau sed by fre e c h lo rin e g en erated in certain fa c to rie s. W auer in 1 9 1 8 139 an d T cle k y in 1 9 2 7 140 su gg ested the term " P em akran k h eit." A nother term som etim es used is h alogen w axacn e. A s the w ord chloracne im plies, part o f the lesion o f this occu p ation al skin disease resem bles ad o lescen t acn e; how ever, it is generally m ore severe and the distribu tion o f the lesion s is not con sisten t w ith ad olescen t acn e, alth ou gh it m ay be su p erim p o sed on ad o lescen t acn e. C h loracn e is d e scrib ed in the literatu re as co n sistin g o f the fo rm atio n o f co m ed o n es w ith o r w ith ou t c y sts an d p u stu les. T h e follicular o rifices are filled w ith se b a ce o u s an d k eratin o u s m aterial. M elan osis an d a secon dary in flam m atory reaction m ay also ex ist. In a d d itio n to th is d e sc rip tio n , sk in ch an g es o'ccur that have been d iscu ssed less frequ en tly in the m ore recent literature. T h ese skin changes are usu ally referred to as cable rash or cable itch . C ab le rash h as b een o b serv ed in w o rk ers w h o have in su lated cab les w ith various, m ixtu res o f chlorin ated naphthalenes and chlorin ated b iphen yls and in w o rk e rs h an d lin g th ese c a b le s. 141 It h as b een o b serv ed a lso in fa c to rie s th at m a k e c o n d en sers.141,14 3 A ccordin g to B raun .143 a few o f January 1974 459 "nSi68 6923 188SGU MOO the o x s w ith m an ifest ch loracn e started w ith ch an ges in the skin o f the face resem bling p h o to sen sitivity and the bearers o f these lesions su ffered severe p u ritu s in th e areas o f the skin lesio n s. T h is lesion term ed cable rash occurred predom in an tly o n the ch eek s b u t also on the skin o f the arm s. T h e o u tco m e o f cable rash varied; it cou ld either d isap p e ar or it co u ld d evelop in to full-blow n chloracn e. In ad d ition to the pustules and com edon es o f ty p ical ch lo racn e, brow n ish keratin ization o f the sk in w as o b serv ed in so m e p atien ts. B rau n 143 p o in ted o u t that the w orkers, particularly those w ith p o o r h ygien e, tran sm itted the disease to their sp o u ses an d children through direct con tact. M any o f the occu p ation al cases o f chloracne th at h ave been rep o rted in the literatu re have been r e v ie w e d p r e v i o u s l y . 1 3 7 *1 4 4 J o n e s a n d A l d e n 1 45 re p o rte d o n e o f th e first o u tb re ak s o f ch lo racn e in a c o m p a n y th at w as en gaged in the m an u factu re o f ch lo rin ated biph en yls. T h ey described one typical case an d m en tion ed 2 4 m en w orking on the m an u factu re o f ch lorin ated biphenyls; 23 h ad had an acn e fo rm eru p tio n on the face an d b o d y . P articu larly du rin g the 1 9 3 0 s and 194 0 s large o u tb re ak s o f ch lo racn e occu rred in a n u m b er o f fa c to rie s. M u ch d iscu ssio n h as b een d evoted in the literatu re to the p roblem o f w hether the chloracne w as prod u ced by external con tact alone or w h eth er it w as cau sed b y sy stem ic ab so rp tio n , p articu larly the in halation o f vapors. O ne qu estio n ask ed w as w hether internal adm in istration cou ld c a u se c h lo ra c n e , a n d i f s o w h at p art it p la y e d in an y particular case. A s we know now from o u tb reak s o f p oison in g d u e to the co n su m p tio n o f rice oil that w as co n tam in ated w ith p o ly ch lo r in ated b ip h en y ls in Ja p a n (see un der " Y u sh o "), ch lo racn e can be p ro d u ced b y the sy stem ic a b so rp tion o f ch lo rin ated b iph en yls. The experim en ts by S h elley an d K lig m an 146 an d P lew ig147 sh ow ed th at the skin lesio n s cou ld also be produ ced by ap p ly in g the ch loracn cgen ic com po u n d to the skin . A fter occu pation al chloracne m anifested itself, the patien ts also com plained o f system ic effects such as lo ss o f ap p etite, n ausea, edem a o f the face an d h an d s, abdom in al pain , vom iting, and burn in g an d so ren ess o f the ey es. P articu larly w ith the ch lorin ated n aphthalenes, h ep atoto xic effects have also been observed. A p plicatio n o f ch em icals to the skin can lead to their ab sorp tion and cau se 460 CRC Critical Reviews in Toxicology sy ste m ic to x icity ; W hether a sy ste m ic to x ic -effect resu lts w ill depen d on the sk in su rface area covered , the am ount o f the to xic substan ce ap p lied , and the ability o f the skin to absorb it. I f derm al ab so rp tio n is p o o r, a sin gle ap p lic atio n w ill u sually n o t be very toxic. H ow ever, if the co m p o u n d is n o t easily m etab olized an d e x cre te d , rep eated derm al ap p lication m ay result in su ffi cien t accu m u latio n in the b o d y to cause sy stem ic to x ic ity . C ro w e144 h as pointed o u t that chloracne due to chlorin ated naphthalenes h as decreased because these co m p o u n d s have been u sed less since the S e c o n d W orld W ar. H ow ever, W eber in 1 9 6 9 148 re p o rte d an o u tb re ak o f ch lo racn e in a c o m p a n y en gaged in the production o f electric coils that w ere co ated w ith a m ixture o f ch lorin ated n ap h th alen es w ith the trad en am e " N ib re n w a x .'1 D etailed exam in ation by several G erm an invest igators in d icated that the cap acity o f 2,4 ,5 trich loroph en ol as w ell as 2 ,4 ,5 -T (2 ,4,5-trich lorop h en oxy acetic acid) 149,150 to produce chloracne w as due to con tam in ation w ith 2,3,7,8-tetrach lo ro d ib e n z o fu ra n or 2 ,3 ,7 ,8-tetrach lo ro d ib e n z o d io x in . Sch w artz et a l.l s l indicated that the m ost p oten t chloracne producing agen ts were the chlor in ated n aphthalenes, chlorodiph en yls, and chlorod ip h e n y lo x id e s. Persons w h o w ork ed w ith chlorin ated naphthalenes usually developed acne after a m onth or m ore o f exposure. Light m icroscopic observation s m ade o f the chloracne lesions o f hum ans varied som ew h at, depen din g prim arily on the len gth o f tim e the lesio n s h ad ex isted at the tim e o f exam in atio n . T h e earliest change en coun tered con sisted o f acan th o tic w idening o f the extern al ro o t sh eath o f the hair follicle. A bove a lay er o f basal cells 6 to 10 layers o f large round cells w ere ob served , follow ed by a layer o f 2 to 3 cell row s o f granular cells. In later skin biopsies, fo ld in g o f the b asal layer occu rred and com ed o form ation w as o b se rv e d , re su ltin g in d ilatatio n o f the fo llic le an d atro p h y o f the epith elium su rrou n din g the co m ed o . T h e con ten ts o f the follicles con sisted predom in an tly o f keratin ous m aterial rather than sebu m . T h e sebaceou s glands also underw ent ch an ges. W ithin the alveoli o f the glan d s the b a so p h il c e lls in c re a se d in n u m b e r a n d b a sa l -cell h yperplasia w as n o ted . The du cts o f the sebaceou s glan ds w ere filled w ith a so lid m ass o f b asop h il epith elial cells. T h e sebaceou s glands gradually cc06i63 disap peared and in Ja te r secretions w ere on ly recogn ized as so lid p rotru sion s o f the co m ed o w all. A sid e from atro p h y o f the sebaceou s glan d s, h yp erp lasia h as a lso been described and m any large seb aceo u s glands form in g huge cy sts filled p ri m arily w ith k eratin o u s m aterial h ave b een observed. H yperkeratosis and acan th osis o f the su rro u n d in g epiderm is h as u sually acco m p an ied th ese lesio n s. Foreign b o d y gran u lom ata w hich arc th ou gh t to result from ru pture o f the follicular cy sts have been fou n d in the sk in .14 7,151 In the Ja p a n e se poison in g incident (Y u sh o ), the p red o m in an t skin lesion w as m arked h yp erkeratosis o f the ep id erm is, cy stic d ilatation o f the h air fo llicles, an d an in crease in the m elan in p igm en t in the b asal cells o f the epiderm is. Foreign b o d y gran u lom ata w ere also observed. T h e con ten ts o f the cysts co n sisted partially o f k eratin . In a stillb orn child the h yperkeratosis w as even m ore pron oun ced, acco m p an ied by atro p h y o f the ep id erm is; cy stic d ilatio n o f h air fo llic le s w as o b se rv e d , e sp e c ia lly in th e skin o f the h ead . A ccord in g to earlier re p o rts151 so m e d ifferen ces exist in the m icro sc o p ic ch an ges ob served in the sk in fo llo w in g ex p o su re to the various chlorin ated cyclic co m p ou n d s that can produce the lesio n . It is, o f co u rse, possible that th ese h isto p ath o lo gic vari a tio n s are in reality o n ly d iffe re n c e s th at e x ist betw een an early, a w ell-developed, and a su b sid in g lesion. M any th eories have been advan ced to explain the developm ent o f ch loracn e. N on e is satis facto ry . T h e concept o f m echanical plugging o f the hair follicles on skin exp o su re to poly ch lor in ated poly cy clic com po u n d s is n o t su p p o rted by the fact that ch loracn e d evelop s follow in g in gestion o f these com po u n ds. It is possible that the skin lesio n s are caused b y a to x ic effect o n the ep ith eliu m o f the skin an d its ap p en d ag es w hich is m an ifested in p ro liferatio n an d lo ss o f n o rm al fu n ction o f the ceil. Sin ce the ch em icals are lipid so lu b le th ey co u ld o ccu r in a m ore co n cen trated fo r m in se b u m a n d th e re fo re a ffe c t se b a c e o u s glan d s m ore than sw eat glan d s o r salivary glan d s. T h e reports on the Y u sh o ou tb reak suggest that proliferation o f the epith elium o f the m u cou s m em branes m ay also occur. T he increased pigm en tatio n o f the skin and m u co u s m em b ran es m ay in d ic a te a fu n c tio n a l d istu r b a n c e re su ltin g in increased m elanin p ro d u ctio n . H y perp igm en tation is also ob served w hen the ad ren als are d am ag ed , as in A d d iso n 's d ise a se , an d ad re n al fu n c tio n sh o u ld be in v estigated in ch lo racn e p a tie n ts w h o m an ifest h yperpigm en tation . * In n on e o f the h um an case rep o rts on ch lo racn e ' have the m ucous m em branes been stu died to an y great e x ten t. V aginal sm ears in w om en an d sp u ta, corn eal, and nasal scrapin gs m ay give h elp fu l in fo rm atio n in th is re sp e c t. S in c e in an im a ls exposed to polychlorinated poly cy clic com pou n d s lo w vitam in A levels h ave b een o b serv ed in th e liver, it is possib le th at the co m p o u n d s m ay affe c t vitam in A ab sorp tion an d sto rag e, an d ad eq u ate u tilizatio n in th e liv er an d elsew h ere in th e b o d y . I f the lesion is p rod u ced b y in ad eq u ate u tilization o f vitam in A , then ad m in istratio n o f vitam in A per se m ay n ot necessarily alter the cou rse o f the disease. Protein d eficien cy m ay also m im ic h ypo- vitam in osis A in th at it a ffe c ts tran sp o rt o f vitam in A . T h e liver sto res 9 0 % o f th e available vitam in A an d th ese sto res m a y be a ffe c te d in liver disease. T h e m icroscop ic ap p earan ce o f the skin lesions resem b les in som e resp ects o b se rv atio n s m ade in skin o f patien ts su fferin g from vitam in A deficien cy. In vitam in A d eficien cy , h y p erkeratosis o f the hair follicles is o b serv ed . M ultiple firm papu les m ay develop as a result o f the keratin plugs in the se b ace o u s glan d s. D ry n ess an d scalin ess as w ell a s fu ru n cu lo sis o f th e sk in m ay also be present. In con trast, night blin dn ess has n o t been reported as a sy m p to m in patien ts su fferin g from ch loracn e. U n fo rtu n ately , chlor acne o u tb reak s are o ften n o t w ell in vestigated an d , b ecau se o f prob lem s w ith litig atio n , ac c o u n ts are often on ly publish ed m any y ears a fter the o u t break h as occurred if they are published at all. C hloracne is a very persisten t skin disease and in the Ja p a n e se o u tb reak o f po iso n in g w ith chloro- d ip h e n y ls, th e sk in ch an ges w ere still p re se n t in a n um ber o f patients three y ears after exp osu re to the ch lo rod iph en yis h ad been d isco n tin u e d . W hat n eeds to be in vestigated is w h eth er this persisten ce o f the skin d isease is cau sed o n ly b y th e fa c t th at on ce the lesion h as d eveloped it regresses very slo w ly , o r w hether it m ay be su stain ed b y the ch loracn e-producin g ch em icals w hich have been a b so rb e d an d sto re d in the b o d y , p a rticu larly the ad ip ose tissu e, and are on ly very gradually e x c re te d . In p atie n ts w ith c h lo rac n e , ad ip o se tissue b io p sies sh o u ld be an alyzed fo r th e presen ce o f the o ffen d in g poly cy clic polych lorin ated com po u n d s. I f ad ip o se tissue is u n o b ta in ab le, the se b u m o f the ear canal o r the con ten t o f th e skin lesio n s m ay represent a good su bstitu te. _ 6925 C +70 January 1974 461 DOW l135882 DOW 1135883 A p paren tly there is n o know n efTective treat m en t fo r ch lo racn e an d th e b e st co n tro l m easu re is prevention. T h e m an u factu re o f chlorinated h yd rocarb on s an d the co atin g o f w ires and conden sors w ith in su latin g m aterials co n tain in g them sh o u ld be don e in to tally en closed areas so that the fu m es o f the in su latin g su b stan ces an d o f the solven ts if an y are u sed d o n o t com e in co n tact w ith the w orkers. T h e w ork ers sh ou ld b e provided w ith clean overalls an d u n dercloth es d aily . T h ese sh o u ld b e la u n d ere d a t th e p la n t an d cle an e d in such a w ay that n o ch lorin ated h yd rocarbon s rem ain on them . Sh ow er b ath s after w ork should be com pu lso ry an d su pervised and there sh o u ld be su fficien t show er facilities provided so th at the w orkers are n ot un duly delayed after w ork. Special synth etic w ettin g agen ts151 seem to rem ove the m aterial from the skin m ore effectively than ordinary soap s. T h e e x p e rim e n tal in d u c tio n o f c h lo rac n e in m en h as also been accom p lish ed w ith 2 ap p li c a tio n s o f 10 ;/g 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p d io x in 107 an d w ith H alow ax 1 0 1 4 , pen tach loron aphthalene, and h exach loron aph th alen e.147 E xperim en tal w ork in an im als h as sh o w n that m o st species d o n ot d evelop the sam e typ e o f d erm atitis. The rab b it's sk in ,151 p articu larly the inner asp ect o f th e ear lo b e, provides a go od test m edium fo r the developm en t o f acn eform derm a titis. T h e rab b its, in ad d itio n to the developm en t o f h y p erk e rato sis o f th e e a r, w ill a lso sh o w a h ep ato to xic e ffe c t.150 M icroscopic exam in ation o f the ch an ges in the rab b it ear w ere d escrib ed b y Jo n e s an d K rizek . 1 53 In agam i an d K o g a 154 w ere ab le to p rod u ce h y p erk erato sis in h airless m ice th at w ere fed rice oil co n tam in ated w ith K an eclor 4 0 0 . T h ese m ice also show ed h ep atom egaly. M icro sco pic exam in atio n o f the skin revealed cy stic dilation an d h yp erk eratosis o f h air follicles an d sw eat glands. H airless m ice m ay be an oth er su itable experim en tal anim al fo r testin g the effect o f these ch em icals on the skin . X -d ise a se in A n im a ls C h lo ra cn e -ty p e le sio n s d o n ot u su a lly o c c u r in o th er sp ecies ex cep t fo r the rabbit an d the hairless m o u se , alre ad y m e n tio n e d . H o w ev er, in 1 9 4 7 O la fs o n 1 55 d e sc rib e d a d ise a se in c a ttle w h ich w as d e s i g n a t e d a s X -d isease o r h y p e rk e ra to sis. S y m p to m ato lo g y in clu ded excessive lacrim ation , diarrhea, poly u ria, m arked salivation , and dis charge from the n o strils. T h e an im als developed a 462 CRC Critical Reviews in Toxicology chronic cough, p o o r ap p etite, n u m erous red m acu lae in the buccal m u c o sa ; in ad d itio n , h y p er k erato sis o f the skin d evelop ed . T h e skin w as h ard , an d fissu red particularly acro ss the w ith ers and sid es o f the n eck. M any w art-like proliferation s on the lips, tongue, and h ard palate w ere also observ ed. T h e ab om asu m w as greatly in flam ed , sw ollen, a n d e d e m a t o u s w ith m a n y s u p e r fic ia l u lc e r s . 1 56 In the cow s that h ad X -disease, a low vitam in A p lasm a level w as also ob serv ed , w hich again in d i cates th at vitam in A m ay play a role in the etiology o f these various disease en tities. T h e disease w as first th ou gh t to rep resen t a virus in fection and oth er facto rs were also suspecte d . A fte r n u m ero u s in vestigation s it w as finally e sta b lish e d th at X -d isease c o u ld be p ro d u c e d in cattle b y the in gestion o f highly ch lo rin ated n aphthalenes, and also by petroleum produ cts such as crankcase oil. B ell157 tested the ability o f the variou s com pou n ds o f the chlorinated naphthalene group to produce X -disease. H e d e m o n strate d in h is stu d ie s th at d ich lo rin ated an d trich lo rin aled n ap h th alen es d id n o t result in X -d isease, w hile tetrach loron ap h th alen e h ad an effe ct an d the h igher ch lo rin ated n aph th alen es su ch as pen tachloron aphth alen e, h exach loro n a p h th a le n e , h ep tach lo ro n ap h th alen e, and octachloronaphth alene caused severe disease. O ctach loronaph th alene w as less toxic than hexaan d heptachloron aph thalen e. W agener158 rep ort e d the occu rren ce o f h y p erk erato sis in cattle in G erm any betw een 1 9 4 6 and 1948. T h e cause o f this o u tb reak w as traced to a w o o d preservative w hich w as used in p o stw ar G erm an y . T h e au th o r p ro v ed e x p e rim e n tally th at an im als h o u se d in cab in s p ain ted w ith the w ood preservative d ev elo p e d ty p ic a l X -d isease. T h e p o iso n o u s in g red ien t in the w o o d preservative ap p eared on ly in certain lo ts o f this p ro d u ct an d later lo ts d id not con tain it. T h e p o iso n o u s ingredient w as never id en tified by chem ical an alysis. A nim al exp erim en ts w ere co n d u cted w ith the to x ic in gred ien ts o f rice bran oil that p ro d u ced Y u sh o in Ja p a n . T h e se to x ic su b sta n c e s c o n siste d o f a m ixtu re o f ch lorin ated b iph en yls con tain in g 4 8 % ch lo rin e, w ith a trace o f 0 .0 1 % o f n ap h th a le n e s.1 59 W hen m ic e w ere e x p o s e d to th e m fo r 3 to 4 m on th s, they show ed .eczem atou s changes o f the skin aroun d the ey elid s, ero sio n , ulceration ar.d p e rfo ra tio n o f the e ar lap s, l o s s o f h air, an d erosion an d ulceration o f the skin aroun d the n eck , fo relegs, and sides o f the ch est. The au th o rs 6926 tsssetliMOfl stated that, at the end of ihe experiment, some changes in the skin similar to those in the experimental mice were seen in some of the control mice also, which makes it difficult to interpret their findings. In 1 9 5 7 a d isease th a t w ill b e d isc u sse d in g re a te r d etail late r in th is article o c c u rre d in a large n u m b er o f chickens. It w as term ed chick edem a disease an d w as fou n d to be caused by certain to x ic fat. A llen and C arsten s160 fed this su b stan ce to m onkeys and found that, am ong other changes w h ich w ill b e d isc u sse d late r, the m o n k ey s developed a generalized alopecia and su b cu tan eo u s edem a one to tw o m onths before death. Sch oettle e t a l.161 p ro d u c e d h y p e rk e rato sis o f the sk in in rats b y feedin g them hexach loron aphth alen e. Y o u n g sw in e 1 4 3 a n d s h e e p ' 63 d id n o t d e v e lo p skin lesio n s as a result o f exp o su re to ch lorin ated n aphthalenes. P k o to c o n tac t D erm atitis a n d Irritatio n o f th e Sk in As was pointed out earlier, the hyperpigmenta tion observed in patients with chloracne as well as the reaction described as cable rash could also be the result of a photosensitive reaction. This photo sensitive reaction is the predominant lesion in the skin produced by a number of germicides, namely, bithionol (Figure 2), tetrachlorosalicylanilide (TCSA), and. a number of other halogenated phenolic compounds including fentidor. Patients who develop photodermatitis complain initially of a burning sensation, and this is followed by itching. A rash develops which in the beginning is erythematous and associated with varying degrees of edema. As it subsides, pigmentation is observ ed .'64 Wilkinson'6* was the first one to describe cases where patients developed a photo allergy which was due to the incorporation of TCSA into popular soaps. He observed that the outbreak of dermatitis was confined to the areas that usually were exposed to light. Most of the cases reported from England were transient and as soon as exposure to the compound was discontinued the reaction subsided. Jillson and Baughman166 and Jillson,167 on the other hand, reported that their cases were more persistent. Willis and Kligman'6* demonstrated that the photocontact allergy of the skin with these various compounds was simply a contact sensitization. The reason that allergic reactions of the skin developed only after exposure to the sun was due to the photodecomposition of the various products to which the skin had been exposed, if various brominated and chlorinated salicylanilideswere irradiated in vitro in an appropriate solvent, the resulting breakdown products caused an aller gic reaction on contact, without exposure to UV light. These studies were performed because of information provided in earlier studies by Coxon et al.,'69 who had irradiated brominated and chlorinated salicylanilides and found that these compounds gave off halogens. With 3,5 substituted salicylanilides. the halogen substituted on the 3 position was lost. Tetrachlorosalicylanilide photo sensitized subjects reacted to 3', 4', 5-trichlorosalicylanilide and to 3', 4'-dichlorosalicylanilide. A reaction to nonhalogenated salicylanilide was not observed. Halogens on the anilide ring were un affected by UV radiation, while the halogens on the salicyl ring were replaced stepwise by hydro gen upon irradiation. Among the patients who developed photosensi tivity to tetrachlorosalicylanilide reported by Wilkinson,163 a group of patients in a shop of a factory was also included. This same outbreak was reported by Jones,170 who found that among 106 employees who used the same soap very frequent ly, 31% were affected by a dermatitis due to contact with tetrachlorosalicylanilide present in the soap. Additional cases of photodermatitis due to the exposure to tetrachlorosalicylanilide were reported by Calnan et al.,171 who pointed out that pronounced edema of the eyelids was also observ ed when involvement was severe. Some cases were persistent or developed acute relapses on further exposure to sunlight without further exposure to the offending chemical. Frank173 reported an additional nine cases of acute contact dermatitis of the hands due to tetrachlorosalicylanilide. Epstein173 described a photoallergic reaction in two patients to tribromosalicylanilide (TBS). With patch tests these patients showed a cross reaction to TCSA. A similar cross reaction pattern to a number of halogenated salicylanilides was also observed by Osmundsen174 in patients who had developed photodermatitis following exposure to tribromosalicylanilide. Because of the photosensitivity reactions and the cross reactions that were also found with bithionol, which is similar in structure to tetra chlorosalicylanilide (Figure 2), the US.* Food and Drug Administration, on October 24. 1967, with drew all new drug applications of all drugs that January 1974 463 i 6927 '/135885DOW co n tain ed b ith io n o i. in th e n otice o f w ith draw s] o f ap p ro v al in th e F ed eral R egister, the C om m ission er o f the F o o d and D rug A d m inistra tio n p o in ted o u t that b ith io n o i m ay in som e in stan ces cause a very persistent ph otosen sitization an d severe derm atitis m ay o ccu r w ith ex p o su re to sunlight w ith ou t fu rth er co n tact w ith the sen sitiz in g articles, and that bith ionoi m ay produ ce cross p h o to sen sitizatio n w ith o th er c o m m o n ly used ch em icals su ch as certain h alo gen ated salicylanilides an d h exach lorophen e. H exach loroph en e preparation s m ay also cause irritation o f the skin if these p rep aratio n s are used frequ en tly. A true allergic reaction to h exach lo ro ph en e is ap p aren tly rare.173 T h e m ore severe reactio n s w ere usu ally con fin ed to the skin o f the scro tu m an d con sisted o f a prim ary irritant con tact d erm atitis.17 6 1 ,7 7 The cases reported by B ak er et a].176 also included a 33-m onth-old C au casian fem ale w ho developed a rash aroun d the b u tto ck s, vulva, an d u p p er thighs follow in g the use o f a h exach loroph en e preparation in her bath w ater. We observed severe skin irritation and ulcera tion in rats th at w ere e x p o se d d erm ally to a 3% h ex ach lo ro p h en e so lu tio n in p ro p y len e gly co l o r a detergen t. T h e skin reaction w as not as severe w hen the hexach lorophen e con cen tration w as red u ced to 1.5% .118 B outw ell e t a l . 178 rep orted con sid erab le skin irritation in m ice th at h ad S m g /m o use ap plied tw ice w eekly fo r 21 w eeks. H arber et a l.179 w ere able to in duce con tact ph o to sen sitiv ity w ith T C S A o r T B S . T h ese au th o rs also fou n d that cross con tact sensitivity and p h o to se n sitiv ity to h ex ac h lo ro p h e n e e x iste d in guinea pigs w ith prim ary p h o to sen sitiv ity to T C S A and T B S . P hotosen sitivity to trichlorocarban ilide (T C C ) w as n ot p rod u ced in guinea p igs. T h e guinea pig m ay represent a su itable anim al sp e c ies to' stu d y c o n ta c t an d p h o to sen sitiv ity o f th ese and related co m p o u n d s. A fter ex p o su re to the ph otosen sitizin g agents has been discon tin ued, h u m a n s m a y still be- se n sitiv e to su n lig h t, a fa c t th at n eed s furth er stu d y . T o a certain ex ten t this co u ld be due to a persistence o f the chem ical c o m p o u n d on o r in the sk in . D ich lo rap h en e u se d a s an a n tim ic ro b ia l p re se rv ative m ay also cause con tact d erm atitis. Crosssensitivity to h exach lorop h en e w as not d em on strated .180 C h ick E d em a S im p so n e t a l.181 an d S an g er e t a l.182 d e sc rib ed a n ew h ighly fatal d isease in ch ick en s. T h e first o u tb re ak occu rred in I9 S 7 in G eo rg ia. L arge num bers o f birds from ap p ro xim ately three w eeks o f age to ad u lt lay in g h en s w ere a ffe c te d . C linical signs con sisted o f d y spn ea, redu ced b o d y w eight gain, stu n tin g, su b cu tan eo u s ed em a, palen ess, and sudden death . In you n g chickens, gasping w as the first n oticeab le sign. T h is w as follow ed b y a w addling, un steady gait. G ross in spection o f the b ird s revealed a pale h eart, fluid w as n oticed in the pericardial sac, an d the livers w ere pale, m o ttled , an d h ad an. irregu lar gran u lar su rfa ce . In ad v an ced ' stages o f the disease the chickens h ad large d isten ded ab d o m en s that w ere filled w ith flu id . E cch y m o tic h em orrh ages w ere presen t in the skin b en eath the w ing?, on the legs, an d over the Q bon e in a few chickens. T h e kidn eys w ere pale and sw ollen. F ollow in g the first o u tb reak , no ad d itio n al cases w ere rep orted un til late su m m er an d early fall o f 1 9 5 7 , w hen exten sive losses o f ch ickens an d turkeys occurred th ro u g io u t the south eastern part o f the U .S . Suspicion soon developed that losses were cau sed by the con su m ption o f ration s co n taining certain lo ts o f an im al fa t.181 T he an im al fats con tain ed tallow s an d greases ob tain ed as by-p rodu cts from h ides preserved w ith ch loroph enols prior to leath er m an u factu re.8 1 T h is m aterial w as exclu d ed from com m ercial poultry rations and no ad dition al cases occurred w here anim al fats w ere respon sib le. Sim pson et a l.181 cam e to the co n clu sio n th at the e d e m a in the chickens w as the result o f vascu lar ch an ges that seem ed to be the prim ary lesio n . T h e vascu lar lesion , or en dotheliosis, con sisted o f proliferation and h yp ertrop h y o f the en doth elial cells o f arterio les an d sm all arteries in m o st tissu es. P ro n o u n ced proliferative ch an ges in en d o th eliu m o f the glom erular capillaries w ere ob served, b u t no sig n ifican t ch an ges w ere n o tic e d in the tu b u lar epith elium o f the kidn ey. N ecrosis o f the paren ch ym al cells o f the liver w as also n o ted . In so m e rep o rts it w as p o in ted o u t th at e x p o su re to to x ic fa t resu lted in a d e cre ase o f the total serum protein and a sh ift on the album in to g lo b u lin ra tio w ith a d ecrease in th e alb u m in ft i'.tio n . T h is c o u ld , o f c o u r se , le a d to e d e m a fo rm atio n , particu larly w hen com b in ed w ith m al fu n ction in g glom erular capillaries. It h as also been suggested that increased perm eability o f the car diovascu lar bed con tribu ted to the edem a form a- 464 CRC Critical Rcvim-s in Tuxicnlony %* t i o n . 1 4 0 ' 1 1,3 In o t h e r s t u d i e s t h e s e r u m p r o te in con cen tration d id n o t d iffe r fro m that o f the c o n t r o l s ' 44 '8 5 a n d in c r e a s e d p e r m e a b ilit y o f th e cardiovascular bed w as advan ced as the sole cause o f the flu id accu m u latio n . In ad d itio n , pulm on ary e d e m a w as o b se rv e d in d ise ase d ch ick en s an d p eriv ascu lar ly m p h o c y tic in filtratio n as w ell as ed em a o f the card iac m u scle w ith interstitial l y m p h o c y t i c i n f i l t r a t i o n h a s b e e n n o t e d . 1 46 E lectron m icroscop ic exam in ation o f the cardiac m uscle revealed degen eration and loss o f m ito c h o n d r i a . 1 44 I t is p o s s i b l e t h a t t h e h e a r t m a y p la y a prim ary role in th e d ev elo p m en t o f chick ed em a, b u t a h ep atoren al sy n d ro m e could be the under ly in g cause as w ell. S tu d ie s that investigate the developm en t o f the lesion m ore closely are needed to elucidate these p rob lem s. T h e type o f diet also in flu e n c e d the d e v e lo p m en t o f th e d ise ase . It co u ld be b ro u g h t on m ore rap id ly w ith a sem ipu rified d iet than w ith a n atural grain ration ; so d iu m ch loride in the diet w as a prerequ isite fo r th e d e v e lo p m e n t o f th e sy n d r o m e . ' 47 I f low ratio n s o f the to xic fat w ere fed to chickens they did n o t develop chick edem a but reproduction and h atch ability w ere drastically red u ced . T o x ic fat w as n o t the only product that caused the chick ed em a sy n d rom e. C h lorinated biphenyls c a u s e d th e d is e a s e in c h ic k e n s 1 44 a n d in B e n g a le se fin c h e s .26 A m ix t u r e o f p e n ta c h lo ro n a p h th a le n e and h exach loron aph th aien e fed to chickens also resu lted in ch ick e d e m a d ise ase .149 C an trell et a l. ' 90 a n n o u n c e d in 1 9 6 7 th a t o n e o f th e to x ic com po u n d s cap ab le o f produ cin g chick edem a fo u n d in to x ic fa t w as 1 ,2 ,3 ,7 ,8 ,9-h exach loro d ib e n z o - p - d io x in . T h e n H ig g e n b o th a m e t a l .S2 show ed that the ch ick ed em a facto r w as produced by tw o principal co m p o u n d s isolated fro m toxic f a t s : 2 ,3 ,7 -tric h lo ro d ib e n z o -p -d io x in , and 2,3 ,7 ,8 -tetrach lo ro d ib en zo -p -d io xin . T h ey also su ggested fats an d fatty acid s that contain com m ercial ch loroph en ols as possible sources o f con tam in ation . W hen crud e fats and tallow s are h eated to prod u ce fatty acid s, chlorophcnol residu es m ay be con verted to a chick edem a facto r. W hether these last three co m p o u n d s m en tion ed are the o n ly o n es that produ ce chick ed em a disease is q u estio n ab le unless the chlori n a te d n ap h th alen es a s w ell a s ch lo rin ated biphen yls that prod u ced chick edem a w ere also co n tam in ated w ith th em . A n o th er o u tb reak o f c h ic k e d e m a d ise ase o c c u rre d in I 9 6 9 .SI In this DOW J13588G in ciden t m illio n s o f b ird s in N orth C aro lin a w ere in v o lv ed . T h e o u tb re a k w as trace d to a v e g e ta b le . . oil refinery w here feed fat b ecam e co n tam in ated w ith " d iio ro p h e n o ls." T h e com pan y fo rm u lated the ch loroph en ols as an tim icrobial w ater treat m en t p ro d u cts. An un dergrou n d pipelin e fro m the " p esticid e" plant led to traps used to collect b y-product fatty acids held as feed fats. F lic k et a l. ' 41 have recen tly show n th at p y ro ly tic p rod u cts o f 2 .3.4.6-tetrach lo rop h en ol w ere to x ic to ch ick s an d p ro d u ced ch ick e d e m a if SO p p b w ere fed for 21 d ay s. The p ro d u ct fed con sisted o f 53% h exa-, 45% hepta-. an d 25% o c ta c h lo r o - p - d ib e n z o d io x in . The pyrolytic p ro d u ct o f pentachloropheno! con sisted o f a m ixtu re o f 5% hexa-. 62% hepta-. and 33% octach lorodiben zo-p-dioxin that produ ced m ild ed em a and increased m ortality w hen fed a diet c o n ta in in g 1 p p m (2 0 0 fig to ta l in g e stio n ) o f th e m aterial fo r 21 d ay s. W hen a m ixtu re o f tri- a n d tetrach lorodiben zo-p-dioxin w as fed to the an im als a t a d ietary co n cen tratio n o f 0.01 ppm (a calc u la ted in tak e level o f 1.9 ;ig ). the b irds d ev elo p ed ed em a an d 8 3 % o f them d ied . F u rth er stu d ies w ith pure com p o u n d s are necessary to con firm these fin d in g s. T h e effect o f PC Bs on the chicken w as again d em o n strated w hen P C B s that w ere used in the U .S . a s h eat tran sfe r flu id s in fo o d p ro c e ssin g plan ts con tam in ated large qu an tities o f fish m eal th at w as p ro cessed fo r an im al feed in a p la n t in W ilm in gto n , N o rth C a ro lin a . 1 91 F r o m a b o u t A p ril 1971 until Ju ly 1 9 7 1 , P C B s leaked in to th e fish m eal an d 16 thousand ton s o f co n tam in ated fish m eal w ere distributed to m ore than 6 0 co m p an ies in 10 state s. O ne o f the purchasers o f th e fish m eal. T h e H olly F arm s, the n ation 's largest p ou ltry producer, had to slaughter 7 7 ,0 0 0 fow ls after discovering con tam in ation o f the ch ick en s w ith P C B s. S o m e o f the lev els fo u n d in th e a d ip o se tissu e o f the an im als w ere as high as 4 0 p p m . T h e H olly F arm s first discovered that the ch icken m eal w as con tam in ated b ecau se th e h atch ab ility o f their eggs w as drastically redu ced to 18% o f norm al. in F eb ru ary and M arch o f 1968 an ou tb reak o f c h ic k e d e m a a lso o ccu rre d in Ja p a n , w h ere m o re than 4 0 0 ,0 0 0 chickens reportedly d ied . T h e disease w as traced to the-con tam ination o f anim al feed w ith K an ech tor 4 0 0 , a Ja p a n ese P C B . T h e ch ick ed em a d isease occu rred sim u ltan eo u sly w ith an o u tb reak o f polych lorin ated biphenyl poison in g in p e o p le that w as du e to the c o n ta m in atio n o f January 1974 6329 46* rice bran oil, also con tam in ated w ith K an ech lor 4 0 0 an d p r o d u c e d b y th e sa m e c o m p a n y .*92 M e tc alfe ' 93 su g g ested that ch ick ed em a fa c to r co u ld b e m in im ized if flesh in gs grease from h id es preserved w ith tech n ical p en tach lo ro p h en o ls co n tain in g ch lo rin ated d ib en zo d io xin s w ere n o lon ger used for fo o d prod u cts. It w as also poin ted out th at the use o f glue em u lsion s con tain in g chlorop h en o ls in ren derin g o p e ratio n s is an o th er p o ssib le so u rce o f the to x ic fa c to r. W hether elim in ation o f th ese so u rces fro m an im al fo o d p ro d u cts w ill prevent chick ed em a epid em ics rem ains to be seen, since polych lorin ated b iph en yls that m ay be con tam in ated w ith ch lo rin ated d ib en zofu ran s have also prod u ced chick ed em a disease. Y usho D uring the sum m er - o f 1968, 13 cases o f ch lo racn e w ere ob served in th e w estern p art o f Ja p a n , cen terin g arou n d F u k u ok a prefectu re. T h e incidence o f the disease seem ed to be fam ilial and it becam e ap p aren t th at all p atien ts h ad used the sam e brand o f co o k in g oil (can n ed K an em i rice oil). T h ese fin din gs w ere picked u p b y the new s p apers an d a stron g dem and w as voiced to elu cid ate the cau se. T h erefo re, a Y u sh o stu d y g r o u p w a s e s t a b lis h e d b y K y u s h u U n iv e r s ity . ' 92 T h e term Y u sh o sta n d s fo r rice oil disease. A fter exten sive in vestigation s it w as fou n d that several illn esses o ccu rred in a n um ber o f h ou seh old s that h ad used co m m o n can s o f rice bran oil. W hen a to ta l n u m b er o f 8 9 m ales an d 100 fem ales that su ffered fro m Y u sh o w ere exam in ed, a v arie ty o f sy m p to m s w ere fo u n d in th ese p atien ts. T h e in ciden ce o f the differen t sy m p to m s th at v aried a g re at d eal is given in T ab le 2 . T h e epid em ic o u tb reak w as fin ally, after very extensive investigation, pin poin ted to K anem i oil, m anu fac tu red on F eb ru ary 5 , 1 9 6 8 . C h em ical an alysis o f th is oil e stab lish ed th at it w as co n tam in ated w ith 2 ,0 0 0 pp m o f K an ech lor 4 0 0 , a chlorobiphenyl that con tain s 4 8 % chlorine. It w as also dem on stra ted th at m ost o f the com p o n en ts o f the chlorob ip h en y ls w ere sto re d in ad ip o se tissu e o f the p a tie n ts for q u ite a lo n g tim e an d passed through the placen ta in to the fetu s o f a few w om en w ho h a p p e n e d to b e p re g n a n t. 194 T h e am ou n t o f oil necessary to produce disease w a s f o u n d t o b e a t l e a s t 2 .5 1. T h e y o u n g e r th e p a tie n t, the h igher th e m orb id ity rate w ith a sm aller total am ou n t o f ingested oil. T h e average am ou n t o f ch lo rob iph en y l con sum ed by individual p a t i e n t s w a s c a lc u la t e d t o b e a t o t a l o f 2 g . ' 95 (V a rio u s rep o rts w ritten on this su b ject give differen t accou n ts o f w hat actually w as consum ed and the am ou n t can at best only be estim ated .) W hen the rice bran oil w as h eated un der red u ced p ressu re, K an ech lor leaked from the old p ip es in w hich h oles w ere subsequ en tly discovered. T h e Y u sh o ou tb reak spread n ot only over F u k u o k a, Jap an , but also over 2 0 other p re fe c tu re s in the w estern part o f Ja p a n . A to tal o f 1,057 poison in g cases resulted accordin g to the latest tab u latio n (A u gu st 19 7 1 , M inistry o f W e lfa r e ) . ' 96 T h e m o s t c o m m o n in itia l s y m p t o m e x p e rie n c e d b y 136 p atie n ts w ith Y u sh o w as in creased eye discharge and sw elling o f the u pp er ey elid s (38.3% ) follow ed by acn eform eru p tion , fo llicu lar accen tu ation (3 3 .1 % ), and pigm entation o f the skin (9 .6 % ). Itch in g o f the skin an d " stiffen in g" o f the soles o f the feet and the palm s o f the Itands w ere also observed. Pigm ented m u cu s m em b ran es w ere n o ticed in m any o f the p atien ts; a sm all percentage o f them developed jau n d ice, m any com plain ed o f a feeling o f w eakness and h ead ach es; occasion ally vom iting, diarrhea, an d fe v e r a ls o o c c u rre d . V ariou s neurological sy m p to m s m an ifested them selves such as transient visu al d istu rb an ces, n u m b n ess an d sp asm s in the lim b s, an d h earing d ifficu lties. M ost p atien ts sh o w ed acn elike skin eru p tion s that resem bled the ch loracn e observed after occupational exposure to ch lo rin ated naphthalenes o r chlorinated biphenyls. When, th e age an d se x sp ecific in ciden ce rates w ere an a ly z e d it w as fou n d th at a sign ifican t se x d ifferen ce w as n o t p resen t, b u t a low er risk fo r both, m ales an d fem ales in the age grou p s over 6 0 y ears w as n oted . T he proportion o f severe cases am o n g th ose age 13 to 2 9 w as sign ifican tly larger than that o f other age groups. D u r in g th e Y u s h o e p id e m ic , 11 w o m e n w ith Y u sh o an d 2 w ives w hose h u sb an d s h ad Y u sh o , b u t w h o them selves did n o t show the d isease, delivered 10 livebo m an d 2 stillborn babies. N ine o f th ese b ab ies h ad unusually greyish , dark brow n stain ed sk in , an d dark pigm en tation o f the gingiva a n d n ails w as n o te d in five o f th em . M o st in fa n ts also h ad heavy eye discharge. H istological exam i n atio n o f a stillb o rn fe tu s 'sh ow ed a m ark ed h y p erk erato sis and atro ph y o f the epiderm is and c y stic dilation o f hair fo llicles, especially o f those o f the h ead . A m arked increase o f m elanin p ig m en t in the b asal cells o f the epiderm is w as also n oted . M ost fetu ses that w ere b om w ere sm aller 466 CVfCCritical Reviews in Toxicology CGOoiVj e DOW 1135888 TAHLK2 Symptoms (complaints) of Patients (89 males. 100 Females, is of October 31. 1968)* Symptoms M%ales Fem%ales Blackening of nails Black spots in all pores Excessive sweating in palms Acnelike skin eruptions Red spots on limbs Itching Gunge in skin color Swelling of hands and leci Hardening of backs and hands Pigmentation of mucous membranes Sebum (gum secretion in eyes) Hyperemia of mucous membranes in eyes Temporary failing of eyesight Jaundice Swelling of upper eyelids Sense o f weakness Numbness of hands and feet Fever Hearing difficulty Spasms of hands and feet Headaches Vomiting Diarrhea 83.1 64.0 30.6 87.6 2 0 .2 42.7 73.3 2 0 .2 24.7 36.2 8 8 .8 70.8 36.2 1 1 .2 71.9 38.4 32.6 16.9 18.0 7.9 30.3 23.6 19.1 75.0 56.0 55.0 82.0 16.0 52.0 72.0 41.0 29.0 47.0 83.0 71.0 55.0 1 1 .0 74.0 52.0 39.0 19.0 19.0 8 .0 39.0 28.0 17.0 'Reprinted with permission from Kuratsunc et al. 1 * ' than the national stan dards and fo u r o f them w ere sm all for gestation al age bab ies. A s they grew older their skin gradu ally b ecam e lighter over a p erio d o f three m onths. O k u m u ra an d K a ts u k i1 97 stu d ie d a g ro u p o f 2 4 p atien ts w hose m ain co m p lain t h ad been acn efo rm skin eru ption s and w ho had been diagn osed as d efin itely having Y u sh o. Eighteen o f these w ere ad u lts and six w ere children. Irrespective o f the serio u sn e ss o f the disease in all p a tie n ts, the initial sy m p to m s w ere eye discharge asso c iate d w ith e d e m a o f the ey elid s, d e fe c ts in v isio n , gen eralized fatigue, anorexia follow ed by com ed o and acn efo rm e ru p lio n s o f the* lace, an d pigm en tation an d flatten in g o f the nails. T h e principal su b jectiv e sy m p to m s w ere fatigu e, n um bn ess o f the low er lim b s, and in toleran ce for sak e, w hich is a Jap an ese alcoh olic beverage m ade from ferm ented rice. T h ree o f tw elve fem ales h ad an irregular m en stru al cy cle. Seven o f nine p atie n ts w ith serio u s disease sh o w ed a low grade fever. M ost laboratory tests con d u cted on these patien ts were norm al, including seru m en zy m e valu es. A slight elev atio n in alk alin e p h o sp h atase w as o b se rv e d in over h a lf o f the se rio u sly ill g ro u p s. A n in crease in the Oj globulin fraction o f the serum protein w as n oted w ith a con com itan t redu ction o f the album in fraction , but the total serum protein s w ere w ithin the n orm al range. T h ese sam e 2 4 p atien ts h ad ab n orm ally elevated seru m tri g ly c e rid e s ran gin g fro m 2 0 0 to 6 0 0 m g %. In 12 o f 2 4 case s, w hile the to tal serum ch o lestero l rem ained unchanged, ph osph olipids ten ded to be som ew h at lo w e r.1 9 * P relim inary an im al e x p e ri m en ts in ra b b its c o n d u c te d b y th e sa m e a u th o rs show ed that lipem ia w as con sisten tly induced w ithin several d ay s. T h e levels retu rn ed to their previously norm al values several w eeks after e x p o sure had been discon tin ued. T h e scru m c o n c e n tra tio n o f trigly cerid e is u su ally e le v ate d in h u m a n s in gly co g en sto rag e d ise a se (V o n G ie r k e 's d is e a s e ). A n in c re a se in plasm a trigly cerid e co n c e n tra tio n is a lso o b serv ed in e sse n tia l h y f> erj(ly ccrid cn iia. an a p p a r e n t c o n - January 1974 G o o Jl r o i <rt s ^ * 2f . DOW 1.135889 gen ital an o m aly . -Essential h yp ergly ceridem ia h as b een o b se rv e d in sib lin g s o f a n u m b er o f fam ilies, but th e m o d e o f tran sm issio n is n ot kn ow n . T h e co u rse o f th is d isease is gen erally benign an d is c o n s is t e n t w ith a n o r m a l life s p a n . ' 99 O b s e r v a tio n o f the fam ilial cases sh o w s that the m ere elevation o f trig ly cerid e d o e s n o t in itse lf in crease the risk o f a r te rio sc le ro sis. A n o th e r in te re stin g fa c t in th is p o iso n in g e p id em ic w as th at o u t o f 23 c ase s w ith o b v io u s Y u sh o, 10 show ed sy m p to m s or sign s o f a sen sory n eu rop ath y that in clu d ed n u m b n ess, pain and h y p o e sth esia. an d , in a d d itio n . 1 case also sh o w ed a r e f l e x i a . 2 0 0 *201 T h i s e f f e c t o n th e s e n s o r y n e r v e s w o u ld n o t be im m e d ia te ly o b v io u s in an im al experim en ts and p rob ab ly should be investigated fu rth e r. S am p les o f sp u ta and ad ip ose tissue collected from patien ts w ho su ffered from Y ush o contained ch lo rin ated b ip h e n y ls o n ch em ical an aly sis.194 T h e co n cen tratio n in ad ip o se tissu e w as m uch h ig h e r t h a n in s p u t a . R e c e n t l y K u r a t s u n e e t a l . ' 96 reported on the presen t sta tu s o f the patien ts w ho su ffered from Y u sh o . M any o f the patien ts w ere still su fferin g from th e illn ess. W hen the fin din gs m ad e in 15 9 p a tie n ts in 1 9 7 0 w ere co m p a re d w ith th o se o b se rv e d in 1 9 6 9 , it w as fo u n d th at h a lf o f the p atien ts h ad . im proved clin ically , w hile the rem aining h a lf sh ow ed no such im provem en t an d m ore than 10% o f the p atien ts had becom e w orse. Im m unosuppression Several reports have indicated that P C B s and possib ly som e o f the ch lorin ated dibenzodioxin s an d fu r a n s m a y a lt e r th e im m u n e re sp o n se in anim als. A troph y o f the th y m u s and reduced total w h ite co u n t w ere o b serv ed in rab b its follow in g th e derm al ex p o su re to P C B fractio n s con tain in g tetraa n d p e n t a c h lo r o d ib e n z o f u r a n .94 In f is h , e x p o s u r e to A ro d o r 1 254 fo r 14 to 4 5 d ays led to e m a c i a t i o n a n d f u n g u s lik e s k in le s io n s .93 H ow ever, n o attem p t w as m ade to isolate organ ism s from th ese lesio n s. W hen 10-day-old m allard d u ck lin gs w ere fed d iets con tain in g P C B s (A ro c lo r 1 2 5 4 ) an d w ere su b seq u en tly in jected in terp erito n eally w ith a d u ck h ep atitis virus, sign ifican tly m ore ducklin gs died than w hen they received th e v iru s a lo n e .202 In gu in ea p ig s, s u b le th a i d o s a g e le v e ls o f 2 , 3 , 7 ,8 - te tra c h lo r o d ibenzodioxin produced severe atroph y o f the th y m u s w ith destruction o f lym ph ocytes, ly m p h o id d ep letio n in spleen and ly m ph n o d es, as 468 CRC Critical Reviews in Toxicology w ell as exten sive h em orrh ages in to variou s o rgan s inclu ding the adrenals. R ed u ced th y m u s an d sp leen w eig h ts w ere also ob served in rats th at receiv ed a sin gle d o se o f 2 5 p g /k g T C D D an d in m ice the cell m ed iated im m u n ity w as fo u n d to be s u p p r e s s e d .203 T h e v a rio u s fin d in g s liste d h e r e d o n ot con clu sively prove that the im m une respon se is specifically altered b y these com pou n ds as we e n co u n ter it follow in g radiation ex p o su re o r th e ra p y w ith alk y latin g ag e n ts. In m o st stu d ie s relatively liigli d o se s o f the ch em ical c o m p o u n d s w e r e g i v e n , resu ltin g in gen eral to x ic ity a c c o m p a n ie d b y w eig h t lo ss. W eight lo ss in its e lf m a y le a d to a tro p h y o f the sp le e n . In h u m an s m ain tain ed on a low caloric intake w ith negative nitrogen balan ce, a general reduction o f lym ph oid tissue resu lts w hich is n ot specifically related to the im m u n e resp o n se an d vitam in A d efic ie n c y is a lso a c c o m p a n ie d b y a red u ctio n in ly m p h atic tissu e. In th is resp ect it is also o f in terest th at w hen m o n k ey s w ere fed " toxic fat" con tain in g polycyclic polych lorinated h yd rocarbon s. T he germ in al ce n te rs in the ly m p h n o d e s an d sp leen as w ell a s islan d s o f h e m ato p o ie tic ceils in b o n e m a r r o w w e re e x tr e m e ly s p a r s e . ' 60 T h is is m o r e e x te n siv e ly d isc u sse d in c o n n e c tio n w ith c h ic k edem a. T h e fact that a com bin ation o f d u ck h ep atitis virus an d P C B s is m ore to xic to 10day-old d u ck lin gs than the virus alon e su ggests a reduced im m une response b u t could also m erely rep resen t an ad d itive d am agin g effect on the liver, m ade m ore su sceptib le to in fection b y the to x ic , e ffe c ts o f P C B s. S o far no convincing evidence o f " w astin g d isease" as a result o f exp o su re to chlorin ated polycyclic com po u n d s h as been rep o rted in ro d en ts or an y o th er sp ecies in the literatu re. . A tro p h y o f the th y m u s in n ew b orn ro d en ts reduces the cap acity o f these anim als to produce seru m a n tib o d ie s, re su ltin g in w astin g d ise a se , a lso referred to as runt disease or h om ologous disease. Su ch an im als are then in capable o f rejecting fo r e ig n s k in g r a f t s ,204 w h ic h p r o b a b ly r e p r e s e n ts the m o st con vin cin g evidence fo r h o m o lo g o u s disease. A sh ift in the im m une respo n se cou ld in fluence the developm ent and grow th o f can cer an d a lte r th e d e fe n se m ech an ism in in fe c tio n s. In ord er to prove th at P C B s a s w ell as ch lo rin ated d ib en zo d io x in s and furan s d o indeed in fluen ce the im m une respon se, the effect o f these su b stan ces o n new born an im als o f several species sh ould be tested w ith ad eq u ate p aram eters su ch as skin (jO O > - * - 6332  grafting an d th e m easu rin g o f the d ev elo p m en t o f a n tib o d ie s . W hether the o th er ch lorin ated p o ly c y lic c o m p o u n d s in th is article also a ffe c t th e im m une response h as n ot been suggested or re p o rte d . B ioch em ical E ffe c ts an d C hem ical T o x ico lo g y Porphyria P o rp h y ria cu tan ea tard a in h u m an s is an acq u ired d e fe ct in h ep atic porp h y rin m e tab o lism , characterized b y uroporphorinuria, ph otosen sitiv ity as m an ifested b y b listers, and m echanical fragility o f the skin . The h epatic porp h y ria (P orph y ria cu tan ia tard a) w hich is respon sible fo r th e in cre ase in p o rp h y rin s an d the sk in p h o to se n sitivity can be produ ced experim en tally b y a n u m b er o f d ru gs; all o f these have the ab ility to stim u late the activ ity o f the in itial en zy m e in th e hem e sy n th esis 5 -am inolevulinic acid sy n th etase (A L A ). In ad dition to this acquired porph y ria, an acu te in term itten t porph yria erth ropoietica an d an acu te in term itten t porph yria h ep atica, w hich are b o th h ered itary , e x ist to geth er w ith a n u m b er o f su b g ro u p s w h ich w ill n o t b e co n sid e re d h ere. In 1 9 6 4 , B leiberg et al.3 rep orted stu d ies o f 2 9 w orkers in a 2,4-D ( 2 ,4-d ich loroph en oxyacetic acid ) and a 2 ,4 ,5 -T (trich loroph en oxyacetic acid ) facto ry . M any o f the w orkers h ad chloracne and 1 1 h a d a b n o r m a l e x c re tio n o f urinary u ro p o rp h y rin s. M any o f the w ork ers w ith uroporph yrin uria h ad h irsutism , h yp erpigm en tation , an d increased skin fragility . L iver d y sfu n ction w as n o te d in tw o h o sp ita liz e d p a tie n ts. P o lan d e t a l.1 reinvestigated the w ork ers o f the sam e plant stu d ie d b y B leib erg et a l.3 T h e y stu d ie d a to tal o f 7 3 m ale e m p lo y e e s; ch loracn e w as fo u n d in 8 0 % o f the w orkers. In th is study six years after the first rep o rt, clinical porph y ria w as n o t observed an d only one w orker had persisten t uroporphyrin uria. In the Jap an ese poison in g epid em ic (Y u sh o ), in creased u roporph yrin excretion w as n ot reported. A p p aren tly p o ly ch lo rin ated b ip h en y ls, as w ell as tetrach lorod ib en zod ioxin s, produ ce h epatic porph y ria in an im als. E xperim en tal h ep atic p o rp h y ria w as observed in Sh erm an strain rats w h en the ra ts w ere e x p o se d to A ro c lo r 1 2 5 4 in th e d iet. T h e porph yria resem bled h exach loroben zen e p o is o n in g a n d h u m a n p o r p h y r ia c u t a n e a t a r d a .3 0 5 W hen fem ale rats w ere fed 100 ppm A roclor. 1254 in th e d iet (a p p ro x im ate ly 7 .9 m g /k g /d ay ) th ey becam e porph yric after a delay o f ab o u t 2 to 4 m on th s. A t th is tim e th e urinary porph yrin e x c r e t io n in c re a se d rapidly. U roporphyrin excretion w as elevated to a m axim u m o f 5 5 0 -fo ld , proph obilinogen 2 7 -fold , an d 5-am inolevulinic acid 1 8-fold . T h e m a jo r p o rp h y rin s fo u n d in u rin e from A roclor treated rats w ere 8-carboxyporphyrin (7 3 % ) and 7-carboxyporphyrin (16% ). Su b stan tial increases in am o u n ts o f 5 an d 6 carboxcylic porphyrins w ere also observed. A 10-fold in crease in th e e x c re tio n o f co p ro p o rp h y r i n s w a s o b s e r v e d , b u t th e in crease in uroporph yrin excretion w as 1 ,4 0 0 -fo ld . In con trol rat urines, cop rop orp h yrin (8 1 % ) rep resen ted the. m ajo r porph yrin fractio n . A go o d correlation betw een urinary p o rph y rin s an d liver p o rph y rin s w as also observed. O nly a trace o f porph yrin w as fo u n d in liver sam p les in c o n tro l ra ts. W hen feeding rats 100 p p m A ro clo r 12 5 4 , 6-am inolevulinic acid sy n th etase w as n ot increased in the first m on th o f ex p o su re. A fte r this tim e 5-am inolevulinic acid sy nth etase w as elevated 3t o 6-fo ld in p o rp h y ric ra ts b u t w a s n o rm al in nonporphyric A roclor treated rats. C ytoch rom e P -450 and m icrosom al hem e w ere also increased through out the stu d y b u t th e increase w as m axim al in on e w eek d e sp ite th e ab sen ce o f an increase o f the rate lim itin g en zy m e in h em e syn th esis 5-am inolevulinic acid sy n th etase. W hen a large single d o se o f A ro clo r 1 2 5 4 (5 0 0 m g /k g) w as given, a 4-fold in du ction o f 5-am inolevulinic acid sy n th etase co u ld b e d em o n strated in the liver 5 hr after dosing the anim als. T hese results suggested th at 5 -am inolevulinic acid sy n th etase in du ction occurred w hen porph yria h ad developed if low er levels o f A roclor 1254 w ere fed over a period o f tim e, b u t w hen high d o se s o f A ro c lo r w ere given it w as induced im m ediately. V o s a n d K o e m a n 95 r e p o r t e d t h a t c h e m ic a l porph yria w as produced b y the tw o European PCB p ro d u cts, P henoclor D P6 an d C lophen A 60, as w ell as d ie A m erican p ro d u c t, A ro clo r 1 2 6 0 . T h e p o rp h y ro g en ic ac tio n w as o b serv ed in ch ick en s, quail, and rats. It has n ot been establish ed so far w h edier only certain isom ers o f the polychlori n a t e d b ip h en y ls o r co n ta m in atio n w ith a chlorin ated diben zofuran is responsible for the produ ction o f h epatic porph y ria cu tan ea tarda. P o lan d an d G lo v e r304 u se d fe rtile ch ick en eggs a t a developm en tal stage o f 15 to .20 d a y s and in je c te d th em w ith 2 5 p i o f d ie h alogen ated d ib en zo -p -d io xin s. T h e y fo u n d th a t 2 ,3 ,7 ,8-tetrachlorodibenzo-p-dioxin prod u ced a dose-related 03 or oo CO o January 1974 469 C006173 6o o bo DOW 1135891 in crease in A L A sy n th etase activ ity . T h e low est d ose tested, 4 .6 6 x 1 0 ' 1 1 m ol/egg (1 .S ng) d ou bled the enzym e activity. T h ese results w ere statistically significant. S tu d ie s w ith oth er h alo g en ated dibenzo-pd io x in s g a v e th e fo llo w in g results: the 2 ,3,7-trich loro an d 2 ,3,6-trib rom o isom ers are p o te n t in du cers o f A L A sy n th etase w hile 2 .3 - d ic h lo r o and 2,7-d ich loro, 2,8-d ich loro, 1 ,3 ,6 ,8 - t e t r a c h lo r o , an d 1,2,3 ,4 -tetrach lo ro isom ers all failed to induce A L A sy nth etase at d o se s up to 2.5 m g/egg. O f the lim ited num ber o f h alogenated dibenzo-p-dioxins that have been tested for fetal toxicity and the ability to produce ch loracn e, those that are to xic at low d o ses also in duce A L A sy n th etase. W hether som e o f the p o ly n e u ro tic sy m p to m s re p o rte d in Y u sh o an d also in severe ex p o su re to .te c h n ic a l 2 ,4 ,5 -T or tech n ical trich loroph en ol th at w ere heavily co n tam in ated w ith tetrach lo ro d ib en zo d io xin are caused by an effect on the porphyrin m etabolism m erits investigation since n eu rotoxic effects have b e e n r e p o r te d in a c u te in te r m itte n t p o r p h y r ia .1 0 7 T h e d io xin s th at induce A L A syn th etase in the liver o f th e ch ick em b ry o m ay also in d u ce it in the m am m alian system . W oods1 9 * w as not able to in duce 5 -am inolevulinic acid sy n th etase (A L A sy n th e ta se ) in the liver o f m ale ra ts u p to 3 0 d a y s a fte r oral d o ses o f 2,3,7,8-tetrach lo rod ib en zod io xin . G uinea pigs and m ice also did n ot show an increase o f A L A sy n th etase follow ing the adm ini stration o f 2,3,7,8-tetrach lo rod ib en zod ioxin . H ow e v e r, it w as p o ssib le to d e m o n sta te an in crease in A L A sy n th e tase an d u ro p o rp h y rin s in the liver in C j 7 b lac k m ale m ice given 25 tg/kg/w eek fo r 4 w ee k s.109 It is p o ssib le th a t A L A sy n th etase in d u c tio n varies in tim e w ith age an d se x an d m ay sh o w sp ecies variation . In d u ctio n in m am m als m ay req u ire longer d osin g, sin ce, for instance, p o rp h y ria an d in d u ctio n o f A L A sy n th etase w ith m oderate d oses o f A roclor 1254 did not develop u n til 2 to 4 m o n th s a fte r d o sin g w as sta rte d .1 9 5 Fem ale anim als and the em b ry o m ay be m ure su scep tib le than ad u lt m ale anim als. E ffe c t on O th er E n z y m e s a n d o n V itam in A A n um ber o f in vestigators have show n that the p o ly ch lo rin ate d b ip h en y ls in d u ce liver m ic ro so m a l e n z y m e s .1 1 9 '2 11 N o r b a c k and A llen1 1 1 correlated an in crease o f h ep atic m icrosom al en zy m e activity w ith sm o o th en d o p lasm ic reticu lum proliferation . L itterst et al.1 11 stu d ied the effect o f fou r P C B m ixtures, nam ely, A roclor 1242, 1 2 4 8 , 1254, and 1 2 6 0 . T h e anim als w ere fed the m aterial for 4 w eeks an d dietary levels em p lo y ed w ere 5 0 0 , 5 0 , 5 and 0 .5 ppm . A t higher d ietary levels an in crease in liver w eig h ts w as ob served. E lev atio n o f trigly cerides in the liver w as seen at 5 0 0 pp m w ith a m axim u m effect occu rrin g at ap p ro xim ately 50% chlorine con ten t. A n increase in cy toch rom e P 4 5 0 w as observed at the 5 0 an d 5 0 0 p p m d ietary levels o f all PC B m ix tu re s. An in duction o f the pentobarbital h yd roxylation and an in crease in m eth y lase activity w ere observed at the higher d ietary levels. In du ction o f p e n t o b a r b ita l h y d ro x y latio n increased w ith increasing chlorine con ten t o f the PC B s. N itro redu ctase activity w as m ark ed ly stim u lated w ith increasing d o se s o f P C B and the in d u ction w as seen a t levels a s lo w a s 0 .5 p p m fo r all m ix tu re s. It is d ifficu lt to evaluate the enzym e in duction at th ese low d ietary levels since the con trol anim al fo o d can be con tam in ated at tim es w ith P C B s. T w o ty p es o f inducers o f drug-m etabolizing en zy m es o f the liver have been rep orted ; on e g ro u p to w h ich p h n o b arb ital b elo n g s resu lted in in creased P -450 con ten t o f the liver and in d u ction o f a variety o f p ath w ay s including b en zpy ren e h yd roxy lase and dm thylation o f eth ylm orphin e. T h e p o ly c y lic h y d ro carb o n s such as 3,4-ben zpyren e b elon g to the second gro u p , w hich resu lted in the fo rm atio n o f an ab n orm al c y to c h r o m e , P -448, and increased benzpyrene h y d ro xy latio n , b u t not ethylm orphine d m th y la tio n . A lv a re s e t a l .1 1 3 re p o r te d th a t P C B t r e a t m en t p ro d u c e d an in crease in c y to c h ro m e P -4 4 8 , an in crease in b en zp y ren e h y d ro x y la tio n , an d N -d em eth ylation . Sin ce the PC Bs represent m ix tu res, it is possib le that the typ e o f in d u ction m ay depen d upon the isom ers presen t. Tire variou s e ffe c ts o f P C B s on liver m icrosom al en zy m es can be b lo ck ed by the prio r adm in istration o f actino- m ycin D . W hen the e ffe ct o f a num ber o f P C B s on the b e e f h eart m itoch ron drial enzym e sy stem w as stu d ied , it w as foun d th at they in h ibited N A D H - o xid ase, su ccin oxidase, and cytoch rom e oxidase activity. It h as not been establish ed w hether these sam e e ffe c ts w ill o c c u r in vivo. B en th e et a l.1 14 in duced liver m icrosom al en zy m es b y in tra periton eal in jection o f a single dose o f A ro clo r 1248 or 1232. ........................ M ost o f the m icrosom al en zym es have been stu d ie d in th e ra t. b u t th e y are a ffe c te d in o th e r s p e c ie s a s w e ll. A lle n e t a l .1 1 s r e p o r t e d liv e r 470 CRC Critical Reviews in Toxicology C u t i t l x ./i frfifiqpT I /wnn h y p e r t r o p h y a fte r feedin g polych lorin ated biphenyls (A ro d o r 1248) or a polych lorinated triph enyl (A ro d o r 5 4 6 0 ) fo r three m o n th s to p rim ates. T h e in crease in liver size w as attrib u te d to the in crease in sm o o th en d oplasm ic reticu lu m and induction o f m icrosom al en zym es w as observed. P ro o f o f the induction o f m icrosom al e n zy m e s in b ird s is th e fin d in g m ad e b y L in c e r an d P eakall216 that livers ob tain ed from A m erican kestrels exposed to either A ro d o r 1254 or A roclor 1 2 6 2 sh o w ed an in crease in th e in v itro b re ak d o w n o f oestradiol to a m ore polar m etab olite. T h is e ffe c t on se x h o rm o n es is also em ph asized b y P latonow et a l.2 ' 7 A roclor 1 2 5 4 given to Y orksh ire b oars redu ced th e urin ary excretio n o f gonadal steroids. B o th th e p o ly ch lo rin ated b ip h en y ls a s w ell a s th e ch lo rin ated n ap h th alen es a ffe c t th e v ita m in A con ten t o f th e liver. T h is w as particu larly stu d ied w ith th e ch lo rin ated n ap h th alen es sin ce h y p erk e r ato sis and oth er ab n orm al keratin fo rm atio n o f the skin is o ften accom p an ied b y vitam in A deficien cy. Low vitam in A plasm a levels w ere o b se rv e d in c a ttle w ith X -d ise ase an d y o u n g sw in e. ' s 5,162 A d e c re a se in v itam in A sto ra g e in th e liver o f Ja p a n e se qu ail an d rats that w ere fed A roclor 1 2 4 2 w as also d em o n strated .2 " 1 Sin ce vitam in A d e fid e n c y affe c ts rep rodu ction , this aspect should be furth er investigated to determ ine w hether the e ffe c t o f P C B s o n re p ro d u ctio n is secon d ary to vitam in A d eficien cy .2 ' 9 E cobich on and Jo h n sto n e 220 stu died the effect o f various PC B isom ers o n the h ep atic en zym e system . B iphenyl itse lf d id not cau se in du ction o f h ep atic d ru g m etab o lizin g en zy m es and n eith er did m o n o ch lo ro b ip h en y l (4-C1). S u b stitu tio n o f ch lo ro g r o u p s a t th e th ree an d fo u r p o sitio n s o f th e diand tetrach lorobiph en yls caused in duction o f m icrosom al m on oo xygen ases. In du ction o f m icro som al m onooxygenases occurred also w hen hexaan d o ctach lo ro b ip h en y ls w ere given to the rats. N itroreductase an d carbo xy lesterase activities w ere n o t affe c te d w h ile all c o m p o u n d s p ro d u ced a m arked in du ction o f the su lfobrom oph th alein c o n ju g a tin g s y ste m . T erp h en y ls a s w ell a s c h lo ri n ated terph en yls increased th e p en tobarb ital m eta b o lism in th e rat w ith c o rre sp o n d in g in crease in sm o o th en d oplasm ic reticu lu m w hich su ggested m ic r o s o m a l e n z y m e i n d u c t i o n .2 2 1 ,2 2 2 L u cier et a l.223 rep orted that a single oral d o se o f 5 o r 2 5 p g /k g b o d y w eig h t o f tetrach lo ro d ib en z o d io x in in d u c e d c y to c h r o m e P -4 5 0 sig n ifi c a n tly in m ale ra ts fo r a p erio d o f u p to 2 8 d a y s. C y toch ro m e b-5 con ten t w as also in creased. H y d roxy lation o f aniline w as in d u ced an d am inopyrin e dem ethylation rates w ere decreased. M icro som al proline w as increased b y ap p ro x im ately " ' 15% . T h e induction o f U D P glucu ron yltran sferase w as m ost striking. O xidative p h osp h ory lation rates in rat liver m itoch on dria usin g su ccin ate as a su b strate w ere unchanged. The enzym e in du ction r w as observed as early a s one day - the p eak in du ction w as reached betw een three and ten d ays after treatm en t. The enzym e induction w as acco m p an ied b y an in crease in sm o o th en d o p lasm ic reticu lu m an d a m ild in crease in rough en d o p lasm ic reticu lu m .224 T h ese ch an ges w ere first ob served three d ay s a fter th e in jectio n o f the tetrach lorod ib en zod ioxin . T h u s, enlargem ent o f the liver during exp osu re to variou s ch lorin ated c o m p o u n d s is a c c o m p an ie d b y an in c re ase in sm o o th en d op lasm ic reticu lu m . F u rth erm o re, i f " in clu sion s" o r " h yaline b o d ies" are seen w ithin the cy to p lasm w ith th e light m icro sco p e, con cen trically arranged m em bran es th at o fte n su rrou n d lipid v acu oles can b e seen w ith the electron m icroscope (Figure 6). H exach lorop h en e, o n th e o th e r h a n d , d o e s n o t increase th e size o f th e liver. ' ' 8 T h e livers show n o u lt r a s t r u c t u r a l changes and probably m icro so m al en zym es are n ot in d u ced . W hether o th e r c o m p o u n d s listed in F ig u re 2 a ffe c t liver m icrosom al enzym es is n ot know n. H exach lor op h en e h as an effect on a variety o f oth er enzym e sy stem s than those m entioned th u s far. C am m er a n d M o o r e 2 2 5 a n d C a ld w e ll e t a l .2 2 6 h a v e sh o w n t h a t h e x a c h lo ro p h e n e u n cou pled oxid ative p h o rp h o ry latio n , increased ad en o sin e trip h o s p h atase a c tiv ity .b u t did n o t a ffe c t th e activities o f electro n transport en zym es w hen com pared to co n tro ls. G ou ld et a l.2 2 7 d em on strated th at h exa ch loroph en e inhibited su ccin o xid ase activity o f rat h eart, liver, an d kid n ey p rep aratio n s. A cco rd in g to un publish ed m aterial cited b y G lu ck ,228 h exa ch lo ro p h en e is co n ju gated to th e glu cu ron id e co n ju gate w hich is excreted in bile. T h e p ro p o rtio n o f con ju gated h exach lo rop h en e to u n con ju gated h ex ach lo rop h en e in b lood m a y b e m o re sign ifican t in d eterm in in g th e to x ic ity o f h exach lorop h en e than th e h exach loroph en e b lood levels as they are presen tly determ ined. M eth em oglob in em ia In the early 1960s o u tb reak s o f m eth em oglob- C rO O U -A -O ui January 1 9 7 4 6 4 ^ 3 5 689611 M O Q in e m ia s w ere reported From h ospital nur se rie s.2 ,9 ,13 0 O n e o u tb re ak involved a to tal o f 18 in fa n ts w ith a level o f m eth em o glo bin from 4 .9 to 30% o f th e to tal h em oglobin . A n epidem iological in v e s tig a tio n tra c e d th e o u t b r e a k to 3 ,4 ,4 'trich lo ro carb an ilid e (T C C ) th a t w as u sed a s a 2% so lu tio n in a rinse in th e lau ndering p rocess, after w h ich th e lau n d ry w as n eu tralized w ith an acid an d th en d ried in an au toclav e. Tw elve o f the eigh teen c a se s in th is rep o rt w ere p rem atu res w ho w ere prob ab ly lon ger exposed to the offen din g ch em ical than term in fan ts since their h ospital stay is u su ally lon ger. It is also possible that ch em icals p en etrate m o re easily th rough th e skin o f prem a tu re s. I n an o th er o u tb reak , m ethem oglobinem ia o c c u rre d a m o n g a g r o u p o f cu sto d ia l p a tie n ts in an in stitu tio n . T h ese p atien ts w ere given so ap su ds en em as w ith boiled so ap that con tain ed 2% T C C .1 3 1 It is possib le that b y heating T C C a breakdow n p ro d u c t o ccu rred w hich p rodu ced th e m eth em o glo b in em ia. P -ch loroaniline an d 3,4-dich loroan ilin e have been suggested as breakdow n products. N one o f th e o th e r co m p o u n d s discu ssed in th is article have thus far been show n to produce m eth em o globin em ia. S to ra g e , D istrib u tio n , M etab olism , a n d E x c re tio n A s w e p ro g re ss fu rth e r in o u r stu d y o n the d i s t r ib u t i o n , m e ta b o lis m , excretion , and ab so rp tio n o f co m p o u n d s such as chlorinated b ip h en y ls, ch lorin ated terph enyls, th e chlorinated d ib en zo d io xin s, an d ch lorin ated n aphthalenes, we w ill p ro b a b ly And th at sin ce th ey are lipid so lu b le th ey are sto red in q u ite the sam e w ay as the m ore stu d ied persistent pesticides such as D D T and dieldrin . F ro m th e evidence presen tly available on th e ch lo rin ated b iph en y ls it can b e assu m ed that the tu rn o v e r o f th ese c o m p o u n d s in th e b o d y , p a rtic u la rly o f th o se th a t h av e a high ch lo rin e co n ten t, m ay be slow er than som e o f the p e rsiste n t p e stic id e s. T h e tu rn o v er rate w ill v ary w ith th e d egree o f ch lo rin atio n . H exach lorop h en e, on th e o th er h an d , is fairly rapidly excreted and p resen tly n o in fo rm ation is available on bith ion ol and related co m p o u n d s that have a germ icidal e ffe c t. A ll o f th ese co m p o u n d s are n ot vety w ell ab so rb ed b y th e gastroin testin al tract o r the skin. A n aly sis o f v ario u s tissu es o f rats given a single d o se o f the PC B A roclor I2 5 4 232 show ed the h ig h e st c o n c e n tra tio n o f P C B s in ad ip o se tissu e and th e least in plasm a. B rain, liver, an d kidney had levels o f the sam e order o f m agn itude but w ere low er. If, for instance, 1 ,6 0 0 m g/k g b od y w eight o f A roclor 1254 w ere given b y sto m ach tu b e , 2 4 h r la te r th e m e an c o n c e n tr a tio n in ad ip ose tissue w as 1,146 ppm . T h e con cen tration s observed in individual an im als varied greatly. Sim ilar levels w ere fou n d in ad ip o se tissu e w hen 3 ,2 0 0 m g /k g o f A roclor 1260 w ere given by sto m ach tu b e to ad u lt fem ale rats. T h e levels o f A ro c lo r 1 2 5 4 fo u n d in the b rain sh ow ed a m ean o f 13 8 p p m an d the values observed for A roclor 1260 w ere 145 p p m . A ll o f the values w ere calculated on a w et w eigh t b a sis. T h e sam e c o n c e n tra tio n s in p p m w ere fo u n d w ith b o th co m p o u n d s even though on ly h a lf o f the am oun t o f A roclor 1254 w as given. T h is is probably d u e to the fact that on ly a certain am ou n t o f A roclor is ab sorbed a t a given tim e. W hen 1 0 0 p p m o f A roclor 1 2 5 4 w as fed to m ale w eanling rats fo r 5 8 days the m ean con cen tratio n o f A ro clo r derived m aterials fo u n d in the fe c e s v aried ro u g h ly b etw een 11 a n d 3 5 p p m . A fter the anim als had been rem oved from the ex p erim en tal d iet fo r a w eek, th e ex cretio n in the feces w as 8 ppm and after 16 d ay s' recovery an average o f 2 .5 ppm w as found in the feces. P C B s at the con cen tration o f about 2 ppm w ere still ob served in th e feces 2 4 d ay s a fte r the an im als w ere rem oved from the experim en tal d iets. D uring th e en tire stu d y the am oun t o f P C B derived m aterial recovered fro m the urine rem ained b elow 1 p p m . W hile P C B s w ere fed to th e ra ts, th e gas chrom atogram obtained from the feces w as superim posable on the on e obtained from the stan dard A roclor 1 254 that w as fed to the rats. O nce the an im als w ere n o lon ger fed A ro clo r 1 2 5 4 the n u m b e r o f p e a k s ob served in th e g a s c h ro m a togram s that resulted from the an aly sis o f th e fecal m aterial decreased. Few er p eak s w ere also o b serv ed in th e g a s ch ro m a to g ra m s o b ta in ed fo r th e tissu es an d fro m urine. D u rin g th e 5 8 -d ay feed in g perio d , a gradu al increase in the con cen tra tio n o f th e P C B d eriv ativ es w as o b se rv e d in tissu e s. T h e h ig h est co n c e n tra tio n w as fo u n d in ad ip o se tissu e: after 5 8 days it reached m ean valu es o f ab o u t 5 0 0 p p m . T h e c o n ce n tratio n in the liver on ly am o u n ted to a m ean o f 1 2 p p m a t that tim e. F ollow in g a recovery period o f 71 d a y s w hen th e a n im a ls w e r e fed plain ch o w , the c o n c e n tr a tio n o f th e P C B d e riv e d m a te ria l in ad ip o se tissue had not decreased ap p reciab ly and 472 CMC Critical Reviews in Toxicology CCOoiSj. HflW U35894 (till sh o w ed valu es in the n eigh borh o od o f a b o u t 4 0 0 p p m an d 4 .7 p p m w as fo u n d in the liver. T h e c o n c e n tra tio n in th e b ra in n ever reach ed very high p ro p o rtio n s; it ran ged fro m ab o u t 2 to 4 p p m . F o llo w in g a 7 1 -d ay re c o v e ry p e rio d th e a m o u n t in th e brain w as still ab o u t 2 ppm . W hen A roclor 1 254 w as fed fo r 2 4 0 d ay s at the dietary level o f 1 0 0 p p m , th e con cen tration o f th e PC B derived m aterial ob served in ad ip o se tissu e h ad reach ed values o f ab o u t 1 ,0 0 0 pp m . If, on the oth er h an d, 1 0 0 p p m o f D D T is fed to m ale rats fo r 2 y ears, the con cen tration in ad ip o se tissue is ab o u t 9 5 p p m .233 In a sep arate stu d y w here 5 0 0 pp m o f A ro clo r 1 2 5 4 w as fed to m ale rats fo r 6 m on th s an d th e e x p o su re to. P C B co n tain in g d iets w as su b seq u en tly discontinued fo r an ad dition al 10 m on th s, th e con cen tration o f th e P C B s ranged fro m 9 2 4 to 1 ,6 8 8 p p m in ad ip o se tissu e w ith an arith m etic m ean o f 1 ,1 9 2 ppm . T h e concen tratio n s in th e liver ran ged from 1 7 .3 p p m to 2 6 .2 4 p p m w ith an arith m etic m ean o f 2 2 .6 5 _p p_m_ . 2 3 4 F u rth er stu d ies are n ecessary to determ ine w h eth er P C B s are sto re d in tissu e s to a certain poin t w hen a stead y state is reached and the co n cen tratio n d o e s n o t in crease fu rth er. T h e given co n cen tratio n at w hich th is eq u ilib riu m is reached in ad ip o se tissu e w ou ld vary w ith th e am o u n t fed to the an im als and the typ e o f co m p o u n d . T h e e ffe c t o f starvation o r illness w hen the am ou n t o f a d ip o se tissu e in th e m am m alian b o d y is su d d en ly redu ced also n eeds to b e investigated further. W ould th is lead to in creased e x cretio n o f the m aterial o r to redistribu tion o r b o th ? D ahlgren et a l.3 3 s w h o d e term in ed P C B tissu e lev els in ph easan ts, found th at a con cen tration o f 3 0 0 to 4 0 0 pp m o f the P C B derived m aterial w as u su ally fo u n d in th e b rain s o f b ird s th a t h ad d ie d , w hile live b ird s th at w ere fed th e sam e am o u n t o f PC B s h ad m u ch low er co n cen tratio n s in their brain tissu e . S tu d ies in b ob w h ite q u ail show ed th at relative p e a k h eigh ts an d ch ro m ato g ram s fo r th e tissue e x trac ts w ere sim ilar to the A roclor stan dard after p artitio n in g w ith aceton itrile-h exan e w hen th e b ird s w ere given a sin gle cap su le o f A ro clo r and their carcasses w ere an alyzed soon after exposure. T h is suggests ab so rp tio n o f all com po n en ts o f A ro clo r. T h e gastro in testin al tract, liver, sk in , w in gs, and feet h ad been rem oved prior to an alysis. W hen b obw h ite quail w ere fed A ro clo r 1 2 5 4 fo r 1 4 d ay s an d then allow ed to recover, the a s ch rom atogram s sh o w ed a g ra d u a l re d u ctio n in the num ber o f 'p eak s an d after 4 2 days on a con trol diet o n ly 4 m ajo r P C B co m p o n en ts rem ain, w hich suggested selective excretio n an d /o r m etabolism o f som e o f the com pon en ts o f the A ro clo r teste d .234 In a re p ro d u ctio n stu d y , H eath e t a l .99 a n a ly z e d 2 s e c o n d - y e a r e g g s o f m a lla r d s fed 2 5 ppm A roclor 1 2 5 4 in their d iet fo r 2 y ears and found 56 and 33 ppm P C B b y w et w eight. T h e gas chrom atogram show ed a pron oun ced dim in u tio n o f the early peaks w hen it w as com p ared to the stan d ard , suggesting again a selective redu ction o f th e co m p o n en ts w ith lo w er ch lo rin e con cen tratio n s. T h is is a lso e v id en t in en v iro n m en tal sam ples. Low er organ ism s su ch as m u ssels and fish con tain ed a higher con cen tration o f PC Bs w ith low er chlorin ation than b ird s.1 3 In m am m als, excretio n o f PC Bs in m ilk and transplacental passage is also ob served. F e e d in g . cow s 2 0 0 m g/d ay o f A roclor 1254 fo r 6 0 d ays2 * resu lted in h ig h er P C B levels in m ilk fa t th an in body f a t A t 30 days' exposure, 4 5 .6 ppm w as fo u n d in m ilk fa t an d 2 1 .9 p p m in b o d y fa t; a t 6 0 d a y s' ex p o su re, 6 6 .7 p p m w as fou n d in m ilk fat and 4 4 p p m in b o d y fa t. H ow ever, o n ce exp osu re to P C B s w as d isco n tin u ed , th e con cen tration in m ilk fat san k b elow that o f b o d y fat. T ransplacental passage o f . P C B s also occu rred.2 3 7 '2 3 * Sh erm an strain rats w ere given A ro clo r 1 2 5 4 in p ean u t o il b y sto m ach tu b e during days 7 to 15 o f gestatio n . A m ean o f 2 0 .6 0 ppm PC B derived m aterial w as fo u n d in their m ilk w hen 10 m g /k g/d ay w as given to th e d am s during pregnancy, w hile th ose given 5 0 m g /k g/d ay ex creted a m ean o f 6 6 .3 4 p p m in th eir m ilk o n a w et w eight b asis. F etu ses taken b y C aesarean section on day 20 o f pregnancy con tain ed a m ean o f 0 .6 3 p p m an d 1.38 pp m o f P C B s, respectively . W ith a fiv efo ld d iffe re n c e in d o sa g e , th e c o n ce n tra tio n in fe tu se s sh o w ed a tw o fo ld d iffe re n c e an d in m ilk a th reefold d iffe re n c e .2 3 * T h e h y p erp ig m e n te d b a b ie s o b se rv e d in th e Y u sh o in cid en ts in Ja p a n rep resen t ad d itio n al circum stantial evidence o f tran splacen tal passage o f PCBs. Polych lorinated biphenyls have also been de tected in h u m an ad ip o se tissu e . B iros et al.239 an alyzed tw o h um an a d ip o se tissu e sam p les w hich con tain ed 2 0 0 an d 6 0 0 pp m PC Bs ranging from pentachlorobiph enyl to decach lorobiph enyl and Price an d W elch 240 fo u n d 1 0 0 p p m P C B s in an oth er h um an ad ip ose tissu e sam p le. A ccording January 1974 473 CG0t>13 :S37 esseri MOQ . to P rice a n d W elch,240. 4 J to 4 5 % o f th e gen eral p o p u la tio n in th e U .S . c o n ta in 1 p p m o r m o re 1 / 3 P C B s in their a d ip o se tissu e. A m o n g P C B so u rce s th ey discussed the fo o d chain an d h ouse du st '" w h ic h , fro m resid en ces in so u th w estern M ich igan , ` con tain ed up to 180 pp m PC Bs. Polychlorinated ~ b ip h e n y ls w ere fo u n d in 3 1 % o f 6 3 7 sa m p le s o f h um an ad ip ose tissue th at w ere collected from the general population o f the U .S ., as part o f the H um an M onitoring Su rvey. O f the sam p les an a ly ze d , 5% co n tain ed m ore than 2 p p m .241 P C B s h ave a lso b een fo u n d in h u m an a d ip o se tissu e s o f the general population o f Scan d in avia242 and Ja p a n ,2 4 4 ,2 4 S an d in h u m an m ilk an d h u m an ad ip o se tissue o f the general p op u lation o f G e rm an y .243 In gen eral, low co n c e n tra tio n s o f P C B s w ere sto re d in ad ip o se tissu e in a certain p r o p o r tio n o f th e gen eral p o p u la tio n . T h is is p ro b ab ly m ore prevalen t in th e highly in d u strial ized co u n tries an d alm o st n o n ex isten t in u n d er developed coun tries unless m ost o f the fo o d w as im p o rted from pollu ted areas. T h ese low levels o f PC B s are h arm less, as far as w e know at the m o m e n t; h o w ev er, th e o c c a sio n a l levels fo u n d in th e 1 0 0 to 1 ,0 0 0 p p m ran ge give rise to co n c e rn . It w ou ld be o f interest to establish w hether average ad ip o se tissu e levels in crease w ith age and w h eth er th o se in certain p ro fe ssio n s su ch as p a in te rs, p rin te rs, te x tile w o rk e rs, w o rk ers in p lastic fac to rie s, an d th o se en gaged in th e m a n u fa c tu re o f tran sfo rm ers an d cap acito rs h ad higher P C B levels than the general population . R ep orts on the ab sorp tion , storage, and m etab olism o f chlorinated naphthalenes, terphenyls, dich loroph en e, b ith io n o ls, fe n tid o r, triclosan , and te tra c h lo ro salic y la n ilid e are a lm o st n o n e x iste n t in the literatu re. Som e unpublish ed results on som e o f th e c o m p o u n d s u sed a s g erm icid es are m e n tio n e d in the su m m ary m in u te s o f the O v er the C ou n ter D rug Panel on an tim icrobials. C op ies can be o b tain ed by w riting to D r. M ary B ru ch , B ureau o f D rugs, R ockville, M d. A p p r o x im a te ly 9 % o f triclo san , a lso called Irgasan D P 3 0 0 an d C H 3 S 6 S (2 ,4 ,4 '-trich lo ro -2 'h y d ro x y d ip h e n y l e th e r), is ab so rb e d w hen it is a p p lie d d erm ally in a s o a p so lu tio n . T h e h a lflife o f the chem ical w as 20 hr w hen ap plied derm ally and 1 0 h r w hen given in trav en o u sly . H u m an s e x c re te 6 5 % in urine an d 20% in feces a fte r' in trav e n o u s in je c tio n . It is e x c re te d e ith e r a s a free c o m p o u n d o r as a glucuronide. In d o g s n early 1 00% o f the in je c ted m a te ria l is 474 CRC Critical Reviews in Toxicology recovered a s th e .glucuronide o r su lfa te in urine an d feces over a 5-day p erio d. It h as been show n that T C C is absorbed through the skin. T issu e levels o f the ch lo rin ated terp h cn y l A r o c lo r 5 4 6 0 w ere d e te rm in e d in c o d fis h .244 In th is stu d y it w as fou n d th a t A ro clo r 5 4 6 0 w as apparen tly poorly absorbed from the gastrointesti nal tra c t; it w as sto re d in all tissu e s th at w ere an a ly z e d . T h e h igh est c o n c e n tra tio n w as fo u n d in the liver an d follow in g a single d ose o f 1 g som e o f it w as still p resen t in the c o d fish after 7 0 d ay s, an in dication o f slow excretion . T h e au th ors pointed o u t th at w ith the m eth od u se d to determ in e the ch lo rin ated terp h en y ls, it w o u ld be d iffic u lt to distingu ish chlorinated b iph en yls from chlorinated terphenyls. A com parison o f the chrom atogram s o f the stan d ard w ith those o b tain ed fro ih ch lorin ated terph en yls extracted fro m tissu e s're v e ale d m ore o f th e earlier peaks in m aterial ob tain ed from tissu es and few er o f th e later peaks. A v ariatio n in th e in te n sity o f th e p e a k s w as also o b se rv e d . T h ere seem s to b e a ch an ge in the ch lo rin ated terph enyls w ithin the living organ ism ; h ow ever, part o f th is cou ld also have been intro duced as an artefact by the an alytical m eth ods or b y differen tial ab sorption from the gastrointestinal tract. C alves b o m to co w s w ith X -disease d eveloped h y p erk erato sis an d it w as fou n d that highly c h lo r in a te d n ap h th alen es w ere e x c re te d in m ilk . 1 56 T h is s u g g e s t s t h a t c h lo r in a t e d n a p h t h a len es m a y w ell fo llo w sim ilar sto rag e an d excretio n pattern s as PCBs. C h lorin ated d ib e n zo d io x in s an d fu ra n s are p ro b a b ly a lso sto re d in tissu e s o f an im a ls an d m en w ho are exp o sed to them , b u t again n ot m uch in fo rm atio n is p resen tly av ailab le. F ir e s to n e . et a l.242 fed th e u n sa p o n ifia b le fra ctio n iso lated from " toxic fat" or 3% " toxic fat" itself to young cockerels. T h e " to xic fa t" con tain ed chlorodib en z o d io x in s w ith 2 to 8 ch lorin e a to m s. W hen th e ex cre ta w ere an alyzed it w as fo u n d that a certain percentage o f the h exach lotod iben zod ioxin s had been absorbed from the gastrointest inal tra c t, w hile all o f th e o ctach lo ro d ib e n zo d io x in s rem ain ed in the g astro in te stin al tra c t, and only very little o f the h ep tach lorod ib en zod ioxin w as ab sorbed . Because o f the presence o f inter fering com po n en ts, the chick tissu es and excreta cou ld n ot be exam in ed for th e low er ch lo rin ated d io x in s. In th e o rgan s, th e h igh est co n ce n tratio n w as fo u n d in th e liver an d an a p p re c ia b le am o u n t CGOoiSG 6938 DOW 1135896 w as also- fo u n d in th e b on e a n d sk in , w h ile th e oth er organ s con tain ed very few d io xin s. So m e evidence for a possible m etabolism o f hexa- and h ep tach lo ro d ib en zo d io xin by the ch ick w as also ob served . Z itk o an d W ildish2 4 " fed di-, tri-, tetra-, an d o ctach lo ro d ib e n zo fu ran to fish an d d etected o n ly o c ta c h lo ro d ib e n z o fu ra n in m u scle an d gu t o f d ead fish . O ne p ro b lem w ith m e ta b o lism , sto rag e , an d distribution stu d ies o f chlorinated dibenzod io x in s an d fu ran s is that not all o f the c o m p o u n d s are readily available an d, if th ey are av ailab le, an y long-term feedin g stu d ie s in large anim als are expensive. The very toxic dioxin co m p o u n d s can be given on ly at su ch low levels th at th ey e scap e d etectio n easily . In o u r la b o ra to ry w e d e te c te d a ch lo rin ated d ib e n z o fu ra n in th e urine o f rats fed A ro d o r 1254. We fo u n d th is sam e c h lo r in a t e d d ib e n z o fu r a n in th e A r o c lo r i t s e l f 40 a s a contam inant. W illiam s et a l.249 fed rats a to ta l o f 2 2 .7 an d 1 2 0 .7 ng o f o ctach lo ro d ib en zo d io x in . T h ey re covered a great deal o f the com po u n d in th e feces. V ery sm all am o u n ts w ere recovered in th e liver at b o th d o sag e levels an d in a d ip o se tissu e a t th e higher dosage level. F u rth er stu d ies are n eeded to establish the ab sorp tion , excretion , and storage o f these com p o u n d s, an d their persisten ce. Stu d ies w ith labeled m aterial m ay even tually give u s so m e in fo rm a tio n on th eir m e ta b o lism . P ip er et a l.150 gave a sin gle o ral d o se o f C 14 lab e le d 2 3 ,7 ,8 tetrachlorodibenzo-p-dioxin to rats and foun d that ab ou t h alf o f the radioactive m aterial w as re covered w ithin 21 d a y s in the fe ce s an d o n ly 13% in urine an d 3% in ex p ired air. M ost o f the rem aining radioactive m aterial w as fo u n d in the liver. In an oth er stu d y 2 11 100 jig o f C l3 6 o ctach lo ro d ib en zo d io x in w as given d aily to rats b y stom ach tube for 21 d ay s. L ess than 0 .3 % o f the m aterial w as ab so rb ed . Su bsequ en tly the rats received a con trol d iet for 7 w eeks an d still show ed 20% o f the ab sorbed m aterial foun d at 21 d a y s in their tissu es. T h e in vivo m e ta b o lite s o f th e v a rio u s c o m p o u n d s u n d er d isc u ssio n h ave in m o st in stan c e s n ot been iden tified. B lock and C orn ish 233 stu d ied the urinary excretio n o f biph en yl an d 4c h lo ro b ip h en y ! in ra b b its. B ip h e n y lg lu c o sid u ro n ic acid and 4-hydroxybiphen yl w ere isolated from urine o f rab b its fed b iph en y ls. T h e ra b b its fed the 4 -c hI o r o b i p h e n y 1 e x c re te d 4 -(p ch lo ro p h en y l)-p h cn o l an d 4 -ch lorob ip h en y l glu- co sid u ro n id e: Tw ice a s m uch 4 -ch lorob ip h en y l as biphenyl w as excreted as the glucosiduron ic acid d eriv ativ e. It is p o ssib le th at o th e r low ch lo rin ated b ip h e n y ls an d n ap h th ale n e s are e x c re te d in urine in a sim ilar m an n er: w h eth er they w ould b e d etected by present gas ch rom atograph ic m eth ods w ould have to be checked by com bin in g the id e n tificatio n o f radio lab eled m aterial w ith gas chrom atography. H u tzin ger et a l.233 fo u n d eviden ce th at so m e ch lorin ated biphenyls are h yd roxy lated by som e species. A pparently trou t is not able to hydroxylate 4-ch lorob iph en y l, 4 .4 '-d ich lo ro b ip h cn y l. and 2 .2 ',5 ,5 '-tetrach Io ro b ip h en y l, w hile the rat an d .lh e pigeon both form ed m on oh ydroxych lorobiph en yl fro m 4 -ch lo ro b ip h e n y l. In a d d itio n , th e rat a lso produced dih ydroch lorobiph en yl. Large qu an tities o f unchanged m aterial w ere excreted b y the rat w h e n 4 ,4 '- d ic h lo r o b ip h e n y l and 2 ,2 *,5 ,5 'tetrach lorobiph en yl w ere given to them an d only a m o n o h y d ro x y d eriv ativ e w as iden tified in their urine. Pigeons follow ed a sim ilar pattern but no d ih y d ro ch lo ro b ip h en y l w as o b served in th e b ird excreta fed 4-chlorobiphenyl. N o h yd roxy m etab o lite s cou ld be id en tified in the excreta o f the th ree sp ecies w hen th ey w ere given 2 ,2 ',4 ,4 *5 ,5 'h exach lorobiph enyl. N o evidence o f reductive d ech lo rin atio n w as o b serv ed in an y sp e c ies. O nly very few stu d ies have been con d u cted w ith radiolabeled h exach broph cn c to stu d y its a b s o r p tio n , d istr ib u tio n , an d m e ta b o lism . Wit an d V an G en d eren 234 gave rats and rab b its h exac h lo ro p h e n e -C 14 (2 ,2 *-m e th y le n e -C 14 b is(3 ,4 ,6 trichloroph en ol). C om plete recovery o f radio ac tiv ity in e x c re ta a fte r a sin gle d o se o f 5 0 or 15 m g/k g o f radioactive h exach loroph en e w as o b serv ed in ra b b its w ithin 5 d a y s. R o u g h ly o n e third w as e x c re te d in urin e an d fe c e s, re sp e c tiv ely , as unchanged h exach lorop h en e and the rem ainder w as e x c re te d in th e fe ce s a s u n id e n tifie d m e ta b olites. The m etabolites cou ld not be extracted fro m the feces w ith eth an o l. T h e b io lo gical h alflife calcu lated from the experim en tal results w as fo u n d to b e a p p ro x im a te ly 1 7 .8 h r. In r a ts, a b o u t tw o th ird s o f th e ra d io a c tiv ity w as reco v ered in fe c e s w ith in seven d a y s a n d o n ly very little in urine. A com p lete recovery o f radioactivity w as n o t o b ta in e d in th ese stu d ie s w ith in seven d a y s. Five co w s given a single d o se o f 15 m g /k g b o d y w eight o f h exach lo rop h en e excreted ab o u t h alf to tw o th ird s in th eir fe c e s w ith in five d a y s. V ery little w as e x c re te d in u rin e an d n on e in m ilk. January 1974 475 CGOoiSi f>S3$ ft DOW 1 135897 M etab o lites w ere n o t id en tified an d tissu e levels w ere not d eterm in ed in th is stu d y . T h e au th o rs cited a stu d y by K o k ,2 S w ho did determ ine h e x ac h lo ro p h e n e in v a rio u s o rg a n s o f rats. C arroll et aI.2S6 stu d ie d th e ab so rp tio n o f C -14 labeled h exach lorop h en e on the tail skin o f the ra t. H ex ach lo ro p h en e in a d eterg en t so lu tio n w as ab so rb ed b y the in tact skin o f the tail at the average rate o f 1.7 p g /e m 2 o f ex p o sed surface area/h r. Im m ediately after the in fliction o f second d egree b u m s, the ab so rp tio n rate increased 2 .S tim es that o f the norm al skin su rface. A fter 2 4 hr the absorption rate d ropped to norm al or sub n orm al levels. Im m ediately after trau m atizing the tail, the ab sorp tion rate o f hexach lorophen e w as even greater than follow in g the in fliction o f a b u m w ound. M ah ow itz an d Jo h n sto n 2 57 fou n d that h ex a ch loroph en e w as d ep o sited on the skin w hen used in vario u s fo rm u latio n s an d co u ld be extracted from the skin by soak in g the'ex p o sed areas (hands an d fo re a rm s) in a lc o h o l. T h e am o u n t recovered in cre ase d in p ro p o rtio n to th e am o u n t ap p lie d . T h ese au th o rs w ere u n ab le to con firm earlier in v estigatio n s w hich su gg ested th at the qu an tity o f h ex ach lo ro p h en e retain ed b y th e skin reached a p la te au level a fte r a n u m b er o f w ash ings an d rem ained relatively co n stan t th ereafter. A m is were w ash ed and rinsed every 2 0 m in. W hen extraction s w ith alco h o l w ere m ad e on d ifferen t arm s after d ifferen t n u m bers o f w ash in gs, it w as fou n d that th e am o u n t o f h ex ach lo ro p h en e recovered in creased w ith the n u m b er o f w ashings an d no p lateau w as reached. A greater am ount o f h exa c h lo ro p h e n e w as also d e p o site d o n the skin w ith in creasin g len gth o f tim e o f a single w ashing. Since w e now kn ow that h ex ach lo ro p h en e is also ab so rb e d fro m th e in ta c t skin an d so m e o f it is p ro b ab ly also lost front the su rface o f the skin, the t im e in te rv al b e tw e e n th e w ash in g s p la y s a ro le in the am ou n t o f h exach lorop h en e found to accu m u late on the skin. A lso o f interest is that bathing w a te r c o n ta in in g 4 m g/1 o f h ex ach lo ro p h e n e d e p o sited ap p ro x im ately th e sam e am ou n t o f the ch em ical o n th e skin as a sin gle w ash in g w ith a 2% h exach loroph en e soap . Sin ce h exach lorop h en e w as used very exten sively and o ften unknow ingly b y the general p o p u la tio n , w e in v estigated w h eth er it w as presen t in the b lo o d o f a few ran dom sam p les o f the general p op u lation . We fou n d that the arithm etic m e an c o n c e n tra tio n o f h ex ac h lo ro p h e n e in ad u lts 476 CRC Critical Reviews in Toxicology w as 0 .0 2 8 ppm (tg/g) o f w hole b lood o f a to tal o f 14 sam p le s. In in fa n ts w h o w ere w ash ed w ith h ex ach lo ro p h en e in the n ursery the am o u n t o f h ex ach lo ro p h e n e fou n d in w h ole b lo o d a fte r the infants had anyw here from 2 to 14 w ashes averaged 0 .1 0 9 pp m (p g lg w hole b lo o d ). B lo o d ob tain ed from the cord at the tim e o f birth con tain ed an arithm etic m ean o f 0.022 ppm h e x a c h lo r o p h e n e .2 5 8 '2 s 9 In the rats th at w ere u sed in a re p ro d u ctio n stu d y an d w ere fed 100 p p m h ex ach lo ro p h e n e in the diet fo r 2 5 8 d ay s, the h exach lorop h en e b lood levels averaged 1.21 p p m , w hile the b lo o d levels o f rats fed 5 0 0 pp m o f h exach loroph en e fo r 5 5 d ay s w ho had severe sy m pto m s o f poisoning am oun ted to 8 .5 pp m .2 I ,,2 6 A t the tim e we reported these b lo o d levels, o u r recovery rate w as only, a b o u t 75% . A ll b lood levels reported a t that tim e w ere d eterm in ed in w h o le b lo o d . (It is a ssu m e d a t the m om en t th at b lo o d levels are equivalen t to h a lf the am ou n t o f that found w hen plasm a is an a ly zed .) A stu d y undertaken at the request o f Sterlin g L ab o rato ries132 illustrated that new born rh esu s m o n k ey s fo llo w in g to ta l b o d y b ath in g w ith a 3% detergent solution for 1 w eek h ad blood levels o f a b o u t 1 .5 p g/m l (p p m ). In su b seq u e n t m onths a plateau effect w as observed and at 9 0 d ay s the b lo o d level w as equivalen t to 1.1 p g/m l. These m o n k ey s show ed statu s sp o n gio su s o f the w hite m atter o f th e b rain , th is in d icates th at at le ast in th e m o n k e y , b lo o d levels o f a b o u t 1.5 p g/m l w hole b lood w ere con sisten t w ith fluid accu m u lation in the m yelin sh eath o f th e cen tral nervous system . L o ck h art132 cited unpublished stu d ies b y G lu ck , w ho fou n d that h exach lorop h en e b lood levels in term in fan ts after w ash in g w ith a 3% h exach loroph en e detergent so lu tio n w ere som e w hat higher than th ose reported b y u s w ith a m ean co n ce n tratio n o f 0 .3 4 5 p p m (fig lg w h o le b lo o d ). T h is differen ce could have been du e to a d iffer e n ce in w ash in g p ro ced u re an d a b e tte r recovery rate in th e an a ly tic al m eth o d in G lu c k 's stu d y , or b o th . Prem ature in fan ts apparen tly absorbed h exa ch lo roph en e m ore readily. In 15 p rem atu re in fan ts that w ere w ash ed w ith a 3% h ex ach lo rop h en e detergen t solu tio n , the average b lood concen tratio n am o u n ted to 0 .4 7 5 p p m (tg/g).2 41 A b b o tt et a l.262 u sin g a d iffe re n t w ash in g regim en fo u n d a m ean h exach loroph en e b lo o d con cen tration 12 to 18 h r after the initial w ash o f 0 .1 5 4 p p m (p g /g 684 CGOuiSCi w hole b lo o d ) and o n the 7 to 9 th d ay o r life, 2 4 to 4 8 hr after the secon d or third ap p licatio n o f h ex ac h lo ro p h e n e , th e m ean b lo o d level a m o u n te d to 0 .2 8 7 ppm O ig/g). T h e am ou n t fou n d in cord b lo o d o f 16 in fa n ts w as 0 . 0 13 p p m (p g /g ). W hen h exach loroph en e w as applied on ly on the d ay o f delivery, the h exach lorop h en e b lood levels gradu ally decreased fro m a m ean o f 0 .1 4 5 ppm o n the first d ay to a m ean o f 0 .0 S 7 pp m on the ninth d a y , follow in g an in crease to 0 .1 9 4 pp m o n the fo u rth day. The au th ors suggested that prem ature in fa n ts m ay ab sorb m ore h exach lorop h en e th ro u gh th eir skin sin ce in on e in fan t 2 9 w eek s o f g estatio n an d w eighing 1.42 kg the b lood level w as 1 .1 7 ppm 2 4 hr afte r th e initial w ash an d 0 .6 5 S p p m a t 4 8 h o u rs. A ld e r e t a l.243 re p o rte d m ean h e x a c h lo ro p h e n e b lo o d lev els o f 0 .1 8 p p m in in fan ts w ho w ere d u sted w ith a pow d er con tain in g 0 .3 3 % h exach lorophen e. A d u lts using h exach lorop h en e preparatio n s also a b so rb e d it via th e sk in .132 T h e b lo o d levels o b tain ed in ad u lts th at e x p o se d their to ta l b o d y su rface to a 3% h exach loroph en e preparation tw ice d aily fo r 6 0 d a y s, 5 m in each tim e, show ed b lo o d levels o f ap p ro x im a te ly 0.68 p p m (p g /m l w h ole b lo o d ) w ith a range o f 0 .2 5 to 1.0 8 ppm (p g /m l w hole b lo o d ). I f o n ly h an d w ash ing w as em p lo y ed fo r a period o f 2 8 d a y s, 4 tim es d aily , a m e an b lo o d level o f 0 .0 7 p p m w as reach ed a n d in a face h an d w ash in g stu d y th e m ean b lo o d level after 2 8 days w as 0 .1 9 6 pg/m l. B lo o d levels o b tain ed in a p atien t w ith b u rn s w h o died betw een 2 4 an d 4 8 h r a fter th e last h exach loroph en e ap p licatio n w ere 2.2 ppm (pg/g w h ole b lo o d ). T h e b lo o d levels that have been rep orted all in dicate th at follow in g derm al hexach ioroph en e exposu re the chem ical can b e found in b lo o d , an d fro m in fo rm atio n th rou gh an im al stu d ie s as w ell a s so m e h u m an ex p erien ce, levels o f a p p ro x im ate ly 1.5 to 2 p p m (p g /g ) in w hole b lo o d in h u m an s are p resen tly co n sid ered to represen t to x ic levels if the an aly sis fo r b lo o d w as p erform ed w ith th e m eth o d s m en tio n ed elsew h ere in this article. P ro b ab ly a b etter w ay o f assessin g a to xic dose w ould b e to m easure th e am ou n t o f h exach lo r o p h e n e in th e b ra in , b u t fo r o b v io u s re aso n s th at is not possib le. F em ale rats fed h exach loroph en e at the rate o f 5 0 0 ppm (2 5 m g/kg b od y w eigh t/day) fo r 5 5 d a y s w ith very d e fin ite le sio n s h ad a b o u t 4 p p m h e x ac h lo ro p h e n e in their b rain an d 3 .9 1 ppm in th e liver. Sq u irrel m o n k ey s given 5 m g /k g b o d y w eight/day o f h exach lorophen e for 38 d ay s sh o w ed a m e an h e x a c h lo ro p h e n e c o n c e n tra tio n in the brain o f 0 .3 8 pp m an d in a d ip o se tissu e the h ex ach lo rop h en e co n cen tratio n w as 0 .9 4 pp m .244 T h e re co v e ry ra te in th is stu d y w as o n ly 75% . T hese squirrel m on k ey s had n o overt clinical sign s o f to x icity , bu t show ed m ild sta tu s spon giosu s o f the w h ite m atter o f the brain. E lectroen ceph alo g r a p h s c h a n g e s h ad a ls o b e e n o b se rv e d in th e se p rim ates. U lsam er et a !.245 fou n d h ex ach lo ro ph en e b rain levels o f a b o u t 1 p p m in n ew b orn rats w ith sign s o f cen tral n ervou s sy stem to x icity . In 1 su sp e c te d case o f h um an hexach lorophen e p o iso n in g in a c h ild , the h ex ac h lo ro p h e n e brain level w as a b o u t 2 .2 p p m .2 4 1 A d u lt ra ts d y in g a fte r the ad m in istration o f a single d o se o f h exach loro phene show ed con cen tration s o f 6 to 9 ppm o f h ex ach lo ro p h en e in the b ra in .244 S o m e v ariatiih o f th e c o n c e n tra tio n o f h e x a c h lo ro p h e n e in th e brain w hich can be related to n eu rotoxic sy m p to m s m u st b e e x p e c te d in d iffe re n t sp e c ies an d p erh ap s a lso w ith age an d sex. W hether th e fluid ac c u m u latio n w hich m a y o ccu r in the m y elin sheaths after repeated exp o su re to h exach loro ph ene w ou ld alte r h ex ach lo ro p h en e brain levels has thus far not been reported. W ith p re se n t m e th o d s, w e are p ro b a b ly o n ly determ in in g free h ex ach lo rop h en e w hil th e con ju gated portion rem ains un detected. T he toxicity o f h ex ach lo ro p h en e w ill vary w ith the a b ility o f th e organism to con ju gate, m etab olize, and excrete this m aterial. We fo u n d in ra ts that h ex ac h lo ro p h e n e is excreted in m ilk o f dam s fed h ex ach lo rop h en e a t the rate o f 2 0 a n d 1 0 0 p p m in the d iet (2.3-1.1 m g/k g/d ay and 11.8-5.5 m g /k g/d ay , respectively, for 241 d a y s). E lectron cap tu re, gas-liqu id ch rom a tograph ic determ in ation s revealed levels o f 0 .0 7 an d 0 .3 3 p p m in th e m ilk. S in ce h ex ach lo ro p h en e did n ot seem to accu m u late to a great exten t sim ilar values w ould p ro b ab ly h ave b een o b tain ed , had th e levels in m ilk in th e se an im a ls b e en m easured earlier. A lth ou gh w e n ow h ave som e in form ation on the ab sorp tion and distrib u tion as w ell as e x c re tio n o f h ex ach lo ro p h e n e , th e p o ssib le storage o f th e m aterial as w ell a s its m etab o lism needs fu rth er investigation. Sin ce h exach lorop h en e w as fo u n d in co rd b lo o d o f in fa n ts, it can b e assum ed that transplacental passage a lso occu rred. M orph ologic E ffe ct o n the Liver S o m e o f th e ch lorin ated n ap h th alen es, chlori- 1j & w i Q 77 noftini(/> 868SG l MOO 6841 -nated d ib en zo d io xin s, ch lo rin ated d ib en zofu ran s, the ch lo rin ated b ip h en y ls, as w ell as the ch lori n ate d terp h e n y ls h ave an e ffe c t o n the liver in C D variou s species, inclu ding h um an s. B en n ett et ^ al.36 7 co m p ared the to x icity o f d ifferen tly chlori- ^ n ated n aphthalenes and a biphenyl that con tain ed 6 5 % chlorine. T ri-ch loron aph th alen e ap p aren tly j w as less h e p ato to x ic than the higher chlorin ated co m p o u n d s and the chlorinated diphenyl w as the m o st to xic o f all. T h e stu d ies w ere don e in rats 5 ^ an d the exp o su re w as b y feeding as w ell as g -- > in h alatio n . T h e fin d in gs described in th e liver Q con sisted o f liver n ecrosis, fat accu m u latio n , and th e p resen ce o f h y alin e b o d ie s in the c y to p la sm o f the liver cells. A m arked d ep o sit o f fin ely divided gran u lar yello w o r yellow -brow n pigm ent w as o b se rv e d in K u p ffe r c e lls, p articu larly in th ose an im als that h ad been exp o sed to the chlorinated biph en yls. T h is pigm en t ap p aren tly did n ot stain ch aracteristically fo r eith er h em osiderin or h em o fu ch sin . O f co u rse, all livers w ere enlarged m a c r o sc o p ic a lly ; in ad d itio n , these au th o rs d e sc rib e d p ro life rativ e ch an ges in o c c a sio n a l bile d u cts, particu larly after feedin g m ixtu res o f chlori n ated naphthalenes an d chlorinated biphenyls. The n u m b e r o f b ile d u c ts w as in creased in certain areas an d th e ep ith elial cells lining the bile d u cts sh ow ed m ito tic figures. In in h alatio n stu d ie s w ith low co n ce n tra tio n s o f P C B s w hich co n sisted o f either a 16-hr d aily exp osu re to average concen tration s o f 0.5 7 m g /m 3 , o r to 8 h r d aily w ith an average con cen tratio n o f 0 .9 3 m g/m 5 , neither group o f rats ap p e are d ill. T h e p e rio d s o f ex p o su re varied from 3 7 to 143 d ay s. W hen the anim als w ere sacrificed the livers w ere pale o r sligh tly yellow an d so m e w h at m ottled . M icroscopic exam in ation o f the livers revealed sim ilar ch an ges as had been observed after feedin g the m aterial, and an in creased vadu olation o f the liver cells. M icro sc o p ic e v id e n ce o f re c o v e ry w as n o t o b se rv e d in th e livers o f rats th at had been e x p o se d to the ch lorin ated diph enyl fo r a total o f 105 d ay s and w ere rem oved from exp osu re for 2 m onths. S im ilar fin d in gs w ere m ad e b y M iller368 w h o stu d ie d the e ffe c t o f a P C B w hich co n tain ed ap p ro x im ately 4 2 % chlorine. T h is au th or observed v ary in g degrees o f liver d am age a fte r su b cu tan eo u s in jectio n as w ell a s o ral ingestion o f the to x ic su b stan c e in gu in ea pigs, rats, an d rab b its. T h e liver d am age co n sisted o f fatty d egen eratio n and a t r o p h y o f th e c e n tra l lo b u la r ce lls. In th e ra t. h y a lin e b o d ies w ith in th e c y to p la sm o f .the liver cells w ere also n oted . M ost o f the liver d am age w as fo u n d in the gu in ea pig, less in th e ra b b it, an d le ast in the rat. N ish iz u m i,369 w h o stu d ie d th e e ffe c ts o f a Jap a n ese biphenyl K an eclor 4 0 0 w ith 4 0 % ch lorin e in m o n k ey s and m ice, describ ed en large m ent o f liver, fatty changes, gran ular cytop lasm o f the h ep ato c y te s, in creases in the size o f K u p ffe r cells an d h e p a to c y te s, an d a b ro w n p igm en t in so m e o f these cells. He also stu d ied the livers under the electron m icroscope and found an in crease in sm o o th en d o p lasm ic reticu lu m , vari atio n in th e ap p earan ce o f m ito ch o n d ria, an d an in crease in th e n u m b er o f m ic ro b o d ie s. In ad d itio n , " m y elin figu res'' w ere o b se rv e d in th e cy top lasm o f hepatocytes. V o s a n d K o e m a n 95 o b s e r v e d liv e r n e c r o s is in ch icken s that w ere d osed w ith E u rop ean P C B s, w hile a M on san to p rod u ct w ith 6 0 % chlorine did n ot p rod u ce this effect. Increased am ou n ts o f iron w ere d em o n strated w ith P eri's iron stain in the livers o f all ch ick en s e x p o sed to th e E u ro p ean as w ell as the A m erican P C B sam p le. V os and B e e m s 96 s t u d ie d th e P C B - in d u c e d le s io n s in t h e liver o f rab b its, follow in g d erm al e x p o su re to A roclor 1260, Phenocior, and O o p h en . T h e m icro sco p ic ch an ges observed in th e liver in clu ded fa tty d egen eration , focal n ecrosis, cen tro lo b u lar liver cell a tro p h y , ceroid pigm en t in K u p ffe r cells, p erip ortal fib rosis, and cy to p lasm ic h yalin e degen e ra tio n . T h e feedin g o f A roclor 5 4 6 0 , a chlorinated terph enyl, A roclor 1254, o r a chlorinated dibenzop -d io x in , n o t fu rth er sp e c ifie d in th e a rtic le , le d to liver h y p ertro p h y o f Sprague-O aw ley rats w ithin eight d a y s to three w eeks. U ltrastru ctu ral alter a tio n s w ere sim ilar in th e th ree g ro u p s an d con sisted o f n um erous m u ltilayered, concentric m em brane arrays and proliferated sm ooth en do p la sm ic re ticu lu m . In m a n y in sta n c e s th e se m e m b ran e array s su rrou n ded lipid d ro p le ts.370 R ab b its given 3 0 0 m g o f A roclor 1 2 4 2 an d 1 2 5 4 o n ce a w eek fo r 14 w eeks developed en larged livers and the o n es given A ro clo r 1 2 5 4 sh ow ed m id zon al n ecro sis o f the h epatic lo b u les. T h e livers o f rab b its fe d A ro clo r 1221 in a sim ilar fash io n did n ot sh o w an y h isto lo gic ch an ges.3 71 Wc con d u cted studies to com pare the toxicity o f A ro clo r 1 2 5 4 an d A ro clo r 1 2 6 0 in Sh erm an strain rats. T h e acute effect o f these tw o m ixtu res on the liver w as n egligible. I f rats w ere given a 478 CKC Critical Kevu-wa in Toxicology 0006137 DOW ;1135S00 FIGURE 3. Section of liver of a rat fed Aroclor 1260 for 90 days. This figure illustrates a mitotic figure and a hyperchromatic nucleus farrows) H & E X 500. sin gle d o se o f 10.000 m g /k g in p ean u t oil b y sto m ach tube, and w ere sacrificed the n ext d ay, the liver w as essen tially norm al m icroscop ically . D oses o f 1 ,0 0 0 ppm (7 2 m g/kg) o f A roclor 1260 fo r 3 m on th s prod u ced on ly an increase in m ito tic figu res (F ig u re 3 ) an d enlarged h ep ato cy tes. W hen gro u p s o f rats w ere fed d ietary levels o f 20, 100, 5 0 0 , and 1 ,0 0 0 ppm o f A roclor 1260 and 2 0 , 100, an d 5 0 0 pp m A ro clo r 1 2 5 4 fo r 8 m on th s, the liver c h a n g e s w ere p r o n o u n c e d .2 72 T h e h e p a t o c y t e s w ere en larged in m a n y o f the rats o f th e e x p e ri m ental grou ps and lipid accum u lation , foam y c y to p la sm , an d a brow n pigm en t w hich stained p a rtia lly p o sitiv e fo r h em o sid erin w ere o b serv ed in th e livers. T h e p igm en t w as p rim arily o b serv ed in m acroph ages and K u p ffe r cells. In clusions (F igu re 4 ) that stain ed sligh tly m ore eosin oph ilic than the su rro u n d in g c y to p la sm w ere presen t in the ceils. T h ese in clu sion s w ithin the cy to p lasm have been d escrib ed fo r m any co m p o u n d s such as m irex273 ( d o d e c a c h lo r o - o c t a h y d r o - 1 ,3,4-m eth en o-2H c y c l o b u t a ( c d ] p e n t a l e n e ) , D D T 2 7 4 , 1,1,1 -<tric h lo ro -2 ,2 -b is(p -ch ]o ro p h e n y l) eth an e d ie ld rin 2 7 * . 1 ,2 ,3 ,4 ,1 0 ,1 0 -h e x a c h lo ro -6 .7 -e p o x y -l,4 ,4 a 5 , 6 , 7 , 7 , 8 a - o c t a h y d r o - l .4 -e n d o -e x o -5 ,8 - dim eth an o n aph th alen e, and piperonyl b u t o x i d e 274 (a-(2 (-b u to x y eth o x y )eth o x y )-4 .Sm eth yIenedioxy-2-propyltolu en e). T h ey are identi cal to the so-called h yaline bod ies that w ere d e sc rib e d in the e arlier lite ratu re b y B e n n e tt et a l.267 a n d M iller.268 A side from these general changes, exten sive, grayish-w hite, firm , glistening areas w ere n oticed in th e livers o f a n u m b er o f the ex p erim en tal an im als, p articu larly at the high d ietary levels. M icroscopic exam in ation o f these grayish-w hite areas show ed that the h ep atic paren ch ym a had been rep laced by glandular pale stain in g epith ilial cells that fo rm ed d u cts an d w ere su rrou n d ed b y pro liferatin g fib ro u s tissue (F ig u re 5 ). L arger lesion s often had extensive fibrosis an d also co n tain ed collagen an d the d u cts that w ere form ed by epithilial pale staining cells w ere m arkedly dilated an d con tain ed n ecrotic debris or m ucus. T h e lesion h as been classified as ad en o fib ro sis for the tim e bein g. It h as been d escrib ed in detail b y E d w ard s an d W hite276 w h o o b serv ed it in rats that w ere fed b u tter yellow (p-dim eth ylam in o-azob en zen e). T h ese lesion s enlarge peripherally b y the form ation o f new glandular stru ctu res. H epatic GGGG1S3 January 1974 DOW 359011 1 FIGURE 4. Section of liver from nit fed Aroclor 1254. A cluster of inclusions is present in the cytoplasm of one hepatocyte (arrow). Usually individual cells contain only one of these formations at a time. Toluidine blue stain of maraglass embedded material X 300. tissu e m ay be trap p ed betw een the glands o f the peripheral area o f ad en oftbrosis. T h e older central p a rts o f the lesions so m etim es contain pigm ented av ascu lar collagen ized stro m a w ith atro p h ic ep i thelial cells lin in g dilated du cts that ap pear atro p h ic an d d ead . T h e glan ds o f actively grow ing ad en o ftb ro sis sh ow aty p ical epith eliu m w ith large pleom o rph ic nuclei. T he epith eliu m can occasion ally b eco m e stratified . P arts o f the lesion m ay be q u ite e x te n siv e , w hile in a d d itio n , in m an y are as o f th e sam e liver sm all foci o f ad en o ftbro sis exist sim u lta n e o u sly in clo se re latio n sh ip to p o rtal areas. It has th erefore been assum ed that these le sio n s arise fro m bile d u cts, b u t th ey can a lso be p rim arily c o n fin e d to th e region o f th e hepaticv ein s. T h e absen ce -of bile d u cts aroun d h ep atic vein s led to the con clu sion 2 77 that a precu rsor, o th e r than bile d u cts, e x iste d from w hich ad en ofib rosis developed. H ow ever som e investigators o f th e le sio n s have assu m ed that it rep resen ted an a ty p ic al bile d u ct p ro liferatio n , a lso referred to as c h o l a n g i o f t b r o s is .2 7 8 In a d d it io n t o th is le s io n w e also observed sm all clu sters o f glandular cells w ith in areas o f ad en o ftb ro sis as w ell as ad jacen t to b lo o d vessels that w ere surrounded b y relatively norm al ap p earin g h epatic parenchym a w ith stain in g ch aracteristics o f salivary gland tissu e. T h ese clusters o f cells very closely resem bled p an creatic tissu e .279 T h e d istin ctio n b etw een a d e n o ftb ro sis an d ad en o carcin o m a is d iffic u lt. T h e glan d s are q u ite a ty p ic a l in ad e n o ftb ro sis b u t it m a y be h elp fu l to re m e m b e r th at in a d e n o ftb ro sis, in con trast to aden ocarcin om a, the irregularity observed is lim ited an d d u p licated w ith con sid er able e x actn e ss from lesion to lesion . M u cu s is alw ay s p resen t in ad en o ftb ro sis, b u t n o t alw ay s in ad en o carcin om a. The strom a o f ad en o ftbrosis is con cen trically arranged, y ou n g and cellular at the periphery an d old and h yalin ized to w ard s the c e n te r o f th e le s io n .2 7 7 O f c o u r se a liv e r sh o w in g ad en o ftb rosis m ay also con tain carcin om ato u s lesio n s. S o far conclusive evidence is lack in g in the literatu re that ad en o ftbrosis gives rise to carci n o m as. H ow ever, it h as been fo u n d to co e x ist w ith c arc in o m a o f th e liver in ra ts an d so m e in v esti gato rs feel th at the lesion is in fa c i a p recu rso r o f c a n c e r.280 C h o lin e d e fic ie n t ra ts a p p a r e n tly , in ad d itio n to oth er, ch an ges in the liver, a lso d ev elo p 480 IRC Critical Reviews in Toxicology DOW t 135S02 FIGURE S. Section illustrates an area of adenofibrosis of the liver of a rat fed Aroclor 1254. &Note the epithelial ceils forming small ducts. The ducts contain cellular debris and are surrounded by fibrosis which can become quite pronounced, H E X 125. a d e n o fib r o s is .2 * 1 H o w e v e r , th e se r a ts w ere fed peanut m eal and soym eaJ and therefore the possi bility exists that the experim en tal diet w as co n tam in ated w ith a m y c o to x in . B on n et et a l.167 also describ ed the lesio n (a d e n o fib ro sis) in rats w hen a m ixtu re o f ch lo rin ated n aph th alen es and chlorodiph en yls w as fed to them . A critical review o f the h isto p ath o gen esis o f this lesion w as m ad e by Stew art and Sn ell.277 U ltrastru ctu ral ch an ges o f the livers o f anim als e x p o se d to the A r o c lo r c o n siste d o f an in crease in sm o o th en d op lasm ic reticu lu m and atypical m itoch on dria. M any lipid vacu oles w ere observed, particu larly at the h igher d ietary levels. T h ey w ere al tim es su rrou n d ed by con cen trically arranged m em branes. T he " in clu sion s" or " h y alin e" bodies o b se rv e d in the c y to p la sm w ith th e ligh t m ic ro sco p e correspon d to these form ation s w hen they are exam in ed u n der the electron m icroscop e. T heir u ltrastru ctu ral ap p e aran ce h as led to their being referred to as " lin ger prin ts" (Figu re 6). The epith elial com p o n en t o f the ad en o fib ro sis outlined co n sisted o f cu b o id al o r colu m n ar cells with the free su rface lined by m icrovilli, granular cy top lasm w ith m any rib osom es, an d a few e n d o p lasm ic m em bran es. So m e o f the cells con tain ed a great deal o f m ucus and resem bled goblet cells. T o n o filam en ts and term inal b ars w ere also ob serv ed . T h ese various ob servation s suggested that the ep ith elial co m p o n en t o f ad en o fib ro sis had all the m o r p h o lo g ic a l ch aracteristics o f bile d u ct epith elium . C h ick en s w ith chick ed em a disease an d cattle w ith the X -disease, alread y m en tion ed , u su ally s h o w e d c h a n g e s in th e liv e r w h ic h c o n s is t e d pred o m in an tly o f liver cell n ecrosis and fat a c cu m u lation . T he h ep ato to xic effect o f chlorinated n a p h t h a l e n e s h a s a ls o b e e n d e s c r ib e d in r a t s .2 -67 In a num ber o f rep orts on the o ccu pation al o c c u rre n c e o f ch lo racn e in w o rk e rs that w ere e x p o sed to chlorinated n ap h th alen es, chlorinated d iph en yls, or 2 ,4 ,5 -T , an d tech nical trichlorop h e n o l, referen ce is m a d e to -the h e p a to to x ic effect o f these com pou n d s. T h is h ep atoto xic e ffe c t can m an ifest itse lf on ly in a decreased to leran c e fo r a lc o h o l; it m a y a lso d e v e lo p in to h ep atic d eficien cy with acu te yellow atro p h y an d jau n d ice. A p p aren tly the e ffe c t on the liver w as 6945 CG0623S'"" 19,4 4,1 I DOW 1135903 FIGURE 6 . Electron micrograph of a portion of the cytoplasm of a hepatocyte. The section shows concentrically arranged arrays of membranes which surround vacuoles containing lipid. Lead citrate, uranyl acetate X 34,200. p articu larly bad follow in g exposu re to halo-w axes, w h ich are ch lo rin a te d n a p h th a le n e s.3 s 2 earlier th at single oral doses o f 2 0 *5 0 #ig/kg b o d y w eight resu lted in fatal liver n ecrosis in rab b its. C o tte r3 *3 rep orted seven cases o f pentachlorin- W eanling rab b its w ere treated w ith 2 ,3 ,7 ,8 - ate d n ap h th alen e p o iso n in g in w ork ers w h o w ere tetrachlorodibenzo-p-dioxin b y ap ply in g a dose o f en gaged in m an u factu rin g w ire cab le d u rin g W orld 1 .6 7 tig d a ily to the in n er asp e c t o f b o th e ars fo r W a r 11 f o r th e n a v y . F o u r o f th e w o r k e r s d e v e lo p - three d ay s. T h e to tal d ose given w as 7 .0 5 jxg/kg ^ ed jau n d ice and tw o died. M icroscopic exam in a b ody w eight. W hen these rabbits w ere sacrificed tion o f the liver o f the tw o w orkers th at died 18 d ay s follow in g the last ap p licatio n o f the sh o w ed co m p le te lo ss o f liver cells in so m e areas, d io x in , their livers w ere sign ifican tly larger th an the cen trolobu lar areas w ere h em orrhagic, and those o f the con trols. M icroscopic exam in ation o f p ro m in en t bile d u ct p ro liferatio n w as seen in the the livers sh ow ed enlarged h ep ato cy tes. S o m e o f p e rip h e ry . the h ep ato cy tes w ere m u ltin u d eated . T h e c y to A n oth er grou p o f co m p o u n d s that have at least so m e representatives that cause a h ep ato to xic e ffe c t are the ch lo rin ated d ib en zo d io xin s and ch lorin ated dibenzofurans. S in g le o ral d o se s o f 1 to 1 0 jag /k g 2 ,3 ,7 ,8 tetrach lo ro d ib e n zu d io x in given to ra b b its p ro d u c e d a h e p a to to x ic e ffe c t. A t th e h igh er d o sage level plasm w as fo am y or vacu olated. A ligh t brow n pigm en t in so m e liver cells as w ell a s K u p ffe r cells w as also o b serv ed . In clu sio n s w ere seen in the c y to p la sm in som e liver cells, sligh t in terstitial fib rosis w as presen t, an d tw o o f three rab b it livers exam in ed show ed foci o f necrosis that w ere su r r o u n d e d b y fib r o s is .3 *4 the an im als died an d the low er d oses caused S tu d ie s w ith " to x ic fa t," the cau se o f ch ick se rio u s liver d am ag e.1 0 * S ch u lz10 1 had reported ed em a d ise ase , sh o w ed th at it h ad a n e cro tiz in g 482 CRC Critical Reviews in Toxicology CG 0ui3i 6246 lOGgpii /mod e ffe c t o n th e liver in ch ick en s. A s w e now k n o w , the toxic agents responsible for chick edem a disease are chlorin ated dibenzo-p-dioxins. T o xic fats also altered liver m o rp h o lo gy in M acacca m u la tta m o n k e y s .385 H e p a to c y te s w ere e n la r g e d , m u ltin u cleated an d fo c al n e cro sis in the cen trolob u lar zon e w as ob served . M any liver cells w ere vacu olated and stain ed positive fo r n eutral fat. In o u r stu d ie s o f ra ts given h exach lo ro p h en c. w e did n ot ob serve an y liver p ath o lo g y w ith the light m icroscop e and n o change w ith the electron m icroscop e at the dosage levels that we em p lo y ed . 118 T h o rp e 131 on the o th er h an d, o b se rv e d p e rip o rtal fa tty ch an g e s in livers o f sh e e p given three o r fo u r d o se s o f SO m g /k g h ex ach lo ro phene and h istoch em ical changes w ere also observ ed in the h ep a to c y te s. P u gh an d C ro w ley 384 also ob served h e p a to to x ic ity in sh eep afte r giving them hexach lorophen e. O f the o th er germ icides d iscu ssed in this article, only triclosan (Irgasan D P -300) (2,4,4-trich Io ro 2'-h y d roxy d iph en y l e th er) h as been rep o rted to have a to xic e ffe c t on the liver at a d o se o f 12S m g /k g and en larged the liver o f rats at a d ose o f 2 5 m g/kg, accordin g to the su m m ary m in u tes o f the O T C Panel, U .S . F o o d an d D rug A d m in istratio n . It w as not stated w h eth er these w ere single or rep eated d o ses. Oral ad m in istration o f triclosan to beagle dogs p rod u ced jau n d ice an d severe liver dam age w as observed. The effect that these com p o u n d s have on the liver w ill h ave to b e stu d ie d fu rth e r. T h e a c c u m u latio n o f fat in the liver in d ica te s th at eith er fat tran sp o rt o r fat m e ta b o lism is im p aired ; if the in su lt to the liver is rem o ved , the liver gradually recovers un less the lesion has b e co m e irrev ersib le. T h e in crease in sm o o th e n d o p la sm ic re ticu lu m as w ell as th e " in c lu sio n s" w ill also d isap p ear a fte r a certain am ou n t o f tim e if e x p o su re to th e m a te ria l is d isc o n tin u e d .387 T h e sig n ifican ce o f the in crease in sm o o th e n d o p la sm ic reticu lu m and the h yalin e b o d ie s w ith a c o n c o m it an t rise in liver m ic ro so m a l e n zy m e activ ity is presently not u n d erstood . These changes are con sid e re d ad ap tiv e an d b e n e fic ia l, b u t in a d d itio n to m etab o lizin g u n w an ted ch em icals at a higher rate, the stim u lated liver also m etab o lizes som e m ed ica tio n s m ore ra p id ly ,388 as w ell as su b sta n c e s p ro d u ced b y the b o d y such as stero id s. W hether the in clu sion s w ithin the cy to p lasm rep resen t the so-called inactive sm o o th en doplasm ic reticu lu m w h ich is d e p o site d in the fo rm o f array s o f m e m b ra n e s w ithin the cell is not k n o w n .T h e fact that sm o o th en d oplasm ic reticu lu m m ay b eco m e h y p oactiv e w as first p o stu lated b y H u tterer et a l.389 F ib ro sis o f th e liver a s w ell as n ecro sis an d the ad en o fib ro sis ob served afte r PC B e x p o su re in rats should defin itely be considered a very seriou s lesion. U su ally ad en o fib rosis occurs con com itan tly w ith h e p a to m a s o r h e p ato c arcin o m a s in ro d e n t livers. A recen t p u b licatio n by K im u ra an d B a b a 3 8 9 * show ed that h ep ato m as can be induced in ra ts w ith K an e c h lo r 4 0 0 . C h ro n ic fe ed in g stu d ie s m ay sh o w that o th e r P C B s w ill also in du ce h ep ato m as in ro d en ts. N eurotoxicity In so m e o f th e rep o rts on ch lo racn e an d also in the Y u sh o in cid en t, peripheral n eu ro p ath y is m e n t io n e d a s w e ll a s p s y c h i c a l t e r a t i o n s 1 *390 a s p art o f th e sy m p to m a to lo g y o f p o iso n in g , b u t in m ost rep orts n o t m uch em phasis w as put on it. In th e Y u sh o e p id e m ic M u rai a n d K u ro iw a 390 p e r fo rm ed m ore detailed exam in atio n s in 21 cases ad m itted con secu tively to K u y u sh u U n iversity H o sp ital in the n o n h em part o f Ja p a n . Seven o f th e p a tie n ts c o m p la in e d o f n u m b n ess o r p ain in the p erip h eral p o rtio n o f th eir e x tre m itie s an d in five c ase s, h y p o e sth esia, h y p alg e sia, an d th erm o h y p esth esia w ere n o ted . Slo w in g as w ell as re d u c tion o f th e se n so ry nerve c o n d u c tio n v e lo c ity in th e rad ial an d su ral n erve w as o b serv ed in a n u m b er o f these p atien ts. H eadach e an d periph eral n erv o u s sy ste m sy m p to m s w ere also re p o rte d in a p o iso n in g e p iso d e w hich occu rred in a F in n ish co m p an y w here paper w as im pregn ated w ith b ip h e n y l.93 T h e on ly chem ical o f the group o f poly ch lorin a te d , p o ly c y c lic c o m p o u n d s d isc u sse d in th is report that has a predom inant central n eu ro to xic e ffe c t in an im als as w ell as h u m an s is h e x a c h lo ro phene. A dult fem ale rats that w ere fed 5 0 0 pp m h ex ac h lo ro p h e n e in the diet fo r 14 w e e k ; (a b o u t 3 0 to 18 m g/k g b od y w eigh t/day) d eveloped leg w eakn ess after 12 to 19 d ay s o f e x p o su re. T h is leg w eakn ess w hich w as only evident in the h in dlegs p r o g r e ss e d to p a r a ly s is w ith in 3 to 5 w e e k s .391 A t au to p sy the brains o f the exp o sed rats w ere e n larg e d ; th ey w eighed an average o f 2.6 g w hile the co n tro l brain s w eighed an average o f 1 .99 g. M icro sco pic exam in ation o f the b rain s revealed n orm al gray m atter th rou gh ou t the brain an d the sp in al c o rd in th e few in stan c e s th at th e sp in al G&06k3 January 1974 483 ' 6947 DOW J 135905 FIGURE 7. Electron micrograph from the white matter of the brain or a rat, illustrating status spongiosus. Note the large empty appearing vacuoles lined by myelin (MY = myelin, A = axon). Lead citrate, uranyl acetate X 34,200. co rd w as exam in ed . T h e w hite m atter on the co n trary seem ed w idened an d show ed m any cy stic sp a ce s th at ap p eared e m p ty and w ere lined w ith stran d s o f m aterial that stain ed positive for m yelin . T he vacu oles w ere very num erous and v aried greatly in size givin g the tissue a sp o n g y a p p e aran c e . T h e b rain s in these stu d ies w ere u su ally fix ed in eith er B o u in 's solu tio n or b u ffered fo rm alin fo r light m icro sco p ic stu d y . Brain tissu e, particu larly that o f roden ts, m ay n o rm ally show sm all vacu oles that are considered t o re p re se n t an a r te fa c t.592 T h ese v a cu o le s are sm aller, usu ally very u n ifo rm , and at lim es con tain a h om o gen eo u s pin k stain in g m aterial. T h ey are u su ally sep arated by a w ider area o f norm al a p p e a r in g w hite m a tte r th an the v a cu o le s in sta tu s sp o n g io su s. B rains that show this artefact d o not w eig h sig n ific a n tly m ore th an th o se 'that d o n o t sh o w it. E lectro n m icroscop ic exam in ation o f brain s o f ra ts given h ex ach lo rop h en e show ed that the vacuolatio n in the w hite m atter o f the brain revealed large v acu o les lined b y m y elin . T h e ax o n s th at w ere present appeared u sually to be quite n orm al; o ccasion ally stran d s o f m yelin h ad separated and traversed the vacu oles. The vacuoles w ere oth er w ise e m p ty e x c e p t fo r so m e e lectro n den se gran u lar m aterial (F ig u re 7 ). N o oth er alteration s were o b serv ed in th e w hite m a tter. T h is lesio n is n o t unique for h exach lo rop h en e; oth er ch em icals that have p rod u ced sta tu s spon gio su s as w e have called th e le s io n in c lu d e t r i e t h y l t i n ,293 i s o n ia z i d ,294 th e h alogen ated benzan ilide, 2-acetoxy-4-ch loro-3,5d iio d o b e n z a n ilid e ,295 an d ra fo x a n id e ,296 w h ich is the 3,5-d iio d o -3 '-ch lo ro -4 '-(p -ch lo ro -p h en o x y ) salicylan ilide. T rieth yltin seem ed to p rod u ce the lesion at a m u ch low er d ietary level an d m ore rapidly than the oth er ch em icals m en tion ed, par ticularly h exach loroph en e. In an ad d ition al stu d y , after feeding rats 5 0 0 p p m h e x a c h lo ro p h e n e in th e d iet fo r 10 w e e k s an d then d iscon tin u in g the exp o su re to h exach lo ro ph en e, fu n ctio n began to return in the h in d q u arters o f p araly z ed rats a fte r 2 w eek s an d w ithin 484 CRC Critical Reviews in Toxicology 0&06133 -} DOW T 135906 H w eeks th e rats h ad alm ost com pletely recovered clinically. H ow ever, m icroscopic exam in ation o f the brain s o f these rats show ed that a few cystic sp aces w ere still present 12 w eeks after the exposu re to hexach lorophene had been discon tin u ed . In w ean lin g ra ts, b u t n o t in ad u lt ra ts, the brain lesion an d sy m p to m s o f paraly sis co u ld be p ro d u ced w ith a single oral dose o f 100 m g /k g o f b o d y w eight given by sto m ach tube in pean u t o il.1 1 * P art o f the reason for bein g able to p ro d u c e th e b rain lesio n in the w ean lin g ra ts w ith a single d o se m ay be related to the fact that the w ean lin g w as ab le to survive a higher sin gle dose than the ad u lt rat. O ther species that have also show n paralysis and oth er n eurological deficits after exp osu re to h e x a c h lo r o p h e n e a r e r a b b it s ,1 9 1 p ig s ,298 a n d c a t s .299 S h e e p b e c o m e b lin d a c c o r d in g to U d a ll a n d M alo n e . 120 H an ig et a l.299 re c o rd e d the n eu ro lo g ical d e fic its p ro d u ce d in c a ts in m ore detail. E arly sym ptom s consisted o f lassitu d e, w eakness and atax ia o f the h in dlegs, im paired righting re fle x , patellar h yperreflexia w hich later developed in to h y p o reflex ia, urinary reten tion , an d eventually com plete flaccid paralysis. The cranial nerve fu n ctio n s rem ained in tact e x cep t p reterm in ally . In these cats an increase in cy stem al cerebralspin al fluid pressure w as observed. In our first stu d ies we continued exp o su re to h exach lorop h en e fo r a period o f 14 w eeks because we had designed the experim en t to con d u ct an ap p ro xim ate 90-day feedin g stu d y . It is n ot n ecessary to feed rats fo r this len gth o f tim e. A s w e determ ined later, m icroscopic changes can be ob served in rats a fte r a feed in g p erio d o f tw o w eeks w hen the an im als w ere e x p o se d to levels high en ou gh to produ ce the lesion . S o m e vacuolation can already be observed 3 to 4 d ay s after o n se t o f e x p o su re to h ex ach lo ro p h en e in som e rats. W hen low er d ietary levels w ere fed in a rep ro d u ctio n stu d y ,1 1 * the dietary level o f 20 ppm ( 2 .4 - 0 .9 m g/kg body w eigh t/day) had no e ffe c t on the b rain . A t the dietary level o f 100 p p m ( 1 1 . 8 --4 . 6 m g / k g . b o d y w e i g h t / d a y ) f o c a l areas o f vacu olatio n o f the w hite m atter o f the brain w ere ob served in som e o f the rats, but they w ere never ex ten siv e. T h e rats in th ese low er dietary levels did n ot show any clinical-neurologi cal sym ptom s. T h e b rain le sio n o b serv ed in ra ts w as also d escrib ed in m o n k ey s follow in g the ex p o su re to h ex ach lo ro p h en e. N ew born m on k ey s w ash ed w ith 3% h exach lorophene detergen t solu tion developed a d iffu se statu s sp o n gio su s o f the w hite m atter o f th e b r a in .3 " " S a n t o l u c i t o 2 6 4 '3 0 1 w a s a b le t o produ ce the vacu olation o f the w hite m atter, the so-called statu s sp o n g io su s. by in jectin g squ irrel m on k ey s daily su b cu tan eo u sly w ith S m g h exach lo rop h en e/k g o f b o d y w eight for 37 days. W hen m on k ey s w ere fe d h exach lorophen e they did n ot develop the brain lesion but show ed electroen ceph alograph ic chan ges w hich need furth er investigation. The m on k ey s that show ed the vacu olation also d em on strated electroen ceph alographic changes. Sh eep have ap paren tly also show n sta tu s spon gio su s o f the w hite m atter accord in g to H ale an d R e id .302 L am p ert et a l.303 w ere ab le to p ro d u c e the lesio n in m ice a s w ell a s rats. T h e se au th ors reported that the m orph ological -changes w ere also o b served in fro zen se c tio n s o f b rain s q u e n c h ed in liq u id n itro g e n . T ry p a n blu e in je c ted in traperiton eally did n ot stain the brain, an in d ica tion that the b lo o d brain barrier w as in tact as far as try pan blue w as con cern ed . T h e brain chan ges co n sisten t w ith statu s sp o n g i osu s due to h exach lorophen e exposu re have also b een d e scrib e d in h u m an s, p a rtic u la rly ch ild ren . M u llick130 exam in ed the brains o f six hum an cases that died from h exach lorophen e poison in g. T h e tw o ad u lts in th is g ro u p d id n ot sh o w vacu olatio n o f the w hite m atter o f the brain . T h ey h ad died less than 4 8 hr after exp osu re to h ex ach lo rop h en e. T h e fo u r children (tw o w ith con gen ital ich th y osis, tw o w ith b u m s) had at least three or m ore days o f exposure to h exach lo ro ph en e b ath s in w hich a 3% h ex ac h lo ro p h e n e p rep aratio n w as u sed. T h e final co n cen tratio n o f h e x a c h lo ro p h e n e in th e b a th w a te r w as n o t k n ow n . All fo u r children sh ow ed sta tu s sp o n g io su s o f th e w hite m atter. T h e b rain w eigh t given in on e o f the cases, a 12-day-old ch ild, w as 4 2 5 g. T h e n orm al brain w eight fo r a child th at age is 3 8 2 g. S h u m a n e t a l.304 in a re tro sp e c tiv e b lin d stu d y w ere able to d em on strate that the in ciden ce o f sta tu s sp o n g io su s in the brain stem 'reticu lar fo rm a tio n in in fan ts w h o h ad d ied fro m a v ariety o f cau ses could be correlated to h exach loroph en e e x p o su re . T h e au th o rs fo u n d 21 case s in a series o f 2 5 0 au top sies o f children. O f these cases, 18 w ere prem ature in fan ts w eighing less than 1,400 g and w ere o f less than 3 0 w e e k s' g e statio n . A ll b u t 2 o f the cases had 3 or m ore total w ash es w ith 3% h exach lo rop h en e. In the gro u p o f cases that w ere stu d ied w ere a n u m ber o f stillb o m s w ho did not G u O u U .3 % January 1974 485 6949 135907DOW 1 sh o w th e lesion an d o f co u rse h ad n o t had derm al h ex ac h lo ro p h e n e e x p o su r e . S h u m a n e t a l.304 m en tion ed earlier cases o f L etterer S iw e's disease in w hich they h ad o b serv ed sta tu s sp o n g io su s o f the w hite m atter. E ach o f the children w ith L etterer Siw e's d isease h ad h ad p ro n o u n ced e x p o sure to h ex ach lo rop h en e b ecau se o f the skin lesio n s w hich h ad been vigo ro u sly scru b b ed w ith a detergent con tain in g the chem ical. The various o b servation s show th at, in p atien ts w ith ab n orm al skin as w ell as th e skin o f the p re m atu re , h e x a ch loroph en e m ay be ab so rb ed in su fficien t am ou n ts to cause statu s spon giosu s. T he fact that b lo o d levels in p re m atu re in fa n ts w ash ed w ith h ex ach lo ro p h en e ten d to be h igher than in m atu re in fan ts also su b stan tiates these fin din gs. A lm ost identical observation s w ere m ade by Pow ell et al.3 0 s In prem atu re in fan ts w eighing less than 1 .4 0 0 g, 7 o f 13 that had 4 or m ore e x p o su res to h exach lo rop h en e w ash es show ed sta tu s sp o n gio su s o f the m yelin ated tracts o f the brain ste m , i f th is gro u p w as fu rth er su bdivided acco rd in g to the n um ber o f exp o su res, 6 o f 9 infants that had 9 or m ore exposu res to h exa chloroph en e w ashes show ed the lesion , and 1 o f 4 that w as w ashed 4 to 8 tim es. F o u r stillb om s and 13 in fan ts w ash ed 0 to 3 tim es w ith h ex ach lo ro phene and w eighing less than 1,400 g did not show the sp o n gy ch an ge. E lectro n m icroscop ic exam in a tio n sh o w ed th at th e sp o n g y ch an ge o b serv ed in the prem atu re in fan ts w as du e to a sp lit in the m yelin sh eath s. In A ugust o f 1 972 the press reported a num ber o f d e ath s o f in fa n ts in F ran ce th at occu rred because a baby p ow der to w hich hexach lorophene h ad accidentally been ad d ed at a concentration o f 6% w as u sed on the in fan ts. T h e final d eath toll a m o u n te d to 41 in fa n ts a n d y o u n g ch ild re n 306 (N ew Y ork N ew s, D ec. 13, 19 7 2). D etailed acco u n ts have n ot been publish ed o f this incident because o f litigation problem s. A s w e pointed out earlier, u n der acu te to x icity , the higher the co n cen tration o f h ex ach lo rop h en e is in a preparation ap p lied to the sk in , the m ore likely it is that skin d am ag e w ill resu lt. S k in d am ag e a lso o ccu rred in the F ren ch in ciden t. In ad dition to this, the p o w d e r w as p ro b ab ly u se d in m an y in stan ces w here som e diaper rash already existed and w as then w ell covered w ith a d iap er, w hich m ay have co n trib u te d to in creased ab so rp tio n in this area. T h e b rain s an d sp in al co rd o f a few in fan ts from th e French po iso n in g ou tbreak that w ere exam in ed m icroscopically show ed statu s sp o n g io su s o f the w h ite m a tte r .306 T h ese various accou n ts show that statu s spon-. g io su s can also be p ro d u ced in h u m an s and p ro b ab ly represents a n on specific reaction w hich can b e elicited b y a num ber o f chem icals. T h e lesion statu s spon gio su s o f the w hite m atter as far as we now know seem s to represent a specific or restricted typ e o f brain edem a w here the flu id accu m u lation is con fin ed to the in side o f the m yelin sh eath and m yelin is n o t actu ally dam aged o r at least breakdow n p ro d u cts o f m yelin have not been observed. The term status spon giosus or spongy change has been used as a descriptive term an d a spongy change o f the gray m atter m ay also o ccu r in certain diseases. S ta tu s spon gio su s together w ith other m orph ological ch a n g e s can b e o b serv ed in the b rain in a v a rie ty o f m any etiologically different d iseases and the reader is referred to K latzo ,301 A rdon ato and L am p e rt,3 0 * an d a recen t ed ito rial in the B ritish M e d ic a l Jo u r n a l.309 F r o m c lin ic a lly o b se rv e d po iso n in g cases as w ell a s an im al exp erim en ts it is eviden t th at th e brain lesio n is to a great e x ten t reversible. H ow ever, in o u r ow n stu d ie s a s w ell a s th ose b y L am p ert et a l.,303 m icro sco p ic ch an ges in ro d e n ts d id n o t co m p le te ly reg ress and th e fa c t that sh eep d o not recover their eyesigh t after h exach lorop h en e p oison in g120 in dicates th at repair m ay not alw ays be com p lete. N o reason able explan ation exists at the m om en t to indicate w hy certain ch em icals prod u ce this very specific ed em a con fin ed to certain stru ctu res o f the cen tral nervous system . Gastrointestinal Lesions O nly tw o typ es o f com pou n d s under discussion h ave p ro d u c e d lesio n s in th e g a stro in te stin al trac t. We o b se rv e d in o u r stu d y w ith S h erm an strain rats th at high single oral d o ses o f 3 ,0 0 0 m g/k g A ro clo r 1254 and 1 2 6 0 o r m ore caused ulceration o f the g a stric an d d u o d e n a l m u co sa (F ig u re 8 ). U lcers in the G I trac t w ere n ot observed in th e long-term feed in g stu d ies w ith 5 0 0 pp m o r less. N o o th er ch an g es in th e g astro in te stin al trac t w ere o b serv ed . R in ger et a l.,10S in their rep ro d u ctio n stu d y w ith m in k, o b serv ed th at A ro clo r 12.54 p rod u ced h em orrh age in the g astro in testin al trac t o f the o ff sp rin g . A llen and N orback310 observed hyperplasia an d d y sp la sia o f th e g a stric m u c o sa in rh esu s m on k eys. S ix o f these rhesus m on k eys w ere fed 46 CRC Critical Reviews in Toxicology C v> G u j l 3 5 DOW 1 135908 FIGURE 8. Section oftheglandularportion ofthestomachofaratgivenasingledoseof3,000 ing/kg ofArodor 1254.This figure illustratesthe focal lossofmucosa and the infiltration by inflammatoryroundcells.H k E X 50. 3 0 0 pp m A roclor 1 2 4 8 an d six w e n given 5 ,0 0 0 p p m A roclor 5 4 6 0 , a poly ch lorin ated triphenyl. T h e glan du lar fo rm atio n s in th is lesio n w ere highly aty p ical; pen etration o f th e m uscularis m u cosae an d invasion o f th e su b m u co sa b y th e m u co sal epith elium w ere observed. A llen an d C arsten s3SS rep orted an e ffe c t o f to x ic fat o n th e gastric m u c o sa in 1 8 o f 2 7 m o n k e y s, M acaco m u latto , w hich con sisted o f h y p ertrop h y o f th e gastric m u co sa an d sm all gastric u lcers w ith in th e m u cosa. T h e to x ic fa t used in th ese stu d ies, a s w e n ow kn ow , con tain ed ch lorin ated dib en zod ioxin s. It has also b een observed that hem orrhaging occu rred into th e gastroin testin al tract o f rat fetu ses w ho w ere ex p o sed to 2 ,3,7,8-tetrach lo rod ib en zo-o-d ioxin in u te ro . lt w as n o t sta te d in the reports w hether th e gastrointestin al h em orrhages w ere caused by ulceration o f the m u co sa.1 10 ,111 U lceration o f the glan dular p o rtio n o f the ad u lt rat stom ach w as also observed.1 13 P atients w ith ch loracn e freq u en tly com plain ed o f lo ss o f ap p etite, n au sea, vom iting, an d ab d o m in al p ain , an d G old m an n 3 11 d escrib ed a w orker w ith a follicu lar d erm atitis w ho died six m on th s after exp o su re to 2,3,7,8-tetrach lo rod ib en zod ioxin . A t a u to p sy a ru p tu red d u od en al ulcer and a gastric ulcer w ere fo u n d . W hether an y relation ship ex isted betw een th e ulcers an d th e exp o su re to th e p o iso n o u s ch em ical w as n ot m en tion ed in the paper. It should be establish ed i f these gastric lesio n s also o c c u r a t low er d ietary levels an d w hether the co n tam in atio n o f the ch em icals w ith various ch lor in ated d ib en zodioxin s an d fu ran s is responsible fo r them . V aluable in form ation m ight b e obtain ed fro m retrospective stu d ies o f w ork ers follow in g o ccu p atio n al exp o su re. W hether th e ulcers are caused b y a direct effect o f the chem ical o n the m ucosa o f the gastrointestinal tract or w hether stress p la y s a prim ary ro le in th eir e tio lo g y sim ilar to u lcers that m a y o c c u r in b u rn ed p atien ts fo r in stan ce (C urling's u lcer) is n ot clear. COMMENTS Th e acute to xicity o f com po u n d s such as the chlorin ated biphen yls, the chlorin ated n aph th a lenes, an d th e ch lorin ated terph en y ls is very low . C G Q o jL B G January 1974 487 606SSTlMOO On th e other hand, th ey are poorly broken dow n in the environm ent an d the m etabolism o f the b iph en y ls w ith m ore than fo u r chlorine ato m s seem s to be negligible. B ecau se o f these character istics, the use, w herever possible, o f chlorinated b iph en yls that con sist o f m ixtures o f com pou n ds w ith on e to fo u r ch lo rin es h as been p ro p o sed . N eith er the ch lorin ated naphthalenes n o r the chlorinated terph enyls have so far been show n to present a pollution problem . W hether th e v a rio u s d ise a se e n titie s review ed in this article are p rod u ced b y all the d ifferen t p r o d u c ts m e n tio n e d in c o n n e c tio n w ith th e m is n ot establish ed w ith certain ty . F o r instance, som e o f th e com p o u n d s th at prod u ce chloracne or chick ed em a have been sh ow n to b e con tam in ated w ith variou s chlorinated dib en zod ioxin s and chlor inated dibenzofuran s. T h e possibility rem ains that th e co m p o u n d s illu strated in-Figure 1 are co n tam i n ated w ith these ch em icals an d the p ro d u cts them selves are n ot really responsible fo r the observed sym ptom s. O n the other hand, a num ber o f stru ctu rally unrelated com pou n ds m ay produce the sam e disease en tity. S o m e o f the chlorinated dibenzodioxin s and th e ch lo rin ated d ib en zo fu ran s are highly to x ic. T h e m o st to x ic is 2 ,3 ,7 ,8 -te trach lo ro d ib e n zo d io x in s, w hile th e d ib en z o d io x in s w ith ou t an y chlorin e and th ose w ith eight chlorines are m uch le ss to x ic . T h e d ifferen ce in to x icity o f th ese com p o u n d s m ay be partly caused b y the fact that th ey are very p o o rly dissolved and ab sorbed . In traven ou s to x icity d a ta are thus far not available on m ost o f these com pounds. We also d o n ot know at present if the porph yria w hich develops alter prolon ged exposu re to a n u m b er o f technical co m p o u n d s such as hexach loroben zen e, ch lorin ated biph en yls, chlorinated d ib e n z o d io x in , an d 2 ,4 ,5 -T is in m an y in stan ces d u e to th e co n tam in atio n o f these p rod u cts w ith chlorinated d ib en zod ioxin s; o f course it m ay be p o ssib le that on e o r m ore chlorinated diben zo fu ran s also prod u ce porph y ria. It h as also n ot been estab lish ed w hether all -com pounds that prod u ce p o rp h y ria in an im als w ill d o so in h u m an s a s w ell. In gen eral, th e ex p o su re o f the various p o p u lation gro u p s to ch lorin ated biphenyls and oth er c y clic ch lo rin ated h y d ro carb o n s is at* to o low a ran ge to in du ce p o rp h y ria. W hether this can also b e said fo r occu p atio n al exp o su re is not know n. W hen pro d u ctio n con d ition s and general hygiene o f the w orkers are poo r, porph yria cutanea tarda can ap p aren tly o ccu r.2 It is also n ot k n ow n if the com bin ed effe ct o f different porph yria inducing agen ts is cu m u lative. W hether th e p h o to sen sitiv ity observed in h u m an s w ith so m e o f th e germ icid es discu ssed in th is paper is related to an e ffe c t on th e porph yrin m etabolism w as not evident from the published literatu re. S o far th e m echanism that cau ses chick edem a, ch lo racn e, X -d isease, an d liver d isease h as n o t b een satisfactorily dem on strated. C h ick edem a disease h as been thought to be caused by proliferation o f the vascular endothelium . T h is m ight explain the flu id ac c u m u latio n ob served in d iseased an im a ls, b u t so far the sequence o f even ts that led to this disease is n o t really clear and facto rs oth er than the em barrassm en t o f the vascular system m ay p lay a role in its etio lo g y such as a* to xic m y o card itis, d ecrease in seru m p ro tein s, an d renal failure. M ore detailed stu d y o f the path ology o f th e variou s d isease en tities is n eeded fo r a b etter understanding o f the developm ent o f the various lesions m en tion ed. The chlorinated polycyclic h yd rocarb on s seem to alter the im m une response o f certain anim al species an d perhaps also o f people. T h ese very im portan t findings need to b e pursued further. T h u s far it h as n o t been establish ed b eyon d d o u b t w hether this is caused b y a general debilitatin g effect o n th e anim al o f w hich atro p h y o f the lym ph oen doth elial system m ay be on e m anifes tatio n , o r w hether this is a specific alteratio n , as w e know it fro m the effects o f radiation and e x p o su re to certain alk y latin g agen ts. In this respect n o t en ough atten tio n h as been paid to the fu n ction o f the adrenal, and several o f the ob served sy m p to m s in h u m an s su ch a s the h y p er pigm entation suggest h ypofu n ction o f the adrenal cortex. D epending on the in form ation ob tain ed fro m th ese stu d ie s, a toleran ce level o f S p p m in som e fo o d p ro d u cts as set b y th e U .S. F o o d and D rug A d m in istration m ay have to be revised p articu larly since n o lon g-term low level feed in g stu d y h as b een re p o rte d in an im als. M any q u estio n s have recently been raised ab ou t th e u se o f g e rm icid e s in v ario u s to ile trie s, c o m m o n so a p s, e tc . T h e o b je c tio n to th e ir u se in th e specific case o f h exach loroph en e is its n eu ro to xic e ffe c t, th at can also occu r in h um an s, p articu larly i f the sk in is defectiv e in so m e w ay . O f co u rse, lim ited localized exp osu re, such a s handw ash ing or treatm en t o f a sm all w o u n d , w ill n ot p ro d u ce n eu rotoxicity. 488 CRC Critical Reviews in Toxicology CCQ6237 .I 6852 ,fl(|W 1 135910 in in fa n t n u rseries`in h ospitals, one o f th e -greatest 'p ro b lem s is th e co n stan t threat o f a stap h y lo co ccu s in fection o f epidem ic proportion s. In order to prevent outbreak s o f staph yioccocus in fe c tio n s, a ro u tin e w as d e v e lo p e d in m an y h o sp ita ls in th e U .S . an d elsew h ere in th e early 1 9 6 0 ' s w here in fan ts w ere w ashed o n ce d aily w ith a 3 % h ex ach lo ro p h e n e d etergen t so lu tio n , an d in ad d ition the d iap er area w as w ash ed several tim es a d ay. T h e detergen t w as su b seq u en tly rinsed o ff m ore o r less vigorously, depen din g u pon the ph ilosoph y o f th e d ifferen t n urses. In som e h o s pitals th e detergent w as directly ap plied to the in fan t, in o th ers it w as first d ilu ted in w ater. S in ce the n eu ro to xic effect o f h exach lorop h en e h as b eco m e kn ow n , m any h o sp itals have changed their policies on the use o f h exach lorop h en e. A noth er d raw b ack to th is rou tin e, in ad d itio n to a p ossib le n eu ro to xic effect, is th at h exach lorop h en e keeps n o t on ly the staph yloco ccu s o f certain phage ty p es from grow ing on the skin, b u t red u ces all G ram -positive b acteria,312 w hich is h igh ly unde sirable and m ay lead to disease. R ecen tly E vans et aL 313 have pointed ou t that a m arked annual v ariatio n e x ists in th e prevalen ce o f several b a c teria in th e n ares as w ell as th e u m b ilical cord o f new born s. A decline o f stap h y lo co ccu s au reu s w as o b se rv e d in the fa ll: th e p rev alen ce o f E sch erich ia c o li an d stre p to co c cu s also varied. S ta p h y lo c o c c u s epiderm is and E n terobacter organ ism s did not show this flu ctu ation . A ny stu d ies o f efficacy u n d ertak en in a clin ical settin g sh o u ld tak e th ese n o rm al flu c tu a tio n s in to a c c o u n t an d it is p o ssib le that g o o d general housekeepin g practices are u ltim a te ly m o re e ffe c tiv e in p re v e n tin g b ac te rial d ise ase in n ew b o rn s than v ario u s g e rm icid e s. U n fo rtu n ately , b reak s in tech n ic d o o c c u r w hich in m a n y h o sp ita ls m a k e s th e u se o f g e rm ic id e s o n in fan ts desirable. T h e in fo rm ation review ed in this article sh o w s that the toxic effects o f the polycyclic polych lor inated com po u n d s are co m p lex. The m ere know led ge o f an L D jo v alu e in an an im al sp e c ie s d o e s n o t give us a great deal o f in fo rm ation ab o u t the toxicity o f a com poun d. T o xicity or toxic effects are a relative con cep t and such h ou seh old item s as table salt, vitam ins, pepper, and ally! p ro p y l disu lfide volatilizing from on ion s to nam e a few are also toxic. N evertheless, w e have to learn not to u se ch em icals in d iscrim in ately, w ith ou t kn ow in g a great deal a b o u t them , p articu larly their long-range e ffe c ts, w hen they are persisten t and p oo rly m etab olized . T h e in discrim in ate use o f these p rodu cts and discharge in to the environm ent in variab ly lead to their restrictio n o r b an , w hich is accom panied b y a great deal o f em otionalism , d istorted facts, an d o ften great econ om ic loss. Prudence, b etter ju d g m en t, and a critical ap proach to the usage o f ch em icals and drugs, particularly an ev alu atio n o f w h eth er in sp ecific situ atio n s they are n ecessary o r n o t. m ay help us avoid these situ atio n s in the fu tu re. P articu lar atten tio n sh ould b e paid to avoidin g unnecessary exposu re o f children to chem icals that w e know little ab o u t. C h ildren m ay react d ifferen tly from ad u lts and as far as long-term e x p o su re is con cern ed , th ey w ou ld b e the m ost vulnerable. ACKNOWLEDGMENT M y sincere thanks g o to m y colleagues and fo rm er co-w orkers w h o have m ade this review p ossib le, particularly to D rs. R . E . Z eh r, J . E. Su ggs, V . W. Burse, R . E . Jennings, R . E. Linder, V . E . Sed lak , J. A. G o ld stein , E . C . V illaneueva, an d T . B . G ain es fo r lettin g m e in corporate som e o f the un publish ed resu lts in to this m anu script. REFERENCES 1. .EPonlvairnodn.. AH.ePa.l,thS,m2i2th,,31D6.,,1M9et7t1e.r, 2.. Bleiberg,J.,Wallen,M.. Brodkin. G.,and Possick,P.,A healthsurveyofworkersina2,4-Dand 2,4,5-Tplant.Arch. R.,and Applebaum,T. L.,Industriallyacquired porphyria.Arch. Dermatol., 89, 3. 4. 3. 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Laug, E. following oPr.alainndgeFstiitoznhufgohr ,peOri.oGds~,o2f ,s2ix-Bmiso(pn-tchhsltoorotpwhoenyyela)-rls,,l/,l-PtrhiacrhmloarcooelthEaxnpe. T(DheDnT, )87in, the tissues 18, 1946. of the rat 234. pKoimlybchrolourgihn,atRe.dDb.i,pLhienndyelrs,iRn.aEd-ip, oBsuersteis,sVue..WAr,,cahn.dEJnevnirnoinn.gsH, eRa.lWth,.,2A7d,3en9o0f,lb1r9o7s3is. in the rat liver with persistence of 235. 236. gDivahenlgpreonly, cRh.loBri,,naBteudryb,ipRh. eJn-,yLlsi,n/dWeri,ldRL.MLa.,naanged..R3e6td,5in2g4e,r1,9R7.2. Residue levels and histopathology Bagley, G. and Cromartie, E-, Elimination pattern of Arodor 1254 components in the bobwhite, / in pheasants Chromatogr.. 237. 75,219, 1973. Grant, D. biphenyls in1_theVriallbebnietu. vEen,viDro. nC. P, hMydcColu,ll1y,, K. A., and 61,1971. Phillips, W. E. J~, Placental transfer of polychlorinated v 238. 239. 240. CShuerrlemya,nAr.a,t,BFuorsoed, CV.o Wm e,, t aTnodxiGcoriLm, , M. E., 11,471 Polychlorinated ,1973. biphenyls: evidence of transplacental CBioronste, nFt. Price, H. TJ o.,xWicoallt,e5r,, A. C 317, A. and Welch, R. , and Medbery, 1970. L., Occurrence o A., Polychlorinated biphenyls in human adipose tissue, f polychlorinated biphenyls in humans. Environ. Health P passage in the BulL Environ. enpect. No. 1, 241. 242. 73,1972. HYoeablst,hAP.eInLp, Bjerlc, J. E^ eRLceets,vteeNlrsoa.ov1f D,p7oD9lTy, c1oh9gl7o2rp.ionlaytkeldorbeirptheebniyfelsnyinleardiipNooserstkisshuuemoafntthme agteenieiarlael,pToipdusslaktri.oNnoor.f the nation. Environ. Laegeforett, 92,15, 1972. 243. Acker, L chlorierten and Schulte, Insektiziden in EH.u, mOabnemr ildchasunVdomrkeonmscmhelinchevmonFecthtlgoerwieerbteen, NBaitpuhrewniytulennschuandftenH.e5x7ac,4h9lo7r,b1e9n7z0ol. neben 244. Curley, A., Burse, V. pesticides and related W., Jennings, R. W,, Villanueva, E. C , Tomatis, L., compounds in adipose tissue from people of Japan, NanadtuArek.a2z4ak2i,, K., Chlorinated 338, 1973. hydrocarbon 245. Niahbnoto, and PCB in hTu.,mUanetafa, tMJg.,aTkaoune,o SA.,yuCmhiik(aPsraowca.,M1eCd,.NSiesht)iw, 8v2h,i,5JI.,S,an1d97K2.ondo, K., Organochlorine pesticide residues 246. dAedpdoissiotnio,nRi.nFt.i,ssFuleestcohfecro, dG, .BLul.L, REanyv,irSo.n,. aCnodnDtaoina.neT,oJx^icAo ln, a8ly(s1i)s, of a 52, chlorinated 1972. terphenyl (Aroclor 5460) and its 496 CRC Critical Revient in Toxicology OOOGtOS 63BO * V DOW 1135918 2 47. 'FiArtetoset.onOef,f.FA.,nFelticCkh, eDtn. .F5,4R, o1s2t9, 3J.,,1a9n7d3.Higginbotham, G. E , Distribution of chick edema factori in chick tissua,/ 248. Zitko, V , Wildish, D. J ,, dibenzofuruni to talmonid fiHshuetsz,inEgnevri,ro0n.., HaenadlthCPheoni,pePc.t.M, N. oK. .3, Acuta and , 187, 1973. chronic oral toxicity of chlorinated 249. Wzo-ilpli-admioix,iDn.inTt,hCe urantn,BinughllamEn, vHir.oMn. ,CaonndteBnltanTcohxflieciodl,. B. J MDistribution 7,57, 1972. and excretion itudias of octachlorodlben- ' 250. Piper, in the Wrat.,NE.n, vRirooana., JH. eQa.l,thanPdenGpeehcxti.n. gN, oP.. J., Excretion and 5,241,1973. tim e distribution of 2.3,7,8-tetrachlorodIbenzo-p-dioxin 251. 252. dNiobrebnazcok-p, -Ddi.oHxi.nainndgesAtilolann.,Ein.viEro,n.BHioeloagltihcaPlernepspecotn.,seNsoo.f5t,h2e33n,o1n9-h7u3m. an primate, Block, W. D. and Cornish, H. H., Metabolism of biphenyl and 4-chlorobiphenyl in chicken and the rabbit, J. rat to chlorinated Biol Cham., 234, 3301,1959. 253. Hutzinger, 0 ., Polychlorinated Nash, D. biphenyls: M., Safe, metabolic E , DeFreitas, A. S. W, behavior of pure isomers Norstrom, in pigeons, R. rats aJn.,dWbrtotdolksht,roDu.t.JS,,cieanncde.Z1it7k8o,,31V2,,, 1972. 254. Wit, J. G. and Van phenol)) in rabbits, rGaetsnadnedredna,iHry.,cSaottmlee, AacsptaecPtshyosfiothl ePfhaatremoafchoelxNaceherloLr,o1p1h.e1n2e3(2,1,29'6-t2n.ethyleo*-bis<3,4,6-tzichloro- 255. (K2o^k`,-fnKe.th(y1l9e6n1e-)bidst(e3d,4,a6f-tterricWhloitr,oJp.heGn.ola)n) dinVraanbbGitesn, dreartsena,ndH.d, aSiroymceatatslep,eActcstaoPf htyhtelo(LatPehoafrmhaecxoacl hNloaraorplh, e1n1e, 123,1962. 256. 257. wCaoruronldl,s,SFu. rEf. Manowitz, M. ,GSyanleacko, lWO. bWste,,t.a,n1d25H,o9w74ar,d1,96J.7.M. and Johnston, V. D , Deposition of et aL, Absorption o hexachlorophene on f antimicrobial agents across experimental the skin, J. Soc. C om et C hen, 18,527, 1967. 258. 259. Cinuirnlefayn, tAs,.,LaHnacwekt,, E 2, E , Kimbrough, 296,1971. R. D., Nathenton, G , and Kimbrough, E D., Review of recent evidence of toxic effects o f Finberg, E , Dermal absorption o hexachlorophene, Pediatrics. 51. f hexachlorophene 391, 1972. 260. Curley, A. and Hawk, E E , Hexachlorophene. I. Analysis in body fluids and tissues of experimental animals, presented at 161st ACS National Meeting, Los Angeles. CaL, March 28-April 2,1971. 261. Sedlak, V,, EPA, 4770 Buford Highway, Chamblee, Ga. 30341, and Kopeiman, A. E^ University of Rochester, Rochester, N.Y. (personal communications). 262. iAnbnbeootnt,atEes,MAu* sBtu. cPkafeledtiadt,r.P/. M-, Ferry, D. G., . 8, 246, 1972. Malcolm, D. S , and McQueen, E < *, Blood lewis of hexachlorophene 263. LAaldnecre,t.V2., C , Burman, 384,1972. D., Corner, B. D, and Gfficapie, W. A^ Absorption of hexachlorophene from infants'skin. 264. STaonxtiociouldAtop,^J.. PAh,arEmleacctorol,en2c2e.2ph7a6l,og1r9a7m2.s and visual evoked potential of the squirrel monkey fed hexachlorophene, 265. Ulaamer, A. G animal tissues, ,,ToVxoidceorl,APp. pDl ,PahnadrmMaacrozlu. l2i,2F,2. N 76 * , Determination 1972. of hexachlorophene in human and experimental 266. Zehr, E D., EPA, 4770 Buford Highway, Chamblee Ga. 30341, personal communication. 267. 268. Bennett, exposure Miller, J. G. A , Drinker, C K , and Warren, to certain chlorinated hydrocarbons, / MIn.d.FH, yMs.oTrpohxoicloogli,ca2l0c,9h7an, g1e9s38m. W., Pathologic changes in animals exposed to a commercial chlorinated the livers of rats resulting from diphenyl. Public Health R ep, 59, 1085,1944. 269. ENnisvhirizounm. Hi, eMal.t,hL, i2g1h,t6a2n0d, electron 1970. microscope study o f chlorobiphenyi poisoning: in mouse and monkey liver, Arch. 270. 271. NeRnoodlrlobeparlc,akLs,mDDic..raenHtdi.cuZalinundmkl:,AcJ.folGnenc.e,, nPJta.rtihcEoml,oegmCyhborlofarnpienolaaytrecrdhaylosar.riEonmnatveairtdoicbni.phHhyeedanrolytchlsaPrinbeornnapbebicnittd,su,NAceomd. 1/m, 1oP3da7if,tihc1oa9lt7,io27.n0s, of the hepatic 363,1973. 27Z 273. bKiipmhbernoyulsg.hA, rEch.DE.n,vLirionnd.eHr, eEalthE, Gaines, T. B. and Kimbrough, E , and Gaines, T. 25, 354,1972. D., Oral toxicity E of , Morphological changes in liwrs of rats fed polychlorinated mirex in adult and suckling rats. Arch. Environ. Health, 21,7, 1970. 274. Kimbrough, butoxide on E rat liDve.,r.GAaricnhe,s,EnTv.irBon,. HanedaltHh,ay1e6s,, W. 333, J., Jr, 1968. Combined effect of DDT, pyrethrum, and piperonyl 275. EKnimvirborno.ugHhe,aElth,D2,2 Gaines, T. E ,460, 1971. , and Linder, E E , The ultrastructure o f livers o f rats fed DDT and dieldrin.Arch. 276. Edwards, tumors in J. E rats feadndpdWimhietteh, yJl,-aPmatinhoolaozgoibcecnhzaenngees(bwuitttherspyeeclilaolwr)e,f/ereNnacetl tCo apnigcmereInntsat.t,io2n, and classification 157,1941.'' o f hepatic 277. UStewart, H. E and Snell, K. G , o f thehistopathogenesis,Acm TnhioelnhtisCtoopnattrhaoCloagnycmofnue,xp1e3r,i7m7e0n,ta1l9t5u7m. ors of the liver of the rat: a critical review CG06S06 6963 January 1974 497 I uthem, G .iU Distribution or chick edema factor! in chick tissue,/. )., 1. aHndealQthioPie, nPp.ecMt.., K., No. Acuta 5 , l 87, and chronic 1973. oral toxicity of chlorinated ItantBlTanocxhicfoiell.d7, B. J ,57, ,, Dlstiftution 1972. and excretion ltudiet of octachlorodlben* J ,, Excretion and tissue diatribution of 2,3,7,8-tetnchlorodIb*nxo-p-dloxin 5,241. 1973. tthicaPlarnepapaocnt.aeNaoo. f the 5,23 non-human 3,1973. primate, chicken and rat to chlorinated iliam o f biphenyl and 4-chlorobiphenyl in the rabbit, J. B io l Cham., 234, Q J* _ GO ^ CO 11 differentiated cholangiocarcinoma in Syrian hamsters nn, 59.239. 1968. fed polychlorinated biphenyls, J. N a tl Cancer Inst. 'SI, mors o f the liver in rats, /. N atl Cancer In st. 15, 1427, >mas in choline deficiency, /. N atl Q ueer Inst, 15,1549, ated naphthalenes on the liver, Proc. Soc E xp B iol Med,, IA.M .A.. 125,173,1944. Highway, Chamblee, Ge. 30341. penonal communication < * : DeFreitaa, A. S. Wn Notstrom, R. Wfldiih, D. J., and Zltko, V ,, lavior o f pure bomen in pigeons, rata and brook trout. Science. 178,312, ucptaecPtahyosfiothl ePfhaatremoafchoelxNacehelrol,to1p1h,r1n2e3(,2129'6-2in.ethylene-bis(3 ,4 ,6 -triclilorom)) dinVraanbbGite!n, dreatraena,nd1Ld, aSiroymceatatlsep,eActcstaoPf htyhseioflatPehoarfmhaecxoacl hNloareorplh, e1n1e, d, J. M. at aL, Abwrption o f antimicrobial agent! acroaa experimental 1967. ntion o f hexachlorophene on the akin, J. S oc C o m et Chern., 18,527, ,, Natheneon, GMand Finberg, U , Dermal abaorption o f hexachlorophene ce of toxic effect! o f hexachlorophene. Pediatrics, 51,391,1972. >phene. I. Analysis in body fluid! and tiaauet of experimental animaii, jo s Angeles. CsL, March 28-Aprd 2,1971. Char , Ga. 30341, and Kopehnan, A. University of Rocheater, u , Malcolm, D. & , and McQueen, E. G., Blood leveb of hexachlorophene nd GBleapie, W. A~, Abaorption o f hexachlorophene from infanta' slrin, md visual evoked potential o f the xjuirrtl monkey fed hexachlorophene, l,F. Determination of hexachlorophene in human and experimental 276. 1972. am blee tso, J,. Mn.d .GPH-a,.yMg3.0oT3r4po1hx,oicplooeglr.isco2anl0ac,l9hc7aon,m1g9me3su8inn.ictahtieo n. livers of rata resulting from exposed to a commercial chlorinated diphenyl. Public Health P ep, 59, e study o f chlorobiphenyi poisoning: in mouse and monkey Urez.Arch. nsalayrtcrehadyiosar.rEinonmavteiardtoicbni.phHhyeednarylotlhcsaPirnbeornanpbebicnittds,u,NAceond.t1Jm,.1oP3d7ait,fhic1oa9lt,7i2o7.n0s, o f the hepatic 363,1973. es, T. B ., Morphological changes in livers o f rats fed polychlorinated 72. xicity o f mnex in adult and suckling rats. Arch. Environ. Health, 21,7, % 'es, W. J ,, h . Combined effect o f DDT, pyrethram, and piperonyi 6, 333. 1968. , R. E., The uitraatructure o f liven of rats fed DDT and dieldrin. Arch. Siet(tbwuitttherspyeeclilaolwre)f,e/.reNnacetltCo apnigcmereInntsat.t,io2n, and classification 157,1941. o f hepatic lsoalor ~tfret,xp1e3r,im77e0n,t1a9l 5tu7r.non o f the liver of the rat: a critical review O O acopic observations in Macaeca mutatta monkeys fed toxic toxicity of hexachlorophene for sheep. Kef. Aec,, 78, 8 6 , i, A^ DDT in the diet of the n t. Public Health Monogr. 43. 1umaCnosnunbejeyc, tAs,.CRU,n.EPfhfeacrtmoafcionlteTnhseivne, occupational exposure 11,724,1970. .di,rdHartiynIp.teSercrtireoinnpcdheui,cc,e1dh6y1bp,y1o0pa1oe7ltyt,ec1h9slm6o8roi.ontahteedndboipphlaesnmyilcs,rGetaicnunl,u6m4:, orobiphenyl poisoning, Neurology, 21, 1173, 1971. hexachlorophene on the rat brain, light microscopy and stholo.gy o f Laboratory Ratsand Mice Cotchin, E. and Roe, fi trimcntal production of edema in the central nervous system ' lr.ou7s3t,i1ss0u7e.,1A9c5t7a.NeuropathoL 19,271,1971. d Kaump, D. H , Toxicologic studies with s halogenated R. E^ Experimental papilledema in the dog induced by thods In Toxicology, G. E. Paget. Ed., F. A. bans C&, un syndrome. Hospitals, 42.63,1968. m3.nation o f CNS toxicity o f hexachlorophene in the tat and the lew York, March 18-22, 1973, Abatr. 971. L,.hmpeensnehoeenneapcl.ecpLohamanlmcoeput,ant2ihc,ya1t.2iAo5nc1,t,a1N97eu2r.opathoL, 23,326, 1973. Neurotoxicity of hexachlorophene in human infants: a publication to Pediatrics. P., Hexachlorophene myelinopathy in premature infants, /. d Drug Administration, personal communication.. aenrvgoeur.sNtiesswueY, Aorckt,a 1N96e7v.opathol, 19,271, 1971. 3.1972. riphenyl- and triphenyMnduced gastric mucosal hyperplasia in ; Trichlorophenolzersetzungsprodukte, Arbeitsmed. Socialmed. mce that hexachlorophene b not effective. Pediatrics, 51,345, Flora in newborn infants, Arch. Environ. Health, 26,275, 1973. CCQGS06 January 1974 497 6863 i 0 ONN -~~8 0 - < i t m o Q - + - rdon W. Gribble ' rtm outh C ollege nover, N M . 03755 T C D D is incredibly toxic. The lethal dose is less than one m illion th of a gram for guinea pigs. In m an. a single exposure to a few m icrogram s o f T C D D m ay cause a severe sk in disease. T C D D has also been responsible fo r the death o f m illio n s o f chicks. T C D D is 2,3,7,8-tctrachlorodibenzo-p-dioxin. In 1899 a Germ an physician, G. Herxkeim er, described a severe skin disease con* tra d e d b y w orkers involved in the electro ly tic production o f chlorine gas usin g carbon electrodes. H e incorrectly attributed the disease to free chlorine. Subsequent reports o f chloracne continued through 1964, when 60 D o w Chem ical Co. w orkers developed the disease. Chloracne, a generalized derm atitis com posed o f com edones (blackheads) and papules w ith inflam m ation so severe it m ay cause perm anent scarring, is m orphologically sim ila r to teenage acne. Although face, neck, and arm s are the m ost com m on sites, areas covered by clothing also m ay be affected. Chloracne is a hazard for industrial w ork ers p rodu cin g chlorinated hydrocarbons such a s polychloronaphthalcnes (H alow ax), poly* chlorobiphenyls (A ro ch lo r), and polychloro* phenols. In 1957, T C D D w as isolated from 2,4,5-trichIorophenol and found to be the cause o f chloracne. Teratogenicity of TCDD In 1969, the teratogenicity (birth abnor m a lity ) o f pesticides w as studied at the B io netics Research Laboratories under contract to the N ation al Cancer Institute. Laboratory an im als fed sam ples o f the herbicide 2,4,5* trichlorophcnoxyacetic acid (2,4,5-T) were observed to* have given birth to offspring h aving fetal defects such as lack of heads F E B R U A R Y 1 9 7 4 C H E M I S T R Y 15 n i i4 t 4 \ 1 t i I \ t t I I i I 6984 0002269 r* n r -, . ^ k k H (1 0V. and eyes, cystic kidneys, cleft palates, ab norm al livers, or other b iological abnorm ali ties. T h is study w as w idely quoted by the lay press and caused public concern since 2,4,5-T and a related herbicide, 2,4-dichlorophenoxyacctic acid (2,4-D), had been w idely nsed dom estically for weed control and in South Vietnam as a jungle defoliant. L a te r it w as learned that the study sam ple of 2,4,5-T w as contam inated w ith 30 ppm of T C D D . Further, other studies o f T C D D re vealed it to be so pow erful a teratogen that it could com pletely account fpr any b irth m alform ations observed in the Bionetics study. Purified 2,4,5-T, containing less than 1 ppm T C D D , w as found not teratogenic. U n fortunately, the lay press in discussing the herbicides 2,4,5-T and 2,4-D continues to overlook the fact that it is the T C D D im purity that is responsible fo r fetal abnorm ali ties. Preparation of TCDD 1^,4,5-Tetrachlorobenzene and sodium hy-' re sid e in m ethanol are heated under pres sure; then the interm ediate sodium 2,4,5trid lo ro p h e n ate is treated first w ith sodium "*y rhloroacetate and second w ith acid to yield '2.4J5-T. The tem perature fo r the sodium hy- A t tem peratures above 160 *C polychlorodibenzofuran and polychlorodiphcnyl ether, m uch less toxic than T C D D , m ay also be r Polychlorodibenzofuran Polychlorodiphenylether produced. C ontrol experim ents demonstrate th at T C D D is form ed when pure 2,4,5-tri- chlorophenate is heated 350-400 *C. .The re lated herbicide, 2,4-D, is not usually contam i nated w ith chlorodioxins since it is .prepared at low er reaction tem peratures b y chlorina tion o f phenol as show n in the follow ing equation: ` ONa. NaOH CMHOgO'CH OH 0H d ro s e reaction is crucial. I f it is not care fu lly :.;:>intained at 160 *C the interm ediate can condense w ith itself to form TC D D . 16 C H E M I S T R Y V O L 4T NO 2 U ,<* o Q jr 'O- ij { ) 0002270 C h em ical P ro p e rtie s o f TC D D T C D D is a ch em icn lly sta b le w ater-in so lu ble, w h ite so lid h av in g a m e ltin g p o in t o f ab ou t 300 *C . T h erm al d eco m p o sitio n b egin s a t 700 *C an d is co m p lete a t 800 *C . T C D D d o c s n o t re act w ith stro n g a c id s an d is in e rt to m o st re a g e n ts. It slo w ly p h o to d c c h lo rin a tc s in m e th an o l to g iv e dib en zo-p -d ioxin a n d , ev en tu ally , 2-ph en oxy p h en ol. A n o th e r d io x in , 1,2,3,7,8,9-h exach io ro d iben zo-p-dioxin (H C D D ), h a s been id e n tifie d a s a to x ic fa c to r in ch ick e d e m a d ise a se . . O th er C h lo rin ated D ib en zo-p -d io xin s O th er ch lo rin ate d dib en zo-p-d ioxin s c a n b e fo rm e d in re a c tio n s in v o lv in g c h lo rin a te d p h en o ls. Iso m e ric tetrach lo ro p h en o ls a n d p e n tach lo ro p h e n o l a re p re p a re d a t h ig h te m p e ra tu re s b y e x h au stiv e ch lo rin atio n o f p h e n ol o r. h y d ro xid e d isp lacem en t o f ch lo rin e fro m p o iy cn io ro b en zen es. C o n se q u e n tly , p r o d u ctio n o f ch lo rin ated d ib en zo-p -d ioxin s m a y b e th erm ally p ro m o ted . F o r ex am p le, h ea tin g p en tach lo ro p h en o l to 300 *C g iv es o ctach lo ro . diben zo-p-dioxin (O C D D ) in 4% y ie ld .... T a b le I. R elativ e T o x icity o f T C D D . . - - ' LDm mg/kr* ip a u a d ' '*. '.?:! ' Coluta pig tu t ' ' Moot* - Dog DD* : - 0.0006 0.04 0 .0 0 1 0 .1 3 -T * D* X .: 470 380 375 ' 500 370 390 100 . 100 y ch n in e \5 0 .5 trao d oto xin '- .j'0.008 rin (n erv e tgen t) "-- ` 0.05 ; 02 ten o n e CN ' F ' 130 ['Clr'r-'y ,y ' .1 0 . 23 20 rath ion . ' 10 TRheefedreonwcere10q.uired to kill 50H of the animali. Reference T h is c o m p o u n d p re su m a b ly a r ise s fro m polych lo ro p h en o ls u se d to p re se rv e an im al h id es w h ich a re su b je c te d to h y d ro ly sis a n d h eat in g d u rin g fatty acid m an u factu re. T h e fatty a c id com p o n en t o f ch ick en feed b e co m e s c o n ta m in a te d w ith to x ic p o ly c h lo rin a te d di b en zo-p-d ioxin s a n d le a d s to flo ck m o rta lity a s h ig h a s 90% w ith d isa str o u s re su lts to th e ch ick en b ro ile r in d u stry . T oxicity o f TCD D T C D D is acn cgen ic, terato gen ic, an d d ead ly to x ic. G u in ea p ig s a r e p a rtic u la rly v u ln erab le -- th e leth al d o se fo r an av erag e size g u in ea p ig is ab o u t 5 x lO -T gram ! T ab le I co m p ares th e a m a z in g to x icity o f T C D D w ith o th e r p o iso n s. T h e m ech an ism o f th e sy ste m ic to xic a c tio n o f T C D D is u n k n o w n . It is kn ow n , h ow e v e r, th at T C D D c o n c e n tra te s m ain ly in th e liv e r a n d to a le sse r ex ten t in th e b ra in an d fa tty tissu e s o f lab o rato ry an im als. O ral ad m in istratio n o f T C D D to lab o rato ry an im als h a s c a u se d e d e m a a n d h e m o rrh a g in g in th e ir su b cu tan eo u s tissu e s, in testin e, an d b ra in v e n tricle s, a n d g e n e ra l liv e r in ju ry . D eath o c c u rs an y tim e fro m o n e to sev en w ee k s a fte r tre a tm e n t w ith T C D D . A so lu tio n o f T C D D a p p lie d d aily in m ic ro g ra m am o u n ts to th e in n er su rfa ce o f a r a b b it's e a r c a u se s se v e re a c n e c h a ra c te riz e d b y p a p u le s, c o m e d o n e s, a n d c y sts. T h e ch lo racn e resp o n se is ju d g e d b y th e fo rm atio n o f co m ed o n es. M icro sco p ically , the sk in sh o w s m a rk e d p lu g gin g o f h a ir fo llicle s fro m an a b n o rm a l in c re ase in sk in k e ratin (h y p e r k e ra to sis), a lso th e b a sic p ath o lo g ical de fe c t in h u m an c h lo rac n c . Chick edema factor or disease periodically kills millions of young chicks in the U.S. It causes a buildup of fluid In heart sac and lungs. HCDD was Identified In the toxic fat of feeds respon sible for the disease 0 1 U <4 ftj (u O Stru ctu re-A ctivity R e latio n sh ip s T h e -e x trao rd in ary b io lo g ical activ ity o f T C D D is alm o st u n iq u e to its p a rtic u la r ar- F E B R U A R Y 1 9 7 4 C H E M I S T R Y 17 G 2 G 6.. 0002271 T a b ic I I . A ctivity o f T C D D an d R e lated C om pounds* .(mL*D/km*h CbtoncM Tgcraleto Cdhtimck* Cooipotoid mt R ip O B M m p o o M ru p o o N .T C D D 0.04 S tr o n g ly S tr o n g ly S tr o n g ly p o sitiv e p o sitiv e p o sitiv e > 1 0 0 0 " N egativ e N egativ e -- HCDD . (m ix tu re o f tw o iso m ers) > 1 0 b P o sitive P o sitiv e P o sitive OCDD > 1 0 0 0 " N egativ e N egativ e N egativ e * ^OsC -- Stro n g ly p o sitiv e --. -- 2 ) -- N egativ e -- -- Reference 3. . . *Largestdosefedatwhich therewereno deaths. * - -- -! f *- '.mm me . - i l - ra n g e m e n t o f h alo g en s. O th er ch lo rin ated d ib en z o -p -d io x in s a r e m u c h le ss a c tiv e o r in * a c t iv e ( T a b le I I ) . I n a d d itio n , th e 2,8d ic h lo ro -, th e 2 ,3 -d ich loro-, th e 1,2 ,4 -trich loro-, a n d th e 1 ,2 ,3 ,4 -tetrach lo ro d ib en zo -p -d io x in s a r e n o t acn egen ic a n d p ro b ab ly n o t terato gen ic. E n viron m en tal S tu d ie s o f TC D D A lth o u g h th e u s e o f h e r b ic id e s 2,4,5-T a n d 2,4-D h a s b e e n p a r tia lly b a n n e d , m a in ly b e c a u se o f th e B io n etics stu d y , the m a jo r con c e rn sh o u ld b e w ith T C D D a n d o th e r d io x in co n tam in an ts. F o rtu n ately , th e su p p lier o f m o s t o f th e h ig h ly c o n ta m in a t e d 2,4,5-T ( ^ 3 0 p p m T C D D ) n o lo n g er m a n u fa c tu re s it. T h e U . S . D ep artm en t o f A gricu ltu re an d o th e r ag en cies h ave su b je c te d TC D D to en v iro n m en tal scru tin y a n d h av e learn ed th at in so il TC D D is n ot read ily d egrad ed , ph otoch e m ically o r o th erw ise; o r b io sy n th esized o r p h o to sy n th e siz e d fr o m 2 ,4 ,5 -trich lo ro p h en o l; o r a b so rb e d in to p la n ts; o r m o b ile in th e so il. T C D D ap p lied to leav es i s n ot tran s lo c a te d w ith in th e p la n t. T h e m o st im p o rta n t o f th e se fa c ts a s fa r a s th e en viron m en t is co n cern ed is th at the so il is a n u n reactiv e sto ra g e co n tain er fo r TC D D . C O R D O N W . C R IB B L E , assistan t p ro lesso r o f ch em istry at D artm ou th sin ce 1968, h as r e se a r c h in te r e sts in n a tu r a l p ro d u c t sy n th e sis a n d n uclear m agn etic reso n a n ce sp ectro s copy. ftHFUISTB Y V O L 47 N O 2 T h e U SD A recen tly te ste d 129 sa m p le s of 17 p e stic id e s p re p are d fro m p o ly ch lo ro p h e n o ls a n d a ls o 20 s a m p le s o f p o ly c h lo ro p h e n o ls fo r d io x in c o n ta m in a tio n . M o st o f the p e stic id e sa m p le s an alyzed {1 6 % ) co n tain ed f r o m n o T C D D to le s s th a n 0.1 p p m , 15/ c o n t a in e d 0.1 t o 10 p p m T C D D , a n d 9 % c o n ta in e d m o r e th a n 10 p p m T C D D . 2,4,5-T s a m p le s w ere sh o w n to c o n ta in m o st o f the T C D D a n d o n ly o n e o th e r p e stic id e , silvex, c o n ta in e d m o r e th a n 0.5 p p m T C D D . A n a ly se s o f ch lo ro p h cn o l sa m p le s sh o w ed m o re th an 0.5 p p m T C D D . H ig h e r c h lo rin a te d d io x in s w e re p re se n t in m an y o f th e ch lo ro p h cn o l sa m p le s. F o r ex am p le, a n a ly sis o f pentac h lo r o p h e n o l s a m p le s s h o w e d 30/o c o n ta in e d m o r e th a n 100 p p m O C D D . 2 ,4 ,5 -T _ cu rrem lv p r o d u c e d . b y .rD o w ^ C h e m i c a l ' C o . c o n t a i n s ew cA K a^O ^ppn ^T C D D ^ T l" H um an H ealth an d TC D D A d irect relatio n sh ip b etw een TC D D and c h lo ra c n e is c le a r . T C D D 's e ffe c t o n la b o r a to ry an im als h as been e sta b lish e d ..B u t no r e la tio n s h ip^. b e tw e c n ^ 2 ,4 ,5 -T _ a n d / o r .T C D D S n d r h u m a n ^ b ir tK 'a b n o h n a litie s' h ^ .'V e e g fou"nd,_d c s p itc 'stu d ic s_ o f_ th e _ 5 jn illio n a c r e s o f'S o u th V ie tn a m sp ray ed jtQ m .-1 9 6 2 Js5 L l2 6 S w ith 60,000 t e a s c f 2 , 4 ^ J . R e search is n eeded to d eterm in e the e f f e c t s o f 2,4,5-T T C D D , a n d r e la te d c o m p o u n d s o n h u m an h ea lth . A s fu tu re stu d ie s u n fold , d e cisio n s can b e m ad e re g ard in g the p rop er r o le o f th e se c h e m ic a ls in o u r so c ie ty . SI Suggested Reading < (1) Aaronson, T.,Environm ent, 1971, 13 (7).21. (2) Abstracts. Chlorodioxin Symposium, 162nd Na tional American Chemical Society Meeting Washington, D.C., Sept. 17, 1971, Pesticide Di vision. (3) CCoolnlitansm,.TT.oFx.icXo.l.. W19i7l1l,ia6.ms5.59.C. H..B ull. Environ (4) FCaolukr,tnHe.y,J.K,.BaDt.e,sG.ayRl.orR..,DM.itWc.h.elHlo,gIa.n,,ScMie.ncDe. 1970,168.864. (5)CFD.rh,ieemAdnm.s,at1ne9,a5d9L,,.4,2MF,.i,1r2e9Ss.thounee,,DG..,,HJo.rwAistsz. ,OWff,,ic.BaAnneas, (6) Kearney, P.C.,presentation before theCoune: on Environmental Quality and President': Cabinet Committee on the Environment Work .ingGroup inPesticides,Washington, D.C.,Nov 5.1970. (7) Pohland. A. E.,Yang, G. C.,J. Agr. Food Chcm. 1972,20,1093, (8)APorlcahn.dE,nAv.irPo.n,.SHmietahl.thD,.1.97M1c,t2t2e,r,316G..,Possick, P. (9) "Report of the Secretary's Commission or Pesticides and Their Relationship to Environ mental Health,*' 1969, pp. 665-674. U.S. Depart ment of Health, Education, and Welfare, Wash ington,D.C. (10) Rowe, V. K., Hymas, T. A., Am. J. Vet. Res. 1954,15,622. (11)CSpoasrrsnceht.uT,oGx.icoL.l.. 1D9u7n1,n9., F. L.,Rowe, 405. V. K- Fd (12) Whiteside, T.. "Defoliation," 1970, Bailantinc New York. N.Y. (13)JW.oAoltsr.onF.ooEd.CA.h,cTmh.,o1m9a7s2.,20R,.3FS.1..Ensor, P. D. J 6 9 Q)'602272 \ rl Ion n mou 4UCU=TS4. 1*14 COMMUNICABLE DISEASE CENTER M orbidity and Mortality Weekly Report A ttacran e^ t 2 299 * t. LOUIS ENCEPHALITIS - Cnniinued house spariowv On June 25 intensified larvacide and adulti* tide of mosquitoes was begun in areas o f Memphis known to be heavily infested. A total of 18.000 gallons of 95~ mala- thion were iprayea over the next 2 months in an attempt to avert or lessen an epidemic. As soon as human cases were confirmed, ground iprav-ng of d f * malathion was initiated in all urban areas of Memphis and Shelby County. (Reported by Alan L Btsnn M.D.. Director. Infectious Dis eases. Department o f Medicine. University of Tennessee'Col lege o f Medicine. Robert C Rendtorff. M.D.. Director. Divi sion o f Communicable Diseases, f K. Musley. Director. Field Services Division. Harotu Carter. Administrative Assistant, and Irene Duncan R .V. Supervisor. Communicable Diseases. Memptus-Sbeiby Count! Health Department. Robert H Hutcheson Jr .A t D M P H . State F.puJemroiognt. the Arbo virus Re/erenre Branch Vertnr-bome Diseases Dirtsmn. and the Virology Division. Bureau o f Laboratories. CDC: a Pub- lie Health Advisor. and an E1S Officer.) TaMel Horn(gltallio Inhibit (HI Then w St. Louis Encephalitis V an m a rawrai* Memphis. Tranci*. A(um I*" Case No. 1 2 3 4 S 6 Serum Specimen Acute Cnvalesccnt -- ns-- 1:40 1:20 1:10 1:10 1:10 1 160 1:160 1:160 1:80 1:80 1:40 1. Sudu WD. Fowmkk E C o ir a i III St Lumi Encephalitis in Mmnphii, Trancsm. >96*. J Mea Esternai 4 r*-'P. 1901 ILLNESS ASSOCIATED WITHTCDD-CONTAMINATEOSOIL - Mhaoori In !972 the Missouri Division of Health and CDC in vestigated a hone arena >neastern Missouri where 54 of 57 bones exposed to the arena had died of an illness charac terized by skin lesions, severe weight loss, and hepatotoxidty. Birds, dogs, cats, insects, and rodents were also found dead in and around the arena, and one 6-year-old girl ex posed developed hemorrhagic cystitis. Urine cultures for trial and vital pathogens were negative. Immediately to the onset of illness, the arena had beensprayed with salvage motor oil fordust control. Simitar hone illnesses and deaths occurred in 2 other hone arenas in the eastern Missouri areasprayed by the same salvage oil com;.. The 2 cranes had beer sprayed within 1month of each other. Subsequent to investigation, sc from all 3 arenas was excavated and disposed. No further problems have occurred since these excavations. Recent results from laboratory analysis of soil simples taken from the initial arena implicate 2.4,5-trichlorophenoi acetic aad. An investigation a underway to determine the source and extent of the TCDDdisposal. (Reported by H. Denny Donnell. Jr.. M.D.. State Epidemi ologist. and Pat Phillips. D. VM.. Division o f Health. Missouri Department of Health and Welfare, the Toxicology Branch. Qbiital Chemistry Division. Bureau o f Laboratories, and the Cancer and Birth Defects Division. Bureau o f Epidemi ology. CDC. ond 2 S IS Officers.) Editorial Note TCDD is a very persistent chemical and a potent contact poison (1). Its acute oral L D -5 0 for rab b its, m ice, guinea pint, aad other spuvio is in the microgram-per-kilogram range. TCDDmaycauseehloracneandliverdamagein humans, duckedema disease in chickens, and porphyria cutanea tarda in animals and perhaps man. It has been shown to be tera togenic in some mica strains and has also a general embryotoxic effect. and2J3.7,8-te:rachiorodibenzodioxin(TCDD) as the probable toxic substances. TCDDis a waste byproduct in thesynthesis of tzichlorophenoi and the herbicide 2,4,5-trichlorophenoxy- R eftreac* I. Kmbrouih RD: Toxicity of cNonaaud hydrocarbons a d related compounds. Arch Eavtron Health 25 123-131.1972 CD cn tO TULAREMl ; -iMICKINGPLAGUE - NewMexico On May 19. 1974. a 39-year-old man from Coolidge. New Mexico, developed rfttnorrhea. headache, generalized malaise, and a fever of I03*F. He was seen by a physician at a Gallup outpatient clinic and given long-acting penicillin intramuscularly. His symptoms unproved until May 24, when he developed a tender swelling at the anterior axillary line. OnJune 6 physical examination revealeda rectal temperature Of 100*F and 2 right para-axillary and 3 right epitrochJear tender lymph nodes. An interview revealed that I day prior to the onset of his Alness, the man had shot a prairie dog near Coolidpc. He tre*4, the carwa to his wile, who skinned and dressed the t 1. Both the manand mis wifeate some of the meat after it nad been cooked for 2 huure over a wr~* ?>.. Bubonic plague was su spected, and 'he patient was .-uurd Adirect smear of lymph node aspirate .i-.it.. by Gram's methodandwith Ycnumpestu fraction I sp. . fluorescent antibody did not reveal suspicious organisms. Cultures of Mood, sputum, and lymph node aspirate were negative for Y patit. Table 2 gives the results of serologic tests per formed on a scrum specimen ootamed on June 6. The patient denied a history of recent insect bites, tick attachments, or carbuncles. He repeatedly denied having skinned or dressed rabbits or other small mammals. The TaMeS Rrealtsaf Serologic Tots lar Nap* aad Tt Junta. 1994 Tube A lt Tiler Fiatar.- Tularemia Negative 1:320 h a r e kenurpwmattai KIA* Titer Negative 1-8192 6969 0002667 6970  < Attachment 2 (contd.) l THE NEW YORK TIMES. WEDNESDAY. AUGUST 2i, 1974 D O W 4 2 5 O86 DEATH0FAN1ALS LAID TO CHEMICAL Dioxin Identifiedas Causeof , Fatalities in Missouri-- 2 Children Became ID M illtn ilK M M JEFFERSON CITY. M&, Aug. 27--Dioxin, tha deadly chemi cal coaUined in a herbicide once used to defoliate forests in South Vietnam, haa beenidenti fied as the agent that killed many horses and scores of oth er animals in Missoni. Two young girls, one 6 years old and the other 10, becameill because of exposure to the sub istance. The younger suffered such severe kidneydamage that. she was hospitalised for four weeks at SL Louie Children's HospitaL Identification by health scientists of the dioxin is one more step in a medical mystery that began in 1971. The exact sourceof the "t**!.i cout minant in the herhidde 2,4J5T is atm unknown. More than 80 horses! 70 rfrfrkena. several dogs, e doaaa cats and hun dreds ofwildbirdswen Wiled Dr. Renata Kimbrough, ntox icologist at the Federal Cantor toe Disease Control at Atlanta, said that the Missouri Incident as far as she knew, was the first In which dioxin had been identified aa the came at ani mal deaths and injury to Inn tmhaenshoerubtiscididee ofwpaslanmtsanwuhfaecre tured. NoOtherIncidents Dr. Barry Commoner, director of the Center for the Biologyof Natural Systems at Washington tUriesnnti.ovwe--rnsmeitdyteihnnavStirtho. naLmokueninsetwwalhosocfiiesnnoa other incident-in which dioxin had been identified in deaths outside the laboratory, al though it had been suspected in some field cases. Atea mat the laboratories in Atlanta identified the highly toxic substance-- danger even ;at a few parts a billion--after almost two years of analysis. The Atlanta center came into tohfetheeastwe oafstmeraltlhgeirlisn.volvement **Ws didn't know what we wera looking for,'* said Dr. Kimbrough. "That is why It took so long. Wa thought at first it might be some kind of a nerve poison and that sent us off onthe wrong track. It was a; needlainthe haystack." The laboratory taam found the needle, known by --ii-- aptaia-2d4io,7x,i8n. taeltsroachklnoorwodnibsesnzdoi- oxin or TCDD. That substance1 sbcaidentbisetasnassuaspcaeuctseedofbbyirtmhadny lfiasctetsd ianncahsioldfrSenoubthorVnieitnnadmef.o-. Uat of Herbicide Halved foLunabdortahtoarty tdeisotxsinwitnhoatnimonallys cauaad birth defects in guinea pigs but also killed the animals at Meals aa low as 0.0006 milli grams for tech kilogram of `!bfionddyinwgsetihgehtusBeaocfanhseerboicfidteheosre ange, which contains 2. 4. S-T ana which is contaminated by dioxin, was suspended in Sep tember, 1971. Its ust in the hUanditebdaenStaptlaecsedasunadehrerrebsitctiideetions in 1970. terDinr.arPiaantriicnkthEe. PMhiislsliopusr,iaDvivei sMioinssooufri Hinecaidltehn,tsbaeidganthiantMthaye and June of 1971. Four stable areas wen sprayed with waste ootuQilaltlionywcwoanhsticirdhoelntdthiuefisetddi.Imt rwiasevtehnis spnWyiitnhgin, tswpaorrodwayss aanftderbathrne tshweaslltoawblsesb.egan to die at one of "Then Iitenlly were bushel bbairsdkse,t"srefuclallloedf tDhro.sPehdileliapds.wild Horsas began to gee sick, their skin erupting in ulcers. They refused to eat Fifty-four hones were affected. "As of! this date, 35 of these horses -have died," the state veterina rian said yesterday. "Then ywoeunng1c2olatbs.o"rtions in mares of theThsoemmmaenrsoflo19st71t.hTehecohlotsrseins began to die in 1972. 2 Giris Stricken Than two yo&g girls who lived at one of the stables be came in with what was then an< oTufnhiedtihernemtiofsittaahbbellereasnkdicdotnmheeyplcaoidn-ioesedwansoeef.r' constant headaches. Soil samples taken inthe area owxeinn wthaesoindeesnitnifiewdhiecahrltyhethdiis month. Preliminary data indi cated that the dioxin was in ocoilncceonnttraamtioinnatoefd60bypa2r,ts4w. a5s-tTe was sprayed has been removed. "Tha man who spnyed the stable areas was not at fault," Phillips said. "Ha simply bought the stuff. We believe he itsheteldliinogxitnheshtrouuthld. Tnheeveorfi hwaivthe gotten into tha handa of the wbthoaaustgttahhtisosowilmaescaaolrilrliieutrnw.daeTsr.h"thise-igdueya Dr. Herbert R. Domke, direc tor of tha Stats Division of .Health, called the incident an '"episode of environmental coniIctahmeminiactailonwwhiicthh ashhoiguhldly btorixnigc bercenaomumvisaresorkmnthamobareleavnhetatruhlymatpraenoailtlaluiltdimitaonnedgn.iedtrsI.st"nooistf 0 Z 6 3 6 i 6971 THE DOW C H E M I C A L C O M P A N Y February 28, 1980 BENNETT BUILDING 2030 DOW CENTER MIDLAND, MICHIGAN 48640 D O W 428192 MN56835 STATE DEPARTMENT OF AGRICULTURE Gent!emen: REVISED SPECIMEN LABEL FOR ESTERON* 245 HERBICIDE Enclosed i s a copy o f the revised specimen label fo r Esteron 245 Herbicide. The re visio n s are noted on the bottom o f the lab e l. Please place th is in your f ile in connection with our registration for said product. Discard previous specimen lab els. Very tru ly yours, ( P o v S + c P ' ' -------- - Conrad D. Woods Regulations/Labeling Sp e cialist Quality Assurance Department sre enclosure *A Trademark o f The Dow Chemical Company PRODUCT NAME: LABEL/CONTAINER SIZE: y g'j 3 0 - ^ 5-" //i-C DATE CODE Old/New EXPLANATION OF CHANGE (Initiator, Reason, Disposal Action) INITIi & DAT! Y o n t o '^ o Gl io m i < \\0-7l >K > g ^72 *% S^7f C /U , A / ( ( T* * /lA A sto ^ O - 0 - } H x IC z Jb j " ^ 6 e r C C U jS L , s o / ' t & t< J ? > f o M J & o < 0 - ( + M '. //UU^{>K. ~Q A t 14 04" CaACL U//^* C * w s r v tf O - U s u i l o C 0 * 6 t / 0 17 ^ 2 ^ * A / q * ~ 9 j * * * - M * & 1 4 ^ e * ^ o .'t y .x '- ^ r I x S L c f e (^ jS()4t * / G -C rV \4 < /fe< ^ w 4" 'i'i'V T i 'J iP H A , cut4 / t w ^ l> tx 4 ~ \ a s 2 JL J . &&U, ,f//z/r *T /&6 C -0 * U w ^ '. u S * * L * !> J s u e d ? ^ x w v O A *> L V 9 ~ a J U ^ u S ^ l J U j lo + f r a **-- Gtyk/ , "3 ^ c- k . ^ 3 / j 2 i i > i r ) i i A v u ^ - i X o O Cfc to CD to <1 00 ^ :.a 'U i i J ,? i m o c i r M fe t tt b r . Aut . I V t T H E T A S H E s' G T O X P O S T U.S. Scientist Disputes EPA on Toxic Herbicide By Iorton Mintz Wstun*ton fasts t i l l w riter Tbe Environmental Protec tion Agency and one of its top scientists have spilt over con tinued use on rice fields, pas tures and rights of way of a herbicide contaminated by the most toxic chemical known to m an " 1 think it should not be used," Or. Oiane Courtney, chief of the EP.Vs toxic ef fects branch, told a Senate Commerce environment sub committee hearing Aug. 9. "We are using a chemical that we can barely handle." EPA officials agreed with Or. Courtney that the contami nant. dioxin, which is invari ably present in the herbicide. SEX. JOHN V. TCNNEX . . . fears dioxin use 2.4.5-T. causes-birth defects in laboratory animals in concen doubt necessary before you trations "so low we cannot get rid of the product." EPA measure them." Deputy General Counsel Rob But EPA officials told the subcommittee Aug. 16 that they not only favor continued ert Zener said. "I don't think we can prop 'use of the herbicide but see erly assess the risk in a public no substantial question of hearing" which would not be safety which would require a pubr.l:ic'"/heaairdintgh. e lack of an ad "based on any data," said win L. Johnson, an agency Ed pes equate method to detect di ticides official. oxin in parts-per-billion ratios Tunney said he feared the leaves the EPA with no data agency will let use of 2,44-T to show that residues get into the food chain. Moreover, they continue "until there is a sub- - testified, the probability of ex-1 stam ial adverse impact upon' posure appears to be minimal j the health and welfare of the because dioxin apparently de population." The EPA said it composes swiftly ence of sunlight. Sen. John V. in the pres Tunney (D- is stressing the need "continued and intensive" for ef Calif.). who presided over the forts to resolve questions ; hearings, strongly disagreed. ! about the use of 2,4,5-T and re-' It is a "crazy kind of logic" .lated compounds, to let a toxic substance con-j The senator, told by Dr. tinuc in use "because we don't! Courtney that dioxin might be have adequate monitoring: in a steak bought at a super data to determine how toxic i t ; market, said, " It makes me Is." he said. : very angry. . . . because I did; "Why shouldn't you have a! not know that l was being ex- public hearing and just let;posed to dioxin, and 1 don't' people express their;like the fact ;hat 1 am being view points?" Tunney asked. " I. exposed without knowing ju st can't understand why you;about it." are so frightened of having a| In Suth Vietnam, military public hearing." pilots defoliated nearly 5 mil- ent. The herbicide, independ ently of dioxin, was reported in 1970 to cause birth defects when injected into mice. Last year. Harvard scien tists reported that dioxin in significant amounts had en tered the food chain in South Vietnam. Conclusive studies have not heart done on whether defoliation has caused birth deformities in hu mans. Dioxin is so lethal that a tcaspnonful is believed suffi cient to kill 1.200 persons. After the 1970 report on 2,4.5- T, made by Department of Health. Education, and Wel fare panel, the Agriculture Department ordered a ban on the use of 2.4.5-T for eertain uses--around homes, in gar dens and recreational areas, or where it could contaminate | water supplies--but allowed it to go on being used in torests and on righis-of-wav. range- land and. later, rice fields. D oes dioxin get into the food chain in this country? Tunney asked researcher Courtney? No one can say for certain because "the food chain is not monitored for dioxin." she re plied. I The new method, which was developed by the Harvard sci entists, chemist Robert Eaugh- Iman and geneticist Mat:hew Meselson. worked in Vietnam. |But it turned out not to work under the much different con- ditions encountered in t h e United States. : Finally, Quarles cor.ver ;d a| meeting with Dow Chen icaL! the Agriculture Departr. ent. ; and the Environmental De-. fense Fund Inc., which long had been battling to hal. all : usage of 2.4.5-T.-Dr. Cour ney, although an expert in birt i de formities and a pharms ;olo- j gist, was not asked to attend-- a point that irked Tunney. i I Then, on Ju ne 24. Quarles ; announced there would be no 'public hearing, because w th- : out a "break-through" in di oxin detection such 2s had .been counted udoii "a hearing would not be productive.' But he acknowledged in. a -letter to William Butler of the Environmental Defense F 'md. ; " The decision to refrain f.om :proceeding at this t.me should. . . . permit no it :er- ence that the doubt at to the safety of 2.4.3-T and related herbicides has been resolve 1." 1 In July, 1973, EPA an At the Senate hearings. Dr., nounced that it would hold a public hearing on whether these uses of 2.4.3-T should' continue. At such a hearing, iCourtney said that cattle . which has crazed on pasti res snrayed with 2.4.5-T were found to have dioxin in tl tir liver and fat. and that it co; se- which was to have been held . quentiy could be in bee: and this month, the sole manufac turer. Dow Chemical Co., would be obligated to demon strate with clear and convinc ing evidence that--in the words of EPA Deputy Admin istrator John R. Quarles Jr.-- ; 2.4.5- T and dioxin "do not: present an unacceptable risk! of adverse environmental ef- 1 fccts." At the time of she announce ment. the EPA expected that a sensitive new method for de tecting dioxin pans per tril lion wuuld be able to pick un dioxin traces, if present, in hu- dairy products. Tunney cited; evidence that dioxin al-o had! been found in coats and sheep' which had cra7cd on land treated with 2.4.5-T. Tunney told the EPA offi-| cials that he " cannot souare"! Dr. Courtney's view tha* 2.4 5-| T "should not be u-cci" with! Vhc agency decision that the^e 'w a s n o "substantial" qut-s- ition of afety. He was told: : that a much'broader scirnthic; i investigation that Dr. Court-i I ney's had been made by the I j EP Vs criterion and evaluation j division. I " H'ell, 1 guess our basic dis-1 lion acres withAgent Orange | man fat and nulk, and in beef, agreement is over the level of jof which H.A.G-T-- jo million 'rice and wildlife. I pounds of it--was an ingredi f> ]s*,t p * 6 C ^ (J 0001994 lio I \ r\ Herbicide Killed Missouri Aniimals GO N v Tarb fim ai |rv K t Jefferson City, Mo.--A letheI concentration of Dioxin; the deadly chemical contained in a herbicide once used to defol iate forests in South Vietnam, 'has b e e n identified as tl\e agent that killed many horses and scores of other animals in Missouri. Two young girjs, one six years old and the other 10, be came ill because of exposure to the substance. The younger suffered such severe kidney damage that she was hospital ized for four weeks at St. Louis Children's Hospital. Tha e x a c t source of the chemical, a contaminant in She said that if the concern thation of dioxin were kept to one-tenth of part per million parts of 2.4,5-T it "would not h e hazardous" to wildlife when used as a defoliant. Within two days after the spraying, sparrows and barn swallows began to die at one of the stables. " There literally, were bushel baskets full of those dead wild birds." re "The case In Missouri Is an called Dr. Patrick G. Phillips. isolated one where the dioxin was much more concentrated than in the 2,4,5-T used for spraying," she said. The 2,4.5- T in Missouri was estimated to | have a dioxin concentration of 0parts per million. "We are not sure why," said D r. Kimbrough. Herbicide manufacturers can control the amount of dioxin in 2,4,5-T, she said. a slate veterinarian. Horses began to get sick, their skin erupting in ulcers. They refused to eat. Fifty-four horses were affected. Thirtyfive died and 12 m a r e s aborted. Then two young girls.who lived at one of the stable! be came ill with what was then an unidentifiable kidney dis ease. Their mother knd the co-owner of the stables com plained of constant headaches. --.I the herbicide 2.4,5-T is still un known. More than 90- horses. DIOXIN had been suspected by many scientists as a cause i| 70 chickens, several dogs, a of birth defects In children , dozen cals and hundreds of born in defoliated areas of wild birds were killed in Mis- South Vietnam. ' snuri. . Laboratory tests with ani THE HERBICIDE 2,4.5,-T is mals/ found that dioxin not Thursday. Aug. 29. '74 DETROIT FREE PRESS planned for use in Michigan only caused birth' defects in U.S. rarest Service to kill 84 guinea pigs hut f Iso killed the . acres of aspen and hardwood animals at levels 'a s low as trees in Ottawa National For 0.0006 milligrams for each kil est, allowing more room and ogram o f . body weight. Be sunlight fqr more commer cause of these findings the use cially valuable spruce and red of the herbicide agent Orange,- pine trees. which contains, 2.4.5-T a n d ' The spraying has been de w h i c h is contamined by layed until at least nekt year dioxin, was suspneded in Sep- | hy federal court suit brought lember 1571. Its use in the i Ky Michigan Attorney General United. Stales as a herbicide Frank Kelley. The Wisconsin had been placed under restric- I attorney general has also filed lions in 1970. suit against the Forest Ser The Missouri incident began j vice. halting spraying of the In May and June of 1971, after j herbicide in northern Wiscon four s t a b l e areas were sin forests. sprayed With waste oil con-' ' Dr. Renale Ktmhrougb, a toxicologist a t ' I h e Federal laining dioxin, Id control the rust. Center for disease control at Atlanta, said that the Missouri Incident was the first in which dioxin had been identified as the cause of animal deaths and injuries to humans oili- M o az l z q L Z side plants where the herbi cide was manufactured. hl s THE DOW CHEMICAL COMPANY BIOCHEMICAL RESEARCH LAB. MIDLAND. MICHIGAN 48640 TCX. SO. : PILE NO. ,i ROUTEOf AOMlNiSTRATKUj 'lXw; LVZ2-*~i lengthoftreatment c SPECIES: / RAT -0 F DRABBIT f "A pig Df d o g C3m f Dm o n k ey D m f Dfham ster D m OTHER SES 0 , o . l J O.OI oJ. 0 .oolAy/ij/6 i CHECK POINT DATE PLE RECEIVED TOXICOLOGY ESTIMATED STARTING TIME ESTIMATED r e p o r t e d d a t e REVISED ESTIMATE REPORTED REPORT DRAFTED REPORT ISSUED FATE OF SAMPLE 1 1RETURNED [ |DESTROYED DATE \ 1 |REFERENCE SAMPLE STORED | |REFERENCE SAMPLE TO K STORAGE NOTES tfffcr t fi DATEPROJECTCOMPLETED FORMC'32790PRINTE0 INU.SJL >M NOTES, CHANGES, ADVERSE EFFECTS HOTEO o n ? ___________________ -2___ i-- ee479i 6979 FRONT! 02 1 SAMPLE INFORMATION I *AMt '> 7 * / W - - L m L ,,a R s 3R*ULA M O LECU LAR EIG H T . 'NC-NT%lS Tc p 5 * . C " - 9 a ! S 3 R M U L A O R C O M PO SIT IO N r_ i_ iO I o - / s V ' ^ - <--- I SAMPLE D EFERENCE. SOURCE a n 6 OR IO f 't lf l it l N " I_ -7 : a TEB p u r it y o p s a m p l e sa m p le a n a l y s is 1r esu lt s *- , 0 R L - j ^ - V 3 & {' <3 t , t j v - ae 5 BCILINO POINT C EXPLO SIVE LIM ITS rnmHG (% BY FLASH POINT VOLUME IN AIR) | IGNITION TEMP. F 1 C M ELTING POINT C VAPOR PRESSURE <^ g C O R R O S IV E N E S S It * c m m ii nU) Q. . U PHYSICAL STATE COLOR ? __________________________________________________________________________________________________ X u I CHEM ICAL R EA C TIV ITY 0 0 0 R (cl* ewctnttnn hi a Ml S1 w i S T A B IL IT Y rm SH cfcnp **. liM mm* 25 Why or tosicoloqicol studi ndd? In d u d P O fa of dlopmnt, ptooo-- d u m , method f handling, nd -- ourtt handled. 0 you know f orty o d w M ffcri from humen leosur? *r> ^ ^ fc rj /ftiwT J- s^ir+JL^JL-Jt______ C .' 3> O UT - J S j J r & i r. ' ! A w iA h T "C /^ ^ ^ 1 - / ^ ____________ i ^ / Z r t J r G04T92 (____O ATE PROTOCOL COMPLETED !____| A N IM A L S ORDERED l/ - 7 (/ l D ANIMALS st a r t e d o n t e st 1 1necropsy done r--1 1 1 STATISTICS DONE 1 1CLINICAL TESTS REPORTED IL-_-l1 HEMATOLOGY REPORTED TESTS 1 1SLIDES STARTED oate _ 1__ 1SLIDES TO BE REAOY 1 1SLIDES READ p1 I__ 1HISTOPATH REPORTED I WORK ALL DONE, REAOY 1 1TO WRITE OATE p O. Q f"i U U V/ Wx DW8S299S % n O 025271 * Cr* s B. A. Schwetz E . E . Kenaga H. L. Gordon J. G. Gleeson J. Kilian - Fre' INDEX 'r<s. --------30 *t r.-. Ag-Organics Dept. 7/3/75 `** w w i/ffa.--s.-16 T s i t o l o g i y a i Genetikn , He. 3, 2 0 2 - 206 (1971*) - ! CYTCGL'JoTIC -jy-i'C? CF Tr.il i-SiiSICIDS 2,<t-D ON KUF/.N AND ANIMAL CHSCM03CMES K. A. Filinskaya 02 Ir.trcauc t Ion C e r t a i n pesticides can make a definite contri b u t i o n to the induced nutation process in humans. Consequently, the problem of a s c e r t a i n i n g potential mut a g e n s among; the pesticides assur.es special u r g e n c y , the accumulation of which mutagens in various atmesrhere objects of the external 5v.\Jt;..vX can have genetic danger r.ct only * fer the individual but also for the population as a w h ole. A. study o f the m u t agenic activity of the w i d e l y used hcrbici.vj.-. of the 2,b-- D group (derivatives cf chlcr c p h e n o x y compcur.us) is fci'hlv oractical on this score. --m The cytcgenstic a c t i o n of the chlcro derivatives of the rher.c::y acids has been studied mainly on various plants /I - 5/ In roccai years sene members of the chlcropher.oxy compounds have been stuii on insects and mammals /6 - 8/. In the present nap or we made a study of the cytogenetic ;n::: .-in of cno of the compounds cf the -iver. group, and s p e c i f i c a l l y 2,^-dichlcrc"h<*noxyrcc t ic acid 1.2,^-12), on the somatic ccl!u of t hu m a n s in vitro and cf mice in vivo. T his ho r o i c i c e :,;.:.!ct.:.: y from a fairly c;i*.r:..;iv? .'roup of c.\3arc d e r i v atives if th** .t-i. h c ids in viev: of t r. f a r t that the residuo.-, rf an;, c f The .. of this frau; on ilr.nis m a i n l y rerrcr.cr.t / r`/ . CC05G20 6SS2 D O W 882.399 id c 203 M a t e r i al nnri ror.-itirr.nl rrcce d u r e . In the in vitro experimente we used the blood of tv/o clinically healthy donors who had a normal k a r y o t y p e and had not b een exposed to any factor capable of a f f e c t i n g tho frequency of the aberrations. The lymphocytes were cultured and the chromosome procurations were prepared as described in /10/. The herbicide 2 , ^-D was added to the culture 3 hr after the start of incubation and a llowed to remain until the end of culturing. r'or the in vivo experiments we used white male mice that weighed 1 - 20 g. The chromosome preparations from the bone n a r r o w cells were prepared by the procedure used in our laboratory /ll/. The hericido 2 , k - Z was given orally as a single dose with tho milk. The animals were killed by decapitation 20 hr after giving the compound. The chromosome analysis was run as described in /10/. The uniforaitv of i* experiments on the frequency of the aberrant ceils was verified cy the x7 nethod. S u b s t antial di f f e r e n c e s were not observed (P > 0.1) between the duplicated experinents and consequently the experimental results were added. The statistical r e l i a b i l i t y of :re differences between experiment and the control and b e t w e e n the different uoses was determined by the m e t h o d /22/, S t u d y r e -] Is .-.no their diserrsion. 'he cytogenetic a c t i v i t y of 2 ,<*-D in the culture cf n t r a t ;OXi** r* iOun tn t !. t !*;:ijos to human lymphocytes was studied at' AI . 0? n nr.ci f> /V! /..1 0G0SG2 6883 OOOSS8MOO 2 C , analysis of the cultures that had been treated v.ith different doses of 2,*+-D, are given in Tables 1 and 2. i'roffl Table 1 it can be s een that the number of metaphases with aoerratior.s at all of the 2,^-D concentrations, except the minimum, differed reliably from the spontaneous level (P <^0.001; P < 0 . C 5 ) . Increasing the 2,^-D conc e n t r a t i o n in the culture abeve 0.2 >/g/ml was not a ccompanied by a reliable increase in the number of aberrant metaphaces (P > 0 . 0 5 ) although a tendency toward a maximum cytogenetic effect was observed at a dose of 2.0 /<g/ml. Evidently, high doses of the compound, which exerted a cytotoxic ac t i o n on the culture, led to a d e s t r u c t i o n of part of the aberrant cells or else they retarded their entry into mytesis, and as a result the total f r e q u e n c y of the a b e r r a t i o n s remained at the same level. A decrease in the h e r bicide c o n c e n t r a t i o n in the culture, beginning with 0.2 ^g/ml, caused a decrease in the number of damaged cells. The obtained data do not permit judging the character of % tho dose relation in the verified dose range. The a berrations caused by the action of 2 , ^ - D were o f the chromatid and chromosome tyre in a * : 1 ratio (Table l). Single acentric x'ragments predominated among the chromatid rearrangements. Symmctrical enre-nntid interchanges were observed in three metaphases at the m i n i m u m e c n c entration of the c o m p o u n d (Fig. 1, see first V insert at end of number), i. c. this c o n c e n t r a t i o n of 2 , ^ - D is not " n e n operntivd '*, as it would s eem if the s p e c t r u m of the d i s t u rbances in neglected. ine enromoseme typo of a b e r r a t i o n s arc mainly represented by pair-u acentric fragments. CG03 G* OOCS8MOC] 20^, From Tablo 2 i t can be seen th a t the i d e n t i f i e d c l e a v a g e s are distributed at random among the chromosome groups (P > 0 . 0 5 ) . However, a nonur.iforn d i s t r i b u t i o n o f the damages was observed along the sh ou lders of the chromosomes, and here Si*, o f the 20* c l e a v a g e s was l o c a l i z e d in the long s h o u l d e r s . The maximum o f the d i s t u r b a n c e s was found on the t e l c m e r i c t h i r d o f the long and s h o r t s h o u l d e r s ( 70.6 and 7**.3>) The c y t o g e n e t i c a c t i v i t y o f 2,*+-D on mice was s t u d i e d a t do ses 0o f 3OO mgA s (LDje), 100 m g /kg ( 1/3 LD5 0 ) , 50 m g / k g ( 1/6 LBj ) , and 10 mg/kg ( 1/30 LE^C) . The d a t a , obta ined in the chromosome a n a l y s i s o f the bone marrow c e l l s o f mice, are given in Table 3, from which i t can be seen that, a t dose l e v e l s o f the compound . o f 10 and 50 mg/kg, which f a i l e d to cause v i s i b l e s i g n s o f ir .t o x i c a t i c the frequency of the aberrant c e l l s did not d i f f e r from that o f the c o n tr o l. After ad m in isterin g 2,*+-D a t doses o f 100 and 500 mg/kg, where e x p r e s s e d s i g n s o f i n t o x i c a t i o n were o b se r v e d , the number c f metaphases with a b e r r a t io n s r e l i a b l y d i f f e r e d from the spontaneous l o v e l (F 4 . O.0 5 ; P < , 0 . 0 0 1 ) . Ar. i n c r e a s e ir. the frequency o f the a b e r r a t i o n s was accompanied by an i n c r e a s e i n the amerare number o f disturbances, beth in a single studied and in a sin g le aberrant metaphase, ma i n l y due to th appenranco o f c e l l s with m u l t i p l e aberratlcTis (Fig. 2, sec f i r s t in sert), ingle acentric fragments predominated among the induced i n j u r i e s . t Consequently, *az a result of the p e r formed study It wo:* four..: that the herbicide 2 ,*-i) is capable of anna r i n g *.ic n.ru- nomes of the somatic cells of animals and humans. Howevcx*, L;.t- c y t o g e n e t i c CC05G23 6 8 8 5 O O W 883002 i effect in the bone a r r o w cells of mice arose only by the action of toxic doses of tne herbicide, on which basis 2,^-D can obviously be rega r d ed as being a w eak nutagen for'mice. In human l ymphocyte s the compound induced chromosome aberrations at concentrations that exerted ana did not exert a toxic action on the cells Consequently, huaan chromosomes in an in vitro culture are more sensitive to the damaging action of 2,^-D than the chromosomes of the bone aarrow cells of mice in vivo These data again emphasize the importance of the specificity of the object when studying induced chemical mutagenesis. From the literature data on the d i s t r i b u t i o n of 2 ,h -D in the organism of mammals it is known that t:.e compound does not metaboli z e , is e x c r e t ed in u n c hanged form, p e n e t r a t e s into the genads and can pass through the placental barrier /13* 1^/t which is probably the reason for its .teratogenic effect / 1 3 - 1 5 / * It is possible hat the studied he r b i c i d e is diet ibutei in a similar manner in* the h u m a n organism. This poctultiion is s u p p o r t e d by the data that were obtained on volunteers who took the herbicide 2 ,*>,3-trichlorc pnenoxyacet ic acid (2,^,5"-) ' compound that differs from 2,^-D by having an extra chlorine atom /lb/ After a single oral a d m i n i s t r a t i o n of 2,*,5>-T the herbicide was de t e r m i n e d in id for threc days was excreted unehanged in tne ilrir.r. c of 2,*- in the human organism is the sp me , tnon the 14* r<prilC C lk X .s a tenetic hazard as a cytorvne tically active n viev MM:ich it rhc u !d not be TiCTtT. itL.cd t0 enter the sm from var \:eus 00jects of tne ex ternai ritmor.pi;>rc C003GS5 D O W 883003 Table 1 2 0 3 rr c cueny and Types of Chror.osone A b e r r a t i o n s in Culture of Hunan Lvrhocytes at Various /-D Concentrt ions 7) xpOMitiuiioro tnna -- /<> i l OXNHQHIlUt C/HW- M>CYpiM- 'pjrntMiof plINNW* O0- hux o6mc* HCin>9 MOS AeppaHHft K3 ICO K.1CTOK /3 ) teero n1afpi/iux ^parwcuioo 9 / xpOKOcrxKoro t h m Hue!nrSp\u. ClIMiMlCT*OHM- Ko^/ tueowx KOO *w\ oOveKO XpOMOCdM 6.2 0.2 6.6 1.4 0.4 0 0.2 ~3 0 0 7.2 1.4 0.2 0 9.8 0 0 9.8 1.6 0.2 0 0 6.8 0 0 6.8 2.6 0.2 0 0 3.6 ,, - 0 0 3.6 0.6 0 03 0 2.0 0. 0.6 2.6 03 0 0 0 U 0 0 U 03 0 0 ' 0 l'V icrrj 2.0 1.6 1.8 2.8 0.8 0.2 03 1 ) Concentrt!en cf 2,^-3 .?' nur/ecr of invent i ;itd r.tnr!!;tses j) ircr:*:.;-. ci r.e:.; ;ith WkM 1 N*11 cits, `t) Outrer ci :.:-crr:. t :cnn p) Cor i r.o tarnne l'or *.o-.rr:.r.t net .,#M r \i . ] .1i il ) % C' */ V % A iJ .......... 4 V*:."* *\.* :c *r ., *;*.**. v; J1. e .... **r , .-- *'../ 3;D .i--*) Total * l'aire'.' :r:-. -- .1 is) ..iccntr: c:. 36) C:rv>r:ti:;r.v:: .1 C003G2J p Oc ^ U ^ <J i DOWS83004 Table 2 Distribution of '.'ur.ber of Clcavares, Cau s e d by 2,^-D, ?y C::ro.~oso.r.e Croups 1 11 /) KoHUfnpjiH . 4-A. A-l A - A-3 D *FO>40C*M co E 3) F Q + r Cerro 50.000 20.000 2,000 0,200 0,020 0.002 3 l t 8 21 2 21 2 3 3 1 C 19 2. 3 1 0 5 8 2 8 15 I 0 1 1 4 5 2 3 7 3 31 4 0 1 1 4 5 2 52 0 2 0 0 2 9 I 1I 0 41 3S 41 32 20 16 B e e r (itaS.iraancMOC 4IIC.10 p a jp u bob) 17 IS 7 31 76 11 14 7 0/K1U3CMOC 4!tC.~0 p a a p u s o s (h.i ochoB3HIII1 .lltHW s p o uocom) X* 1 6 ,1 9 15 ,1 5 12,71 2 2 .6 4 7 1 ,9 9 18 ,7 6 1C .07 8 ,4 6 0 ,0 4 0 ,5 3 2 , 5 0 3 .0 9 0 .2 2 3 ,2 1 0 ,2 0 0 ,2 5 7 6 .0 4 0 ,1 5 183 1S5 10.31 n p it Me s a h it e . x* 1 0 ,3 1 ; m i c i o crencHcft cboSouu p a a i i o 6; P > G ,0 5 . **) Trtul (observed nunoor of elea v a r - :5) 5) Cxpeoted tc-r of cleavages (on basis cf chrs.tenore length} 6 ) T -.s = 10.31 nur.ber of errees of frie ioti = r i: CGOoG .* . c r? o O V..; w Tabic 3 Free.tency and Tyres of Aberrations for Mice .. poisoned by Various D o s e s of 2 , ^ - D Had been * i c r c t f i k .t c t o k s1) " i r m m H) Co M O A c c i * e i i * x u y h n o n jc ^ . 6) Miic .to i C c p p a u n A T l i n u rfrp p .it{M A MX 100 sS Acnhiim m x p o * o * KJtClOK f tf A ' ** COM 31 M i e j o n M r T j. $13X * a c C cp t.i. i ) 7 ) 2 f. 5r 2 * IS > * IXHMMII. % I 3 S 2 O !rj *2 * *5 ; * <u rz 'i s * v= sr2 5 ewuu Vi SV i_ *S SA S S -& ! *1 AU ij - >S A> ^B * s-ir 10} )3 az II II \!i) 0e* 32 C2 1 '> | 1 10 50 100 300 6 1 2 0 0 0 .3 3 3 0 ,1 6 5 0 0 6 1 2 0 0 0 .8 3 3 0 .2 W 0 .0 3 1 0 ,0 1.004 0 . 0 0 3 3 0 ,2 5 0 0 .0 8 3 0 1.0001 4 0 . 0 1 1 7 1 , 4 0 0 ,0 3 3 0 .0 3 3 6 1200 1 .7 5 0 0 ,3 7 S 0 .1 6 9 .5 2 5 0 ,0 2 0 8 1 ,1 9 1 .8 3 3 0 .2 5 0 0 6 12 0 0 3 .0 S 3 0 ,4 9 9 1 .2 8 4 3 .3 114 0 ,0 9 5 0 3 .0 3 8 .S S 3 0 ,7 5 , 0 ,1 6 7 /t ) K o"' 1.00 y r p a i k 10 100 0 0 .7 0 0 0 ,2 6 4 0 0 7 0 ,0 0 7 0 0 ,7 0 0 0 0 1 ) f>oso of 2,^-0, a:/kg 2) Number of animals 3) Number cf studied metaphases *0 Frequency of me t a r h a s e s with a b e r r a t i o n s , .J ?) Frequency of cells with multiple dasarss cf chro.-oscr.es u) In to number of studied cells 7) In ;<j to number cf aberrant cells S) Number of a berrations 9) Total 30) Per invest i.-ated mutarnaoc 11) Per aberrant metayhasa 12- Tyrrs cf a b e r r n t :rns/iVO cells 13) oir.-lc fr.--.-r.entn 1*0 Faired fra--.cats 1 3 ) f r a r . s lr . c n t : oar. o 00 00 CO o o cn ~''rr'C.27 i.' 900C88^oO 2 0 > Conclusions, A study was made of the cytogenetic action of . the herbicide 2,4-D in both in vitro experiments (on a culture of the lymphocytes of peripheral human blood) and in vivo experiments (on bor.e m a r r o w cells of white male mice). It was s h own that the human chromosomes in the culture are more sensitive to the damaging action of the compound. The o b t a i n e d r e s u l t s make it possible to classify the herbicide 2,4-D with the compounds that are potentially dangerous in a genetic sense. LITERATURE CITED 1. J. Uhrau, Can. S eed Growers Assoc. 1952 - 1953 Ann. Report, 1953. 2 5 - 28. 2. J. Unrau, Can. S e e d G rowers Assoc. 1 9 5 3 - 1954 Ann. Report., 195*+. 3 7 - 39 5 H. Kallak and L. Yarvekylg, A c t a Biol. Acad. Sei. H u n g . . 22, 6 7 - 73 (1971). 4. H. H a k e e m and a . Shchab, A s t r a c t of paper d e l i v e r e d at First Egyptian Congress of Botany, Cairo, 1972, pp. - 35* 5. 3. Ii. Kulikov, A. A. Encrzha, M. I. O v c h innikova, and A. I. Mochalkin, Fiaiolcgiya Hastenii, 17, 1205 - 1208 (1970). 6. G. Berin and S. u. Busovich, First Monograph of Fifth Convention cf -hitc Russian Hygienists, Epidemiologists t and InfeetianoJegists / i n Russian/, Minsk, 1971, pp. 7-5-79. 7. L. C a rving ahi*M. unner, ".crditas, 68, 1 1 5 - 1 2 2 (1971). 8. A. E. Ku3n!:ov, in .ponograph " H y g i e n e of Application, T o x i c o l o g y of Pesticides, anti C l i n i c a l P o i s o n i n g " / i n Russian/, cy, I-?-, ... iw -iss. CG05G23 6330 DOWS83007 XU 205 206 9. S, Yu. 3uslovich, Ya. E. K n i g s b e r g , F. D. Koldobckaya, G. I, Moskovko, V. M. S k l y a d n e v a , I. V. Voinova, and K. G. M i l 'china, Ibid., pp. 3 ^ 1 - 3 5 1 . 10. M. A. Pilinskaya, Genetika, f>, 158 - 163 (1970). 11. A. I. Kurinnyi, Authors a bstract of c a n d idatal d i s s e r t a t i o n /in Russian/, Kiev, 1972. 12. Ji. A. Plokhinskii, 3 i o n e t r y / i n R u s sian/, Izd. MGU, Moscow, 1970 1 pp. 143 - 145. 13. D. Clark, J. Young, R. Younger, L. Hunt, and J. McLaran, J. Agric. Food Chen., 12, 4 3 - 4 5 (1964)., 14. A. Kurt-Zrne, A cta Vet. S c a n d . , 7_% 2 4 0 - 256 (1966). 15* L. V. Martcon, in sionccraph ''Hygiene of Application, T o x i c o l o g y of Pesticides, and Clinical Poisoning" /in Russian/, Kiev, 1971, p?. 2 5 8 - 261. 16. A. Matbuxura, Japan J. Ind. Health, 12, 446 - 4 5 1 (970). T r a n s l a t e d by: John Livitk June 26, 1975 CG05G23 6991 i p y abjuuti T . V I I I . -Vt 3 U H T O .io m n n rn iiK T iiK A 1974 T (T LO<SI/As o C s .f i / V f c / ik a 8 (z) Z02-6, U'nv) V A K 615--099--0S6.7 m i T O r n i l E T H H E C K l i n 3<t>l>E K T T E P D i m i l H A 2 .4 -A H A X P O M O C O M b l 4 E J1 0 B E K A II H C U B O T H b lX AL A. nilJIHHCKASI B b c a c iu ic . B n u jy m ip o n a im u ii M yTam iom ibiu npouccc y McnoncKa onpcA M C H H u f i BK.1 3 A .voryT DiiociiTi. H CKO TO pue n c c T m n u iJ . TIO STO M y o c o C y io aKTya.ibiiocTb n pn oopcTacT 3a.iaM a BbiHD.iciuin cp o an nccT iim iaoB no T cum u LibiitJX M N Tarcuon, n aK o n n eiu ic K O T o p u x n p a 3.niHm>ix o o b C K T ax b u c u iHeft cpo.ibi m o x c c t npcacTaBA JiTb retieTH M ccKyio o n a c n o c T b He To.ibKO a a h OTAC.IbHLlX U U A IIR IU y V M O B , HO II A.1 H n O liy .U im ill B UC.7 0 M. C S T iix n o 3 im iifi B ecb.M a u c n e c o o Q p a 3 !ibiM h b a h c t c h ii c c A e a o s a H i ic m v T a r c m ic fi aK T iiu H o cT ii un ipoK O n p iiM ciiscM h ix r c p o im iu o B rp v n n w 2 ,4 -X l (n pO H 3B O A H blX X .30piteH O KCH CG O .U lH CH IIw ). lIlITOreHCTHHCCKO e ACiiCTBlIC X.1 0 p n p 0 II3 B 0 A H U X tpCHOKCHKIICnOT B o c - HOBHOM HCCneAOBa.lOCb IIa p a 3.1 HHHbIX paC TIITC.IbllblX OObCKTaX [ 1-- 5 ] . B nocACAHiie roAbi HcxoTopuc npcACTaBiiTcm i x-iopijiciioKciicocaintcHiiH H3yva.uicb Ha naccKOMbix u v.ACKoniiTaiomiix [G-- S]. B 3 aaaH>* HacTOHiAcii p a 6 oTbi b x o j h a o uccACAOBaiuic m iT orc.icT iiu ccK or o AefiCTBiiH O A H o ro 113 n p c iia p a r o B A a iu io ii r p y m ib i -- 2 ,4 -Aii.\.-iop<+>uiiGKciiy K c y c iio fi k u c a o t w ( 2 ,4 - A ) -- n a c o m b t h 'ic c k u c k a c t k i i H C A O B cxa in v it r o it u b iu ic ii i n v i v o . 3 t o t r c p o m u u . G u . i B b io p a n 113 A O D o n b iio o u i n n p i i o f t r p y u Dbl X n o p n p O II3 BO AHUX (pCHOKCJIKIIC.IOT B CBH3 H C TCM, HTO OCTaTKH .IIO U U X npenapaT on bt o u rp yr.n u na pncTCHiiax b o c h o b iio m npcacTaunjiioT c o o o A 2 .4 -A [9 ]. MaiepHa.i h Mcroaiiva. B MUTax in vitro itcno.ib3 0 3 3 .nt jcponb jsy x k.i ;iiiii<icckii 3.10- posux ii.vetoimix iiopiia.ibiiuii .jpuoinn aoiiopon, xoTopue nc noaiicprs.iucb .'.-"ctuiiio xa- xoro-atiGo ({taxTopa, cnocoGiioro Kon.iunTh 11a vacroTy aGcppamtii. Ky.iiT::3 t:pocamtc .him- 2i^,c4i-ihAtoBHbO1C11Ln3pHiirboTxoyr..i.bnTc!yi:p:cy nvpcepnc3ap3aT<otnnoxcpnoev.oHcaovjtannapoimooxry.ioi.aiuiinniio11xcorco7.amn.xiae.ii[i 10|. cro repC::u::; ao xouua Ky.ibTHBiipooatmn. K oH uenT pauua S .4 -A ja u fja M i* M aciO Ta k Tim ia xpo m o co m iiux aocpp am i'i x y .it.T y i K o .iu w c c tc o aCvpiM H iift X o atm c iw w e.A f)iu H iiin n e ia :Ja i V .ic io ia M era- < u i e a^-cppa* U U . % K ero N3 1 NCC.1t!A im tm yK } :ta ! nyo a f c r r mT n c ta Jjjy 50,000 20,000 2,000 0.200 0,020 0,002 K o iap aib 202 2^ X I 0 -4 10- 10- to- 10 - 1 0 - 500 500 500 500 500 500 800 8.00-7 JA J3 8 ,4 0 1.2-U 9 .4 0 ;*: 1.313 8.C0.U 1.253 4A 00.iK )7 2 .8 0 i 0 .7 3 5 I.7 50.1G 'J 43 O.OiG 44 0 .0 5 3 53 0 .110 43 O.O'.'G 22 0 .0 11 14 O .O J3 14 0 . O I 7 1.075 1.018 1A31 1,1 IG 1.013 t.OOH 1 .0 0 0 k j o -7"v O Q W 883009 Jl.m onuon in vivo iiciio.iiootin.ni ficciiopo.iiiux c.iijx Muiiieii-caMUon nrroM 18--20 .\ llpoii.ipniu Nj'.'MocoM in K.ifTOK KOCTimro Miiirn ruroiin.m 0 MCTO.iy, njniiiiiTOMy n ii.i :::i-h j;cGi>>.uo'iui [1 I|. IVpKuii.i 2,4-A 11nn.311.111 o.iiioi;p;iTio niiyipiiiKc.iy.iO'tno c mo.iokom. >:0i- OOIiii.a j.if.iirj.iii .ii'KJ tiilin itucti 'topen 2U 'i noc.ie niioaciii:* iipcnnpnTn. ............... ... .XpoMocnviiM.i niin.iiu iipono.iiMit t.ik * e . k.ik 11 cooftiiiciiiiii [10]. O-tiiopoiiiocTi. omutoii no mcTOTc nficppnimiux k.ictok iiponcpn.m no mcto.iv %*. Me*:- jty nonii'pitocTwMii iKcncpiiMcnion tic unfi.iio.in.iii cyutvcir.enitux pai.nt'iim (l'>0.1). no.To- My pcj.n.T.iTU nonnipiiocicii cyM.Miipoiin.ui. Cmmcrii'iccKyio .locToncpimcTb ot.iiihiw or.um or konipo.m h pjj.iifiiiu.i A03 Me>Kay co6on onpcac.in.in no MeTO.iy 9 [12]. Pc3y.11.TaTU iicc.ic.ioBaiimi 11 hx oflcy/Kflcmic. tliiTorciicTHHCCKyio axTjimtocrb 2,-1 b Ky.ibTypc .iiim(}ioiuitod 'ic.ioucxa cc.ic.iona.in b kohiichtpaiumx. oG.ia.iniomnx uiitotokchmcckhm 3(|>(Jicktom (50 11 20 m k c /m a ), b K0.1UMCCTBa.\, HC OKa3UB3IOmnX TOKCimCCKOrO ACHCTBIIH Ha K.1CTKH (2.0; 0,2; 0.02; 0,002 m k c Jm a ). J U iihuc, no.iyMciniuc npn xpoMoco.MOM aita-iaae KV.li,Typ, oOpaOoTauiiux pa3.11mHu.MH A03aMit 2,4-, npimcACHbi b TaC.i. 1 11 2. II3 TaC.i. I Diano, m t o Miic.io MCTatj)a3 c a e p p a m w M i i n p n b c c x k o h - UCHTpaiUlHX 2,-1v L KpOMC MHHHMa.lbHOH. AOCTOBCpiIO 0T.1HMa.10Cb OT CnOHTaitnoro yp o u H H ( P < 0 , 0 0 1 ; P < 0 , 0 5 ) . yBC-iimcinic KOiiuciiTpaiiini 2,4-H B xy.ibTypc 0.1CC 0.2 m k c Jm a h c conpono/KAa.iocb AOCTOBcpitUM i i o b u u k h h - cm KoaimccTBa a G e p p a i m i u x MCTa<|>a3 ( P > 0 , 0 5 ) , x o t h HaG.uo.ia.iacb t c k - JICHHIIH K MaKCHM3.1bHO.My UHTOr C H S T H M C C K O M V StJX^CKTy lipil 303e 2,0m k c / m a . O h c m u h o , BucoKite a o 3u npenapaTa, oKa3binaiointtc u h t o t o k - CHHCCKOC ACHCTBHC Ha KV.IbTypy, IipitBOAILIH K rilC.IH M3CTH aCppaHTHbX K.1CTOK .in 3a.icp:Kc BCTyn.icHiin h x b m i i t o 3, C.iarojapx m c m v oGiuas nacTOTa a c p p a m i H coxpawi.incb na o a h o m 11 t o m x e ypoBiie. C h h sccHue xoimeiiTpauiiH rcpGi:mi.ia b Kv.ibTvpe, H a c i n a n o t 0.2 m k c /m a , Bbt3usa.io ysieuhnieimc KO.iiiHccTBa noBpcHc.icmiux k -i c t o k . no-iynemibie a a m i u e He n03B0.1H10T CVAHTb 0 XapaKTCpC A030B01 3aBIICH.M0CTH B n p O B e p C H H O M HH- Tcpna.ic A03. I lp ii .1CHCTBHH 2,4* B03HHK3.1H a e p p a m m Kan x p o M a T ia iio r o , T a x h xpoM occMHoro Tima b coothchiciihh 4 : 1 (T a.i. 1 ). C p c an x p o v .a r iu x b ix n c p c c ip o c K n p co G .ia .ia .in o .im io m u jc alien tp iiie cH iic titp a rM c im j. u p m h HiiManhiioi KOHHCHTpaium n p e n a p a T a b T pex M C T a*a 3 a x naG.itoA a.iH c;:.mMCTpimilUC XpOMaTH.lllUC OMCHU (pilC. 1, CM. I BK.1C1KV B KOHliS KOV.ep a ), T. C. 3T3 KOHUCIlTpaUUH 2; 1-H HC HS.1HCTCH HCAeiiCTByromCH:, K ax X3- 3aA0Cb, 6 c3 yxcTa cncxT pa H apyuiciiiiii. A G cppaiu n i xpoMOcoMiioro T an a a OCHOBHOM G u .in n p c a c T a s.ic H u * n ap iiu M n aucii-piiM ccKiiM i^ parM K T aM ii. H 3 TaC.1. 2 BH.lHO, MTO llACIITI!(|'lU!lpOBaiI!UilC pnjOUBbl paCHpCAC.1H.lHCb cnyHafiiio no rpvniia.M xpoMocoM (P > ,0 5 ). Oahoko OTMCMa.iocb nepasHOMepiioc pacnpcAc.icimc nonpoKACHiifi no n.icsaM xpoMocoM-- 81,4 pa3- Timm I flHM({)ouMio M c.ioBcxa npn pn i.iiiH itu x K oiiiiciiipn u iiiix 2 . 4-A xpOMrtu'*nrotuna A6cppni|Hft M3100K.-UTTOK XpOMOCCMKOroTMfia n^SuNTHMOHcHmMeYopiWMtMHtDt nutnoqn6mc* cero itipfairaMpHCIMJXion UCKHoarpH* enHvUMN\toftaoiia- KXpOrt.MifOcCt;OfaMm cero 6A OA 0,2 6,0 1.4 0.4 0 0.2 7^ 0 0 7.2 1.4 OA 0 . '0 9.8 0 0 9.8 1.6 0.2 0 0 C.8 0 0 C.8 2.6 OA 0 0 3.C * 0 0 3.6 0.6 0 0.2 0 2.0 0 0.6 2.6 0.2 0 0 0 U 0 0 1.1 0.5 0 0 0 2.0 1.6 1.8 2.3 0.8 0.2 0.5 G G 0363 203 N T a .1 h il .i 2 Pacnpc.ie.icHiie siic.ta pnipunon. nwinaiiiiwx 2,--Jl. no rpynn.iM xpomik-om fpynnu KomucicN 'Ih* jetUJLt 0A t A-2 A-3 It C E F G + r Beer 50.000 20,000 2.000 0,200 0,020 0,002 3 l 1 8 21 3 3 1 6 19 5 8 2 8 15 4 52 37 0 1l 45 O* 0 0 2 9 2 21 2 2 31 0 1 01 1 3 31 4 2 52 0 1 l1 0 41 38 41 ( 32 20 16 B erro (ita.iioaac- 7M oe ihc.io p a jp w - bos) 17 18 7 31 7 6 11 14 7 ISS O a tiu a c M o c nic.to p a a p u o e s ( 11a ochosan ti;! am iliu xpomocom) X* O 4O 1G .I9 1 5 ,1 5 12,71 2 2 ,6 4 7 1 .9 9 I S ,76 1G.07 8 ,4 6 0 ,5 3 2 .5 0 3 ,0 9 0.22 3,21 0 ,2 6 0 ,2 5 6,0 4 0 ,1 5 ISS 10. 3 f n Pn M e t a h n e . x ; = 1 0 ,3 1 ; ih c .i o crencH C ii c s o o j u p a s i t o 8; P > 0 . 0 5 . pbiBOQ .Toxa.Ttt30Ba.TOCb b x i h h u x nrcMax. M a x c i n i y M Hapyuieniift npitxo- AH-iocb na Te.ioMcp'iyjo TpcTb x t h h i i u x it x o p oTxnx oren (70,6 h 74,3c/o)UinorcncTimecxyio axTtiBHOCTb 2.4-.H na vbtiuax iiccncxosanii n A03ax 300 mc!k2 (.liljo ), 1*00 xz!k2 (Va Jlilsa), 50 .ua/xa ('/g lUzz) it 10 mzk (Vas JI 50). ainiue, nonyneHKNc non xpomocomhom ana.iirce x.ictok KOCTnoro Mosra vuiiic, npcACTaBaciiu b Ta.i. 3, OTxyza bii.iho, '-to npii ACiicTBi:;t npcnapaTa s jioaax 10 11 50 Meine, hc BW3binaiomnx BtiziiMbtxnnit3iiaxoB iiirroxciixaiutn, xacTora aeppaHTitux k.tctox ho oT.inna.Tacb or TaxoBoii b xoHTpo.ie. iiocac bbc.ichhsi 2.4-A b ao3ax 100 n 300 mks, npii KOTopb-x ua.Tto.ia.Tiicb Bupa/xciuibic npiiaitaxit tuiTOKcuxauiiii, mtc.io mcTa4>a3 c acppannnMH aocroocpno oT.TtiMa.Tocb or cnouTaintoro ypoBitn (P cO .05; P < 0 ,0 0 1 ). noBuuiciiue nacTOTu aOcppaumi conpono/X.ia.Tocb yBcniiHCiutCM cpcmcro u:c.ia itapyiucnitn xax b oanoit hcc.tc.ivcv.oh, Tax H B oahoi aCcppatiTiion vcra({)a3c b ochobhom 3a chct noin.Tcmin k.iotck CO MHOSKCCTItClUIMMIl aOeppailHilMH (pitC. 2, CM. 1 BK.ieftKV). CpC.lII ntI.1VmipoBatntwx noopcxucinin npco6.Ta.ia.in oimionnuc ancHTpnuccxne <pa"rMCHTU. TaxiiM o6pa30M, a pc3y.ibTaTC n p o n c j c m i u x nccjicjioBamift oGitapyjxe- HO, m o rcpimn.1 2,4-A cnocoocn nonpc>K.inTb x p o v o c o v u coMani'iccxtix KJICTOK XvimOTHUX II `IC.TOncxa. O.lltaKO IlUTOrCllCTItMCCXIl 3(J>(|>CXT b k .t c t - K a x KOCTHOrO M03ra MUlIlcii 11031111X3.1 T0.TI.K0 npii .ICflCTUtlll TOKCIl'tCCXIIX 0 3 rcpimit.ia, na ocitonnmnt tero 2,4-A, o m c b h .i h o , m o /Kiio cmiit.tti. Bc n a u M MyTarciioM x t h vutitcn. .Tn:.n|>otinTax j kc m c .i o b c k .t npcriap.iT HiiAyuHpor.a.T x p o v o c o M t i u c a c p p a n n n b xoimciiTpaminx, OKaaunatottutx il hc OKa3iJn3iomttx Toxctntccxoro a c h c t d i i h tra k ^t c tk i i . C.TC.10n.TTC.TI.It0. XpOMOCOMIJ IC.TOI1CK.T It Ky.TbTypc ill vitro O.TCC IVUctbhtc.ti.iii4 k nntipoJK.inioiHCMy .icficTitmo 2,4-A. mom xpovocoMU k.tctok *y KOCTiioro MOira muiiich in vivo. 3 th .laimuc cmc pa3 no.i'icpKiiiiaioT tii.tmchiic ciictnt'l'ii'iitocTii oGi.ckt.t npii tiayicttint un.iyiinpouatimiro xiiMinccxoru M>Tarcnc3a. II3 .TiiTcpaTypni4x .laiinux o pacnpc.ic.Tcnnn 2,4-A opranii3MC m .t c k o miTmoiunx louccmo, m t o iipcnapaT h c MCTnOomianpycT, bij.i c .tiictcji b iic- DOWSS3011 , T a 6a mi a 3 `lac io ra m iiium xpOMocOMiiux aGcppauiiii y mmiiich, aaT pacacim ux pannuwiuMii A 013M II 2 .1 -/1 Maerora a ti m 5 Ma X 1lXb 2 Cr XO r*a SVi A3--w^* lz fXt K co MiiOArciani* huvii ACHHIOdH COII M4*a0cTeora hcts- *4 M S% Xx ryT** rS i >cw. Z:=X% 59 5 3< .*-OC ZO X Mnc.rj j6cppiKA Thhu na 100 jk io k obVm1. <o o* ZAa_ * --q sn--p>-n_ nc. n- *--1> *t 5 >r.* a-.3= IS 2*1 <CTr z9u xz cr. sv A 3u1 5m 10 50 100 300 KouTpo.it 6 1200 0.3.*0.tC 5 0 0 4 0,0033 1.00 0,250 0 ,0 5 3 0 6 1200 0.533= 0 .2 5 4 0 ,0 3 10,0 14 0,0117 1.40 1,000 0 ,0 3 3 0.033 6 1200 1,750 0 .3 7 8 0 ,1 6 9 ,5 25 0.020S t .1 9 1,833 0 ,2 5 0 0 6 1200 3 ,0 8 3 ^ 0 ,4 9 9 1,23 4 3 ,3 114 0,0 9 5 0 3 ,0 3 3 ,5 3 3 0 ,7 3 , 0 ,1 6 7 10 1000 0.7000.264 0 0 7 0,0070 1,00 0,700 0 0 H3MCHHOM cocTonium, riponiiKacT b roiiaA u ii mojkct n p o x o jirrb iepc3 n.iaueHTapnuu 6apb cp (13. 1-1], c neM, B cpoim io, h CBS3an ero TepaTorciiHuii 3<J)<|>CKT (13-- 15]. B o3MO/iciio, hto b o p r a iu i3 M e K e .io B C K a n c c n e a y e M U H r e p o m i m p a c n p e A e.iH CTCs n o .io C n u M a c e 000330M. B n o .ib 3 y 3Toro n p e an o .-iO /K em :?! r o B o p s x a a m i b i c , n o .x y H c in iu c iia A o o p o n o .ib u a x , n p iiiiiiM a B U Jiix r c p 6 u m u 2,4,5T p .\M o p y K c y c n y io khc.iotv (2, 4. 5-T) -- n p c n a p a i , o x .n iH a ic tu iiiic j ot 2.4-H .tuuihiim aTOMOM x .x o p a [ 1G ] . noc.xc o a H O K p a m o r o n e p o p a .x b iio r o n ? ;:c M a 2,4,5-T r c p o n m u o n p c .x e .iii.ic jt b K p o B jr b tc'iciikc T p c x cvtok B bi.xe.iK .xca B H eiU M ciicuiiO M B l u e c mo'ioi'i. E c .u i c y j b C a 2,4-H b o p ra tm a .M e s c .io n e K a T a xoaa jkc, to A a im u f t u p c i t a p a T x a K m iT o r c m r n m c c K ii a K T iiB iio c ' bciuccteo n p c iiC ia B .ir ic r lc iiu iim e c K Y jo oiiacH O CTh, u c b h jii c wcm h cu o m U um u iil k . ik j- iaT b n o n a .m m i c c r o b op*raim 3.M mc.iobck3 ji3 p a 3 .u p iH b ix o f o c x x o B bhcui- HCH CpCAU. Buboau. HccneAOBaiio UHTorciieTiwccKoc achctbhc rep6m uaa 2.4-H B onuTax in vitro (na Kv.ibType .xiimcIjouhtos ncpn6cpiiMccxou Kpcmii ko.xoBCKa) h in vivo (na kactkox kocthoto M03ra CccnopoAninX -uc.xux Mbiiuefi). noKa3ano, hto xpo:.:ocoMu ic.ToncKa b Ky.TbTvpe Co.tcc MyBCTBine.ibitbi k noBpc'HviiaiouicMy jcftcTmno npcnapam. rioAvncuubic pesy.TbTaTu no3Bo~ft!OT othcctii repomuu 2,4-H k BcmccxQaM, noTcmuiaAbiio onacuuM b rencTimeckom OTHomeinm. JIHTEPATVPA 1. J. U n r a u , Can. Seed Growers Assoc 1952--1053 Ann. Report., 1953. 25--28. 2. J. U n r a u . Can. Seed Growers Assoc. 1953--1954 Ann. Report.. 1951. 37--39. 3. H. K a 11 a k. L. Y a r v c k y 1c. Acta biol. Acad. Sci 1 22. 67--73. 1971. 4. H. 11 a k e e m , A. S h eh a h. Abstr. Pap. 1 Egypt. Congr. Uot. Cairo, 1972. 24--35. 5 . B . 11. K y n i i K o a , A. A. n . i e p j u a , M . 1 1 . O o <i m i ii ii k o d a , A. I I . M o M a . i K n t i , OioiioaoniH pncTcintii. 17. 1203-- 1203, 1970. 6. E. T. I i c p i m , C. 10. D y c . i o n u >i, 1-3 eflopiiiiK V ci.c3.in rnrucmiCToa, sniiACMito.ioroB H iimfcKtiitomiTon Ec.iopycctiii. .Miiiick, 1971. 73--79. 7. L D a r v i n g. M. S u n n c r, IIereJitns. 68. 115--122. 1971. * 8. A. E. Ky. i.i Koii. c<V -riiriieiia npii.Meiieiiii*, TOKciiKonorn iicctiiiiii.tob ti K-niinika ot- panat'iiitfo. K.. 1970, 351--353. 9. 5- K). Ii y c a o n n <i, Jl. 3. K e ii ii r c 6 c p r. C1. A. K o .1.1 o 6 c k a a. I*. II. M o cko b- ' k o. B. M. C k a ii ,i ii c a a. II. B. 11o it it o n a. M. T. M .i i. mii ii a. c6. rnntcm npuMciiciiint, T0i.ciiK0.iorm n cctm illion ii k.iiiiiiiu.t OTpati.ieiinu, K ., 1970, 311--351. 10. M. A. il n a ii ii c k a :i. IVncTiiKa. 1!, 158--163. 1970. U . A .. I I . K y p ii Hn u ii, A u io p o l * . xaii.i. Alice., K .. 1972. GG05G33 205 69n . : \ ? T n r *00 12. H. A. IJ .1 o x il il CKil ii. HnoMoipmi, .M., Il i.vno MI'V. 1970. 113--145. 13. H. C I a r k, J. Y o n g. R. V o u u g e r. L 11u I. J- M c L j r a n, J. Agric. Food. Cliem 12. 43--lf>. l'.Vil. 14. A. K u r t- r n e. Ada vct. scainl.. 7, 240--230. 19G. 15. .1. H. M n p t t o i i b . c. r m a e i i a npiiMciieiimt, T O K C iiK O .io n iR nccTimii.ioii h KniimiKn OTp.iiinciiiiit>, K.. 1971. 27c*--2GI. 16. A. M a t a u m u r a , Jap. J. Itni. Health, 12. 4 IC--451. 1970. Bcecoioaiiuii ii.-ii. hh-t ru m e n H TOkciiKo.ionui nocTiiKinon. Rivmwpnux u n.iacTHMccKiix mocc M3 CCCP, Khco norrynn.aa 21.V1 1973 r. CYTOGENETIC EFFECT OF THE HERBICIDE 2,-I-D ON HUMAN AND ANIMAL CHROMOSOMES M. A. P I L I N S K A Y A Sum m ary The frequency of chromosome aberrations was studied in the culture of human peri pheral lymphocytes in vitro and in the mouse bone marrow cells in vivo treated with the herbicide 2.4-D at different do>es. The human lymphocyte chromosomes in culture are more sensitive to the injurin'.; effect of the preparation. These results emphasized potential gonetical hazard of 2.4-D, that should be taken into account in hygienic regiamentation oi the herbicide application. CG05S34 2oL 69 9*. H3 t DOW 281058 MN0 2 2 7 7 7 jrTr + ':; r - -f-' MB The Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Guppies ( Poecilia reticulatus Peters) by Locan A. Norris and Richard A. Miller Pacific Northwest Forest and Range Experiment Station Forest Service, U S. Department of Agriculture Corvallis, Ore. 97331 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a contaminant of the commercially produced herbicide 2,4,5-trichlorophenoxyacetlc acid (2,4,5-T). TCDD is immobile and only slowly degraded in soils, not readily taken up by plants, subject to photodecomposition, and low in water solubility (0.2 ppb)l/ (ISENSEE and JONES 1971, KEARNEY et al. 1971, 1972). It is highly toxic and teratogenic in mammals (COURTNEY ec al. 1970, COURTNEY and MOORE 1971). The effects of TCDD on aquatic organisms have not been previously reported. 2,4,5-T is an extremely Important tool for reforestation of brush-dominated forest sites. During 1969, 358,800 pounds of 2,4,5-T were applied to forest and rangelands managed by U.S. Department of Agriculture and U.S. Department of Interior (MacLEAD 1971). 2,4,5-T probably presents minimum hazard to organisms in the forest environment, but the effect of TCDD must also be consid ered (NORRIS 1971). Small quantities of 2,4,5-T are found for short periods in streams after aerial application on nearby forest land. TCDD is presumably also present because most of the herbicide in the stream results from drift or direct application of spray formulation to the stream surface (NORRIS 1967). Ue are determining the toxic characteristics of TCDD in aquatic organisms and report here some of these characteristics in guppies, P o e c ilia r e tic u la tu s (Peters). MATERIALS AND METHODS The TCDD used in these tests was part of lot 851:142-22 from the Dow Chemical Company, Midland, Michigan.-- ' Based on mass spectral analysis, the chemical was 98 percent 2,3,7,8-tetrachlorodibenzo-p-dioxln and the balance was 1.3 percent trichloro- and 0.7 percent pentachlorodibenzo-p-dloxin. Stock solutions (166.6 ppm) were made in chloroform and subse quent dilutions in acetone. Cupples of various sizes (9 to 40 mm) were placed in 1-gallon ' glass jars (20 to 22 fish per jar) and received one of four treat ments (three replications). The fish were exposed to 0, 0.1, -- ppb - parts per billion. --2 / Mention of a company does not imply endorsement by the U.S. Department of Agriculture. Purchased by the Forest Service, U.S. Dept, of Agriculture, for official use. Bulletin of Environmental Contamination Toaieolofy. Voi. 12, No, 1 1974 by $prinf*V*rUf No York lac. 76 0003039 6398  ; ":lr. ' -.J. *.* ' -...,,,,.... A 1.0, or 10.0 ppb TCDD in 3 liters of well water. WILSON (1968) reported the composition of the well water. All jars received 1.2 ml acetone and were maintained at 20 C for the duration of the experiment. After 120 hours exposure, fish were transferred to clean jars with well water containing no dioxin. Water was changed at 14-day Intervals. Dead fish were removed each day and their length recorded (DOUDOROFF ec al. 1951). Fish were fed live tubifex worms ad lib daily except during the exposure period. RESULTS The effect of 120-hour exposure to 0.1, 1.0, or 10.0 ppb TCDD on guppies was irreversible, and all guppies eventually died. Some of the treated guppies died during the 5-day exposure period, but most fish died after they were placed in clean water. All treated fish were dead 37 days after the beginning of the exposure period, and in general, the smaller fish died first. There was no mortality among control fish (Table 1). TABLE 1 CUMULATIVE GUPPY DEATH AFTER 120-HOUR EXPOSURE TO TCDD Days since start of period Mean cumulative death (percent)-- ^ TCDD (ppb)-- ^ 0 0.1 1.0 10.0 5 08 10 0 24 15 0 34 20 0 45 25 0 61 30 0 72 35 0 97 37 0 100 Days to 50 percent mortality 21.7 11 38 65 80 96V 100^' 11.6 18 32 42 56 83 97 100 18.2 -- Mean of three replications. --2 / Parts per billion. -- Last fish died in this treatment on 28th day. The data were expressed as survival time in days and body length in mm for each fish and subjected to analysis of covariance Survival time was significantly different at the 5 percent proba bility level for fish exposed to different concentrations of TCDD (F2 s 11.818). The analysis showed the effect of body length on survival of fish exposed to TCDD was significant at the 1 percent 77 0002040 8S99 DOW2810C0 level (F] " 5 " 65.896) (Figure 1). Using the regression equation in Figure 1, we adjusted the mean survival time of fish exposed to each concentration of TCDD for the effect of body length on survival (Table 2). Figure 1. Effect of body length on survival time of guppies exposed to 0.1, 1.0, or 10.0 ppb dioxin in water for 120 hours. TABLE 2 GUPPY SURVIVAL TIME AFTER 120-HOUR EXPOSURE TO TCDD Mearr^ TCDD (ppb)-- ^ 0.1 1.0 10.0 Fish length (mm) Survival time (days) Survival time adjusted for fish length (days) -- Mean of 3 replications. -- Parts per billion. 20.1 21.3 21.9 78 20.7 14.1 13.7 20.6 17.2 17.0 0002041 7000 A tgiia y i i U y W ^ .-M -' I i DISCUSSION Our results show smaller fish are considerably more sensitive to TCDD than larger fish. Similar results have been reported for several other toxicants. Many organisms increase their tolerance to environmental stresses with increasing age and body mass. Toxi cant uptake, storage, and detoxication patterns probably change with fish age as lipid levels increase and the ratio of gill sur face area to body mass decreases (COPE 1971, MURPHY and MURPHY 1971). We are uncertain about the reason for the apparent decrease in toxicity between 1.0 and 10.0 ppb TCDD, but suggest it may be the result of working beyond the normal limits of TCDD solubility. Fish in the 1.0-ppb treatment may have been exposed to supersatu rated levels of TCDD, possibly mediated by interaction with fish skin mucus in the water. The solubility of DDT in water is enhanced in the presence of some large organic molecules (WERSHAW et al. 1969). Approximately 1 week after initial exposure to TCDD, guppies exhibited a declining interest in feeding and swimming. All treated fish surviving more than 10 days showed fin necrosis which became more pronounced with time. Necrosis of the maxillary carti lage appeared in two large females surviving more than 25 days after exposure to 0.1 and 10.0 ppb TCDD. None of these behavioral or pathological signs of distress were observed in control fish. The site of action of TCDD in guppies is not known; however, exper iments with other animals suggest the liver may be involved (BUU-H0I et al. 1972a, 1972b, CUNNINGHAM and WILLIAMS 1972, PIPER and ROSE 1971). The delayed mortality we observed with guppies is consistent with the hypothesis that TCDD induces liver dysfunction. Static water bioassays are not particularly satisfactory for determining chemical toxicity in terms of field exposures (JUNTUNEN and NORRIS 1972). They can, however, provide informa tion on the toxic characteristics of a particular chemical and on the relative toxicity among chemicals. Most static water tests are terminated after 96 hours of exposure. The degree of TCDD toxicity in guppies was hardly apparent in our tests even after 120 hours of exposure. We suggest all tests of chemical toxicity to aquatic organisms provide for observation of delayed mortality. The concentrations of TCDD used in our tests were much higher than those likely to be encountered in forest streams. Concentra tions of 2,4,5-T in excess of 0.1 ppm have not been found in North west forest streams (NORRIS 1967). Current production grade 2,4,5-T contains less than 0.1 ppm TCDD: therefore, streamwater containing 0.1 ppm 2,4,5-T should contain no more than 0.01 part per trillion TCDD. Experiments are now in progress to determine the threshold-response level of salmon fingerlings and guppies to TCDD in water. 79 0002C42 7001 D O W 281062 > SUMMARY Exposure of guppies to 0.1, 1.0, or 10.0 ppb TCDD for 120 hours caused complete mortality In the next 32, 21, and 30 days, respectively. Duration of survival was significantly and positively correlated with body length. The concentrations of TCDD used in these tests were considerably greater than expected to occur in forest streams after aerial application of 2,4,5-T. The thresholdresponse level for TCDD in fish has not been reported. LITERATURE CITED BUU-H0I, N. P., PHAM-HUU CHANH, G. SESQUE, M. C. AZUM-GELADE, and G. SAINT-RUE: Naturvissenschaften 59,173 (1972a). BUU-HOI, N. P., PHAM-HUU CHANH, G. SESQUE', M. C. AZUM-GELADE, and G. SAINT-RUF: Naturwissenschaften 59,174 (1972b). COPE, OLIVER B.: Ann. Rev. Entomol. 16,325 (1971). COURTNEY, K. D . , D. W. GAYLOR, M. D. HOGAN, H. L. FALK, R. R. BATES, and I. MITCHELL: Science 168.864 (1970). COURTNEY, K. D. and MOORE, J. A., Toxicol. Appl. Pharmacol. 20,396 (1971). CUNNINGHAM, H. M. and WILLIAMS, D. T.: Bull. Environ. Contam. Toxicol. 2.45 (1972). DOUDOROFF, P., B. G. ANDERSON, G. E. BURDICK, P. S. GALTS0FF, W. B. HART, R. PATRICK, E. R. STRONG, E. W. SURBER, and W. M. VanHORN: Sewage and Ind. Wastes 23,1380 (1951). ISENSEE, A. R. and JONES, G. E. : J. Agr. Food Chem. 19.,1210 (1971). JUNTUNEN, E. T. and NORRIS, L. A.: Special Report 354, p. 26 (1972) Oregon Agr. Exp. Sta., Corvallis. ' KEARNEY, P. C., A. ISENSEE, C. S. HELLING, E. A. WOOLSON, AND J. R. PLIMMER: 162nd Natl. Meet. Amer. Chem. Soc. Div. Pestle. Chem., Wash., D.C., Abstr. 9 (1971). KEARNEY, P. C., WOOLSON, E. A. and ELLINGTON, C. P., JR.: Environ. Sci. Technol. 6,1017 (1972). MacLEAD, C. M. [Chairman]: Report on 2,4,5-T. Report of the Panel on Herbicides, President's Science Advisory Committee. U.S. Govt. Print. Off., Wash., D.C., p. 68 (1971). MURPHY, P. G. and MURPHY, J. V.: Bull. Environ. Contam. Toxicol. 6,581 (1971). MORRIS, L. A.: p. 103, in Herbicides and vegetation management (1967) Oregon State Univ., Corvallis. NORRIS, L. A.: J. For. 69,715 (1971). PIPER, W. N. and ROSE, J. Q.: 162nd Natl. Meet. Amer. Chem. Soc., Div. Pestle. Chem., Wash., D.C., Abstr. jt (1971). WERSHAW, R. L., BURCAR, P. J. and GOLDBERG, M. C.: Environ. Sci. Technol. 3,271 (1969). WILSON, DENNIS C. : The effects of the herbicides diquat and paraquat on pond Invertebrates, MS thesis, Oregon State Univ., Corvallis (1968). 80 0002043 7002 UM- IlM- 7003- 7_ to tk CoasuweA r * ^g|! R / b0X5449:1 P ^ - V - " ' * ^ ' ".-'^:.'-!!?f-':-'W^ . DIRTY TRICKS `:r " V` SHERMAN OAKS, CALIF. 91403 ^ Voi. V No.113/114 (Nov 75) 5^ This special 8-page newsletter deals with two. dastardly and possibly-illegal .'t C: deeds. 1) Deliberate falsification of facts by the L.A.County Chief Adminis- trative Officer (CAO) or. a vital health issue, aided and abetted by the Los g Angeles County Health Services, the Forester and Fire Warden find the Agricul- . v tural Commissioner! 2) Violation of Federal Law, the Federal Insecticide,- Fungicide and Rodenticids Act (FIFRA) by the United States Department of Ag- riculture Forest Service, w i t h the approval and blessings of the C a l i f o m i a ^'-^-- -; Agricultural Conmiissioners!'<-; ->7V`":-Av:r X #1. Ever since 19^5, charitable donations have been doled out to the Nation- al Forest Service by the L.A. County Board of Supervisors under "Aid to Other . Governments and Non-Profit Organizations" to maintain "recreational" services ^ for persons living in the Coun t y . The amount ranged from $20,000 to the cur- . rent $90,000. The Sept.2, 1975 agenda item (for another hand-out) was chal- q ^; lenged by this Consumer Activist, charging that "the continued use of herbi- oo" ! cide/defoliants (2,4-D, 2,4,5-T, 2,4-DP,etc.) by the Forest Service through- cri-! out the U.S. Is a hazard to the entire community, especially to pregnant - .jcmen and women of child-bearing age, due to the teratogen DIOXIN that is present in all. phenoxy herbicides - the very reason that the U.S.Surgeon Gen-,.. er in 1970 ordered them banned from military use...Since the market for neruicides was lost when the war ended in Vietnam, the chemical companies .-" :"'0/\ nave accelerated the big.push to unload these deadly defoliants on US." The $3? million government "Bionetics Study" (commissioned by National Can cer Institute) revealed that these herbicides caused cancer, birth deformities 2nd genetic damage. Th e y were developed by the military at Fort Detrick, Md. v- zo be used as Chemical Biological War Weapons, and only recently our Senate ;v renounced their use as weapons of war. It is immoral that their use contin-i? m-ies, especially since evidence has shown that they, accumulate in the food- :hain^ presenting a long term danger to all living things. The herbicides fa are able genetic atomic bombs timed to outcome" said Senator Gaylord explode in Nelson and the future with an unpredicthe Justifiably Insisted that - - ?o m- i Tunds sought b y the National Forests to spray 2,4,5-T be deleted from the *: budget of the U.S .Forest Service .;.Vr ?vJr ?he National Cancer Institute (NCI) statistics for the first 7-months of '75 showed that CANCER MORTALITY has increased 5-2# this year, higher than it's >een since they began gathering mortality data in 19331 The recent Library )f Congress report "Effects of Chronic Exposure to Low-Level Pollutants in :he Environment" documented the inadequacies of-:our current research and mon-'--v. -toring activities, and reported that environmental problems might account 'or as much as 70 to 90# of, causes of cancer, and were responsible also for ' ieart diseases. 1 It is no'surprise that the only pollutants naaad were a s b e s - :' -os, chlorines, fluorides, nickel* and mercury. ' Ichlorcphenorcyacetic. acid caused cancer xn 22 laboratory animals, focal .o f neuaichltTs"in l ! "animals and metamorphosis of the liver. ..2,4-DP caused cen- er in 7 laboratory animals and focal pneumonitis in 6 animals. 2 4,5-T caused ;V: ancer in 9 animals, folicular hyperplasia of the spleen, lymphoid hyperplasia f the lungs, focal pneumonitis, lymphoid nodules, pulmonary edema, lymphoid nfiltration in. the lungs, and monstrous birth deformities. They are all oxic chlorinated hydrocarbons in addition to being hormones. There are no r.own fir**ldotes. for pni r.h*v v ,,-- i-- > . ':'' T h e :;iten. ($90,000 to Forest Service) appeared, on the agenda .'for. 6 ;.consecutive^^; weeks and was finally heard on Oct. 30th., Just prior to Halloween, and a s . ^ ^ S |j! < g g '/hif usual, the CAOs office was ready with their hag of tricks. I n ^ ^ his letter to the Board of Supervisors, CAO Harry Hufford con-^fe' veyed the following misinformation: "Herbicides which produce toxic dioxins have not been used by the Forest Service in 1973/v?? 1974, or to date in 1975. This i3 confirmed by use permits to.^; the Forest Service and the County monitoring program which has 7? discovered n o trace of dioxins, (emphasis added) in any of the q 250 samples of soil, water and air examined in the County p e s t i Q cide laboratory over the past 12 months." THIS STATEMENT IS A < 1 BLATANT FALSEHOOD 1 The County does not have facilities or equipment to test for dioxin - THEY DID NO TESTING FOR DIOXIN i 00 In addition to testing "soil, water.and air," the CAO stated that they had tested horses, rabbits, coyotes, squirrels, .deer, fish and 2 lions for resi- dues of phenoxy herbicides and dioxin. Since the issue raised was c o n t a m i n a - tion within the forest service, one would assume that the animals tested had come from within the Angeles National Forest. Under questioning they admitted^- 1 that one of the rabbits came from Downey, the other from the Long Beach Kaval i Base - one of the lions came from "Lions, Inc." owned by John Marshall Farms, nobody knew where the other lion came from, or whether lions still roam in .7* our forests I . One coyote came from Malibu, another from Antelope Valley - a deer from William S. Hart Park - a horse from La Canada, another from Altadena/ and the fish from Rio Hondo 1 If one has integrity... in order to determine j residues of toxic chemicals in wild animals that live in the national forests | one would go directly to the scene of the crime (the forest). It is highly [ Irregular and downright dishonest, but very convenient for the agencies named,( I to secure specimens from Tim-Buck-Two. . C October 30th. Acting Board Chairman, Baxter Ward, instructed the CAOs of- W t '| fj.ce to get an immediate response to the questions I had raised,, and a m e e t - | ing v/as held with Dr. John Affeldt (County Health Services), Paul Engler (Ag- ricultural Commissioner), Dick Baird and Jack Michaels (CAOs office) and your 'f : Consumer Activist. ~ After one hour of dickering with Affeldt, Engler and Baird^ ; who finally admitted that they knew little, if anything, about dioxin, we ; 'awaited the arrival of their expert, Dr. Naresh Jain (Toxicologist,Rancho L o s-' Amigos Hospital/Downey) who was unaware that 2,4,5-T is not the only phenoxy u | herbicide that contains Dioxin. I pointed out that scientists Edwin A.Wilson,; ! Ronald F. Thomas and Peter D.J.Enser with the USDA Plant Research Division at ^ i Beltsville, Md. on 8-19-71 had proven that hexachlorodibenzo-p-dioxin was present in 2,4-D up to 1 part per million - that 13 dioxin Isomers had been. isolated in the phenoxy herbicides, and that there were reams of government ; 2 publications showing 2,4-D, 2,4-DP, 2,4,5-T, 2,4,5-TP,etc . contain dioxinl The County "experts" treated the information with disdain and even though they had been their skulls. told to answer my questions, Hours of valuable time were there v/as no way wasted and since of pe Baird netra had ting : ' trouble -0 formulating my questions, I wrote them for him, but neither the questions nor > the answers were submitted to the Board. I charged the CAOs office with de- liberately lying to the Board (Hufford should;have verified the facts before submitting such misinformation to the Board). Dr. Affeld defended the CAO . saying "It was an honest/mistake - they weren't able to test for dioxin, and so they didn't find any." Dr. Affeldt holds one of the most responsible and highest paid Jobs in our County - Our health rests on his "educated J'*^gement." We finelly agreed that it was Imperative that there be routine 1 ting for dioxin, but since the equipment would cost around $150,OCC. the suggestion was cade that the Environmental Protection Agency (who new has the equipment).be contacted to see whether they could do the testing for the County. The recommendation was never made to the Board. ... v. 7005 The following was submitted to the Board of Supervisors:. "The controversy ' jT.-V In addition, the herbicides presently being used by the Forest Service have c i approved by theEnvironmental Protection Agency. While we will be happy^|i^:. to work with Ms. Honorof further on the matter, we feel the Forest Service's' use of herbicides is proper, and.-recomend the Board sign the policy budget contract with the Forest Service.". The Board approved the recommendation 1 T h e three Supervisors (Ward, Edelman and Schabarun) admitted that they knew -'-f? nothing about the herbicides, or about dioxin, at the same time that they re- fused to listen so they could learn. The question we must raise is HOW MANY : TIMES HAS THE CAO's OFFICE SUBMITTED TOTALLY FALSE AND MISLEADING INFORMATION WHICH WAS APPROVED 3Y THE BOARD'0? SUPERVISORS? If these people had been un- der oath, any statement they submitted would be under penalty of perjury, t h e y j would be nighty careful with their recommendations. The issue on hand isn't just one of total waste of taxpayer's money - it is a serious health problem. Now the Board of Supervisors will be held liable for perpetuating a program that is detrimental to health and l i f e M / . - . . ... v- " .<-.* O In a personal letter to Supervisor Baxter Ward I pointed out that I had cor- O respondence from Drs. Granville Knight (Santa Monica), Terry Lash (Environ- <! / mental Defense Fund/Palo Alto) and E.W. Pfeiffer (Univ/Montana- Missoula)with F the warning that PREGNANT WOMEN AND WOMEN OF CHILD BEARING AGS SHOULD NOT 3E ' PERMITTED TO ENTER ANY AREAS WHERE PHSNOXY HERBICIDES HAVE BEEN SPRAYED, due 00; to the birth deforming contaminant that is present in all of the phenoxy her- cicides.' Four years ago statistics showed that of all live babies are corn with some birth defect in the U.S.'* This figure is nothing compared with lT' j what will happen in the future. How will our scientists be able to pinpoint . ; the cause of* increased birth deformities? Babies bora with gross deformities . ! w< t have finger-prints" or a label attached saying: "THIS DEFORMITY WAS [ CAUSED BY 2,4-p, or some ether phenoxy herbicide. ' ' Dr. Gerhard Grummer (Rostock Univ/Germany) toured both South and North V i e t - . ' nan and returned .with documented evidence proving that these herbicides should NEVER AGAIN*BE USED, either during periods of war or periods of peace. 25:;j of ail mothers that came in contact with' "Agent Orange" (50^-2,4-D and gave birth to deformed babies suffering from a most serious heredi tary disease -"Down's Syndrome". (They had 47 chromosomes instead of the normal 4c). Children with this surplus chromosome will at best attain t h e ' t physical constitution of a 10-year old when they are adult. They remain ; small in stature, their limbs are short and stunted and their bodies are clumsily structured. The neurocranium is not infrequently smaller than nor- ' ual. At best they will acquire the mental status of a normal 7-year-old ..'hen fully g r own. The harm dene to their minds is even graver than the dam age acne to their bodies. MUST WE WAIT FOR THIS SAME TRAGEDY TO OCCUR IN THE J.3. BEFORE W E WAKE U? AND STOP THE U SE OF THESE DEFOLIANTS? o .] =Irth deformities are on the increase in the U.S., *it there isn't a single 'cvernment agency that has an on-going monitoring system to determine what :ausec the increase. I spoke to one of the people with Department of Health in Sacramento (who asked to remain nameless) who stated: "The data we have . '% * I I lr l isn't worth a damn - there .s no standardisation anung doctors. Sometime ago -hey used to put down congenital*birth defects on the certificate, but the ioctcrs are lax because it is not demanded cf them...We don't have the hard :ata to collect - WHY MASSAGE IT?" Another call to Berkeley netted: "There is on-going monitoring program, simply because many times doctors don't :now what to look for, and often the birth deformity will show up after the icctor has signed the birth certificate, so it isn't included on the certifcate. 'The information is sketchy," said Mitch Tashirc, a statistician with 21 nI | * he'Kealth Department ih Sacramento, "fragmented - trying to monitor off of ir-n certificates would be enormous - birth certificates are poorly kept - There^'is* no program'geared `'to identification end reporting birth deformities..'! The Pesticide Chemical New3 of 9-10-75 revealed the information that had been' ^ suppressed for over 6 months, that "TCDD (tetrachlorodibenzo-p-dloxin) had been found in cattle tissues in levels up to 100 parts per trillion (ppt)` p: f that dioxin is present in our food chain. The Department of A g r i c u l t u r e ^ ' ana the chemical companies were aware that the phenoxy herbicides remained / and bio-magnified in our food chain in the early 70s (Sen.Kart Hearings). San- pies are now being taken by the EPA from small mammals who were exposed, to TCDD, and samples-will be taken from mothers milk as well, to determine res "v : idues of TCDD, and the Hearings that were postponed on June 2 4 ,1 9 7 4 , "to per mit the EPA to set up a monitoring program to determine if TCDD is bioaccum ulating in man and the human food chain," will resume soon. The EPA must also include monitoring for everyone of the phenoxys and their metabolites - 0 DIOXIN IS AN INTEGRAL PART OF THE PHENOXY HERBICIDES. 1The Board of Supervisors have allowed themselves to be lied to - by the CAOs office --the Health Services white-washing and covering up (which makes them equally guilty) - with the inadequate monitoring and non-existing Mtesting" . for dioxin. I suggested a public hearing in L.A.County to-expose the insan ity of herbicide use - working tov/ards the State of California enacting a total ban. The herbicidas are too toxic to be used by the military - how ... CO o o co can they Justify their safety on our forests, rights of way, schools, agri r o ^ cultural use, etc; I also suggested that legislation be enacted whereby . heavy fines and Jail sentences would be Imposed on government officials, pub lic health service personnel and scientists, who deliberately make false ;>- statements concerning data not borne out by proper analysis and evaluation. Jail sentences would be the only way of curing the problem, especially since these deadly compounds Jeopardize the health, welfare and life of the entire community. We pray that our public servants will rise to the occasion. . Ir he Environmental Impact Report (EIR) that was prepared by L.A.County Ag- _ ricultural Commissioner for their "Alligatorweed Eradication Project in '73 AND WHICH NONE OF THE COUNTY SUPERVISORS HAD BOTHERED TO READ, the facts .'re presented proved that "2,4-D and 2,4,5-T have mutagenic potential as demon- ... '4 strated in tests with several plant systems (Mrak,1969)> Unrau and Barter . (1952), Unrau (1953/1954) found `highly significant1 abnormalities of chrorn- osome behavior in rapidly dividing cells of wheat and barley sprayed with 2.4- D ethyl ester. Muhling et al (i960) also found chromosomal effects in I i peas treated with 2,4-D. 2,4-D isoctyl ester, 2,4-D butyl ester as well as -v-- 2.4- D isopropyl ester produced statistically significantly higher incidences of congenital malformation in mice or rats. Innes, et al (19o9) reports 2.4- D isopropyl ester and 2,4,5-TP yielded an increased carcinogenic tumor; incidence in comparison to negative controls. Mrak (1969) suggests these compounds need more testing but the priority for testing is not high in com parison with some other pesticides." The Agl. Comm, should review this E I R 1 TCDD(dioxin) is so lethal that only 6 parts of dioxin per 10 billion parts (bodyweight) was lethal in laboratory tests. It is the wor l d 's most lethal synthetic agent. The_Llbrai^r_c>f Congress lists its acute oral_toxicit.v- for humans as 42 micrograms7~~~0he drop can kill 1200 persons- 30 gallons could kill every llvlhg~t5ing on this earthl Records-:show that millions of chickens died in the U.S. in 1957 from pericardial edema, which was traced to the in clusion of fat in the diet.,which contained hexachlorodioenzo-p-dioxin (Can 0 trell, Webb & Mabis,1969)/ Structurally similar compounds, tetra and penta o chlorodibenzofurans are contaminants of polychlorinated biphenyls that cause hy-'-opericaralum In chicks and hyperkeratosis .of the rabbit's ear (Vos < Beems, IS Vos & Koeman, 1970; Vcs Kceman, van der Maas ten Noever de Brauw & deVos, 1970). Other studies have shewn that dioxin is a potent teratogen and foeticidal agent for rats and mice (Courtney & Moore,1971; Neubert 8: Eill- mann,lS72; Sparschu, Bunn & Rowe,1571). Since the 1930c, chloracne has been recognized as an occupational hazard asspclated.with the p r a d u a t ^ n of mental ability, as well as many physical manifestations. It was this sane.:-^^, contaminant that forced the closing of Dow's Midland Michigan plant in the "/U- te 60s, until they could overcome the problem. Since 1950 Dow had been keenly aware of the possibility of a highly toxic impurity formed, in 2,4,5-T.':^ Workers developed chloracne and their bioassay program showed that chloracne.,uV potential of the waste-oil from 2,4,5-T process was building up to a danger point - lieither the FDA nor the U5DA were notified in 1965, because no-effect " levels were ever reached even at the lowest dose tested. The Diamond Alkali plant stopped manufacturing 2,4,5-T. (Julius Johnson at Hart Hearings, 1970) Despite the fact that the L.A. County expert Naresh Jain, PhD, stated that .. the only toxic dioxin he was aware of was tetrachlorodlbenzo-p-dioxin in 2,4,5-T. Page 29 of the L.A.County EIR reads: "Dioxin content of phenoxy herbicides is Important.. Dichlorodibenzo-p-dloxin would be the major species of dioxin in 2,4-D and tetrachlorodibenzo-p-dioxin would be the major species O Oof dioxin in both 2,4,5-T and 2,4,5-TP." Page 107: "The highest tissue levels of 2,4-D and 2,4,5-T were found in liver, kidney, lung and spleen, the levels sometimes exceeding the plasma level...In blood cells, 10-20# of the plasma level was found. Penetration of 2,4-D Into adipose tissue end Into the cen a tral nervous system was restricted, whereas a ready placental transfer was CO demonstrated in swine. (K.Erne,i960)." o There are additional facts that the County experts omitted. The fact that many plants have accumulated lethal quantities of nitrates after being CO CO sprayed with 2,4-D. The County EIR p-143 states: "The accumulation of ni- > trates in the leaves of treated sugar beets is well known (Savage 1949). In creased levels of nitrate in the leaves of this crop as a result of herbicide ( a^nlicatea have been reported by Willard (i960), S t a h l e r & Whitehead (1950), - Whitehead et al (1956). Isolated incidents have been reported of nitrate . poisoning of cattle in America as a result of feeding on sugar beets that had previously been sprayed..,N.Dakota, the nitrate content of sugar beet leaves after spraying was found to vary from 1.8l to 8.77# of the dry weight, as against 0.22# for, untreated plants and a toxic level of 1.5#. (Stahler and Whitehead (1950). p-144: "Studies on forage crops (Berg & McElray, 1953)and J on a range of weed species (Frank & Grigsby,1957) have shown which of these may contain high levels cf nitrates after auxin application...Nitrate poi- . sonir.g in stock does occur from time to time and it is possible for the haz-'_ : ara to be increased by application of auxin herbicides to nltrate-accumula- . ting plants. ?-1^5: "2j4-D and MCPA were toxic to bees, not only from visit ing the flowers but also as a result cf drinking contaminated water trapped -W xn^tres plants. (Wahlin, 1950) 2,4-D might have seme effect on nectar which made it toxic to bees. (Palmer-Jones, 1964; Antoine,1966). It is a pity that the Board of Supervisors did not read the Alligator Weed EIR. They would not have approved the project. In the recommendations made by the Agri. Comm, the following appears: "Herbicide monitoring of the An- geies Forest area could be increased. The Agri.Comm- Health Services herbi cide monitoring program has thus far been concentrated in mere highly popula ted areas and comparatively few samples have been taken from the Angeles Natl. Forest. It should be noted that testing.samples Is a laborious costly process as is the collection of-samples. To gather a greater number of samples*from . relatively inaccessible areas in the forest would add appreciably to the time demands upon the monitoring employee." This recommendation will be closely 00 f' 'owed, inasmuch as I have a solemn promise that I will accompany whoever S0-.3 out to the Forest Service to collect specimens for analysis. o o + t- -r + + -r /?2. . Seme months ago I requested information from the Ar.geles Forest Service pertaining co*`what~herbicides had been used- by the Forest Service during the years 1973, 1974, 1975f and what "do they have planned for 1 9 ? 6 , e ^ ^ ^ 1 j s ^ Q - 6 1 -- v,?' 'The cheerful reply-1 received'from Robert R e e s e / ^ ^ ' v ^Watershed & Resource Management Officer) was ...The amassing of the data you have requested ^ . u .;' exceeds the time that I have to allocate - concid- I' ering my other heavy duties In the management of the Angeles National Forest." He Invited me to -IIIV call for an. appointment - he would provide the w o r k space - the charge was "0.10/page for large amounts of zerox copy work." . ~- I had also communicated with the San Francisco office and received a report from John A.Vance. (Chairman Pesticide Use Coordination Committee) - and T.W.Costella (Deputy Regional Forester in San O oFrancisco). This report listed those herbicides that had been used in the California National Forests in 1975. Based on their information and .-'ti-q*--.. the information shown on the Monthly Pesticide ' Use Report received from L.A.County Agricultural si 00 Commissioners office, I. learned that the California Forest Service as well as O the Agricultural Commissioners (who approve pesticide use) had violated the Federal Insecticide, Fungicide and Rodenticide Act, inasmuch as the herbi cides had been used contrary to the instructions on the label. Some of you will recall the expose in RTC #60- Sept.'73i and the near fatal;;":,^ i incident where a L.A.City Park employee, Harry Fales, had been applying pest- % : Icides without ever being advised of their toxicity or their identity. The herbicide PARAQUAT had been transferred from the original container to an u n - ': - marked one. Protective attire was not provided - and other serious violations -. I f FIFRA - - - then there was the death of a student at El Dorado High School,-;! 0 v_rry Maes, after drinking what he thought was fruit juice in the principal's ':;:; office. Jerry Maes died from drinking PARAQUAT, despite the fact that ALL . Paraquat was to have been removed from schools, universities, playgrounds, : golf-courses, etc.,etc. It was being used in violation of the label precau- j tion. (See RTC #Sr) The label specifically reads "KEEP PESTICIDE IN ORIGINAL . CONTAINER - DO NOT PUT CONCENTRATE OR DILUTE INTO FOOD OR DRINK CONTAINERS,. i KEEP OUT OF REACH OF CHILDREN, DO NOT USE AROUND HOME, GARDENS, OR OTHER AREAS' i LIKELY TO BE CONTACTED BY CHILDREN OR PETS." On Feb. 2, 197^, in an unprecedented action, the E?A formally charged three ' government agencies (Los Angeles County, Los Angeles City, and Thousand Oaks School District) lvith violation of FIFRA, and warned them that future viola- "7 tions would be punishable with fines up to $25>0C0. and prison terms ud to 1 year for violators. PARAQUAT HAD BEEN IN USE FOR SOME 20 YEARS, BUT'NOBODY,' NO AGENCY, HAD EVER TAKEN THE TROUBLE TO READ THE LABEL WARNINGS, UNTIL R.T.C. PUBLISHED IT IN 1973.' In a Certified-(return receipt requested) letter to Russell E. Train, Admin istrator of the Environmental Protection Agency, I informed him' that there has been a violation of FIFRA "on the part of (1) the USDA Forest Service on a State (California) level; The Individual Forest Services at Angeles, Los Padros. Cleveland. Mendocino and Sierra. (There may also be others throughcut the U.S., but I- only Wave the report for California) By the Los Anneles County Agricultural Commissioner as well as the Agricultural Commissioners o f 'SarTljieno C o unty, Santa Barbara (or Ventura} County, etc.,etc. and other * a icies who have been responsible for concealing the violation. The above ^ named Forest Services each used AMCHElI's Weedone-170 (butoxy ethanol ester ^ of 2,4-D and 2,^-DP - 31.S'/ of 2,^-Dichlorophenoxyacetic acid, butoxyethanol e s t e r ar.d 31.1$ of 2,^-Dichlorophenc:cypro?ionic acid butoxyethancl ester) in addition to-using Dow!s Isteron-99 The Issue of violation is with the use '- of Amchen's Weedone, prohibited from cejr.g used In r e c r e a t i o n a l a r e a s . (Many of th ese pesticides are us^a in direct violation of*"trie lacei, where they are ` ? ; / ^ 5 4 - - ^ ^ i ) :'::- ;: , ; , } ';... ; : ' . ^ y ^ ^ " t - ;; -::'r ; * '` - * p i r g , ~ ' : i restricted if the pesticide gets into v/ater supplies,etc., but we were ' c o n i ^ J ^ . cerned with a stark violation that even a child can understand: "DO HOT USE AROUND THE HOME OR J RECREATIONAL A R E A S ." The Forest Service are a mu Iti-:' r *e use natural resource. Despite the fact that there has been a ban on the v a of 2,4,5-T in "recreational" areas, and hundreds of people enter OUR f o r - ^ v ests for recreational purposes, the Forest Service harbored the thought that.-- ' they were above the law - immune from regulations, The Anchen Weedone-170 label reads: "CAUTION, Harmful if swallowed. Avoid contact with skin, eyes or clothing. Avoid spray drift to susceptible plants , such as cotton, beans, tomatoes, tobacco and ornamentals as this product may cause injury...Under very high temperature of use, vapors from the product may injure susceptible plants in the immediate vicinity. This product is ' toxic to fish.' Keep out of lakes, streams or ponds. Do not apply where run- off is likely to occur. Do not apply when weather conditions favor drift .V. from treated areas. Do not contaminate any body of water by washing or spray v/- equipment or disposal of waste...Do not contaminate water used for irrigation; or domestic purposes. Do not use in a greenhouse. Do not use around the-;..y. home or In recreational areas." SG008 M O O Our National Forests ARE recreational areas I It is inconsistent and outra geous that government agencies do not conform to laws. There are 43 National Forests ir. our country (17 of them are in California) and millions of persons are exposed to these toxic defoliants. According to thi^^port I received from the Regional Forest Service in San " Francisco, Angeles National Forest, in addition to using 2,4-D Ester, 2084 lbs. a.e. on 521 acres, also used 1076 lbs a.e. of Amchem's Weedone-170 on 269 acres. Cleveland National Forest used 3 3 ^ Ids a.e.(on 03b acres) of Am chem's weedone-170. Los Padres National Forest used 2528 lbs a.e. on t-32 ' js ss of Amchem's "Weedone-170. Mendocino National Forest used o400 lbs, a.e. on lbOO acres of Amchem's Weedone-170. Sierra National Forest used 1200 lbs, a.e. on 300 acres of Amchem's Weedone-170. The Amchem Weedone-170 carries the "EPA Reg. No. 264-222 AA, ERA Est 264-PA-l Form No.3757-30, 10/74-MP." The date stamped is May 1975- The label is specific in cautioning that the product (propionic acid mixed with phenoxy- acetic acid) is not to be used in recreational areas. The Pesticide Use Re port (dated June 1975) for Angeles shows that, 226 gallons were used in 1975, and the July 1974 report shows that 175 gallons were used in 1974. The res-' pense from Robert Reese was a violation of Freedom of Information Act (l8- ... U.S. Code Sect. 1001). Under this Act I am entitled to the information on request, but I imagine Mr. Reese is still smarting from L.A.City Council's historic'motion of September 20, 1972, censoring the USDA Forest Service for using 2,4-D, 2,4-DP and 2,4,5-T. At that time he stated "Passage of the motion need have no effect on the operations of the Angeles National Forest - the effect of such passage however would tend to place an undeserved stigma on the use of these sophisticated tools of land management...such stigma is undeserved." And Mr. Reese and all of the other Resource Management Officers continue to rain down these teratogenic, mutagenic and carcinogenic hazards on the populace. . ,^ 7010 <$05322: Ser.etor Gaylord Nelson recently stated "We are closer now to the truth on 2,4,5-T than we were when we so blindly dumped one hundred million pounds of herbicides on South Vietnam. W e ere closer to the truth than we ever were wl'K'-' the Grant County rangeland spraying occurred two years ago. Eut al.1 the fa s are not in. Knowing what we do, however, it seems to me the height of folly to have the U.S. Forest Service shutting Its eyes to the EPA pre-trial brief while .they routinely spray in the.U.S. National Forests." Senator Nelson was referring to the E P A 's statement indicating .that they were having technical difficulties'In the use*of its'method for detecting dioxin (July 1374) and noting new uncertainties about the persistence of 2,4,5-T." The-EPA brief<summarized the potential dangers of 2,4,5-T.. Hazards of.birth defects from 2,4,5-T and TCDD - TCDD was mutagenic - There was careInogenic potential - a pernicious, little understood toxicant, capable in minute quon-x-X:- titles of inducing a variety of chronic illnesses and, perhaps of causing - c th as a delayed response to exposure - TCDD poisoning may be cumulative because the effects of long-term exposure to low levels of TCDD remain u n d e - ';' termined, an acceptable level for man cannot be set. If TCDD exposure causes delayed lethality, or if continuous impingement of TCDD on human organisms otherwise causes cumulative effects or if TCDD concentrates in human tissue, a level of exposure v/hich would be safe for the general population may not exist. Even residues below the current level of detection may be unsafe... The capacity of TCDD to penetrate, persist, to move and to bio-concentrate %O in the acquatic and terrestrial, environment, given the incomparable toxicity of this small molecular compound, and given the practical nonexistence of facts about its ecological effects..there cannot be a reliable conclusion that TCDD is not causing serious environmental injury. oo00 "The polychlorophenal in 2,4,5-T may decompose into dioxin when exposed to high temperatures such as might occur with incineration or even cooking of CD 05 food. TCDD can be generated by the thermal stress of 2,4,5-T and some of its ' metabolites, raising the potential for the generation of additional dioxin under environmental conditions...People may be exposed to a latent destruct ive force - accidental or unknown triggering of the thermal release mechanism by which 'harmless' amounts of 2,4,5-T, its esters or salts, convert to letnal,; tetra-dioxin...dioxin and 2,4,5-T could build up to dangerous levels within ? man and animals - possibly in the food chain as well.(This was proven 9-10-75) "Model ecosystem studies suggest that TCDD bioconcentrates more than DDT... acquatic ecosystem showed catfish accumulated tetra-dioxin in only 3 days by a -factor of l4,000.. .Analysis of residues in Vietnamese shrimp and crusta- c .is detected significant levels of tetra-dioxin following defoliation in regions draining into the area from which the shrimp were collected...Resi dues have not declined between 1970 and 1973, although defoliation -ceased in 1969. Wildlife in the vicinity of"Agent Orange"application at Eglin Air Force 3ase (Florida)-retained measurable levels of TCDD several years after ; use of the herbicide (2,4-D and 2,4,5-T) was stopped...25# of the dietary in- ' take of tetradioxin may be stored in body tissues...withdrawal of cattle from a diet contaminated with dioxin for as long as one week may have little effect in decreasing TCDD residues...Current label provisions requiring 'feed o f f ' periods on dioxin free food to assure absence of dioxin residues in the m e a t - . are not likely to be effective in reducing tetra-dioxin residues...Monitoring : wildlife collected along rights-of-way in the U.S. demonstrates as does the Vietnamese aquatic residue data, that 2,4,5-T related TCDD can enter the food chain from 'n en-food' uses." The above holds true for all of the phenexy herbicides. These facts will be brought out during the upcoming Hearings. PS: Russell Train was also advised that after talking with back-packers, we have been advised that the Angeles Forest Service has violated a contract they had with L.A.City Attorney's Office*to post areas in Angeles prior to treatment with any herbicide, warning the public that such treatment will take place, by either stationing of guards or posting of signs. THERE IS NO ALIBI FOR VIOLATIONS OF LAW! THE ONLY ALTERNATIVE IS A TOTAL BAN! Sda `Sfcno&oji R e p e a t t o t h e Consuweft 1 P .O .B O X 544 9 SH ERM A N O A K S. C A LIF. 91403 Please notify us-o f your change of address //L J!-, '\Ja % bulk rate U.S. P o m o PAID /tA Sherman Oak, Calif. 91413 A w *3/3Permit No. 372 /- 0005323 SO'U O L I S H E D O I M O N T H L V S U B S C R I P T I O N R A T E S 00 PE R Y E A R ^ Copynghi >97$ b y I d s H o n o i o t A l l ngnti i r u m t P a r m n n o n to P r o d u c t t n v poition of trut publication m a y b a o b t a m a d b y a n t i n g 8 1. M o n o r o f R . o m r to in. Caniuffiir P O 5 4 4 9 . S h a r m a n O a ti. Calif --< 5^ / (q Sn 210 L Su Removal of 2,4-Dichlorophenoxyacetic Acid (2 ,4 -D) Formulations from Noncombustible Pesticide Containers by T. E. Arche* Departm ent of Environm ental Toxicology Univertity of C alifornia D avie, C alif. 95616 d c .: 4 * * 4 / e Retained p e s tic id e s in d isca rd e d used con tain e rs can be a , se rio u s environmental p o llu t io n problem. I f 1, 5, 30 and 55 gal metal p e s tic id e drums are d efine d as noncombustible packaging mate r i a l s , then the la r g e number o f these con tain e rs th at accumulate yearly entail special disposal considerations. Published reports have d iscu sse d the decontamination o f organophosphorus in s e c t ic id e s on and around a i r c r a f t and spray equipment (W0LVERT0N et al_. 1970); edaphic asp e c ts o f the d isp o sa l o f unused p e s t ic id e s , p e s tic id e w astes, a n d 'p e s tic id e con tain e rs (ST0UAN0VIC e t a l . 1972); chemical and thermal aspe cts o f p e s tic id e d isp o sa l IKENREDY et a l . 1972); en gineering aspects o f the d isp osal o f un used p e s t ic id e s , p e s tic id e s w aste s, and p e s tic id e containers (SHUMAN et_al_. 1972); and decontam ination o f noncombustible a g r i c u lt u r a l p e s tic id e co n ta in e rs (HSIEH e t al_. 1972 and ARCHER and HSIEH 1973). The pre viou s in v e s t ig a t io n s , p a r t ic u la r ly in regard to d e to x i c a tio n o f metal drums, were d ire c te d toward organophosphorus in s e c t ic id e s . The present work was undertaken to In v e s t ig a te procedures f o r the removal o f retained amounts o f form ulations o f 2,4-d1chlorophenoxyacetic a c id 1n a water m is c ib le (non-volat11e weed k i l l e r ) and a p a r a f f in ic ( lo w - v o la t ile weed k i l l e r ) so lv e n t base from 5, 30, and 55 gal metal drums so th a t the d e to x ifie d metal c o n tain e rs could be d isposed by conventional methods or reclaim ed f o r non food usage. M a te r ia ls and Methods P e s tic id e fo r m u la tio n s . (1 ) Dow Esteron R 99R Concentrate Low- V o la t i le Weed K i l l e r c o n ta in in g propylene g ly c o l butyl ether e ste rs o f 2,4-D; acid equivalent: 4 pounds per g a llo n ; active In gred ie n t: 2,4-dichlorophenoxyacet1c a c id , propylene glycol butyl ether e ste rs, 2%.72.8X, 2,4-d1chlorophenoxyacet1c a c id e q u ivale n t 44.9X; In e r t in gredients, 27. (2 ) Dow Formula 40 N o n -V o la tile Weed K i l l e r con ta in in g alkanolam ine s a lt s o f 2,4-0 ; acid equivalent: 4 pounds per g a llo n ; a c tiv e in g re d ie n ts: alkanolam ine s a lt s (o f the ethanol and isopropanol s e r ie s ) o f 2,4-dichlorophenoxyacetic a cid , 59.7X, 1Throughout t h i s paper 1 gal 3.8 l. o O' sO -M >o *0 cn cn ro CT Bilctla of E a r ir a iN a u l C ix riiiM llw A ToxiaaJafr, Vat. 15, Na. 1 1973 by 3p rtf*r*Y ertaf New York la c . 7013 OOOloSM 2 4, -d ich lo ro p he n o xyace tic a cid eq u ivalen t 38.6%; in e r t in g re d ie n ts, 4 0 . 3X. C o n ta in e rs. The co n tain e rs were fla t-h e a d ste e l drum types commonly used f o r a g r ic u lt u r a l p e s tic id e s and represented 5, 30, and 55 ga l s iz e s . Washing s o lv e n t s . The aqueous so lv e n t was deionized water, and the ethanol was f r e s h ly d i s t i l l e d a b so lu te ethyl a lc o h o l. Diazomethane p re p a ra tio n . Diazomethane was prepared by the method o f ZWEIG e t a l . 1964, and the etheral s o lu tio n was adjusted to 25 ml volume. One ml o f t h is s o lu tio n was added to 1 ml o f the drum wash e x tra c ts and allowed to stand a t room temperature f o r 15 min. The excess reagent and so lv e n t were evaporated by a warm a i r stream to d ryn e ss, the volume adjusted and an a liq u o t in je c te d in to the g a s - liq u id chromatograph (GLC) f o r a n a ly t ic a l measurement o f the 2,4-dichlorophenoxyacetic acid equivalent. C o n ta in e r re te n tio n volume. Excess spray form ulation was added to empty, d ry , weighed c o n ta in e rs. The con tain e rs were c lo se d , shaken, and sw irle d so th a t the inner su rfa ce s were wetted with the fo rm u la tio n . The co n tain e rs were opened, in ve rte d , and the excess form ulation allowed to drain Into a graduated cylin d er u n til d rip pin g stopped ( a t le a s t 5 m in), and the co n tain e rs were reweighed. The fo rm u latio n re te n tio n in the c o n tain e r was expressed as net re te n tio n volume o r w eight o f a c tiv e in gre d ie n t. The re te n tio n volumes were determined in t r i p l i c a t e f o r each c o n tain e r s iz e and the mean re te n tio n volumes were added to the c o n tain e rs f o r the washing experiments. Container w ashing. Adequate volumes o f washing so lv e n ts, determined by v is u a l o b se rv a tio n s, were added to each s iz e con tain e r to r e s u lt in e f f ic i e n t washing o f the In n e r su rfa c e s. These volumes were 250 ml f o r the 5 g a l, 750 ml f o r the 30 g a l, and 1000 ml fo r the 55 g a l c o n ta in e rs. The washing so lv e n t was added to the con t a in e r , the c o n ta in e r was c lo se d , and i t was shaken, s w irle d , and tumbled repeatedly. The co n tain e r was opened, the wash m ixture was drained into a graduated cylin d er u n til dripping stopped (at le a st 5 m in ), and the s o lu tio n was measured and stored f o r e x tra c tio n and a n a ly s is . Each co n tain e r was washed repeatedly with each wash s o l vent, and the data are shown in Tables I I through V. Washing m ixture e x tr a c tio n . Ten ml a liq u o t s o f the aqueous wash m ixture c o n ta in in g the propylene g ly c o l butyl ether e ste rs o f 2,4-0 were a c id if ie d w ith 5 ml o f 5N HC1 and extracted fo u r times in a se p a ra to ry funnel w ith 25 ml d ie th y l ether. The ether e x tra c ts were f ilt e r e d through a Whatman #1 f i l t e r paper, combined and ad ju ste d to ap p rop ria te volumes. To 1 ml o f the ether e x tra c ts was added 1 ml o f the eth eral diazomethane f o r m ethylatin g the 2,4-0 p re se n t, and the excess reagent and so lv e n ts were evaporated to d ry ness a f t e r the s o lu tio n s remained a t room temperature f o r 15 min. D ilu t io n s were made to approximate 1 ng m ethylated 2 ,4 -0 per pi hexane CCCIc&L5 DOW 1376526 and the samples were analyzed by GLC. The aqueous wash m ixture con ta in in g the alkanolam ine s a l t s o f 2,4 -0 were extracted as described above. Each t o t a l ethanol wash was concentrated in vacuo a t 50-60C to approxim ately 1 ml a f t e r the pH was adjusted t c T le s s than 2 w ith 10 ml o f 5N HC1. The s o lu tio n was adjusted to an ap p rop riate volume with d ie th y l ether and 1 ml o f t h i s so lu tio n was added to 1 to 3 ml o f the eth eral diazomethane ( u n t il the c o lo r remained ye llo w ) and allowed to stand a t room temperature f o r 15 m1n. The excess reagents and so lv e n t were evaporated by a warm a i r stream and the methylated 2,4-0 was adju sted with hexane to approximate 1 n g /y l f o r a n a ly s is . Detection and determ ination o f the methylated 2 ,4 -D . Gas-11qu1d chromatography (G Lt) procedures were employed r o u tin e ly . A ll chem i c a ls used, w ith the exception o f the p e s tic id e , were reagent grade and the so lv e n ts were r e d i s t i l l e d s h o r t ly before use. The gas chro matograph was a V arian Aerograph (Model 1200) equipped w ith an e le ctro n -ca p tu re d e te cto r and a Leeds and Northrup Speedomax W 1-mv recorder w ith a c h a rt speed o f 1 /2 -1 nch per min. Areas under the peaks were measured with a p o la r planim eter. The chrom atographic column, made w ith a 6 -fo o t x 1 /8 -1 nch g la s s tube packed with 60-80 mesh s ily la t e d Chromosorb W, a c id washed, was coated w ith 5X SE-30 s ilic o n e gum rubber and 5X Dow 710 s ilic o n e f l u i d . N itro ge n c a r r ie r gas (50 p . s . i . , 30 ml per min) and a column temperature o f 190C gave the b e st r e s u lt s and were used 1n these experim ents. Results The re te n tio n volumes o f emptied metal drums o f v a rio u s s iz e s are shown in Table I . C o nsid erab le amounts o f 2 ,4 -d ich lorop h e n oxya c e tic a cid (2 ,4 -0 ) e q u iv ale n t in the form ulation in terms o f ml re te n tion volumes were found a c t u a lly retain ed in the drums when they were drained as com pletely as p o s s ib le . However, these re te n tio n volumes represented approxim ately 0 .1 2 o f the 5 ga l co n tain e r volume and ranged from 0.05X to 0.07X f o r the 30 and 55 ga l c o n ta in e rs. Tables I I to IV and V to V I con tain data on removal from the emptied containers by water and eth an o l-acid o r ethanol washing o f the 2,4-0 a c id e q u iv a le n t 1n the form u latio n s c o n ta in in g the a l k anolamine s a l t s o f 2 , 4-D and the propylene g ly c o l butyl ether e s te r s. The i n i t i a l high removal e f f ic ie n c y f o r water washing 1s probably due to the presence o f e m u lsifie rs or other adjuvants in the form ulations which were d ilu te d r a p id ly d u rin g the repeated c o n ta in e r w ashings. The rem aining 2 ,4 -0 was removed by the e th a n o l-a c id o r ethanol wash in g s 1n which the 2 ,4 -0 1n the re sp e c tiv e fo rm u la tio n s i s so lu b le . Washing the variou s sized containers with eigh t water washings follow ed by three e th a n o l-a c id o r ethanol washings (T ab les I I to V) removed 95 to 99X o f the re tain e d form u lation s 1n the supposedly empty c o n ta in e rs. When the 55 ga l drum c o n ta in in g 83.6 g o f the 2,4-0 acid equivalent o f the propylene glycol butyl ether esters form ulation o f 2 ,4-D (Table VT) was washed w ith fo u r water w ashings follow ed by three ethanol w ash ings, approxim ately 96X o f the 2 ,4 -0 contaminants were removed from the con tain e r. 46 0006816 DOW 1376527 TABLE I Retention Volume o f 2,4-D Form ulations in Various Sized Metal Containers Drum S iz e Retention Volume* Grams A ctual %of Container (g a l) (ml) 2,4-D Total Volume Alkanolamine S a lts o f 2,4-D 5 24 30 83 55 146 11.5 39.8 69.9 0.12 0.07 0.07 Propylene Glycol Butyl Ether Esters o f 2,4-D 30 72 55 109 55.5 83.6 0.06 0.05 *Rententiori volumes were determined in t r i p l i c a t e fo r each sized con tain e r and mean values are reported. TABLE I I lF ive G allo n Metal Drum Washed w ith 0.250 Solven t 2,4-D Removed Per Wash ( g ) * %Present Before Removed Total t Total Wash Removed Washed** in Wash Removed Recovered Water 1 2 3 4 5 6 7 8 10.200 0.624 0.060 0.025 0.008 0.007 0.008 0.007 Ethanol-Acid 1 2 3' 0.031 0.004 <0.001 11.500 1.300 0.676 0.616 0.591 0.583 0.576 0.568 0.561 0.530 0.530 88.6 48.0 8.8 4.0 1.3 1.2 1.3 1.2 ' 10.200 10.824 10.884 10.909 10.917 10.924 10.932 10.939 88.6 94.1 94.6 94.8 94.9 94.9 95.0 95.1 5.5 0.7 <0.1 10.970 10.974 10.974 95.3 95.4 95.4 * 2 ,4 -0 a c id e q u iv ale n t 1n alkanolam ine s a l t s o f 2,4-D. **Tw enty-fou r ml (11.5 g 2,4-D ) added to drum before washing. 47 DOW 1376528 0006817 TABLE I I I Thirty Gallon Metal Drum Washed with 0.750 l Solvent 2,4-D Removed Per Wash (g )* Wash Present Before 2 Removed Total 2 Total Removed Wash** in Wash Removed Recovered Water 1 2 3 4 5 6 7 8 34.900 3.200 0.682 0.175 .089 0.067 0.039 0.028 Ethanol-Acid 1 2 3 0.088 0.032 0.019 39.760 4.860 1.660 0.978 0.802 0.714 0.647 0.608 0.580 0.492 0.460 87.7 34.900 87.7 65.8 38.100 95.8 41.0 38.782 97.5 17.8 38.957 97.9 11.0 39.046 98.2 9.3 39.113 98.3 6.0 39.152 98.4 4.6 39.180 98.5 15.1 39.268 98.7 6.5 39.300 98.8 4.1 39.319 98.8 *2 ,4 -0 acid equivalent in alkanolamine s a lts o f 2,4-0. Eighty-three m] (39.8 g_2,4-D) added to drum before washing. TABLE IV F ifty-F iv e Gallon Metal Drum Washed with 1 l Solvent 2,4-0 Removed Per Wash (g )* Wash Present Before 2 Removed Total 2 Total Removed Wash** in Wash Removed Recovered Water 1 2 3 4 5 6 7 8 59.900 8.520 1.070 0.150 0.041 0.021 0.012 0.009 Ethanol-Acid 1 -2 3 0.009 0.007 0.005 69.940 10.040 1.520 0.450 0.300 0.2S9 0.238 0.226 0.217 0.208 0.201 85.6 59.900 85.6 84.8 68.420 97.7 70.3 69.490 99.3 33.3 69.640 99.5 13.6 69.681 99.6 8.4 69.702 99.6 5.0 69.714 99.6 3.9 69.723 99.6 4.1 69.732 99.7 3.3 69.739 99.7 2.4 69.744 99.7 *2 ,4 -0 acid equivalent 1n alkanolamine s a l t s o f 2,4-0. **0n e hundred fo r ty - six ml (69.9 g 2,4-0) added to drum before washing. 48 D(W 1376529 0006818 TABLE V Thirty Gallon Metal Drum Washed with 0.750 l Solvent 2,4-0 Removed Per Wash (g ) * Wash Present Before X Removed Total X Total Removed Wash**- 1n Wash Removed Recovered Water 1 2 3 4 5 6 7 8 Ethanol 1 2 3 30.500 9.800 5.540 3.540 1.500 1.040 0.390 0.380 1.340 0.550 0.390 55.500 25.000 15.200 9.660 6.120 4.620 3.580 3.190 2.810 1.470 0.920 54.9 30.500 54.9 39.2 40.300 72.6 36.4 45.840 82.5 36.6 49.380 88.9 24.5 50.880 91.6 22.5 51.920 93.5 10.8 52.310 94.2 11.9 52.690 94.9 47.6 54.030 97.3 37.4 54.580 98.3 42.3 54.970 99.0 *2 ,4 -0 acid equivalent 1n the propylene glycol butyl ether e ste r s of 2,4-0. **Seventy-two ml (55.5 g 2,4-0) added to drum before washing. TABLE VI F ifty -F iv e Gallon Metal Drum Washed with 1 l Solvent 2,4-0 Removed Per Wash (g ) * Wash Present Before X Removed Total X Total Removed Wash** 1n Wash Removed Recovered Water 1 2 3 4 Ethanol 1 2 3 58.850 12.200 4.110 1.650 2.060 0.860 0.160 83.600 24.750 12.550 8.440 6.790 4.730 3.870 70.3 58.850 70.3 49.2 71.050 84.9 32.7 75.160 89.9 19.5 76.810 91.8 30.3 78.870 94.3 18.1 79.730 94.1 4.1 79.890 95.5 *2 ,4 - 0 acid equivalent 1n the propylene glycol butyl ether e ste rs o f 2,4-0. **0ne hundred nine ml (83.6 g 2,4-0) added to drum before washing. 49 DOW 1376530 0006S19 DOW 1376531 G e n e ra lly the 2,4-D 1n these form u latio n s was more p e rsiste n t a g a in s t removal by the aqueous and o rga n ic so lv e n t washing tr e a t ments than as p re v io u sly d isc u sse d f o r emu1s1f1ab1e concentrate 99%form u lation s o f parathlon (ARCHER and HSIEH 1973). However, 95 to o f the retain e d fo rm u la tio n s were appare ntly removed from the co n tain e rs, and con sid erin g the low manmallan to x ic it y o f 2,4-D, t h is 1s probably an adequate e f f ic ie n c y . D isc u ssio n Supposedly empty noncom bustible metal con tain e rs retained as much as 24 to 146 ml o f form u lation f o r 5 and 55 gal s iz e s , r e s p e c t iv e ly , a f t e r the co n tain e rs were drained as com pletely as p o s s ib le . Although 2,4-D 1s harm less to mamnals a t phytotoxic con c e n tr a tio n s , 1 t has been w id e ly used as a h e rb ic id e toward most broad-leaved p lan ts since i t is r e la tiv e ly nontoxic to monocotyledonous p la n t s . I t has a ls o been used f o r the prevention o f pre h a rv e st f r u i t drop, the production o f se e d le ss f r u i t , and the re gu latio n o f the growth o f p la n ts. These e f f e c t s are useful when 2,4-D 1s ap p lie d to p lan ts in t e n t io n a lly . However, what r e s u lt s would occur 1f 2,4-D were to appear in and p o llu te the environment by un in ten tional a p p lic a tio n such as contam ination o f su rfa c e and ground waters as well as s o i l ? The Improper d is p o s a l o f noncom bustible used metal containe rs which previo u sly contained form ulations o f 2,4-D could contribute to such environmental p o llu tio n . I t has been shown by previous stu d ie s (HSIEH et a]_. 1972 and ARCHER and HSIEH 1973) th a t the e f f ic ie n c y o f paratEion removal from noncombustible used p e stic id e containers by solvent washing i s Independent o f the chemical p ro p e rtie s o f the s o lv e n ts, and t h is a ls o appears to be tru e f o r 2,4-D fo rm u la tio n s. The important fa c to r s to be considered are s o l u b i l i t y o f the p e s tic id e 1n the s o lv e n t, o r p a r t it io n between the inner su rfa c e o f the c o n tain e r and the s o lv e n t, and the presence o f adjuvants o r e m u ls ifie r s . The la t t e r i s p a r t ic u la r ly Im portant when water 1s used. Form ulations o f 2,4-D can most e f f i c i e n t l y be removed from a used noncombustible c o n ta in e r, f i r s t by fo u r water washes, follow ed by three washes with a water m iscib le organic solvent such as ethanol or ethanola c id depending upon the fo rm u la tio n 1n the co n tain e r. The aqueous and a lc o h o lic washes can be added to the spray tank and disposed with the spray mixture provided th at th is does not adversely a ffe c t the properties o f the spray liq u id or co n stitu te a flam m ability hazard. The decontaminated drums could then be disposed in a con ven tional manner or reclaim ed fo r p e stic id e use 1 f th e ir co n d ition warrants it . T h is procedure 1s a convenient and economical method f o r r e moval o f p o t e n t ia lly hazardous p e s tic id e s rem aining 1n emptied non com bustible metal co n ta in e rs, and 1s a p r a c t ic a l s o lu tio n f o r the disposal o f the containers as well as fo r the prevention o f environ mental p o llu t io n with unwanted chem icals. so 0G06820 7019 References ARCHER, T .E . and D.P.H. HSIEH: P e s tle . Sc1. 4, 69 (1973). HSIEH, D .P .H ., T.E. ARCHER, D.M. MUNNECKE and F .E . McGOWAN: . En viron. S c i. and Technol. 6, 826 (1972). KENNEDY, M .V ., B.J. STOJANOVIC and F.L. SHUMAN, JR .: J. Environ. Q u a lity 1, 63 (1972). SHUMAN, J R ., F . L . , B .J . STOJANOVIC and M.V. KENNEDY: J . Environ. Q u a lity 1 , 66 (1972). STOJANOVIC, B . J . , M.V. KENNEDY and F.L. SHUMAN, JR.: J. Environ. Q u a lity 1, 54 (1972). WOLVERTON, B .C ., R. RICHTER and S.M. LEFSTAD: Sc1. and Technol. 26, 7 (1970). ZWEIG, G ., D .L. GUTNICK, R. GULLI, T .E . ARCHER and H.T. HARTMANN: J. A gr. Food Chew. 12, 59 (1964). Si 0006821 7020 MW 1376532 Wb, WU, 7021 ' ('SOITT/WOCI 18.373 . L. Delvaux, J. Verstraete and A. Hautfenne, in collaboration with F. DeSart and G. Goffin*: Polychlorodibenzo-p-dioxins [Les Polychloro Dibenzo-p-Dioxines]. * -Catholic University at Louvain - I.A.G., Dekenstraat 2, B-3000 Louvain (Belgique} Received on May 24, 1974 t t 0001279 Translated from French by the Ralph McElroy Co., Custom Division 2102 Rio Grande, Austin, Texas 78705 USA 7022 Code: 101-5778-1 POLYCHLORODIBENZO-p-DIOXINS a O Summary Polychlorodibenzo-p-dioxins (chlorodioxins) are produced most frequently when chlorophenols are heated in an alkaline solution, particularly in the preparation of chlorophenol derivatives. Con sequently chlorodioxins may contaminate a great number of products which are widely used, such as certain pesticides for example. Occasionally, chlorodioxins are also synthesized from chlorophenols or their derivatives in situ, outside of industrial processes, when all the required conditions are accidentally met. It has been shown that chlorodioxins are the cause of chloracne as well as of chick edema which killed thousands of chickens in American farms. Furthermore, chlorodioxins are suspected to cause congenital malformations, possibly even in humans. It is thus undeniable that the toxic effects of chlorodioxins must be studied, and the importance of analytical detection methods becomes understandable. ; In this first part of our study we survey the research work which has led to the discovery of chlorodioxins and their proper ties. In the future, we shall publish a review of analytical methods and the results of our experiments. Introduction The designation and numbering of the atoms of substances pres ently called polychlorodibenzo-p-dioxins (or possibly chlorodioxins, an abbreviated and unspecified designation) have changed in the course of time. The paper by Gilman and Dietrich [1] seems to represent the starting point of our present designations, at least as far as the numbering of the atoms is concerned. "Chemical Abstracts" uses 7023 00017,30 Page 2 this numbering system. Some authors, generally predating Gilman DOW117056 and Dietrich, use other names for chlorodioxins or other numbering systems. In such cases, we kept the earlier designation and put it in quotation marks. To the degree that the indications of the authors permit an identification, we give the present designation in parentheses. In a table included as an appendix we list the formulas and the current designations for all the polychlorodibenzo- p-dioxins cited in this paper. Chlorodioxins had already been discovered a century ago as the products of the pyrolysis of phenochlorinated compounds or as secondary products which form during the synthesis of such chlori nated derivatives. Workers who handled chlorodioxins were afflic ted with chloracne even if they had handled only traces of the sub stance and they rapidly identified the responsive agent. Later the extremely acute toxicity of the chlorodioxins was again dis cussed when several thousand chickens died in American farms. The substances acquired the name "chick edema factor". Their im- portance has not decreased since. At the present time, chloro dioxins receive priority in toxicological research as a result of tiie harmful effect they seem to have on human heredity factors and oh the enzymatic system (in this respect chlorodioxins can be com pared to carcinogenic substances such as benzpyrene. The importance of the problem is increased by the fact that many products of the phytopharmaceutic industry (pesticides and defoliants of the chlor inated para-phenoxyacetic*acid type) contain chlorodioxins. Furthermore, there is the undeniable possibility that chlorodioxins can form and concentrate by pure chance. It seemed of interest to us to conduct, parallely to our laboratory work, a bibliographic research to determine the present level of scientific knowledge with respect to polychlorodibenzo-p-dioxins. This publication is the first part of our work and it consists of a bibliographic survey of chemical and toxicological studies on-chlorodioxins. In the future we shall publish a survey of the analytical.. methods and the results of our experimental work. 7024 0001231 DOW11757 Page 3 We c e r t a in ly r e a l i z e t h a t ou r paper i s a modest c o n tr ib u tio n to the study o f p o llu tin g a g e n ts, an enormous, world-wide p r o je c t. N e v e r th e le ss, th e work c o n ta in s a g r e a t number o f s a l i e n t t r a i t s which marked th e d isco v e ry o f th e in sid io u s and o fte n v io le n t e f f e c t s o f th e ch em ical s u b sta n c e s which we use'. May t h i s c o n t r i bution in crease the in te r e s t in to x ic o lo g ic a l research and in id e n tific a tio n methods. Chemical asp e c ts F ir st studies The f i r s t s tu d ie s d e a lin g w ith p olych lorod ib en zo-p-d ioxin s were p u b lish ed in th e l a s t cen tu ry . D en iv elle [2] has review ed these publication s. At th at time octachlorodibenzo-p-dioxin c a lle d "perch lorodioxydiphdnylfene" in French and "P erchlorphen ylen oxyd" in German was formed a s a re a c tio n product from chlorocyclohexadienones or s im ila r products heated to tem peratures between 210 and 3 0 0 . 2- c h lo r o 2 ,3-dichloro 2 ,7-dichloro a 2,8-dich loro 2 ,3 ,7-dichloro 1 ,2 ,3 ,4 -tetrach lo ro 1 ,3 ,6 ,8-tetrachloro 2 ,3 ,7 ,8 -tetrachloro 2,3 ,4 ,6 ,7 ,8 - .iexachloro 1 ,2 ,3 ,4 ,6 ,7 ,8 ,9-octachloro Principal formulas for syntehsized polychlorodibenzo-p-dioxins Page 4 DOWU 7058 In 1872 Merz and Weith [3] announced th a t th ey h eated "Kalium Per c h lo rp h e n y la te " C.O .O K OK o a a and observed the form ation o f "Perchlorophenyloxyd", a su b stan ce which was sp a r in g ly so lu b le in most con ven tion al so lv e n ts (eth y l a lc o h o l, eth y l e th e r, chloroform ) and whose w hite n eedle-shaped c r y s t a l s m elted a t 3 2 0 . The found t h i s compound's r e a c t i v i t y to be low and, although unable to determ ine i t s m olecular w eigh t, they a ttrib u te d a formula to i t : /N* vy The su b sta n c e was o c ta c h lo ro d ib e n z o -p -d io x in . ' S e v e r a l y e a r s l a t e r , in 1889, Hugounenq 14] h eated "h e x a c h lo rphdnol" to produce "perch lorodioxydiph enylene" whose p h y sic a l and ch em ical p r o p e r t ie s le d him to b e lie v e t h a t th e form ula sh ou ld be a m u ltip le o f C^Cl^O. He proposed th e fo llo w in g fo rm u la s: c ,o 4o One y e a r l a t e r he [5] produced th e same compound by s u l f u r i c a c id a c tio n on p en tach lo ran iso le c .a ,e cH , 7026 0001233 DOW11705!) according to the re ac tio n s: Page 5 c ,a ,o c n , .a .o - 2 0 4 ,a c,a ,o cH , c ,a 4o In 1894 Zincke and Schaura [6] succeeded in producing a C^jClgC^ compound by h e a tin g "h e x ach lo ro k e to d ih y d ro b en zo l" coa. or "heptachlorketotetrahydrobenzol" C.OOtH 1 Cm a^ a In th e f i r s t case the re a c tio n path w as: 2C6OCl 200~2il (CjOCU), +2a,; in the second case: 220* 2 C6OHCl,--(C6OCl)a+ 2 H Q + 2 a, In 1904/ th e f i r s t r e a c t io n was con firm ed by B i l t z [7] who produced what he c a lle d ."P e rc h lo rp h e n y le n o x y d ". In 1895 B a rra l [8] c l a r if ie d the stru c tu re o f "hexachloroph6nol C .0.0 o DOW117060 ' Page 6 using the known fact that heating of this molecule would produce perchlorodioxydiphenylene" whose formula, according to him, could be: A or p fo fT T Y Furtherm ore, in 1900, B a rra l and Johnson [9] d iscovered during the preparation o f "pentachlorophenol" OH aa C.C1.0H aa a by c h lo rin a tio n o f phenol the form ation o f a s i g n if ic a n t amount o f "perchlorodioxydiphenylene" in the presence o f excess o f c h lo ri n atin g su b stan ce s, e s p e c ia lly SbCl^ and SbCl^. T his was the re s u l t o f th e c o n v e rsio n o f th e "p e n tach lo ro p h e n o l" form ed. In some c a s e s th e co n v e rsio n reach ed 50%. F in a lly , in 1908, Zincke and Broeg [10] d iscovered th e forma t io n o f a compound w ith th e e m p ir ic a l form u la C^2Cl g 2 h e a t in g to 240 "aldehydoheptachlorketotetrahydrobenzol": lOL O CJ ,I T h is compound was id e n t ic a l to th e one Zincke and Schaum [6] had produced e a r l i e r by a method d e sc rib e d above. In these f i r s t stu d ies octachlorodibenzo-p-dioxin has u su ally been considered a re a c tio n by-product or even an im purity. 00012:5 . 7028 First syntheses Page 7 oX) It was not until the work done by Ueo [11] in 1941 that chlor- odioxins became the deliberate target of organic syntheses. Meanwhile, Ullman and Stein [12] as well as the chemists of CD C5 Bayer [13] had succeeded in 1910 in synthesizing "Diphenylendioxyd", the unchlorinated dibenzo-p-dioxin nucleus. Ueo [11] succeeded in producing "2,6-dichlorodiphenylenediox- yde" (2,7-dichlorodibenzo-p-dioxin) via catalytic reduction of "2 ,6- dinitrodi-phenylene dioxide" followed by a denitration with CuCl. In this manner he obtained a light yellow compound with a fu sion point of 207C and a dipolar moment of 0.62. The paper by Julia [14], in 1953, mentions the formation of the same "dichloro- 2,6-dibenzo-p-dioxine" (2,7-dichlorodibenzo-p-dioxin) by two dif ferent paths. In the first reaction path the formation takes place during certain condensations between p-bromoacetophenone and 2,4- dichlorophenol, but it also occurs in the absence of p-bromoaceto- phenone which led Julia to conclude that the dichlorophenol was the responsible substance. The second possible reaction occurs during the heating of sodium dichlorophenoxylate in the presence of copper powder. The path is as follows: . j O T X i' - X O O ' -- ' sodium 2,4-dichlorophen- "2,6-dichloro dibenzo-p-- oxylate dioxine" (2 ,7-dichloro dibenzo-p-dioxin) These works by Julia also make mention of a preparation of dibenzo-p-dioxin from o-chlorophenol. They are a sign of a new emphasis on studying the biological effects of chlorodioxins. In tests on weevils-and Drosophil a "dibenzo-p-dioxine, 2,6-dichloro" (2,7-dichlorodibenzo-p-dioxin) had no effects. Gilman and Dietrich's 1956 study [1] is strictly chemical, but it is nevertheless of great use. It is devoted to the propor tion of halogenated derivatives of dibenzo-p-dioxins and describes o c\i P O W i i 7 06 Page 8 the .synthesis procedures for 2-chlorodibenzo-p-dioxin and 2,7-dichlorodibenzo-p-dioxin. In addition, this study presents the numbering system for the dibenzo-p-dioxin nucleus mentioned in the introduction of this pa:per. The principle is diagramed below It has been used by "Chemical Abstracts" since Volume 74 (1971) *. 4a> in the "Subject Index", p. 1137S. In 1957 Sandermann et al. [15] published the results of their search for pentachlorophenol derivatives which could be used as wood protection agents. They showed that the pyrolysis at 300 of pentachlorophenol or potassium pentachlorophenate resulted in the formation of "Oktachlor-Diphenylendioxyd" (octachlorodibenzop-dioxin). This compound was of no interest to them and this par ticular synthesis was already known since it was at the origin of the discovery of chlorodioxins. The chlorination of dibenzo-pdioxin lead to the formation of "Tetrachlorodiphenylendioxyd" (tetrachlorodibenzo-p-dioxin) which was an effective agent against sjibstances which damage wood. But, after noticing that even traces oif this compound produced "mild chloracne", the authors decided that it should be researched further. Toxicological Aspects Chloracne This fortuitous medical observation by Sanderman and his con clusion to study which dealt basically with chemical issues had served for years as the starting point of medical contributions to the study of chlorodioxins. As soon as 1899, Herxheimer [16] had described the symptoms of chloroacne (appearance of pustules, comedones and lesions of the parenchyma of the liver) without knowing exactly the nature of the responsible agent. The symptomatology of the disease and the rco 7030 b O W U 70G Z Page 9 evolution of its etiology are presented in a work written by Bauer et al. [17] in 1961 after a study of a hundred cases of severe poisoning in workers who had been making or handling chlorophenols As the number of chloracne cases increased in industrialized Countries all over the world it became certain that neither the chlorine nor the chlorinated hydrocarbons alone were the responsi ble agents. Kimmig and Schultz's papers [19, 20], published in 1957, rep resent the last step toward the final recognition of the role whic the chlorodioxins played. The starting poin.t of their investiga tions was an observation of chloracne patients who had worked in plants where 2,4,5-trichlorophenol was manufactured by alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene at high pressures in the so-called "Druck-Phenolprozess" [21, 22].- The chloracne was not produced by the main reaction products in pure form, but by secon dary products. Since the authors were at first unable to identify these products they conducted chloracnogenic tests on rabbit ears and even on one of themselves (Schultz) using synthetic products which could be produced in possible secondary reactions of the "Druck-Phenolprozess". The active substances tested were: "trichlordibenzofuran", "tetrachlordibenzofuran" and "2,3,6,7-tetracixlor-dibenzodioxin" (2,3,7,8-tetrachlorodibenzo-p-dioxin) . It was not proven that the first two compounds are actually formed during the "Druck-Phenolprozess". 2,3,7,8-tetrachlorodibenzo-p-dioxin however corresponds to the reaction discovered by Julia [14]: the decomposition of two molecules of sodium trichlorophenoxylate molecules according to the path: This chlorodioxin which turned out to be the most active of the tested compounds was detected in the unrefined products of the "Druck-Phenolprozess". Chemists who had been working at the syn thesis of the compound were afflicted with severe chloracne. CO o CJ C_J rz> o 7031 Page 10 Kimmig and Schultz [20] suggested also that other chlorodioxins could form in reactions similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, for instance octachlorodibenzo-p-dioxin during the heating of sodium pentachlorophenoxylate. Thus a link between medical and chemical research was estab lished. The toxicological potency of chlorodioxins did not fail to impress. Indeed, three drops of 2,3,7,8-tetrachlorodibenzo-p-dioxin diluted in polyglycol with concentrations from 0.01 to 0.005% were sufficient to cause chloracne when applied to a rabbit's ear. Oral doses ranging from 0.05 to 0.1 mg/kg weight of body caused the death of test rabbits within one to two weeks as a result of necro sis of the liver [17]. In 1962 Jones and Krizek [23] suggested that 2,3,7,8-tetrachlor odibenzo-p-dioxin be used as a standard by which to measure various substances' power to cause chloracne. These authors used the preparation method of Tomita [24]; it consists in heating sodium 2 ,4,5-trichlorophenate. Most of the information concerning the pathological effects on humans have been collected on patients who worked in plants where chlorophenolated products were made. 9 out of 31 workers who had contracted chloracne were still being treated five years after the outbreak of the disease for acne, permanent weakness of the leg muscles, for vasovegetative fragility and especially for psychopathological trouble [17]. Recent bibliographical surveys on chloracne have been gathered by Poland and Smith [25], in 1971, and by Kimbrough [26], in 1972. The- chick edema factor As mentioned above 1957 was a year in which chemical and med ical researchers-had joined their efforts to confirm the role chlorodioxins played in the "chloracnogenesis". In the same year an avian disease killed several million farm chickens in the United. States. The autopsies of the chickens revealed in most cases ah DOW 1 1 7 0 6 4 to (1 co o o 7 G 32 Page 11 accumulation' of fluids in the pericardium and in the peritoneal cavity (for this reason the responsible agent was called chick edema factor) as well as swollen livers and damaged kidneys [27, 28]. The course of the studies which permitted the identification of the factor has been discussed in Nutrition Reviews in 1968 [29], by Brion in 1969 [30] and by Parsons in 1970 [31]. These studies, including the most recent ones will be surveyed again below. However, -the emphasis will lie on the discovery of the chlorodioxins rather than on the biological implications of their effects. The first authors [27, 28, 32] to mention the disease con cluded that the noninfectious affliction was caused by an extremely toxic chemical agent which was heat-resistant and remained stable over an extended period of time. This agent, it was concluded, must be contained in the fatty substances included in the feed ration of the chickens. Friedman et al. [33] isolated the factor from the nonsaponifiable content of tar by-products of the preparation of oleic and stearic acids from different manufacturers. Wootton and Alexander [34] isolated the factor from animal fit, and although their analyses were based on products of doubt ful purity, they correctly assumed that the toxic factor had an aromatic structure and that it had a high molecular weight. Brew et al. [35] separated the factor from the particularly high nonsaponifiable content of toxic animal fat. They found that the molecular weight was between 366 and 360. Separation chromato graphy and a study of the infrared and ultraviolet spectra led them to attribute a steroid structure to the factor. In 1960 Harman et al. [36] submitted the factor which they had isolated from the nonsaponifiable part of tallow to silane paper chromatography and to an analysis of its IR and UV spectra. But due to the small isolated quantities, they could not draw any conclusions. p>0w 117065 C"! c~ o Page-12 Ames and Swanson [37] found the toxic agent in samples of oleic acid and in some commercial glyceric acids. However, these were cases of accidental contamination. In 1961 Firestone et al. [38] published the results of their reserach for the chick edema factor in several fatty substances. After having analyzed a great number of samples they concluded that the toxic factor must have a cyclic structure. It is as tounding that they found the factor in distillates and distillation residues which had been produced during the preparation of commer cial fatty acids, in hydrogenated and nonhydrogenated inedible *L tallow, in cotton oil, in acid oils, in saponified palm oil which had been recovered from the production of tin plates and in numer ous samples of commercial oleic acid and stearic acid. Yartzoff et al. [39] isolated the factor from triolein which has toxic effects on monkeys and which causes chick edema. They obtained a crystalline substance which was quite similar to that discovered by Harman [36]. The spectra were reminiscent of highly substituted naphthalenes and the presence, of chlorine - a new and important factor - led to the assumption that the chick edema fac tor was a chlorinated naphthalene. Chlorinated naphthalenes, are ih fact known toxic agents, but they do not cause chick edema. Microcoulometric gas chromatography showed that the retention time of the isolated substances was 37 to 38 min as opposed to approxi mately 10 min for the chlorinated naphthalenes. Yartzoff et al. [39] formulated the hypothesis that the molecular weight of the chick edema factor should be higher than that of the chloronaphthalenes or that it would contain an additional substituent other than chlorine. For this reason the solubility and the polarity of the chick edema factor would be different from those of chloronaphthalene. In 1962, Wootton et al. [40] isolated three active compounds, two of which in their pure form, from the nonsaponifiable content of -commercial fat which had caused chick edema. They succeeded in crystallizing the two pure substances. The fusion temperatures were: 313.5-314.5 and 246-246.4. The molecular weight was 391 in both cases. The two compounds had 6 chlorine atoms per molecule O O O oen o Cl o o o 7 DQW11706? Page 13 in addition to an aromatic nucleus. Two years later Wootton and Courchene [41] concluded that the substances were 2 isomers with the empirical formula C14H^0Clg and they felt justified to asstime that they were hexachlorohexahydropheanthrenes. Meanwhile# in .1962, McCune et al. [42] had discovered by chance that chlorinated biphenyls are toxic for chickens and that they cause symptoms which are quite similar to those caused by the chick edema factor. Flick et al. confirmed this in their paper [43]. In 1963 Firestone et al. [44] designed a method for detecting the chick'edema factor in fatty substances and in fatty acids by microcoulometric gas chromatography. The use of gas chromatography with an electron-capture detec tor became later on a helpful tool in the research on the edema factor [45]. In 1965, this method made it possible for Flick et al. [46] to separate 8 compounds from a crystalline substance which had been extracted by the Yartzoff method from the nonsaponifiable content of.a toxic fat [39]. One of these compounds corresponded to one of the active substances isolated by Wootton and Courchene [41]. In 1966, Campbell and Friedman [47] detected 9 compounds by applying this chromatography method after a purification by thinlayer chromatography. The detection limit was 0.2 pph. The publications by the crystallographers Cantrell, Mabis and Webb added the final conclusion to the clarification of the nature of the chick edema factor. The results of their studies had been announced since 1967, but they were not published in extenso until 1969 [49] . According to this study the chick edema factor is "1,2,3,7,8,9-hexachlorodibenzo-p-dioxin" (2,3,4,6,7,8hexachlorodibenzodioxin) as shown by an analysis of x-ray diffrac tion patterns obtained with a monocrystal of one of the toxic fac tors isolated by Wootton and Courchene. The molecule is almost flat and has the formula: 0001232 7035 D O W 117068 Page 14 The authors also report that once this chlorodioxin was synthesized it was submitted to toxicity tests by Wootton. It indeed caused chick edema. In 1972, Kimbrough [26] published a bibliographic survey of the work which had been done on the chick edema factor. Questions of present interest The origin of the chlorodioxins The question of the origin of the chlorodioxins remained un answered.- In 1969 it gained renewed interest in the U.S. following another outbreak of chick edema. This time soapstocks, by-products from alkaline refining of a vegetable oil, were suspected to be the source of the toxic factor. Campbell and Firestone [50] summed up the situation at the International Symposion on the Identification and Measuring of Pollutants held in Ottawa in 1971. Their lecture was based on the most recent studies such as those by Higginbotham et al. [51] pub lished in 1968 and those by Metcalfe [52] which were published in 1972. It dealt with the presence of chlorodioxins in fatty sub stances, in pesticides which seem to be one of the most important - vectors and in the atmosphere in general. The formation of chlorodioxins seems indeed to occur in hightemperature alkaline reactions of chlorophenol according to a reaction path which has already been mentioned repeatedly. This formation occurs particularly in the preparation of chlorophenols or during their conversion into certain derivatives which are often pesticides or defoliants. Chlorodioxins which are preformed in this way contaminate the derivative and accompany it. But in situ reactions of this type occur in cases where all the prerequisites are met by accident, for example during the al kaline refining of vegetable oils which have been contaminated with chlorophenols or closely related compounds. In addition, Firestone et al. [53] have discovered that all chlorodioxins, with the exception of octachlorodibenzo-p-dioxin, remain and concentrate in the chicken tissues into which they have 0001233 ; 7036 6 .9 0 'TT MOo Page 15 been absorbed. This may possibly trigger a dangerous cumulative process in the food chain. However, Zitko [54] reported in 1972 that he failed to detect any chlorodioxins in marine animals, but his studies dealt with the population of a very small geographic area. In the case of chick edema it has been established that the toxic tallow which was responsible for the chick edema came from tanneries in which chlorophenols were used to conserve the skins. As far as the soapstocks are concerned, they had been con taminated by chlorodioxins contained in pesticides produced nearby. Campbell and Firestone [50] showed in their lecture that chlorodioxins are formed and accumulate during the manufacturing processes of certain pesticides which are used all over the world. In addition, these compounds can be produced accidentally in the food industry and they can contaminate animals which are consumed by humans. Their discussion was a message of warning recommending prudence and reflection. While the spectacular effects of the chick edema factor received the most attention, chlorodioxins had been identified as the responsible factor for chloracne. In spite of this the chemical operations which involve the use of chloro phenols or its derivatives had not been discontinued. In fact the use of the toxic agent had increased significantly and several severe cases of poisoning had occurred in chemical industry workers of the chemical industry who had been exposed to chlorophenolated products. In Vietnam, where defoliants have been used intensively since 1964, an increase in congenital malformations has been ob served. The works published until today can in some respect be divided into two categories. The first category contains works which deal only with the chemistry of dioxins and the second, which accounts for most of the papers written, covers research work which has been prompted by the extraordinary toxicity of the chlorodioxins. These works deal often with observations and studies of the many effects chlorodioxins can have, in addition to and sometimes to the ex clusion of chemical studies. As a result the distribution between ' chemical and toxicological studies becomes more and more tenuous. - Page 16 The links which unite the two disciplines became increasingly numerous and it is not always an easy task to classify the pub lications in one category or the other. The classificatory system we adopted to group papers published after the discovery of the role chlorodioxins play in the develop ment of chloracne does not account for the above-mentioned research work on the chick edema factor and it attempts to circumvent the difficulty by collecting the publications under three headings: 1) new chemical properties of chlorodioxins, 2) the toxicological importance of chlorodioxins, 3) chlorodioxins in the environment, using based on the general purport of the text. New chemical properties of chlorodioxins In 1959 Tomita et al. [24] published preparation methods for several polyhalogenated dibenzo-p-dioxins such as 2,7-dichloro-, 2,3,7,8-tetrachloro- and octachlorodibenzo-p-dioxin. In 1960, Denivclle [2] described preparation methods for octachloro- and octabromodibenzo-p-dioxin. In 1961, Kulka [55] studied the conversion of pentahalophenols into octohalogenodibenzo-p-dioxin using halogens or halogenated compounds. He suggested that a pentahalophenoxy rad ical forms during the reaction and acts as a reactive intermediate. By this method he had succeeded in synthesizing octachlorodibenzo- p-dioxin. More recently, in 1970, Darsow and Schnell [56] of the Bayer A.G. developed the synthesis of 2,3-dichloro- and 1,2,3,4-tetrachlorodibenzo-p-dioxin to improve the fire-resistance of polymers and polyesters. Their invention was patented. In 1971, Buu-Hol et al. [57] published a preparation method for 2,3,7,8 tetrachlorodibenzo-p-dioxin in its pure form. This distinguishes their work from that of Tomita et al. [24]. They also list the physical properties of this chlorodioxin and the re sults of mass spectrography of tetrachlorodibenzo-p-dioxins. These last mentioned results were full of mistakes due to catalytic -de compositions in the spectrometer. They were corrected shortly after 158]. JI Dow-117071 I Page 17 In 1972, Pohland and Yang [59] published a systematic study of the preparation and characterization of chlorodibenzo-p-dioxins which contain from 1 to 8 chlorine atoms. They review the various preparation techniques, discussing the yield and the ease of the -reactions as well as the degree of product purity obtained. They list chemical and physical properties and mention the stability, the coloring reactions, the IR, UV, NMR and phosphorescence spec tra. They define a relation between the wavelength of the phos phorescence and the life of the triplet on the one hand, and on the other, the number of chlorine atoms and their position on the nucleus of the dibenzo-p-dioxin. Boer et al. [60-62] published in 1972 the results of their preparations and studies of crystals and molecules of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,7-dichlorodibenzo-p-dioxin. According to them 2,3,7,8-tetrachlorodibenzo-p-dioxin can be prepared in a reaction of chlorine and a heated chloroform solution of dibenzo-p-dioxin or 2,7-dichlordibenzo-p-dioxin in the presence of a catalytic amount of ferric iodide and iodine. It crystallizes in the triclinic system and the molecule is almost planar [60, 61]. The 2,7-dichlorodibenzo-p-dioxin is prepared by heating sodium 2,4-dichlorophenate in the presence of copper as catalyst for several hours at temper atures from 180 to 200C. The crystal belongs to the triclinic system and the molecule can be considered planar [60, 62]. Neuman et al. [63] continued this research showing that the octachlorodibenzo--p-dioxin crystal belongs to the roonoclinic system. With the exception of slight deviations the molecule is planar. Finally, in 1972, Boer et al. [64] published a study dealing with 2 ,8-dichlorodibenzo-p-dioxin which crystallizes' in the or thorhombic system. It is prepared by pyrolysis of the potassium salt of 5-chloro-2-(2,4-dichlorophenoxy) phenol. The molecule is not entirely planar. j ^^ i.: ju 7039 D O W 117072 Page 18 The toxicological aspects of chlorodioxins The publication of works focusing on a better understanding of the toxic properties of chlorodioxins goes back to 1969-1970 when congenital malformations were observed in animals which had ' ` the herbicides 2,4,5 T and its esters and to SilIt is very likely that many of these research projects were triggered by the controversial and alarming reports observers brought back from Vietnam where they had witnessed the damages caused by massive use of defoliants for strategical pur poses [6S, 66, 69, 70, 71]. At that time, the American Government^ proceeded to cutbacks in the use of 2,4,5T [65, 66, 68, 72], The program of the first research steps has been retraced by various authors in publications which report on the danger of the contaminants. The work by Golberg [73] generally speaking deals with food contamination. Clegg [72] looks at the question of toxicity to the embryo, especially the toxic effects of 2,4,5T. Somers and Smith [74] deal with environmental contaminants focusing parti cularly on chlorodioxins. It was in fact a chlorodioxin, specifically 2,3,7,8-tetrachlorodibenzo-p-dioxin which had caused malformations in new-born animals who had come into contact with 2,4,5 T and Silvex-2 . The toxins in these herbicides which are derivatives of phenochlorinated products were impurities which had probably formed during the preparation according to a mechanism which has already been mentioned. It did not take long before this chlorodioxin was suspected to be responsible for the anomalies. Dow, the main producer of 2,4,5 T had thought so from the beginning on [66, 68]. Nevertheless, the very first works dealt with the action of the herbicides as manufactured, partially since no pure chlgrodioxins were available, and the hypothesis of a combined action was not excluded [67, 68, 75, 76]. Sparschu et al. [77] studied the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the rat. Courtney and Moore [76] also studied D O W 117073 Page 19 the -effect of 2,3,7,S-tetrachlorodibenzo-p-dioxin on mice and rats. A work by Collins and Williams [78] dealt with the teratogenic effect of 2,4,5 T and 2,4 D (2,4-dichlorophenoxy acetic acid) on the hamster. Diether and Imke [79] studied the embryotoxic effect of 2,4, 5 T and 2,3,7,8-tetrachlorodibenzo-p-dioxin on mice. The import of the conclusions depends on the type of test animal, on the ingested doses and on whether the experiments have been conducted.with pure or technical 2,4,5 T or with pure 2,3,7,8tetrachlorodibenzo-p-dioxin. The research work is still in progress. In 1971, Firestone et al. [53] studied the distribution of hexa-, hepta- and octachlorodioxins in the tissue of chickens. In 1972, Cunningham and Williams [80] published a study dealing with the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the growth rate and the synthesis of lipids and proteins in rats. Williams et al. [81] studied the distribution and excretion of octachlorodibenzo-p-dioxin in the rat. The effect of chlorodioxins on human embryos is less well, known, but the congenital malformations observed on-Vietnamese who lived in zones which had been the target of massive defoliant raids cannot be ignored [65, 69, 71]. Several authors report these observations [65, 66, 71, 72]. The danger of chlorodioxin contaminations produced a 1971 publication which dealt with health protection problems of workers in the 2,4 D and 2,4,5 T manufacturing industry [25] and in a 1972 article which reviewed the toxic effects of chlorinated hydrocar bons and their derivatives, including chiorodibenzo-p-dioxins and chlorodibenzofurans [26]. The dangerous biological effects cited above, the chloracne and related diseases, the chick edema, the embryotoxic, feticide and teratogenic effects (possibly affecting humans) together with the cumulative effect contribute to make of 2 ,3,7,8-tetrachlorodi- .. benzo-p-dioxin on of the most dangerous toxins known. This has r ft L* o l J i !0 Page 20 been pointed out by /Buu-Hoi et al. [82], According to these authors, the extreme toxicity is due to the fact that this chlorodioxin causes malfunctioning of the enzymatic system affecting the liver, the thymus, the heart and the lungs [82-84], They made the same observation in experiments on rats and mice. This led them to attribute carcinomimetric properties to 2,3,7,8tetrachlorodibenzo-p-dioxin similar to those of benzo[a]pyrene and hepatocarcinogenic substances. Chlorodioxins in the environment The extraordinary toxicity of certain chlorodioxins has given rise to a trend in research which studies their identification, their photochemical reactions and their stability in the atmosphere. In this respect, the article by Buu-Hoi et al. [57] is a very valuable contribution. The authors describe ways of detecting 2,3,7,8-tetrachlorodibenzo-p-dioxin in the pyrolysis products of defoliants (2,4,5 T and butyl 2,4,5T the base of the defol iant "Grange") or in a substance which is impregnated with these defoliants. According to the same authors the pyrolysis of such defoliants at approximately 500 results in a significant produc tion of 2,3,7,8-tetrachlorodibenzo-p-dioxin. The yields are re ported to reach 5 to 15%. Very recently, in 1972, Saint-Ruf [85] has shown that the pyrolysis of sodium 2,4,5T at 500 during 5 hours also resulted in the formation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. For this reason he condemned the use of these products. It is to be noted that the experimental conditions of the pyrolyses mentioned above occur during the combustion of- plants. The humidity facilitates the hydrolysis and the alkalinity of the ashes facilitates the transformation of chlorophenolated products into chlorodioxins. Thus populations whose domestic combustibles are vegetal material and wood are in serious danger if the burnt material was exposed to defoliants such as 2,4,5 T. This applies to the population of Vietnam (cf. the alarming reports of the first observers) and also to the populations of most developing countries 'PloijjMoa C5 C c\i o o 7042 Page 21 where the use of organic chlorinated products is becoming more and more widespread for fighting parasites and for increasing the agri cultural output. Finally, this shows that there is a real risk that chlorodioxins may appear at every point of the globe, since chlorophenolated products play a role in the production of paper and paint as well as in the production of pesticides [68]. In 1971, Crosby et al. [86] studied the reactions of 2,7dichlorodibenzo-p-dioxin, 2,3,7,8-tetrachloro- and octachlorodibenzo-p-dioxin under the action of light. An irradiation with sun light or with a lamp which puts out an equivalent radiation produces identical results. The photolysis observed in alcohol solutions depends on the number of chlorine atoms. It is weak in the case of octachlorodibenzo-p-dioxin and faster in the case of 2,7-dichlorodibenzo-p-dioxin. No photolysis occurs in an aqueous medium or if the chlorodioxins impregnate a sandy soil directly without sol vents. The influence of a hydrogen donor organic solvent seems decisive. Since such donors are part of the leaf surface of plants, the photolysis of chlorodioxins could take place there, while it is unlikely that it could occur n the open ground, in water or on stone. According to Plimmer and Klingebiel [87] there is a possibil ity that chlorodibenzo-p-dioxins form in water by photoxidation of 2,4-dichlorophenol due to the influence of riboflavin. The fact that they have discovered dimers under such conditions, the most frequent dimer.was 4,6-dichloro-2-(2,4-dichlorophenoxy)-phenyl, and the fact that chlorodioxins can be formed by the closing of a ring of a 2-phenoxyphenol derivative speak in favor of their hypothesis. However, the authors were not able to verify their theory in their experiments, but according to them the photoly sis of chlorodioxins which contain few chlorine atoms is so rapid that their presence could not have been detected. Another field of investigation opened by the study of the toxic effects of chlorodioxins is the study of their stability in the soil and their absorbtion into plants. DOW 117075 o o ro o o o 7043 Page 22 OVH17076 Experiments carried out by Isensee and Jones [88] on oats and soybeans show that these plants accumulate neither 2,7-dichloronor 2,3,7, 8-tetrachlorodibenzo-p-dioxin. The chlorodioxins intro duced were either dissolved in the food, or the soil of the cul ture was impregnated with them, or they were applied onto the leaves, or dissolved in an aqueous solution with a surfactant, Tween 80. No migration of the chlorodioxins which had been applied to the leaves toward the root system was observed. Helling [89] studied the mobility of 2,7-dichloro- and 2,3, 7,8-tetrachlorodibenzo-p-dioxin in soil. Tests conducted by thinlayer chromatography on clay plates revealed no mobility. Most likely this is due to the insolubility of chlorodioxins in water. However, it is possible that chlorodioxins are carried by the wind. The studies by Kearney et al. [90] showed, in 1972, that the stability of 2,3,7,8-tetrachlorodibenzo-p-dioxin is such that one year after its application 60% of it is still present. According to these authors it seems that no microorganisms can transform 2,4-dichlorophenol or 2 ,4,5-trichlorophenol into 2 ,7-dichloroor into 2,3,7,8-tetrachlorodibenzo-p-dioxin. Research on chlorodioxins is obviously a subject of current interest. In 1972, Jensen and Reuberg [91] found chlorodioxins, especially octachlorodibenzo-p-dioxin, in pentachlorophenols used as fungicides for the preservation of wood. They called the ethers of 2-hydroxydiphenyl "predioxins" and the ethers of 3-hydroxyor 4-hydroxydiphenyls "isopredioxins" and detected some "octachloroisopredioxin". (ether of 2hydroxydiphenyl ) OH (ether of 3-hydroxy- dipheny1 ) C~3 ro o o o (ether of 4-hydroxydiphenyl) DOW117077 Page 23 According to these authors the late discovery of predioxins was a result of the compounds' instability. In 1972, Woolson et al. [92] published the results of their analyses of chlorophenol derived pesticides. They detected various chlorodioxins at different concentration loads. Finally, in 1973, Edmunds et al. [93] published the results of their analyses of 2,4,5 T and the alkyl esters of this acid. They were trying to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin. The highest concentration found was 28.3 ppm. o I 24 Page References 1 H. Gilman and J.J. Dietrich, J. Am. Chem. Soc., 79 (1957) 1439. 2 L. Dnivelle, R. Fort cl P. Van Hai, Bull. Soc. Chim., 5 lin e (1960) 1538. 3 V. Merz et W. Weith, Chem. 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Sesque, M.C. Azum-Gelade et G. Saint-Ruf, Natur $0 84 swNe.inPsss.cehnBasucfuht-eaHnfot,ei5'n,9,P(5.1-9H9(7.12C9)h71a27n)4h1.,7G3.. Sesque, M.C. Azum-Gelade et G. Saint-Ruf, Naturwis 85 G. Saint-Ruf, Naturwissenschaften, 59 (1972) 648. 86 D.G. Crosby, A.S. Wong, J.R . Plimmer et E.A. Woolson, Science, 173 (1971)748. 87 J.R . Plimmer et U.I. Klingebicl, Science, 174 (1971) 407. 88 A.R. Isensee et G.E. Jones, J. Agr. Food Chem., 19 (1971 ) 1210. 89 C.S. Helling, Soil Sei. Soc.-Am. Proc., 35 (1971)737. 90 P.C. Kearney, E.A. Woolson et C.P. Ellington, Environ. ScL Technol., 6 (1972) 1017. 91 S. Jensen et L. Renberg, Ambio, 1 (1972) 62. 92 E. Woolson, R. Thoms* et P. Ensor, J . Agr. Chem., 20 (1972) 351. 93 J.W. Edmunds, D.E. Lee et Christine M .L. Nickels, Pestic. ScL, 4 (1973) 101. v.;../vV.Vs V .w;-.< V-it*c.. t-:rt,>. Vm r L *. i/... _*A''*e.i-ri_- r N --T Or V-- o 3. co C5 ro o o o -1 TV I 1)1 \\*-l DOW L E P E T I T - U S A U6MI * TCO P. J. Gehring CHAMCl 411-0000110 DC 2/9/76 9 tWft K HUMltN NBK 113 "-002372 THE FATE OF 2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) . .FOLLOWING ORAL ADMINISTRATION TO MAN M.W. Sauerhoff, W.H. Braun,-,;'*1 reported' b y G . E . - 3J.au and P.J. Gehring\ 1" cmeckeo b y : J.E. LeEBeau ------------------------- I. j i c e p c :j --------------------------------------------- "Nl IT The pharmacokinetic profile of 2,4-D is defined in man. Five male human0 volunteers ingested a single dose of 5 mg/kg 2,4-D without detectable clinical effects. Concentrations of 2,4-D were determined in plasma in three of five subjects and in urine in all subjects at intervals after ingestion. The elimination of 2,4-D from plasma in all subjects occurred by an apparent first order rate process with an average half-life (t..,) of 11.6 hr. .All subjects excreted 2,4-D from the body in the urine with an^average t./2 or 17.7 hr. 2,4-D was excreted mainly as 2,4-D (82.3%) with smaller amounts excreted as a 2,4-D conjugate (12.8%). Essentially all of the 2,4-D is absorbed from the gastrointestinal tract in man. There is no evidence that clearance of 2,4-D from the plasma or excretion from the body involve non linear kinetics. D0W 0628036 DISTRIBUTION ATTACHED 0003338 0W0628037 INTRODUCTION 2.4- Dichlorophenoxyacetic acid (2,'4-D) is a chlorophenoxy acid herbicide. Following oral administration to rats, pigs, calves and chickens, 2,4-D is readily absorbed, dis tributed and eliminated in the urine (Erne, 1966). The half-life values for the apparent first order clearance of 2.4- D from plasma were reported to be 3 hr for rats, 8.hr for calves and chickens, and 12 hr for pigs. The fate of 2.4- D following ingestion by man has not been reported. Human male volunteers ingested 5 mg/kg 2,4-D. Samples of blood and urine collected at intervals after dosing were analyzed for 2,4-D. This report presents results of the fate of 2,4-D following oral administration to five human male volunteers. 7050 0003339 D0W0628038 -2- MATERIALS AND METHODS Subject. Five male human volunteers age 29 to 40, weighing 70 to 90 kg, participated in the study. The medical history of each individual was reviewed and a physical examination conducted prior to the study. Included in the examination were blood pressure, pulse rate, pulmonary function, electro cardiogram, chest x-ray, hemoglobin, packed cell volume, red and white blood cell counts including differential, and sedimentation rate, serum concentration of total calcium, cholesterol, triglycerides, glucose, inorganic phosphate, albumin, total protein, uric acid, bilirubin, glutamicoxaloacetic transaminase, lactic dehydrogenase, alkaline phosphatase activities, as well as urinary protein and sugar. Dosage and Sample Collection for Analysis. Five subjects ingested 5 mg/kg analytical grade 2,4-D (sample AGR 30653C, Dow Chemical Co.). Two subjects ingested the free acid of 2,4-D as a slurry in milk and three ingested it directly from a weighing paper followed by a few swallows of water. All subjects ingested 2,4-D between 8:00 and 8:05 AM. Subjects were not fasted prior to ingestion of the dose. Blood samples, 10 ml, were collected from three subjects in tubes containing 4 mg of potassium oxalate at 1, 4, 8 , 12, V .7051 0003340 D0W0628039 >i -3- 24, 36, 48, 72, 96, 120 and 144 hr after ingestion. Fol lowing collection, the whole blood samples were centrifuged, the plasma separated and placed in tared vials and frozen for subsequent analysis of 2,4-D. All the urine voided by each subject during successive 12-hr intervals post-dosing was collected and the volume recprded. The urine collected during each 12-hr interval was thoroughl mixed and aliquots were frozen until subsequently analyzed for 2,4-D. Samples of plasma and urine were analyzed by gas chromatography-mass spectrometry. Urine and plasma samples (5 ml) were acidified with 1 ml of 1.0N HC1 and extracted twice with 5 ml of diethyl ether. To determine whether hydro lyzable conjugates of 2,4-D may be present, additional samples of urine (5 ml) from the 0-12, 12-24 and 24-36 hr collection intervals were acidified with 1 ml of concen trated hydrochloric acid and heated at 80C for 1 hour. Subsequently these samples were extracted twice with diethyl ether and analyzed for 2,4-D together with non-hydrolyzed urine samples. The ether extracts were methylated with diazomethane, evaporated to dryness and redissolved in 0.5 ml of hexane. A 2 ul aliquot of the hexane was injected 7052 0003341 U0W628C40 -4- into a LKB 9000 gas chromatograph - mass spectrometer (GCMS). The GC column was glass, 6 ft long x 2.0 mm (I.D.), packed with 10% OV-1 on Chromosorb W 80/100 mesh. The m/e 234, 236, and 238 peaks were monitored for sample quanti tation. Recorded peaks were symmetrical; therefore, peak heights, not peak areas, were used for sample quantitation. Standards of 2,4-D prepared in plasma and urine were stored with the experimental samples. No decay of 2,4-D was observed in my of the samples or standards during the storage period. 7053 0003342 OW62804 t -5- R ESU LTS No untoward effects associated with ingestion of 5 mg/kg 2,4-D were detected in any of the subjects. The concentration of 2,4-D in plasma, ug/g, is shown in Figure 1 as a function of time following adminis tration. The concentrations found in the plasma of each subject are presented. The data suggest that subjects 2 and 3 can be characterized by a one-compartment pharma cokinetic model while subject 1 may be characterized by a one or two-compartment model. Pharmacokinetic parameters for the three subjects were calculated using a non-linear parameter estimation program. Parameters for a two-compart ment model were calculated for subject 1. They are reported in Table 1 with their linearized standard devia tions. Comparing the parameter estimates reveals the importance of characterizing each subject individually. For example, the absorption constant (k ) for subjects 2 and 3 are significantly different at the 95% confidence level as are the elimination rate constants Ke . A lack of fit test was performed for all compartmental models based on the known analytical error. Each model was shown to be inadequate to describe the data at the 95% confidence level. 7054 0003343 D0W0628O42 -6The urinary excretion of 2,4-D expressed as a percent of the dose excreted during each collection interval is presented in Table 2 and represented graphically in Figure 2. Subjects were modeled individually using a one-compartment linear pharmacokinetic model. The pharmacokinetic parameters determined using a non-linear parameter estimation program are presented in Table 3. The half-life of elimination } ranged from 10.2 hr to 28.5 hr for subjects 2 and 4, respectively. Recovery of 2,4-D ranged from 87.6% to 106.3% of the administered dose. 7055 0003344 -7- DISCSSION These data demonstrate that orally administered 2,4-D is well absorbed in man and excreted in urine as 2,4-D. A fraction of administered 2,4-D is also excreted in urine as a conjugate. The small fraction of 2,4-D not recovered from some individuals may represent lack of absorption, excretion of 2,4-D and/or conjugation in feces, formation of a metabo lite which was not detected or a combination thereof. C3 CD O CD ro CO o After ingestion of 5 mg/kg 2,4-D plasma concentrations Co increased and reached a peak of approximately 25 yg/ml at 4 hr. Clearance of 2,4-D from the plasma of subjects 2 and 3, as well as the urine from all 5 subjects, was monophasic. The plasma data for subject 1 exhibited a biphasic clearance phenomenon. The explanation for this is unclear. It is interesting to note that this biDhasic clearance in subject 1 is accompanied by the delayed peak in the urine excretion data. The slow clearance phase parameter for the plasma data of subject 1 is only slightly slower than the monophasic clearance for subjects 2 and 3. This suggests that the over all clearance characteristics of subject 1, 2 and 3 will not be markedly different. Statistical tests performed to determine the suitability of the compartmental models to characterize the 2,4-D plasma 7056 0003345 -8 - UUW0628044 d a ta b ased on a combined a n a l y t ic a l and sam pling e r r o r o f 10.2%, in d ic a te s th a t th e re was a s ig n if ic a n t lac k o f f i t between the m odels and the exp erim en tal d a ta . A s ig n if ic a n t la c k o f f i t im p lie s e it h e r th a t a more complex model ( i . e . , one having more param eters such a s a two-compartment model) i s req u ired or th a t the d ata are s u ffic ie n t ly p re c ise to r e v e a l d e v ia tio n s from the lin e a r approxim ations in h eren t to compartment m odels. In th is c a se , the l a t t e r situ a tio n i s o ccu rrin g . I f , fo r exam ple, the experim ental e rro r had been 15% r a t h e r t h a t 10.2% , no s i g n i f i c a n t la c k o f f i t would have been d e te c te d . T h is means th a t the d iffe r e n c e s between d ata p o in ts and th e lin e drawn through th e plasm a d a ta in F ig u re 1 a re r e a l d iff e r e n c e s and not random s c a t t e r . M oreover, th ese d iffe r e n c e s re p re se n t r e a l d ep artu res from the assumed f i r s t order rate processes. The volume o f d is t r i b u t io n was c a lc u la t e d f o r a l l s u b je c t s ( r e f e r t o T ab le 1 ) . The f o r s u b je c t s 2 and 3 were 238 and 294 m l/k g , r e s p e c t iv e l y . The fo r su bject 1, and Vj were 83 and 218 m l/k g . T h is in d ic a t e s 2,4-D i s n o t exten sively d istrib u ted to tissu e s. Excretion data are presented fo r fiv e su b ject in gestin g 5 mg/kg 2,4-D . I t i s not re aso n ab le to pool the in d iv id u a l su b je c t data because of the d ifferen ce in the pattern of 7057 0003346 W0B28C45 e x c r e t io n . One o f the f i v e s u b je c t s d id n o t form th e con ju g a te o f 2,4-D w hile oth er su b je c ts excreted between 4.8 to 27.1% o f th e ad m in iste re d dose a s 2,4-D c o n ju g a te . M odeling o f th e d a ta was b ased on the e x c r e tio n o f 2,4-D and not the con jugate. The d a ta p re se n te d in T ab le 2 and F ig u re 2 show s i g n i f i c a n t between su b je c t v a r ia tio n in the ex cre tio n o f 2,4-D from th e body. The slo w e r e x c r e tio n o f 2,4-D in s u b je c t s 4 and 5 may be a t t r i b u t a b l e to f a c t o r s in c lu d in g in c r e a s e d p r o te in bin d in g, decreased c a p a b ility o f the kidney to ex crete 2,4-D or d iffe r e n c e s in re a b so rp tio n from the tu b u lar f i l t r a t e . Data presented in th is rep o rt in d icate f i r s t order excre tio n o f 2,4-D . The p o o led d a ta h a l f - l i f e v a lu e s f o r c le a r a n c e o f 2,4-D from plasm a i s 1 1 .6 h r . I t may be u s e f u l to compar t h i s d a ta w ith p o o led d a t a from human 2 ,4 ,5 - T p h a rm a co k in e tic s t u d ie s . Gehring e t a l . , 1973 have shown th e :plasm a c le a r a n c e tl/2 2 ,4 ,5 - T was 2 3 .1 h r. D ecreasin g th e number o f c h lo rin e s from 3 to 2 on th e phenoxy rin g a p p are n tly in c r e a s e s the r a te o f clearan ce o f th ese chlorophenoxy a c id s from the body. In con clu sion , 2,4-D i s w ell absorbed follow in g o ra l adm inis t r a t i o n to man and e lim in a te d a s unchanged 2,4-D (82.3%) and 0003347 -1 0 - UOW0628046 2,4-D co n ju g ate (12.8%) in u r in e . C learan ce o f 2,4-D from plasm a and e lim in a tio n from the body occur by apparen t f i r s t o r d e r r a t e p r o c e s s e s . No e v id e n c e o f n o n - lin e a r k i n e t i c s were observed fo llo w in g the 5 mg/kg o r a l dose o f 2,4-D . WRITTEN BY: Pharm acokinetic/M etabolism Dow L e p e t i t , U .S .A . W. H. Braun S r . Research Chemist Pharmacokine t i c/M etabolism Dow L e p e t i t , U .S .A . G. E . B lau , Ph.D. C entral Research 1707 B uilding CHECKED BY: P. J . G e h rin g ,/.V .M ., Ph.D. D irecto r, Toxicology Res. Lab. H ealth and Environm ental R esearch Group Leader P h arm acokin etic/M etabolism Group Dow L e p e t i t , U .S .A . 7059 0003348 . U0W0628047 -11- REFERENCES E rne, K. 1966. D istrib u tio n and e lim in atio n o f c h lo rin ate d phenoxyacetic a c id s in an im als. Acta Vet Scand., 7:240-256 Boxenbaum, H. 6 . , R iegelm an , S . , and E l a s h o f f , R. M. 1974. S t a t i s t ic a l E stim ations in Pharm acokinetics. J . Pharm acokinetics Biopharm. , 2 (2 ):123-148. G ehring, P. J . , Kramer, C. G ., Schwetz, B. A ., Rose, J . Q ., Rowe, V. K. The F a te o f 2 , 4 ,5 -T ric h lo ro p h e n o x y a c e tic A cid F o llo w in g O ral A d m in istra tio n to Man. T o x ic . A p p l. Pharmacol. (26), 352-361 (1973). 7060 0003349 TABLE 1 I Rate Constants and Half-Life Values for Absorption and Clearance of 2,4-D From Plasma in Three Male Subjects Given a Single Oral Dose of 5 mg/kg 2,4-D Subject 1 _______ Absorption________ Rate Constant Half-Life ________ Elimination_________ Rate Constant Half-Life Model k^(hr *) tl/2 ^ r^ ^ ( h r ' 1) fcl / 2 ^ - Two Compartment 0.165+0.055 4.20H.40 k^-0.013 hr_1l0.009 k a-0.048 hr~110.007 21 t k -0.145 hr" 10.049 ac -0.163 hr-1Al0.048 4.2511.25 3C -0.043 hr-1l0.012 16.214.31 Distribution Vj(ml/kg) V 1d-83.6128.4 V.2e- 218115.7 2 One Compartment 0.20210.047 3.4310.80 0.06210.002 11.210.36 283142 3 One Compartment 0.41510.082 1.6710.33 0.09510.003 7.2910.23 294139 ak ' ^12 ^^r8t tder distribution constants 12 between the plasma and slow exchange compartments b.|Q First order rate constant for overall elimination e from the central compartment a and 3 hybrid rate constants for a and 3 portions of plasma clearance curve Volume of central compartment 0 Volume of slow exchange compartment. 0003350 8 >08290M0(] <1 O 05 TABLE 2 Percent of 2,4-D (Free and Conjugate) Excreted In Urine During Successive Time Intervals Following a Single Oral Dose of 5 mg/kg 2,4-D I SUBJECT Free l Conjugate 0-12 14.3 2.50 12-24 13.9 8.80 24-36 31.6 10.8 36-48 15.9 48-60 5.07 60-72 1.10 72-84 0.86 84-96 0.93 96-108 0.27 108-120 0.11 120-132 0.08 132-144 0.07 TOTAL 22.1 Free 2 Conjugate 29.9 - 33.9 - 9.18 - 8.78 3.91 1.10 0.49 0.22 0.06 0.05 0.03 0.01 87.6 0.00 Free 3 Conjugate 40.6 10.5 17.3 11.8 8.64 4.76 2.33 0.68 0.31 0.16 0.12 0.02 0.04 0.01 0.01 70.1 27.1 Free 4 Conjugate 21.0 - 17.1 3.10 14.1 1.70 11.0 8.98 7.15 4.04 2.97 86.3 4.8 Free 5 Conjugate 24.7 5.70 13.8 3.00 10.7 1.10 10.0 8.99 7.90 6.03 1.25 83.4 9.8 <? 6 * 0 8 2 9 0 M 0 n O cn> to CN GO 0003351 Sublect 1 2 3 4 5 TABLE 3 I Rate Constants and Half-Life Values for the Elimination of 2,4-D in the Urine of Individuals Following a Single Oral Dose of 5 mg/kg Estimated Fraction of Dose Eliminated Absorption Constant t 1/2 (hr) Elimination Constant (2) Kt (hr-1) Absorption Ke(hr *) 71.4118.9 0.10810.052 6.4213.09 0.056610.0060 84.3114.5 47.8115.5 0.19210.098 - 3.6111.84 1 0.067810.0023 0.063810.0029 96.514.67 88.4118.5 0.52214.67 1.3311.44 1 0.024410.0020 0.029010.0065 t 1/2 (hr) Elimination 12.2+1.3 10.2+0.44 10.910.50 28.412.38 23.915.42 1 ^Meaningful estimate of absorption constant could not be obtained from these two subjects. SCCOO oso8s90Ainn si O CD CO Figure 1 Figure 2 LEGEND Plasma levels of 2,4-D in man following a single oral dose of 5 mg/kg 2,4-D. Excretion of 2,4-D in urine expressed as percent of dose excreted during successive time intervals following a single oral dose of 5 mg/kg 2,4-D. 00W062805 1 7064 0003353 FIGURE 1 7065 000335 2,4 D Plasma Levels (/ ZsoZQOMOn o o. Percent of Dose p 0003355 O Cl c S0890M0fl y ) cxvJ O < o ^ r O ^ ~ , 2020 Dow Center E. H. Blair K . 0. Groves 2030 Dow Center H. L. Gordon T. R. Torkelson 2040 Dow Center S . Brown DISTRIBUTION LIST 607 Building R. D. Olson H. C. Scharnweber CRI - (6) Zionsville C . F . Thompson R. Fike C. W. Hinman Freeport C. V. Johnston D. J. Kilian T . Pullin R. L. Daniel 9008 Building C. A. Goring R. E. Hefner M. L. Leng D. D. McCollister W. R. Mullison A. E. Shober L. -.Southwi'ck 9001 Building Davidson Dishburger M. E. Getzendaner D. J. Jensen A. J. Watson 1803 Building W. H. Braun P. J. Gehring J. F. Heeg *H. R. Hoyle/R. Langner *C. G. Humiston *G. C. Jersey * R. J. Kociba J. E. LeBeau M. J. McKenna * F . J. Murray *J. M. Norris *K. J. Olson * J . F . Quast L. W. Rampy J. C. Ramsey * D . E . Rapp *V . K . Rowe M. W. Sauerhoff *B. A. Schwetz * L. K. Scory M. G. Swank P. G. Watanabe M. A. Wolf J. D. Young 0003356 WS \\6 T-age 20 M ay 5, 1976 PESTICIDE CHEMICAL NEWS JEFFERSON SERVICE COMPANY'S PESTICIDE SALES IN N .C . HALTED BY STATE BOARD Jefferson Service Com pany, Baltim ore, M d. , h as been barred from se llin g p e stic id e s for an indefin ite period in North C a ro lin a. The S ta te 's P e sticid e Board and the firm have agreed that: i O o-- P e stic id e s a le s by the company are prohibited in the S ta te "until such time a s the Board may reconsider application for registratio n . -- The firm w ill refund d is s a t is fie d farm ers' m oney. $ 5 ,0 0 0 . -- Farmers are to initiate refund re q u e sts. Total refund not to exceed ^xx- *o -- The company w ill tak e back unopened con tain ers of p e s tic id e s from farm ers who have not paid their invoices and clo se the accoun ts. o o h-* -- Unopened p e stic id e s can be returned to the company freight co llect. The agreem ent, the Board noted, w as in lie u of a sch ed u led hearing for the firm. William B. B uffaloe, Secretary, North Carolina P esticid e Board, in a letter sent la s t week -o the P resident o f Jefferso n Serv ice Com pany, ad v ise d "th at should evid en ce constituting a breach of this agreem ent, or a failure on the com pany's part to abide by all of the terms and conditions agreed to, the Board w ill, upon reapplication by the company for registration , consider all allegation s of violation s, and evidence pertaining thereto, which occurred prior to the date of this agreem ent" (See Feb. 18, Page 5 and April 21, Page 13). POSSIBLE TOXIC EFFECTS OF TCDD IN HUMANS LISTED BY DIOXIN GROUP A summary of the January 21 m eeting of the Toxicology Working Group o f the Environ mental Protection A gency's dioxin p ro je ct (See D e c . 24, P age 14) included a l i s t of 13 p o s sib le to x ic e ffe c ts o f TCDD in humans agreed to by the group: "C hloracne, porphyria, lethargy, anore-xia, liver alteratio n s, diarrhea, h eadache, alo p ecia, thrombocytopenia, lo ss of libid o, neuropathy, lowered hemoglobin, and hyperpigmentation. " According the the summary, "chloracne and increased excretion of porphyrins are probably the m ost common findings a sso c ia te d with TCDD overexposure . . . The group agreed that the other item s may be a sso c ia te d with TCDD but that the relationsh ip between cau se and effect is ill-d e fin e d ." n -vn \ EPA l a s t w eek d ecid ed that future m eetin gs of the dioxin p ro ject and it s w orking group w ill be open to the p r e ss and p u b lic, an ag en cy o ffic ia l s ta te d . EPA o f f ic ia ls a ls o # decided that a s soon a s research data and resu lts came into the project m anager, they 7089 .1 M a'/ 5, 976 Page 21 PESTICIDE CHEMICAL "NEWS w ijj be given to p ro ject p articip a n ts and to the EPA' s O ffice of G eneral C o u n sel, i the o fficial sa id . O At its January 21 m eeting, the group d is c u s s e d certain animal study re su lts which Q appeared to in d icate few m ajor ad v erse e ffe c ts of dioxin in the te s t an im als. - 171002 The D irector of the N ational ducer for T ox icolo gical R esearch (NCTR), Dr. Cranmer, indicated la s t December that in his opinion, the teratological risk to humans of 2 , 4 , 5-T with le s s than 0 .1 p .p .m . dioxin was n il. The meeting summary included stu d ies relevant to exposure to low le v e ls of TCDD. The summary noted that the NCTR "h a s studied the teratogenic potencies of both technical (< ^ 0 ,0 5 p .p .m . dioxin) and purified ( ^ .0 .0 0 5 p .p .m . dioxin) 2 , 4 , 5-T in various strains of m ice. There w e re n o sta tistic a lly sign ifican t differences for cleft palate incidence, emb ry oleih ality and fetal w eigh t. The ab se n ce of d iffe re n ce s in p o ten cies between the two 2 , 4 , 5-T preparations su g g e sts that dioxin contaminants are not the teratogenic agents in such preparations or that the increased adverse effects due to dioxins are insignificant in relation to effects produced by 2 , 4 , 5-T at d o ses stu d ied ." Dr. B. A. Schw etz, Dow Chem ical Company, review ed teratology stu d ies on TCDD in rats and m ice, the sum m ary, dated M ay 3, noted. } It seated that the "co n sen su s of the stu dies (reviewed by Dr. Schwetz) is that TCDD is fero to xic but not terato gen ic in r a t s . " The summary continued; "A study w as undertaken to determine a non-teratogenic d o se le v e l of TCDD in m ice , The n on -terato gen ic d o se level of TCDD given to CF-1 m ice by gavage on days 6-15 o f gestation w as found to be 0 .1 ug TC D D /kg/day . . . R esults to date indicate an interference with the reproduc tive capacity at a dose level of 0 .1 u g/k g/d ay ." Dr. R. J . K ociba, Dow C h em ical, USA, the summary noted, presented resu lts of a 13 week oral to x icity stu dy in r a t s . Tne summary sa id that from th is study " ' the data in d ica te that no d isce rn ib le il l e ffe c ts occurred in ra ts g iv en 0 .0 1 or 0 .0 0 1 ug T C D D /kg 5 d a y s/w e e k for 13 w e e k s .' He also d isc u sse d a presently ongoing 2 year chronic toxicity study of TCDD in rats . . . As of January 1976, (the 16th month of the study) a d o se of 0 .1 u g /k g /d ay of TCDD affected body w eight, m ortality, urinary porphyrins and hem atological parameters but no e ffe c ts were noted at a dose of 0 .0 1 u g /k g /d a y ." Dr. Renate Kimbrough, Communicable D ise a se C enter, according to the meeting sum mary noted that "TCDD has been involved in a large horse poisoning episode in t M isso u ri. This incident w as a c a se of g ro ss accid en tal overexposure to TCDD and is not indicative of the e ffe ct of TCDD a s it may e x ist elsew here in the environm ent." 0 7 0i 00017 3 < J .'ay 3, 1976 PESTICIDE CHEMICAL NEWS The group heard a report on re su lts of a rat stu d y , the summary s a i d , which sh o v ed that " sin g le oral d o se s of 0 ,1 ,3 ,1 0 and 30 u g /k g TCDD did not enhance porphyrin excretio n , in dicatin g porphyria o ccu rs oniy after chronic dosing v.-hereas 16 w eeks after sin g le d o se s of 3, 10 and 30 u g /k g , enzyme activ ity w as sig n ific a n tly in c r e a s e d ." R esults of another rat study d iscu sse d in the summary indicated "that TCDD approaches O stead y sta te le v e ls in the body within 13 w e e k s. The rate constan t defining the ap o proach to steady state le v e ls is independent of the dose level of TCDD over the dose range of 0 .0 1 to 1 .0 ug T C D D /k g /d ay . This m eans that the re su lts found in the 90 days feeding study may be indicative of the resu lts of the 2 year feeding stu d y ." 171803 The summary noted that the group rejected ad v ise d te rato lo g ical stu d ie s of TCDD in prim ates. There may be additional follow up on industrial workers exposed to TCDD, the summary indicated. It stated that "D r. Cranmer at NCTR is requesting the data for the l a s t 25-30 years on cleft p alate and cleft lip in the State of A rkan sas. This will be compared with the u se h istories of 2 , 4 , 5-T to see if there is any correlation between cleft p alate and high 2 , 4 , 5-T u sa g e ." Dr. John Sp au ldin g, Agriculture D epartm ent, presen ted a brief report to the group on "The Im pact of Dioxin R esid u es to A gricu ltu re." The summary said " if and when EPA d ecid es to set an action le v e l, the methodology for testing and econom ic co st of en forcement must be considered." Dr. Spaulding said: "The increased production expen ses and any changs in the prices at the market p lace w ill be the respon sib ility of the agency estab lish in g the tolerances or ad visin g on action le v e ls . In those in stan ces where the Department b eliev es the projected benefits do not outweigh the proven risk s, they will utilize the admin istrativ e and leg al options av ailab le under the law to a ssu re the American public is fully informed concerning the b a s is of the d ecisio n and the potential c o n se q u e n c e s." H is report sta te d that the USDA "w ill not encourage the u se of any to x ic m aterial in a manner that is known to create an unacceptable level of a toxicant in the food supply at the time of m arketing. " D r. Cranm er, NCTR D irector, gave h is opinion of the relativ e risk s to man and the environment of 2 , 4 , 5-T with le s s than 0 .1 p .p .m . dioxin in a D ec. 17, 1975 memorandum to EPA's dioxin project m anager. "I p erson ally feel that at ju diciou s u se le v e ls , the teratological risk is n il, " his memorandum a sse r te d . 030173S Page 23 M l y 5. .'976 PESTICIDE CHEMICAL NEWS o He added that " 2 ,4 ,5 - T is teratogenic and must be dealt with a s su ch . NCTR O decided that research onTCDD w as im portant, but not in the context of 2 ,4 ,5 - T and. therefore, we Junked plans for su c h ." His memorandum said : "1 b eliev e that d io xin s in the environment are important, but I feel that p esticid es w ill contribute little , if control over the quality of production is m aintained. The problem lie s not with the p e stic id e , but with industrial chem icals escapin g into the environm ent." fee O C ** Dr, Cranmer stated that "At NCTR we proved, I b e lie v e , that the currently av ailab le 2 4 ,5 -T is terato gen ic in sev eral d o se -re sp o n se stu d ie s and th at the e ffe ct is not due to a g en eralized n o n -sp e c ific e ffe ct on the m aternal an im al, and that TCDD p lay s no discernible ro le ." PBB CONTAMINATION OF LIVESTOCK BRINGS PRESSURE FOR TOXIC SUBSTANCES BILL Toxic Substances Control legislatio n w as called for by Bobby D. Crim, Speaker of the M ichigan H ouse of R epresentatives in testim ony la s t week before the House Agriculture C om m ittee's Subcom mittee on C on servation and Credit (See April 21, Page 15). The Subcommittee is reviewing the current situation regarding the toxic contamination of liv e sto ck . M ost d iscu ssio n centered on the problems resulting from the mixing o f polybrom inated biph enyls (PBB) in anim al feed in 1973 in M ichigan and the need for some form o f fin an cial re lie f for farm ers. Crim told the Congressm en that "in order to know what we are dealing w ith, we needed rigorous pre-market testing of toxic substan ces before the contamination occurred. Information on the effects of toxic su b stan ces is our most powerful weapon in com batin g th e m ." He c a lle d for thorough te stin g of the e ffe c ts on humans and an im als o f a ll to x ic su b sta n c e s bein g marketed where such information is now la c k in g . A lso n eed ed, he s a id , are .minimum stan d ard s fo rlab elin g and packagin g o f to x ic su b sta n c e s. a s well a s p recise records of where and when each container of toxic material w as distributed. Stringent requirements for the labeling of toxic substan ces are needed throughout the country, Crim a sse r te d . Many toxic su bstan ces are potentially lethal to humans and anim als and they should be m anufactured, p ack aged , distributed and marketed with appropriate knowledge and cau tion , Crim told the C on gressm en. Little or no le g isla tio n now e x is t s which tak es th ese into account, he added. He warned the Congressm en that sim ilar incidents could take p lace in other sta te s and that is why it is v ital for C on gress to approve le g isla tio n to d e a l with situ a tio n s such a s the M ichigan PBBs befo re they happen. The N ational Milk Producers Federation called on the H ouse Subcommittee to include a p ro v isio n , sim ilar to one alread y in the S e n a te - p a s s e d v e rsio n , to provide for loan s to processors whose products are destroyed a s the result of such toxic ch em icals. i\q j 5.105 'i MN05 3305 f 3,7,8-Tetrachloroclibenzo-p-dioxin environmental] contaiminant and molecular probe'42 / t*> \ i-- , ALAN POLAND' AND ANDREW KENDE Deftartmnih o f Pharmnrolngy m id Toxicology m id o f Chemistry, I'uiirruly ofRochester, Rochester, Xnr York J-/6-I2 nXv'S' -jru it1i J \ j T hc chlorinated dibcnzo-/z-dioxins and dihenzofurans arc among the most potent toxins and terato gens known. The present concern, about these compounds and the po tential human health hazard they pose is a result o f a growing aware ness o f their extraordinary toxicity uid their inadvertent dispersion in he environment as trace contamilants o f important commercial hem : -als. O ver the past two tecs . hundreds o f chemists, iologtsts, and clinicians have made nporiaru contributions to our unerstanding of this problem. Several xcellent symposia (3, 38), reviews !6), and government-sponsored reorts (13. 52, 53) have appeared on ie subject. T h e present paper akes no attempt at a comprehcne review; rather it presents the nrk d o n e in o u r lab orato ry , efneed by a brief .review o f pertint investigations o f others. DRMATION AND OCCURRENCE OF THE CHLORINATED ABSTRACT T Jic chlorinated dilenzo-/i-dioxin. an d d ib ritzofiiran i are form ed a s trace con tam i nants d arin g the synthesis o f a num ber o f com m ercially im portant chem icals. T h e p ro to ty p e co m p o u n d o f this g r o u p . 2 .3 ,7 .it-ictrachliirodilicn/.o-p-dioxin (T C D D ). is o n e o f the m ost potent low m olecular weight toxins an d terato gen s know n, an d its inadvertent d is u n io n in the environm ent has caused concern about the potential h a z a rd to h u m an h ealth . In stu d y in g the bioch em ical e ffe c ts o f T C D D . it w as fo u n d to be extraordinarily potent as an inducer o f two hepatic enzym es: / ) o-aininnlevulinic acid synth etase, the initial an d rate-lim iting enzym e in hem e synthesis. a n d 2 ) ary Ih ydm earbon hydroxylase, a cytochrom e P -450-m ediated m icrosom al m onooxygenase. A m ong a series o f h alo g cn aicd dihen/.tv/>-dii>\ins there is an excellent correlation betw een their toxic potency and their potency, as inducers o f these two enzym es. T h e adm inistra tion o f polycyclic arom atic h yd rocarbon s le.g., 3-m eth ylch olantbrene (M C)) to i r e tain inbred strains o f mice induces aryl hydrocarbon hydroxylase, while otltcr in bred strain s fail to resp o n d : an d the trait o f a r sl hvdrocarlxw i respo n siven ess is inherited as an au tosom al dom inant. T C D D . about 30.0 0 0 tim es as potent as M C. induces all strain s w hether rcsjm nsive or n onrcs|xinsive to M C: how ever, the res|>onsive strains arc m ore sensitive ( D y > -l a 10"* m nle/kgi to T C D D than are the nenresponsivc strain s (ED W2 I x 1 0 '* m ole/kg). T h e resu lts su ggest iluu the m utation in the nonresponsive strain s results in a ligand binding site Ian in duction receptor) that has a d im in ish ed affinity fo r MC. an d T C D D . T h e correlation am o n g the h alogeiin tcd dibenzo-pdioxins. between their potency as toxins and their potency as inducers o f aryl hvdrocarbon h ydroxylase, is discussed in relation ship to various p ro p o sed m ech an ism s n f toxicity. -- P o lan d , A ., an d A. K endo. 2.3,7 .S-T etraeh lorodibcn zo-f-dioxin : environm ental c o n ta m in a n t a n d m o le c u la r p r o b e . Frdnntttm Pent. 3 5 : 2 4 (1 1 - 2 4 1 1 . 197fi. 05 O hChi Co CO 1l (TCDD), is formed by the condensa formed as unwanted contaminants tion o f two trichlorophcnatc mole in the production o f the polychlori cules (31). nated biphenyls (PCBs) that arc f \ DIBENZO-P-DIOXiNS AND D13ENZ0FURANS e chlorinated dihenzo-^-dioxins formed from the condensation o f orthoehlorophenaics. T h e parb r dibenzo-/>-dioxin formed deds on the ehltirnphcnol.s pres- In the (nmnitTcial synthesis of herbicide 2.-1..VT. ilte first step lie conversion o f I.LM.o-tetraroh -tie to 2,-l.:Vtrichlnrou le ,. it*. 1). At hi^h Irniprr;< an unwanted nutatuiuaui. '.S-iet ra liloi otlilu-n/o-^ -tlioxin In the commercial synthesis of pentachlorophenol. a widely used wood preservative and hactcriostat. octa-, hepta- and hcxachlorodibenzo-/-dioxin are formed (23, -13). Chlorinated dihenzofurans are 1Komi dir Xyin|xiMuin mi tin H m t t h /tiwiWni lii'ln itil ht I.ir. ttfntfu-tiSil t>!\ jnr- vuiii'il by tin' Ami'in.iii Simiimv Ini I'b.iiiu.i- inliigr ami Kx|WMimMii.il I in i.i| uni * ,n ilu- .VHti Aaiiii.il Mri-iing ol ilu- tiili i.iiimi >( Ami'liiaii Vminn's I,,i l'\|,iMiiiuMil.il Untlugs. Allaiun (ir, N.J., A|>n! 17. I`.)7.*>. 1 S u p p o r t e d by N a tio n a l In stifu ir o f F.n- vironm enial H ealth Sciences G ian t l-W H -hS- IMft)li.M-02. J Rcujikmii n f ( a r e r f D cvrlnjim cm A w ard. I KIM-K.S00IM7-0I. AlJirwimioiis: 2.1.5-T. 2.t.ri-lrilili*rijihenoxyamu- avid: 2.1-0. 2. l-dirii!nmjilirinxvacriir mid: TGOO, 2.3.7..H-ieir;uhlurtidil>rii/i-/i-dioxin: MG. 3-iiumIu Ii Imlaitiliietu': AI.A ivntlui.iw. A-;iiiiiimlrvuliiii< arid svmlielasr: l . l K f iiii'iiu tellial di.sr-- tin- iIiim* ill a drug tlul wIhmi ailiimuMrird kills 50' 1 llir animals; K.Ov.. iiu-aii 'Ill'll iUim*--Ihr il*isr 1 a ding dial wliru aditiinisirird i-lims ball thr in.ixiin.il ii'njkiiiw 1lull iluu ilmg ian |iihiIiii r: and.4/r.aiiniialM iiyrlow.ilium ir\|nHisivriiess. 7074 0003440 FEDERATION PPOCCEOINCS V O L 35. NO. t! OCTOBFn mzr  CI OM* WYV F i g u r e 1. O rig in id l . ' J i ' I r l r . Iil<mwl il K / f >- synilirin/>-rlioxiit in lilt' nl 2.4.5-lrtililiiro- p lic n a tr. In ilir com incrci.il M iiiIicms nl tlic lirrliiridv 2 . 1-VT. the Ins step is the conver sion o l 1.2. IJj-ic ira c h liiriilK 'ii/i'iie In 1'. L i - iriclilornpliciitiie. At rlex.u rri icm|N*raturrs I n iritliloioplirnatem olecules can con den se to y ield T C D D (i Ihiwii a i b o tto m ) in trace am ounts. widely used as plasticizers and heat exchange fluid (54, 62). Historical background N O ur current understanding o f the chlorinated dibcnzo-/>-dioxins and dibenzofurans arose from several seemingly unrelated lines o f investi gation, each stimulated by a poison ing epidemic or concern about the pot ai for poisoning. Chick edemafactor In the southeastern and central United States in 1957 there were several o utbreaks in d om estic poultry o f a disease characterized by hydropericardium and gross kid ney and liver dam age which pro duced substantial economic losses '37). T he etiologic agent 'was traced :o a toxic impurity sporadically present in the nonsaponifiable fracion o f fats added to commercial ccd. Intensive investigations o f this chick edema factor" eventually led o the isolation and identification o f .2,3.7.8.9 - hexachlorodibenzo - p lioxin (7, 12, 66). The probable ori;in o f this compound was as a conaininum formed in the synthesis o f lilorophcnnls that were used as ueservatives for hide curing operainns (11). T h e contaminated fat tripped from the hides was then Id as a by-product and used in iticken feed. CJdornrnc > C.eriiiain in 1951 there was an iithicak o f severe acne am ong the em p lo y ees o f a chem ical plant TA BLE t. Comparative toxicity of manufut tin i:ig 2.4.5-T. In a pioneer selected poisons ing investigation. Kimtnig and Scholl/ and cn-workcrs found that acne could be produced in rabbiis MolniiLir MiitimiuN in4'vk^ by direct dermal application o f die technical grade tricldorophenol which was contam inated with a number o f chlorinated dibcnzo-/idioxins and diben/ofurans (27. 5K). T he most potent acnegenic com pound was TCDD. llnllllillllx to sili A Tt-iunns loxin llip liih e i toxin TCIU Saxillixitl TtlnxIiH oxiii B llfo to x ilt* y x in 1 1 x n r* 7.2 x M>322 372 319 757 3 .3 x 0 1 x in 4 .2 x ill 11 3.1 x M r* 2.1 x M r* 2.5 x M l5.2 x in ; In the late I{150`s and early 1960`s C u rare 696 7.2 x M l- several outbreaks o f cltloracne were Sirv rh iiiitc 331 1.5 x M l- reported am ong workers in 2.4.5-T M useanti* 210 5.2 x M l- factories in Germ any. France, and D ii'o |> ru |n in u o ro - the United States: manufacturing phosphatc 184 1.6 x M r* conditions were altered to minimize S o d iu m cy an ide 49 2.0 x M l- the formation o f TCD D and the con taminant concentrations were moni tored during production and in the final product. Ttic*c data v r rt compilcrt bv Mmhrr rt ai. :**> and ih r ra lim in d irair nU rctalitr m xiriu. Ii vhnulil b r nuird that wir t ro n ip jiiu c d illrrm l y ic tir'. rnnte* o f idm iniuraiH in. u iry t.il u m . and in ,nr n v ih r m ran leilu l dittr raiT.cr than ihr mimmum T he most recent poisoning at tributed to TCD D occurred in 1971 in eastern Missouri, where waste oil Irthai d o v . Exce|x ttlirrr nmrd. adminitiraimn v u bv d ir intraprriiunral route in nnre. * L D ^ nral adminiurauon in Ihr guinca pig. * IniravahMit injectiun in the cat. DOW 670434 contaminated with TCD D was sprayed for dust control in riding areas (8). This mishap was responsible for one human illness and for the death o f 48 horses and numerous small animals. dioxin (TCDD). TCD D is a nearly planar tricyclic aromatic molecule with twofold svmmetry. It is remark able for its lack o f reactive functional groups and its chemical stabilitv. Vietnam war and X C I study A mixture o f the -butyl esters of 2,4-D and 2,4.5-T (Agent Orange) was used extensively in the Vietnam war from 1962 to 1969. Besides the ecological effect o f this defoliation, which has been the subject o f sev eral reports (13, 52, 53). application o f Agent O range contaminated the environment with large amounts o f TCD D , which was present as a trace impurity. A final stimulus was a National Cancer Institute study on the car cinogenicity and teratogenicity pro duced by a number o f insecticides and herbicides in which a 2.4.5-T sam ple containing a significant am ount o f T C D D was shown to he teratogenic (40). Subsequent work has shown both the herbicide anti TCD D to be teratogenic and TCDD to be one o f the most potent terato gens known (9. 60). T C D D is an extremely lipophilic molecule, notably insoluble in water (6 x 10- , m) and only sparingly solu ble in most organic solvents.'1 2) The most striking fact about TC D D is its potency: lethal potenev. and potency as a teratogen and as an acnegen. In guinea pigs the oral LD S0 is approxim ately 1 tg/kg (3 x 10~ molc/kg) (59). TCDD is per haps the most potent toxin o f low molecular weight known (Table 1). Almost all the other poisons listed in Table 1 act rapidly and kill by im pairing the physiologic function o f the nervous system. TCD D . in con trast, is a "cellular |>oison." In the rat. death appears to result front hepatic necrosis, and ensues weeks after a single dose. ?) T h ere are large differences in species susceptibility to TCDD (Ta ble 2). T h e d og is almost 1.00(1 times less sensitive than the guinea pig. T h e cause o f death is tint ertaiu in all rases. In the Kit. rabbit, and Chemical and biological properties mouse, hepatic necrosis seems to In* I ) Chemistry. T h e prototype com the cause o f death. However, in the pound o f the dihe!i/o-/;-dio\ius is 2.3.7,S lelrat blot odibett/o - /> * llmv (.Th'IiinmI Cntii|Miiy. 7075 \SS HfALIH DlSASTrnS nEl/VTEOTO f NVinONMCNTAL CHF.MICAI.S 24205$ * 4 4 t T a B L E 2. M ean leth al d o s e o f TCDD in various spec-es \j*nits 1Sc* i.n.-.nK'kK K.it (itlilMSl JHg Hog C h irk rm liryii M .ilr Krm .de M ;ilc triiK ilr M ixed M ixed M ixed M ixed it XVI >130 0.3-2.0 -- 30 1.000* <! T h e tnWc w toinpiU'tl laii'rlv fr<mi <hu nlMainnl In Sihttet/ rt itl. i.VJ), T ( il) I) oralli in all iaM> exrrfK llu* d ik L rm ltryi (111). in whirl <asc it a rtm iiiN c rd |umticraHy inn ilic }lk tar. " KMiiiiaird. guinea pig, one o f the most sensi tive species, liver dam age is mini mal. 4) TCD D produces a ntimhereof specific toxic actions in dilTercm sp ecies: teratogen esis occurs in several species, edem a in the neo natal chick, and dermal contact pro duces acne in rabbits and man. Am ong the chlorinated dibenzo-/d '^xins studied, those that produce t o f these responses scein to evoke all o f them; and changes in the number or position of thechlorine atom s produce large changes in potency (59). 5 ) TCDD is a remarkably stable com pound in biological systems. T h e whole body half-life o f TCDD in rats is about 24 days (42, 56). and despite a rather thorough search, no metabolites have been found over a 20-day period following in vivo ad ministration. nor after incubation with liver microsomes in vitro (61). T h is suggests that if T C D D is metabolized, the rate is very slow. In contrast, the parent ring, dibenzop-dioxin, is rapidly converted to polar metalM>Iiles by hepatic microsom es (15, C l). TCD D is also quite stable in soil, and microbial degra dation is limited (25). 2,4-D and 2,4,5-T factory study T he starting point o f our work was a stud} of !llcil>crg et ai. (I) in 1961 whf >wd that o f 29 workers in a 2.1... s' factory, all o f whom had industriallv-:u qiiitcd acne, ! 1 had por phyrinuria and .several had overt rliuieal porphyria cutanea tarda. K :i)I> was known to Ik- a coulatni- uanl in tlx* 2. !..*>-1 and was know n in Ik.* the causative agent ol the acne: however. Uu* cause o f the puiplivria was unclear. Porphyria cutanea tarda is an ar<|iurctl delect o f he patic |M>rpltM'in ineialtolisiii charac terized hy an overproduction o f |>orpliyriiis hy the liver, increased urinary excretion o f porphyrins, me chanical fragility and photosensi tivity o f the skin (blistering in areas exjmsed to sun light). Jiyperpigmentation. and hirsutism (33). We rest tidied the same factory 5 years later and found the acne was reduced in severity and there was no evidence o f porphyria among the employees (50). T h e fact that this syndrome abated following meas ures to reduce the formation o f TCD I) and minimize exposure o f the employees to this contaminant suggested that TCD D might have been the causative agent o f the por phyria outbreak originally reported by Blciberg et at. (4). Laboratory investigations Porphyria A variety o f xenobiotics produce hepatic porphyria in animals, and most if not all have in common the ability to induce the initial and ratelimiting enzyme in the heme biosyn thesis pathway, 6-aminolevulinic acid synthetase' (ALA synthetase). T o test whether T C D D was porphyrigenic, we adm inistered the com pound dissolved in /t-dioxanc to chick embryos (17-day gestation); 48 hours later the embryos were killed and ALA synthetase activity was assayed in their .livers (44). As seen in Fig. 2.4. TCD D produced a dose-related increase in enzyme ac tivity. As little as 4.66 x ]0~,s mole/ egg (1.5 ng) produced a doubling o f enzyme activity and at the highest level tested. 1.55 x 10 '" mole/egg. there was a 35-fold induction. TCDD is more potent. hy at least three or ders o f magnitude, lliau any other inducer o f A1.A synthetase yet rc|>orted. and unlike many other porphvrigenic chemicals, the duration o f induction is prolonged (most likclv a result o f the long biological half-life of TCDD). We next screened a series o f 15 halogeiiatetl diheti/o-//-dioxin.s lot their ability to im bue A IA synthe tase (15). As seen in Figure *2/1, the live isomers tb.it weir imlmers had two roiiumm propel tics: /) Jrubtgen atoms occupy at least three o f the /bur lateral ring positions (2. 3. 7. ami S). and 2 ) there is at least one tree, nonhalogeiiated ring position. T he inactive com|mumls were tested at 200 to 400 times the molar con centration if TCD D that produced a significant response. Woods ((>5) reported that adminis tration o f TCD D had no effect on Al.A synthetase in rats. mice, and guinea pigs. Goldstein et al. (17) found that while TCDD produced only a modest induction (twofold) o f ALA synthetase in C571iI7()J mice, a gross accumulation o f hepatic por phyrins (K'Cttrred (30). Thus, it seems likely that TCD D vyts the causative agent o f the porphyria cutanea tarda reported in workers in a fac tory manufacturing 2.4.5-T. Aryl hydrocarbon hydroxylase N um erous investigators have noted that many, but not all. com pounds that induce ALA synthetase also induce microsomal mixed func tion oxygenase activity in the liver (also called " drug metabolizing enzymes"). (For a more extensive discussion o f the relationship be tween heme synthesis and drug metabolism, sec refs 11 and 18). H epatic m icrosom al oxygenase activity is produced by a membranebound enzyme complex requiring XADPH and molecular oxygen, which is responsible for the oxida tive metabolism o f many foreign compounds. Reconstitution experiments o f microsomal oxygenase activities in vitro have shown that the enzyme complex consists o f three com ponents: a phospholipid, a flqjaiprotein (XADPH-cytochrom e P-450 re ductase). and cytochrome P-450 (32). T h e cytochrome is the enzyme active site am i determines the reactivity o f the microsomal oxygenase towards any drug. T h ere is a large body o f evidence to suggest there are al least two types o f cytochrome I*--130. ami recent evidence reveals four or live dif ferent hands o f the cytochrome on gj*l electrophoresis (1. (54). The a d ministration o f polyeyclic hwlrocaibons selectively increases one type o f cyiixhrom c l'-4.">0. termed 1*,-13*. which is spent ally distinit (31). .yjv* a ^ social cd with the in* reuse o f this yrrzz U 'le&OW M ( 20 "I DOSE TCDD per egg (moles) ALA SYNTHETASE ACTIVITY (mpmoles ALA/gmliver/hour) , -11, 20 60 noUi*tO /egg . u ......!.. >77 rnqi, "> 100 U0 i ..A 180 220 26i0 ,30i0 a3 ci P " o l O T ""! Cl 3 ? a h m 4 aisO^T^ F i g u r e 2. Iiich irtiw i o f A I-A -syn tlietaso by lia lo g n u iic tl <iil><.-ii/<>-/j-<Jinxitis. C h ir k r n e g g s o f S17 d a v s 1 fo ia W H i w ere injcrM fl with 2 filtil so lv e m (c 'liiiin l) o r sn lv cm (o iitu in in g v a rio u s concern ration* o f T ( 'J)D o r m lier (iiliciiso-/j-dioxin*. T h e em bryos were killed 4K h our* later a n d assayed fo r lie|M tk' A I.A -sy n ilirtJsc a r m tv (H ). .4) log tio v -r e *|> o ii*c relation ship o f T C I ) 1 ) : rat'll |kiint r e p r e se n t* llir m ean s. M am lard e r r o r o l th re e tie lo u r g r o u p * o f |m olerl liv er*. ID stru rtu ie -a n iv iiy telatiouvliip; eat h liar represents a single g ro u p o f 3 to 3 {m oled liv e r* except th e u n iu jetletl control.* (no. il) an d the solveul-injected to n tio ls too. * 12). chrome I' ,-150 are a limited number til microsomal oxygenase aeiiviiies. ih 'inst si udirti heing aryl liydro- ca it hvdioxvlnse (-11). Because TC D D resembles ihe |tolyt velie h v t lr iH 'a r iio n s , we clitise to study it* effect on arvl hydrocarbon hydrox ylase activity. As seen in Fig. 3.-/,TCI)l) produces a dose-relaied iiirlttr tion til' aryl hydrotarhon hydroxylase in chick einhryo liver ( 15). At the lowest dose tesietl (1.55 X 10 15 mnle/egg (0.5 ng)) (here is a nearlv iwnlnlri ini tru st- in eti/yine activity, and maximum in duction is produced liy 1.55 X 10 jn o Jc /e g g <50 ttgl. Wv-screened 15 iiafogenau-d dilM *n/o-/>-<lioxins lo t their ability to induce arvl IiviIi .hui- Ijoii hydroxylase activity- at three tlose levels. -I.7. 17 and -170 x ]() 11 mule/ egg. The structure-activity relation ship is the same as that seen with in duction o f AI.A synthetase (Fig. 'Mi): I ) coni|x>nnds that are |M*iom in ducers (induciionoi'aryl hydrocarbon hydroxylase at -1.7 or 47 x 10-11 molc/egg) have halogen atoms at at least three o f the four lateral ring positions and 2 ) they have at least one nonhalogenatcd ring position. The potency o f the halogcnated dibenzo-//-dioxins congeners to in duce ALA synthetase anti aryl hydro carbon hydroxylas! corresponds pre cisely with their'lethal, teratogenic, and acncgenic potency, to the extent that toxicologic data areavailabic (50). T o date we have tested over 4U di- benzo-^-dioxiiis and 20 dihenzo- furans; the basic requirement for halogens in the laterai ring positions holds in all cases. The striking corre spondence between the potency o f these compounds as inducers of L.A synthetase and aryl hydrocarbon hy droxylase and their whole animal toxicity, and the sensitivity o f the biochemical response, prompted the suggestion that enzyme induction he used as a screening bioassay for the presence o f these compounds in en vironmental samples (45). Bradlarv and colleagues at the Food and Drug Administration are currently explor ing this possibility, measuring the in duction o f aryl hydrocarl>oii hydrox ylase in ceil culture (5). T he most commonly used inducer o f aryl hydrocarbon hydroxylase activity is -3-nicthylcliolanthrcne (MC). A comparison o f and T C D l) on the induction o f hepatic hydroxylase activity in the rat showed that these drugs evoked the same maximal response: simultaneous ad ministration o f maximally inducing doses oflxilh drugs evoked no greater response than that produced by either d n ig alone: and the s Io j k -s ol the log- dose resjmnse curves were parallel (Fig. -I) (Mil. TCI)1). hoyvcver. is 30.000 times as potent as MC. I lir*c rcMills suggest that the drugs sliaie a common me lianisui for the indu lion DOW 670436 n ^>*1 i IJ S M A S S HEALTH D IS A S T E R S R E L A T E D TO ENVIHONA1ENTAL C H E M IC A L S 'JW3443 BOX MHAClMtT |M0l(>IO^lGC) DOJI AHMACinntT |MOU*W^(CO lUtilVuvrii DOW 670437 F ig u r e 3. In d u ction o f ary l h yd rocarb on h y d ro x y lase by h alo g cn a te d d ib cn z o ^ i-d io x in s. C h ick en e g g s o f 18 d a v s' g e statio n w ere in jected with 2 3 fil o f/r-d iu x an e o r th e lest com [tuund d issolved in /-d io xan c. T h e em b ry o s w ere killed 24 h o u rs later a n d assay ed fo r h epatic aryl h yd rocarb on h y d ro \ik ix *:i(iiv iiy .W ) lo g d (tsc -r i-.|x m sc rckitiutiNl)i|><il TC .D 1): each poin t is the m ean -- sta n d a rd e r r o r o f lo u r g r o u p s o f pooled livers. B l in n tun.--iioiviiy tela iio n sh ip : each point is the m ean -- sta n d a rd e rr o r ol' lo u r groii|>s o l poo led livers e x c e p t the solven t-in jected ciuitruls a n d TC'.DO. w liere no. 12. r ryl hydrocarbon hydroxylase and p . -iaps a a at the same receptor. T he administration o f TCDD in duces hepatic aryl hydrocarbon hy droxylase activity in the chick embryo, rat, and mouse, and the dose o fTCDD that elicits half-maximal induction is very-similar: F.D = 4 x 10- , mole/ kg in chick embryo: 8.5 x 10-,# molc/` kg in the rat, and 4.0 to 12.0 x 10~' moie/kg in various strains o f inbred mice (46). Genetic expression of aromatic hydrocarbon hydroxylase When 3-methylcholanthrene is ad ministered to wild type and certain inbred strains o f mice, there is induc tion o f aryl hydrocarbon hydroxylase activity and the formation o f cyto chrome P|-450 in a number o f tissues (16). In contrast, other strains o f inbred mice do not respond to ad ministration o f MC. In crosses be- F ig u re 4 . L o g d o se -re sp o n se curves fo r th e in duction o f aryl h yd rocarb on h ydroxylase in rats by T C D I) an d 3 -M C Y ou n g Spn tgue-D aw ley m ale rats w ere in jected in traperiton cally with t-tiuixunc (0.3 m l/kg), T C D I) iii^-tlioxaitc. corn oil (2 m l/kg). n r M C in corn oil. an d killed 21 h r later. T h e livers w ere rem oved an d assayed fo r aryl h ydm carlm n h ydroxylase activity. T h e initial |xiiut on each cu rv e is the enzym e activity fo r con trol an im als treated only with the vehicle ( ./ d ioxan c: a . corn oil). Each value rep resen ts the m ean stan d ard e rro r o f fo u r o r five rats. tween the prototype strains C57BL/6 (responsive) and DBA/2 (nonre sponsive), the phenotypic charac teristic o f aromatic hydrocarbon re sponsiveness is inherited in a simple autosomal dominant mode. Extensive studies o f crosses among 12 different inbred strains have shown that aromatic hydrocarbon responsiveness is controlled by multiple alleles at at least two, and possibly more loci (55). T h e important point is that in nonresponsive mice, MC fails to in duce hepatic aryl hydrocarbon hy droxylase activity. Because o f the greater potency o f T C D D , we administered it to inbred responsive and nonresponsive mice. As shown in Fig. 5, TCD D induced hepatic aryl hydrocarbon hydroxylase activity in all strains regardless o f whether or not they responded to MC (49). T h e aryl hydrocarbon hy droxylase activity and cytochrome P,-450 level, characterized by dif ference spectra, cleciron paramag netic resonance, and in vino enzyme* inhibition studies, were iptalitatiwlv the sam e in all induced mice, whether o b tain ed from resp on sive mic r treated with MC or T C D I). or I 'm tn ^ n nonresponsive mice treated with * { T C D D . T his implies that the 2400 FPnrnATiriM pnnrernmfie u*** --  *'* for MC and TCDD. Thus, die non- tion in until es|niive mi c. Then- j> !'" 5 *'<] r v>. -t %&* v11i ?. " i t>- i1 ' *: i . # W ri ~ ! ! ** i`1t s * T'.'u* * ri! mm ii r *sJd>L" * " 'I | #^ I1 y* * M F igu re 5. li'|o iic aiy l hwtiucurlioii hsdrnxy lase an iv itv in " res|m iiiie" am i "n oiire*| m usile- in tinsi struins ni m ire. M ite w ete ad iin iU -ietl an iiitiajK-vitiiiic.il d ose n* / e lio x a iic <0.-1 m i I n . m i m i *1 . MC. in i n n i til (K(l m j'-'lg . A l. ir T C D l) in /-d in x an c ( I I ) ( n ' i j , s i . Km li- c i" I n Im iti lau-r. ili.an tiii.il u-rc kilh sl a n d tlu-ir li s u c i r 4>xi\itl tur cn/ynic ac liti. uct(s) of gene expression arc (he same for MC and TCDD. What is the defect in nonresponsive mice? Clearly they do respond to a potent stimulus, i.e.. TCDD. There fore, nonresponsive tttice do possess the necessary regulatory and struc tural genes for the expression of aryl hydrocarbon hydroxylase activity. We postulated that the mutation in nonresponsive mice that resulted in a failure to respond to MC was a failure recognize MC. In biochemical .ns, we suggested that the mutation resulted in a recognition site for MC (an induction receptor) with a dimin responsive mi e should lie nniiornilv less sensitive to TCDD than the res]xmsive mice. As -shown in Pig. I), the dose of TCDDthal produces hall-maximal in duction (KD:rfl) in three responsive in- bred strains of tnice is ahotn I x It) 9 mole/kg. and the KD,VI in the three nonresponsive si rains is equal to or greater than 1 x JO-'* mole/kg (-17). As one might anticipate, hybrid mice o f responsive ami nonresponsive parents-- (C:>711I7(>J x D1)A/2)P,-- show a sensitivity intermediate to that o f the parent strains. AH the data are consistent with the hypothesis that: /) the Ah locus is a regulatory gene whose product is the receptor that binds TCDD and MC and initiates the events that ultimately result in the expression of aryl hydro carbon hydroxylase, and 2) the muta tion in thcW/t locus in nonresponsive mice results in a receptor with a diminished affinity for these inducing drugs. This hypothesis proved useful in suggesting further experiments on the nature o f the induction receptor. Wc reasoned that after ad ministration of low doses of radiolabeled TCDD, insufficient to saturate the receptor, hepatic uptake o f TCDD might be determined at least in pan by the affinity o f the receptor: one should an implicit assumption th.il TCDD. an extremely ii|x>|>liilit molecule. is handled in pavsive ililliiMon. Pol!liv ing iutru|>enioMeal administration of "C-labeled TCDD at li x 1 0 * mole/ kg. the C.'iTlWAi) (responsive) mite concentrate more 1'CDD in their liver than do D15A/2J mice (mm- responsive) lor all time periods ex amined (15 hr to 2 wk: Fig. 7). The hvbrid mice. (C'i7B17(iJ x D15A/2|tF,. show he|xiiic uptake ititermetliate to the two parental strains. Our recent research ha been directed toward idcutilication and characleri/ation of the induction re ceptor in vitro: that is. the isolation o f a macromolccular fraction from liver that binds TCDD and that has the binding properties in vitro would be predicted from the in \ ivo hinlogv. We have identified and begun to characterize a liver protein fraction that has the following properties: 1 > TCDD is bound with a high affinity, 2)and the binding is saturable and re versible: congeners of TCDD that do not induce aryl hydrocarbon hy droxylase do not bind, and the bind ing alfiuity of active congeners of TCDD corresponds to their in vim potency: and Si the protein fraction from the liver o f responsive mice ha a greater affinity for TCDD than the 670438 ished affinity for the ligand. Why see greater hepatic uptake in re com parable fraction from noti- then can TCDD induce aryl hydro sponsive mice and a lesser concentra resjxmsive mice. carbon hydroxylase and cytochrome Pi-450 in these nonresponsive micer Two hypotheses come to mind: I) TCDD acts as a receptor she different F ig u re 6 . D i g d o s e - r e s p o n s e c u r i e s fo r tin- in d u ctio n o i ary l h y d m c a rlm n h y d ro M la sv activitv l>y T C D D in six in b re d strain >!' m ice. M ice w ere in jected iiu ra tie riio n e a lli with /- d iu x a iic (O.-t m L k g ) ir v a ry in g <loes o f T C D D d isso lv e d in th e i c h i d e . T u e n t v - lo n r h o u r s la te r the front that o f MC, a site not affected by a n im a l w ere killed a n d h cjiatic a r i l h y d ro carb o n h y d m x y la sr activity'w as assayed . T h e a i*o lu e 2)the nunaiion in.nonresponsive mice, cn/yiiK- a n il ities h ave keen inverted in fn irtin n ai resjw m ses tcnntrol ( * * ;. inaxini.illv im lu reti or TC D D acts at the same reee|>- IlKl'.i) lo r e a se o f c o m p a riso n (F o r raw d a ta se e re f-i~ ). E ach {m int is ilir m ean 1 1 o r 5 atiiitials. tor site as MC, btit because o f its T h e re sp o n siv e stra in s a r e th ose with o |ie n sy n ilm k : the iiotircsjK in sive strain s a r e sh o w n with greater potency (and presumably b b e k svinlwils. greater receptor ailinity). it is able to saturate this site and initiate the process that results in the induction o f aryl hydrocarbon hydroxylase. One can design experiments to differen tiate between these two hyjjotheses: 1) if TCDD acts at a site different from that of MC, a site tiuaffceied by the mutation in nonresponsive mice, then responsive and nonresponsive tniie should not differ in their sensi 2)tivity to TCDD. and if. however. T C I'1'* acts at the same indmiion ree i as NIC., then the diminished allium of this leieptor in notire- Mo t U i. >_ spuiisiie mice should lie diminislied 0003445 MASS XL ALI H OlSASICIIS RELATED TO ENVIRONMENTAL CHEMICALS 2409  *77 ._ ... . 7 / o>. es IM * lire 7 . l l i jiiuir u ptake n f "C-kilK'Ictt T (II)I) C r7U U U am i D H .V 2J m io*. (3 7 H I./)i| (rc- aii'l D I W JJ (iniri->|KH tvr) m ire trn- a < tm m i\ic r c t- a * in g lr nlr.ipeiitiiiH.-al e nI" ,4(M ilrk < i T C D I ) (( x H r * m olivi, n: ici fu .<tit i n . I l l iiiCi'iiinH ilt'l. A l t lir in d i, irti liiiu-N a lt e r atlm iniviratM iii. lite an im aU e ie kilti-il. lite lite rs tterv u u u i n f l . tteivjltetl. m ifii'en i/erl.an aliqu ot Miltiliili/eti with l'roto>1"" an <l c o u n te d by liq u id sc in tilla tio n enr o s e o T h e results rep resem the per m ane o f the ad m in istered dette concetu n ned the lisc i, flach point rep resen ts the m ean standard error o f 5 o r 6 anim als. DISCUSSION s apparent from die data presented u TCD D is a powerful probe in loot ing the.mechanism o f gene ev asion of aromatic hydrocarbon Jroxylase. A model to explore a ;ulatory gene in eukaryotic cells of r sidcrable interest to basic cart But one might justly ask, . how does this help us with our ;inal intent to explain the mcchan by which TCD D acts as a poison, craiogcn, and an acnegenr The etic expression o f aryl hydroion hydroxylase and the toxicity chlorinated dibenzo-/t-dioxins n to be related: induction o f this me is one o f the earliest, most itivc, and most universal (in difnt tissues and different species) o f f ents produced by TCDD. and .structure-activity relationship ng the halogenatcd dibenzo-/;ins for induction o f hydroxylase isely matches their potency as is. How then is hydroxylase inon related to toxicity? ries of toxic action nechanism by which TCD D cxts toxic action is unknown. A >er o f proposed mechanisms bservniums that suggest mediao f lovieily arc sumtiiari/ed he ist, tosals iltal relate toxiriiv ha Hon o f microsomal oxy- aditiiies. iollotteil by a more geuewtis group. DOW(>70439 Overstim ulation oj though this speculation does uoi pro- microsomal oxygenase activities I*isc a mechanism nl toxiriiv. it docs Since one o f tlie most sensitive anti jH'olouged responses ii TC1)I) is in duction o f aryl liytlroearltou Itvdroxylase (anti oilier cyitxhinmc I*,-- suggest a reason lor the rnrrrlaiioM ol toxic potency of dihen/o-/-dioxins and the potency as inducers ol ar\! hydrocarixm hydroxylase. mcdiaied microsomal tixvgeuasc ac tivities). it has been .suggested that this Intercalation prolonged enzyme stimulation might TCDD is a planar tricyclic aromatic result in excessive inactivation o f compound similar chemically to many endogenous substrates (e.g.. steroids) polvcydic Itytlrtxatbon carcinogens o r produce an excess o f harmful and tricyclic compounds that hind to metabolites o f endogenous enm- DXA. directly or through the forma pounds (20). It should lie noted that tion o f a inctalxiiitc. and produce in addition to induction o f the micro mutation and consequent tenitogenesomal m onooxygcnascs.TCDD is also sis or carcinogenesis (2). L'sing specific a potent stimulator o f L*DP-glu- mutant strains o f Salmonella txphicuronyl transferase (33) and ligandin murium Ames was unable to demon (glutathionc-.V-transferase B) (29). strate reversion o f mutants by TCDD Reactive metaboliteformation (personal communication. B. X. Ames and E. Yamasaki). At present, the We suggested that perhaps T C D D is available evidence su ggests that metabolized to some reactive inter TCD D is not a mutagen. mediate^) and that this metabolite binds covalently to an essential macromolecule (48), analogous to the Delayed hypersensitivity mechanism o f hepatotoxicity pro posed for bromobenzene (6) and phcnacetin (24, 36. 37, 51). I f the rate-limiting step is the formation o f the arene oxide ofT C D D . then those congeners that induce aryl hydro carbon hydroxylase would stimulate Vos and Moore (63) found that TCDD produces atrophy o f the cortical cells o f the thymus, a reduction in the general lymphoid mass, and an ac companying depression of cellular immunity. They suggested that this may be related to toxicity. their own metabolism and flood the system with an excess o f toxic metabo Inhibition of mitosis lites. (For a review o f the proposed mechanism o f arcnc oxide toxicity, see refs 10, 22.) T o date, there is little evidence supporting metabolism or covalent binding to TCDD. Jackson reported that TCDD in hibited mitosis o f the dividing endo sperm o f the African blood lily (21). No similar effect has been reported in animal cells. Tissue localization as a result of the induction receptor Our data on the hepatic uptake o f radiolabeled TCD D in responsive and nonresponsive strains o f mice sug gest that hepatic uptake and reten tion o fT C D D arc in part determined by the genetic constitution of the ani mal. in particular by the affinity and perh aj the concent nition o f the induc tion receptor. One might spondaic that the halogenatcd diltenzo-yi-dioxins that induce and thus hind to the induction receptor arc thus concentrated in the liver (or other parenchyma! organs containing substantial receptor con cen tration s) w hereas d iben/<>-/<dioxins that do not induce hydrox ylase ac tivity anti do not bind to the ititInti ifit receptor base more uptake in less critical organs (e.g.. fat). Al We have not yet exhausted the list o f theories proposed for the toxicity o f chlorinated dilrcnzo-/)-dioxins. At present our knowledge is inadequate to tentauvely accept or reject any one o f these. There are two sources o f encouragement on this search for a -- mechanism o f toxicity. First, there is an ever-increasing hotly o f data con cerning the dil>cnzo-/> -dioxins and dibenzofurans. and any proposed theory must account fora) differences in species sensitivity, b) different histological patterns in different species, r) the structure-activity rela tionship. and d) the correlation of the various types <(' toxiriiv. Second. TC.DDisam ong the most potent small moleeulr toxins known. If otic reex amines Table 1, vittually every toxin o f comparable |xitenc\ has a wellestablished mechanism o f action, and 7080 0003446 FEDERATION POOCHCDINGS VOL. 35. NO. 12 OCTOBER 1976 t.. n . o s i I i ;i \ t * I k t o i i h * i i m -Ii i I l i m l s 11 j> .v ||,, .t i w .m i I i . fj*3 and J. P n lh .J . (. V ii, ,J. t/ii;n9. 1 9C-o9m2m8o. n1. 97t'3.nem. 13. G r a o ifk . 8 ., au il fi. N assa. J u : M.liiLJn U 'f Miniti lik f In f | i i v im i a p p t r * ialin o In l>i. |.ni-> W .ide .uni M .uk D ft-iu ip Im Ih gulalino. \n |. a . f i l i i f i l li* II. J. V n gcl. pp. 77-111. 19. H ig g in b o th a m , ( i. IL , A . H u a n g . I). l-'irr- l l i r i i kill a m i t l l m l in iiu h f i/ iiig i Ih* iiu iiv sto n e , J . V e r r e ll.J . U c a n d A . 1). Cinip. m i i g r i i f l n i IO D I), unti In Mi'. K dlvatd ( i lm c r tur In m rlK ululi film i. in la n v in g b e ll. Snttne il.niiiliiiil 2 2 0 ; 7 0 2 --7 0 3 . Unix. fini iinil ni llir liitilngir:il c \|ieriiiiriiis. In ad ditim i w gralclully ai k n m ilfd gr lite ini* InlM irjtim i ni O r. D .m ifl Nel ieri am i Ili c o llc .ig n e ni ltk ` N aliim .il ln iu u if ni C Jiild I l c a li li a m i H u m a n l)f\fl< i|in ifiil in iiu iiv ni t li f g c iie lic c x |ic i in c ili*. am i D r. I)a \iil hi n sln iif. Alliert rn h lam l. am i (m in l'niiH Taui/. 20. Ilnnk, 0 . E. R ., T . C . O rton. J . A. M oore a n d C . 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A u s t . //<inhem. Biofdtys. Res. Common. 3 6 : 8 9 8 --*.88i, . 1973; 6 5 . W o o d s. J . S . Envitim. Ihnllh Pnsfnit. 3: 2 2 1-226. 1973. 6 6 . Yart/.idT, A -. 1). F irr sto n e . I). H ave. W. I liir n w it /, 1 . F r ir tlin a n a n d N. N e h r iin . J . Am. Oil (Jilin. 000344, :S; ikl -62. 1961. 70 ? ]<?> y V *sj, r;-1 A c * V ,41 i V- t i -I ~r-- vi'S'V \ ' ' l-..* r vA/i-fi-r iv;-- ;.r >* >. 7 f %.* vv '-..fc''!-.'-. hr-*. * A.-'* .? .V L-I^Vf'vV- '-y*1---*-** *:vvr V*' }.-9' ,* "r* v ^ % 2^*' `A I ?**v,1>--w.*&V**-:*iJI|I 1 .vTi.V*. 4 'tC-*- 'IV' '-^\v-,, M ouse Teratology Studies with Chlorodibenzo-P-Dioxins K Diane Courtney Health Effects Research Laooratory Environmental Protection Agency Research Triangle Park.N.C. 27711 Recently, much attention has been focused on the poly chlorinated dibenzo-p-dioxin c la ss of compounds. The extremely high to x ic it y of the 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) isomer warranted an examination of additional isomers and members of th is c la ss of compounds. TCDD was produced as a contaminant or by-product in the manufacture of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a herbicide which was widely used in th is country. Awareness of the to x ic it y of TCDD occurred as the re su lt of an in du strial accident that produced chloracne in the workers (BLE1BERG et a l . , 1964} as well as porphyria cutanea tarda (POLAND et a l. , l`57lTI In 1968, chlorodibenzo-p-dioxins were found to be the causative agent of chick edema disease f i r s t observed in 1957 (HIGGINBOTHAM et a l . . 1968). More recently TCDD was found to be teratogenic and fetotoxic in mice (COURTNEY and MOORE, 1971; NEUBERT and DILLMAN, 1972) and rats (SPARSCHU, et K , 1971). The dosage le ve ls for these effects are in the range o f microgram per kilogram of body weight. Since TCDD affected fetal development so markedly and at such low dosage le v e ls, th is compound ranks as one of the most b io lo g ic a lly active chemicals studied in mammalian fetuses. The uniquely high degree of to x ic ity of TCDD has generated inte rest in other tetrachloro-isom ers as well as other members of th is c la s s o f compounds containing more or le ss chlorines. Two current reviews of the wide range of toxic and pathological e ffe cts has been presented by Kimbrough (1972, 1974). P ossib le mechanisms of actions of TCDD were suggested at a symposium on dioxins (MOORE, 1973). I t was not known how many of th is class of compounds shared the extremely toxic and teratogenic proper tie s of TCDD in the mouse. Thus, th is study was undertaken to evaluate the teratogenic potential in mice o f other members of the c la ss of chlorinated dibenzo-p-dioxin compounds. MATERIALS AND METHODS Female CD-I mice and pregnant CD-I mice with known insemi nation-dates were procured from Charles River Laboratories, Wilmington, Massachusetts. Detection of a vaginal plug indicated day 1 o f pregnancy. The mice were randomly selected and assigned to control or experimental groups. The various chlorinated dibenzo-p-dioxins were prepared and supplied by Dr. A. Pohland o f the Food and Drug Adm inistration (POHLAND and YANG, 1972). The abbreviation TCDD is reserved fo r the 2,3,7 ,8-tetrach lo ro dibenzo-p-dioxin compound. 674 VBealll.e1imC. .V*i.LCauCf)im9TwCetayuSl fOiMwfinu*mVimrviliim| KInTeYtmirkNlltatri., -/> C3 00 ICC oo kU I-- o 00 V I ro 7083 1 Conpounds were administered o r a lly or subcutaneously. Oral adm inistration was by means of g a s tr ic intubation using a volume of 0.1 ml/mouse/day. The follow ing solutions were used for oral adm inistration. Dibenzo-p-dioxin was dissolved in corn o il. The octachlorodibenzo-p-dioxin was dissolved in 15i anisole in corn o il. The remaining compounds were dissolved in 5Z anisole in corn o il. Compounds administered subcutaneously were dissolved in DMSO employing 0.1 mg/mouse/day. In order to se le ct dose le v e ls for the teratology studies, the various chlorinated dioxins were administered by ga stric intubation d a ily to female CD-I mice for 14 days. They were observed for an additional 7 days. For the teratology stud ie s, compounds were administered from the 7th through the 16th day of gestation. These mice were sacrifice d on day 18 except those receiving octachlorodioxin and their respective con trols. They were sacrifice d on day 17 of gestation. Upon s a c r ific e , the fetuses were weighed, examined and stored in Bouin's solu tion u n til necropsied. The follow ing conventions were observed in compiling data. I f a fetus was either dead or resorbed, i t was regarded as a dead fetus. Only liv e fetuses were examined for physical abnormalities. A fetus was c la s s ifie d normal i f i t was a liv e and had at le ast one type of anomaly, regardless of type. A fetus was said to have abnormal kidneys i f at le a st one of it s kidneys were affected. In calcu la tin g the r a tio s of liv e r to body weight in the mother, maternal body weight was defined as the difference between the weight of the mother on the day i t was k ille d and the gravid uterus weight'. Maternal weight gain was defined as the difference in the cor rected maternal weight on the day i t was k ille d and i t s weight on day 6 of pregnancy. Averages were calculated for each li t t e r , then across li t t e r s . S t a t is t ic a l analyses were performed using the Student " t " te st. RESULTS-AND DISCUSSION The re su lts from tre atin g female CD-I mice with various doses of the d iffe re n t dibenzo-p-dioxins are presented in Table 1. Adm inistration of the compounds fo r 14 days at the doses in d i cated produced no le th al dose values. The dosage, fo r the dibenzop-dioxin, dichlorodibenzo-p-dioxin, and octachlorodibenzo-pdioxin are expressed as m illigram s per kilogram of body weight per day while the others are expressed as micrograms per kilogram of body weight per day. TJie change in body weight re fle c ts the difference in body weight on the f i r s t day of the study to the la s t , which was seven days afte r the la s t dose. The only unusual value was from those mice which received 50 mg/kg of dibenzo-pdioxin. Further study is needed to determine i f th is increase of almost seven grams was real or not. Lim itations o f the supply of these compounds precluded further exploration of dose studies at th is time. In general, none of the dibenzo-p-dioxins studied 675 ^ ^ -- ^ r n T n r r ^ '-'" *yi>n ^fc.t*i. k > Table 1. Oral Administration of Dioxins to Female CD-1 Mice for 14 Days Compound Dlbcnzo-p-dloxln 2,7-Dlchlorodlbenzo-p-dloxln Combination of: 402 2,7-Dlchlorodlbenzo-p-dloxln 60Z 2,3,7-Trichlorodlbenzo-p-dloxln 1,2,3,4-Tetrachlorodlbenzo-p-dloxln 2,3,7, B-Tc traclilorod Ibenzo-p-dioxin Octachlorodlbenzo-p-dloxln No. of Mice Dose/ Day No. Dead/ Change Body Doses Wt. (gms) 6 1 mg/kg 6 10 6 50 0 0 1/14 6 0.5 mg/kg 0 6 1.0 1/8 +0.6 -0.3 +6.8 -0.1 +1.5 6 6 6 6 6 6 6 10 6 6 6 10 Mg/kg 50 100 5 Mg/kg 10 50 100 10 Mg/kg 0.5 mg/kg 0.75 1.0 1/9 0 1/13 0 0 0 0 0 1/11 0 0 +1.2 +0.8 +1.6 0 -0.2 +0.8 -0.9 +0.1 +0.1 +0.7 -0.4 oa T a b le 2. T o x i c o l o g i c F .v a lu a t lo n o f C h l o r i n a t e d n iC k r t liif Y r t iF ^ f p Com pounds In P re g n a n t Cl>-1 M ic e (Compounds A d m in iste re d Ir n n I f i j 'M l M l t ( G e sta tio n ) A v. 7. v< W . i-'.. ` .^V'*i1<*Vr*.v;`l 'VxV* 1 < -rS<*|v ,1 VV^Jr- ' *i\3' * 5 ` ;*i? 7, ' f^Wv.fa pr*S--v Ai.'W'* . frtfr'V- < -.V* i Wfcv 1^ - ; 3 Na ;. !*-&*:. fstv"/ r r-Vj* V ' .?,. ^ k => ^ o00' Tabic 2. Toxicologic F.valuatlon of ChlorInated Dtbenzo-p-dloxln Compounds In Pregnant CD-I Mice (Compounds Administered from Day 7 to 16 of Cestatlon) 4 Dlbcnzo-p-dloxIn Compound 5Z anislecorn oil * Routo Dose/kg/day oral 0.1 ml/mouse No. of Litters 15 Av. Z retal Mortality/ Litter 6 Fetal Height (p.ms) J SD 1.02 0.16 Hatcrnnl Height Can (P.) X SO Maternal L1ver/Body Weight x 100 X sn 3.5` 1.93 7.8 0.59 2/3 mixture" oral oral 100 pg 200 Mg 6 3 1.36 0.13*** 4.6 1.10 8 . 0 0.84 5 5 1.25 0.19** 4.4 1.59 7.5 0.66 1,2,3,6-tetrachloro oral oral oral oral sube sube 100 pg 250 pg 500 Pg 1000 pg 500 Pg 1000 pg 6 10 1.09 0.09 4.8 0.51* 7.8 0.47 6 20 1.10 0.12 4.8 1.64 7.7 0.37 5 5 1.08 0.08 4.5 1.56 7.8 0.43 5 10 1.03 0.09 3.1 1.31 8.3 0 . 5 8 / 5 7 1.24 0.07 3.4 0.82 7.6 0 . 4 0 6 2 1.31 0.07 2.3 0.57 8.1 0.44 DMS0b sube 0.1 ml/mouse 6 14 1.19 0.17 3.1 1.96 7.5 0.94 2,3,7,8-tetrad: loro oral oral oral oral oral 25 Pg 50 pg 100 Pg 200 pg 600 pg 7 6 1.13 0.13 3.4 2.20 8.6 0.68** 7 13 1.01 0.13 3.4 1.07 9.4 0.44** 6 14 0.95 0.12 2.1 3.57 7.8 1.00 6 87 d dd 5 97 d dd sube sube sube sube 25 Pg 50 Pg 100 pg 200 Pg 5 36 1.25 0.07 3.0 0.86 9.7 0.42** 6 56 1.20 0.15 2.3 1.17 9.0 1.09* 6 72 d dd 6 76 d dd 1SZ anlsolv:cnrn ollc oral 0.1 ml/mouse 5 8 0.61 0.05 0.8 1.47 6.2 0.22 octachlo?oc .oral oral 5 mg 20 mg 6 14 0.60 0.06 0.3 1.40 6.6 0.72 6 12 0.53 0.05 -0.1 2.31 7.2 0.60* ' mixture * 40Z 2,7 dlchlorodlhenzo-p-dloxln and 60Z 2,3,7 trlclilorodlbenzo-p-dloxln b DMSO * dlmcthylsulfoxldc C sacrificed day 17 of gestation: all others sacrificed day 18 d marked edema p values: * * 0.05; ** 0.01; ** " 0.001 ' ^ or,u 1 v M o a A * I "v *i .r .: 1 ' -< r >m\> 'U; '".yJtr*--i .v v 1 :i <> J jmfV 7*** wV : 1*.Vw - 't "" were as toxic as TCDD, the 2,3,7,8 tetrachlorodibenzo-p-dioxir. isomer, and some of the compounds could be considered r e la tiv e ly non-toxic. Toxicologic results of administering the various dibenzo-pdioxins to pregnant CD-I mice are presented in Table 2. The diluent control solutions of 5 anisole in corn o i l , DMSO or 15S anisole in corn o il did not adversely affect fetal m ortality or fetal weight. The mixture of the d ichloro- and trichlorodibenzo-p-dioxin administered o r a lly at doses of 100 and 200 mg/kg/day had no adverse e ffe cts on the fetal and maternal parameters. The fetal weights of these two groups are higher than those of their respective controls. They are a lso higher than values which might be anticipated for a gestational day 18 mouse fetus. I t was assumed that these mice were very near term because the weights r e fle c t a gestational day 19 mouse and one member of the experimental groups litte re d (omitted from these data). The maternal weight gains of the experimental groups were s lig h t ly higher than the values of the control groups, but these d i f ferences were not s t a t i s t ic a l ly s ig n ific a n t. This s lig h t increase in maternal weight gain was also seen in mice treated with the 1,2,3,4 tetrachlorodibenzo-p-dixoin isomer administered o r a lly at doses ranging from 100 to 1000 mg/kg/day. Whether or not th is increased weight gain was a true increase in weight or a m anifestation of edema needs to be explored since studies with TCD0 produced maternal edema. In contrast, the subcutaneous adm inistration of the 1,2,3 ,4-tetrachloro-isom er produced a s lig h t decrease in maternal weight gain compared to co n trols. The difference in these values was not s t a t i s t ic a l ly s ig n ific a n t. In marked contrast, TCDD adversely affected fe ta l weight and su rvival and maternal weight gain at a dose of 100 mg/kg/day. Dose le v e ls of 200 and 400 ug/kg/day produced generalized edema in the mothers and vaginal bleeding a fte r the six th dose with some mice aborting shortly thereafter. The increase in the ra tio of liv e r to body weight seen in the mice receiving the lowest dose either o r a lly or subcutaneously was p rim a rily due to an increase in the liv e r weight. The dose of 50 ug/kg/day produced a decrease in maternal weight gain, and at the two highest doses the obvious edema made i t d if f ic u lt to interpret maternal weight changes. The subcutaneous admin is t r a t io n of TCDD produced more fe ta l m o rtality at lower doses than oral adm inistration. This suggests that the b io a v a ila b ilit y of a subcutaneous dose is greater than that o f an o r a lly admin istered dose. Treatment o f pregnant mice with octachlorodibenzo-p-dioxin at 5 mg/kg/day had no adverse effects on fetal or maternal parameters. At a dose of 20 mg/kg/day, there was a s lig h t 678 Tabic 3 . Teratogenic Evaluation of Chlorinated Dlbcnzo-p-dloxln Compounds in Cl-I Mice MOO V ;.J .'- " L i i ' T 1,. ** v V Ulipj ; .*/f::U * ,.' $ ' u- K * K v'.'. mS] A* il;i W .-rt C&j % 1 . ^1 V l*Jv * :v.V.- :h,,r.-v~!``i> i Table 3. Teratogenic Evaluation of Clilorlnntcd Dlbenzo-p--dioxin Compounds in CO-1 Mice 1 Dibenzo-p-dloxin Compound 32 anisle:corn oil 2/3 mixture 1,2,3,4 tctrachloro- i DHS0b 2,3,7,8 tetrachloro- 15Z nnlsole:com oilc octachloro-c Route . Dose/kg/day oral oral oral orai oral oral oral sube sube sube oral oral oral oral oral sube sube sube sube oral oral oral 0.1 ml/mouse 100 ug 200 pg 100 pg 250 ug 500 ug 1000 ug 500 ug 1 0 0 0 ug 0.1 ml/mouse 25 ug 50 ug 1 0 0 ug 200 ug 400 ug 25 ug 50 pg 1 0 0 ug 200 ug 0.1 ml/mouse 5 mg 20 mg No. of Litters 15 6 5 4 4 5 5 5 6 6 7 7 6 6 5 5 6 6 6 5 6 6 Av. No. Live Fetuses/ Litter 11.0 12.3 12.8 11.B 11.5 11.6 11.8 11.6 12.2 11.3 10.9 11.0 9.7 1.5 0.4 7.7 5.3 3.5 3.3 11.2 11.2 11.6 Av. No. Abnormal Fetuses/ Litter 0.8 3.2 3.8 0.8 0.5 0.2 1.0 5.4 1.0 0.2 4.6 8.1 8.3 1.5 0.4 6.7 5.0 3.5 3.1 0 0.2 0 X Anomalles/Total Fetuses Cleft Palate Kidney Clubfoot 01 4 0 10d 9 0 l 22 20 01 00 00 2 3 0 0 00 14 2 3 00 1 3 34 19 72 66 71 100 100 100 50 3 7 13 14 50 82 53 79 58 85 95 100 38 11 17 0 18 0. 0 0 10 00 0 0 a mixture * 402 2,7 dlclilorodlbcnzo-p-dloxin and 60Z 2,3,7 trichlorodlbcnzo-p-dloxln b DMSO dlmethylsulfoxide c sacrificed day 17 of gestation: all others sacrificed day 18 d 9/10 from one litter 1; V I-." I - * V T. \ > ` 1 :' * :V'* " : . - r -, 1 1 *. 1 ! ` i Ai ,e V ", ;-v .\ .V *j : t v *- < a : ,* T * f-v reduction of fetal weight which was not s t a t i s t ic a l ly sig n ific a n t. Also, the reduction in maternal weight gain was not s t a t is t ic a lly s ig n ific a n t due to a very large standard deviation. The increase in the ratio of liv e r to body weight was prim arily due to a loss in body weight. Fetal m ortality was not affected at either dose. A ll of the liv e fetuses were examined for malformations. These resu lts are shown in Table 3. The mixture of dichloro- and trichlorodibenzo-p-dioxin pro duced a s lig h t increase in the number of abnormal fetuses. At the lower dose th is was partly due to an increase in kidney mal formations which were a mild form of hydronephrosis. Since most of these fetuses (9/10) were from one li t t e r and kidney malfor mations were not observed at the higher dose, i t is doubtful that th is malformation was produced by the compound under study. At both dose levels there was an increase in the incidence of club foot. This may re fle ct both a natural incidence of the malfor mation and uterine crowding, since these fetuses weiyhted s lig h t ly heavier and the lit t e r s were s lig h t ly larger than the controls. However, th is does not negate a possible compound effect. The 1,2,3,4 tetrachloro-isomer did not increase the in c i dence of malformation at any dose level by either oral or sub cutaneous adm inistration. Since th is stra in of mouse has a tendency to d isp la y clubfoot, the 8 incidence of th is anomaly observed at the 1000 mg/kg/day dose level needs further sub stan tiatio n before being accepted as a compound effect. In contrast TCDD produced many abnormal fetuses at a ll doses studied and by both routes of adm inistration. The majority of the malformations were c le ft palates and hydronephrotic kidneys, both u n ila te ra l and b ila t e r ia l. A few other anomalies such as hydrocephalus and open eye were o ccasio n ally seen. TCOO administered subcutaneously produced a greater teratogenic response at a lower dose than adm inistration by the oral route. Administration by the subcutaneous route at the lowest dose produced about 67-abnormal fetuses per li t t e r . This made i t d if f i c u l t to demonstrate a dose related response since th is was close to being a maximum response. At the higher dose with both routes of adm inistration many fetuses were observed with marked edema and petechiae. The oral adm inistration of 5 or 20 mg/kg/day of octachlorodibenzo-p-dioxin to pregnant CD-I mice did not affe c t fetal development m orphologically. The only malformation detected in th is group of fetuses was a sin g le c le f t palate a t the low dose. In conclusion, TC00, the 2,3,7,8 tetrachlorodibenzo-p-dioxin member of th is c la ss of compounds was the most feto toxic and teratogenic of the compounds studied. The related compounds were , re la tiv e ly non-toxic and were not teratogenic at the doses studied. 680 DOW 410330 ! ACKNOWLEDGMENTS I gra te fu lly acknowledge the technical assistance of Mrs. J. Putnan and Mrs. M. Ebron. I also thank Dr. A. Ponland of the Food and Drug Adm inistration fo r the generous supply of dioxin compounds. REFERENCES BLEIBERG, J ., K. WALLEN, R. BRODKIN, and I. APPELBA'JM: Arch. Dermatol. 8, 793 (1964). COURTNEY, K.D. and J.A. MOORE: Toxicol. Appl. Pharmacol. 20, 396 (1971). HIGGINBOTHAM, G.R., A. HUANG, D. FIRESTONE, H.J. VERRETT. J. RESS, and. A.D. CAMPBELL: Nature 22, 702 (1968). -- KIMBROUGH, R.D.: Arch. Path. 94, 125 (1972). KIMBROUGH, R.D.: CRC C r it ic a l Reviews in Toxicology, 445 (1974). MOORE, J.A ., ed.: Chlorinated Dibenzodioxins and Dibenzofurans: Environ. Health Perspectives 5, 1 (1973). NEUBERT, D. and I . DILLMAN: Haunyn-Schm Arch. Pharmacol. 272, 243 (1972). POHLAND, A.E. and G.C. YANG: J. Ag. Fd. Chem. 20, 1093 (1972) POLAND, A .. D. SMITH, G. METTER, and P. POSSICK: Arch. Environ. Health 22, 316 (1971). SPARSCH'J, G.L., F.L. DUNN, and V.K. ROWE: Fd. Cosmet. Toxicol. , 405 (1971). A1 I 1 r '.- v 'V * r ; .. . . . - . . \ * ' ' ; i. . . / ' * r " 1 ' ; . r . *' * ** -w 'f l ii uvrit.]itp\xunuii its a im no sccvv m y . P3.uro<>Cp3jii3ii K.iniiH*K*ci3a xapnnia am ty. veimip.>aaii3 pvjoM i|*.'itorpa$.iii. Jn c n a .liia tk m cip a.iasiu iix ite.io ncaii non-un c tipita- u k 3m porphyria cut.inca lartla. a y ncvom piix uaimviitoD Cu.m oGuapyxciiu nepsHue .a aenxiHecKae pactpoucraa. O x ila .w u n o xnajiiuc n nCumo tmnpeixjcmm Cu.nt ausaanu 2. 1 6. 7 -- rcrpa- x-iopmCctooiuoMMiioM. Koropuu MMiiHkacT iipu aik-a.iitMccKtii: rii.ipm uac rcip a xio p . V nx'iu s ijm x.X 'p > 'iiU T cn .iitcru ii. Pcskim * npono.vi.i.ick m -ntuxu.iaiiti.'ii rcxnaxirueu ipn lo m rp jtip o ISO 'C h a a a x'in iii 43 . Ha nenoiK' paOur lU v ix n Kintunra 3. aoctiio. <ito yrii M3WMM3.U3H) TuxCMivrKtv actiuvrnn no. oG pa:ycrca. can <xc auoic> iK313HH3a pcaKiins epnananrcs npii ifviu-payjpi- nuxe re v 133'C riopaa>oiuuvii 6u.ni ax' ToakMi paterswiuu'* npn MenrepctcraiTiiiOM nponsanocrac. no u paflomic no pvMOirry. a T3k*c nayim w.iv.X'.loajTr.ikCKiK* paCornnxii n paCrnno -- x itie x o n u . Konamanc u * u-iiuaimomtuu xana.x ( V 8 m anx naea.ioek ia6n.icB3niie cerpuu civtapiiun ai-pMarnmno. v ncTa.iv- m ix -- ttM.vrrmuv pacccaimcK xaHcjonoa h nx ncpcxojnu a kuctu. Boc.-jicH im it .erau nain o .ia.in rk a uau6n.xv ib m m u x c.iynaax. .lia a .ia jjin m h k iiiiiih c k h c npuaiiaan cooTncTCThoaun to xoTopuc luaccm u u i cespcMCiinux .TiiTopaTypnux iic tm h iik o i. 3* 17 fo itn u x, n.in3iin. arnn chlorina nana-iack m-nCuKiioacniiu cnncoCosi -- nany.Ttv ayrrt.iaitn aa xotvsttivTax. V 20 6a iw iu x saw p av ytm .icck oOnnpyxHTk aoKa.iiiuunw a a xu iax. n o r $airr ii-j flu.1 onionon e cotpcMcnimii .inrcparypc. 3a6aicaaHHo lie npo- aiacrc* na ttx jx c ra x . u c pacrvr a a io ru . a v oanoro C a.iw irrn coavpuicmio hcvcxio a tcx n.x'Tax -mua n men. r je aupnc.ni yew n Cnpnja. npiraov a o c ti.iw u x *0CT3\ ja> -Cax-asuix- rp o .vo A a.ttcu C aiutuo c nrpjHhimaMn Ji.'raOo.itnjxa nop^mpnna hmc.th tn rm iy m ni'piwrtitT.niinu n nn!opri|Tixo]. npnno arnn m mix n sc.i bullos> actlnlcn at mechanica. O aniw i asropu oOHapyaimn nnx'pTpiixo] n mnrpnnr.Hcirtauuio n j 19 vanm iToa. 'nomnx acnn Oca .laOopaTopitux npnaiiaxna nop^npim. B ncpmu aopoa aaCax-nannni v 6 a iu iu x nc (h<.i Hafacn npcaonpcjc.inmttnA ho- w cciBrnr.aitJulanrno n uniataC .nOnaua.ia.uckp a xtiucamoi.oiuiio. B txknropux noapcAOcuna c n xa a x n nocac acckua nopontoft nan xnxM uc, Tax n inyipenm ia. B jp vritx u i u i i n a v iv o k n a * n jisccm uaa Hicnoinim auaaaia Taiu to ncaHaurrc.ik- ru r Goapoxaonna. a Coiuuoii CTiTvnn Ikaxa-axmro-TiipTnctnaaxacnrcoanauautnMp.u<k yacpo u ut iiu. np nro itnToncnanocTk r.ipacnaioM riinn. Y aaCatoannn* Saikn u x ua> Cmoa.Hick cyOu xinaiiu o om>monnx a cinuTniccKaii. 3\Tnc-anefi n ecKcyaikiioii o C u rra x . a y arxoTiipu.T a in*Top acca. Jla a vnop-ia ot nCutou nnm iicnKauiin. ap\Tno ja a o r paxa .x-rn ix. ajanvooniotuniix- voToporo x apn poccanua-iumii tKceoanuiiix xjtom nnc tk-aoivoauio aupaiitTu Paxiim v aviicTBiic x.m piipoaaiiiiux yr.x'aojopoaoa. na ocnoae rm caiinaro npiw iooj.'tea aan-n nno a ra u o JOKaaaru B jp y rn x paflnrax asropu a i T mupuOnei; wiMcainx- anyTpoiiHiix n noepniorimociiiix npnaitaKoa canux S aikn u x. Bee Caikinim naxo.iorra nna nocmmima naCHoaouncM TvpancaTa a uoapaiora. H oc.v n cm xru aii Haxcirvyna -- oCutho nonay ! n 2-um roan -- aaCax-sannc ocTcnonidt ynonkmarrea. 3* noxnTopux C a iu iu x bo a iiu c .ia Caressmm n cnunm nia i<ca'8 aiteM-NKamni 0]>c oxiataicTca aanpuTaairnan 8 .X T . Bee apnaicvaenuc Korean m taia.Hiei^m 'anrrarm M UHii. 3'.iyniuettnc aiH n .ir.io ck apn pcry.iapnniV um peccnn oen.aonoi^ii npa cayiic. Bee m crp-iaaaiuix' npoxtun-ia .vaonac n an 8u .ia aojMnnenii- ; ----------- j n~niii " Ci.D tm * 48. m 3 , e. 5, 3(5-377, K m. I l r C s t k L, K ^ l e n s k y K u b e c K.: Acnt Chlorina and Porphyria Cutanoa Tarda during the Manufacture of Herbieidaa Tha author submit after nn evxliutlon o f data reported in the llicrature resulfa I'- of the long term follow up of 7 patients w ith an occupational disease caused by manu facture nf berWcldcs -- penuchlorphm ol and derivatives o l 2.4.3-triehlnrophenoxydacr- lic acid. Of this numtmr 76 subiucts suffered from acne chlnnrui m anlfeslcd In m ild cases "y i only by comedones nnd In severe cases by largo costs, abscesses and m utilating scars on the whole Indy. The varied c lin ica l plrture Is dvnm iiilralud on photographs. Twelve of ihe patients had a hepatic Insion with symptom;, ot porphyrin cutnuea tarda and some patients had nervous and mental disorders. 315 anm m m m m m m m m m ur 1 8 .3 5 7 -- INDEX Tf. ~B. "Seward "W. R. Mullison hl O 1 PROM: J. H. Davidson 1 /2 4 /7 7 - O . .Ay .v ,._>... -.I - _ ji' . @ &k PS rt.-. ` .. ; --- .... . P'W<y<MU<A5': DOW101798 ICINE FOR PURPOSES OF STUDY OR RESEARCH IN LIEU OF OF LENDING THE ORIGINAL [ 0 0 C IU 5 70.92 ) i ... il i 1 ; f i A -J 5 11 .f It was revealed that the skin and general etfrrtlnns were caured by 2.3.6.7-lrtra. chlnrutlilicnzndifwtn which If formed, during alkaline hydrolysis of ictrachlurolicnrene Into at a tseomdipuemratturtrceholnfroUpHhI cXmialnaide.pTrehsesurreeacntifoInS was performed alni. From the. by an unsuitable procedure wnrlt of Schulz and Kimm.g It lx known that this highly nine substances is not formed when the above reaction Is carried out at a lower temp'iraturc of 153 "C. JCoi only workers engaged in produr lion of herbicides were affected but also maintenance workers, research workers and workers digging the sewage channel. In six the disease began a s acute solar dermatitis, (n all the remainder It started by a slow eruption nf comedones and thetr development into cysts. Inflammations and scarring were found only in the musi spier and extensive affections. The site and ciinu-al manifestations corresponded tu data rc|Kirte<i in (he literature. In 17 patients, hmvcver, acne chlorina started In a unusual wav by papulnpuslulous eruptions on the extremities. In 20 patients the aut>\orj observed localization In the axillae which wav not reported previously. The disease does not affect the hairy part of the skull and In one pa(!e::tit disappeared evun from those sites In the fare and on the neck where he grew a beard, while the condition periste! at other sites. Patients with an impainni porphyrin metabolism sufrred from typical hyprrplgmentntinn and hvpertrichosis and only one of them from bullosis actlntca et mechanic.!. Hypertrichosis and hype(pigmenta tion was observed alsc in IP subjects nlth acne without laboratory signs of porphyria. In the period prior to the disease no significant predisposing factors were noted. The lutency differed. In some instances severe dermatnlngieat and internal damage de- vclot'.d after short exposure. In other Instances apparently long and massive exposure caused only mild symptom;. Despite this the authors are convinced that the Intensity of .the disease depends more on exposure than predisposition. The patients had various complaints-somatte. mental and sexual and some of them hae considerable weight losses. Tiro died from general intoxication, two from lung cancer but so far it Is nut possible to ' state reliably whether It was related to (he occupational exposure. The cancerngenie , effect of chlorinated hydrocarbons produced In the particular plant w as not proved so far. The medical and neurological symptoms will be discussed by the authors in subse quent work. All patients are being followed up In collaboration with specialists In Internal * medicine and a neurologist. After the peak has been reached -- usually afler 1--2 years duration -- the disease recedes very slowly. In some patients, however, the manifesta tions and symptoms of general Intoxication persist already for 8 years. All therapeutic methods proved little effective. A certain Improvement was obtained by regular expres sion of comedones and sauna. In all patients secondary prevention was ensured, treat ment and damages in accordance to legal provisions. ` o. Ci- Dmt, 48, 1973,t. J, X. 308--317. Literatura 1. Baader. E. W , Bauer, H. Industrial mann, o.: Pentachlorphenol -- Vergiftung. intoxication due to pentaehlorphenol. Ind. Vortrag. GewiirhJrztf. Tagg, ln Hamm i. W. Med. Surg- 20, ISSI. s. 28S. -- 2. Bellmans: 1950 -- cit. Baue' -- 49. B. Flinn, F. B,, Chlor-Akni-. eine besondere Form von pro Jarvik, D. E Action of certain chlorinated fessioneller Hnuturkrnnkung. Dtsch. med. naphtahtnes on the liver. Proc. Soc. esper. Wschr, 27 ;S31. s. 437. -- 3. Bleiberg. J , Biol. Med, 35. 1038. s. 118--120. -- 9. Wallen, M., Hrodkln, R.: Industrially acquir Fuchs, F.: clL Breun -- 5. -- 10. Gavrilova, ed porphyria. Arch. Derm., 89, 1964. s. V. M.: O patogeneze fotodermatltov ot chlo793--737. -- 4. Bowen, S. S.. Moursund. M.'< , rirovannych naftallnov. Vtstn. Derm. VeFa Chiortcne In the manufacture of DDT. n er, 1949, t 3. s 32. -- 11. Gold mane, ). .Arch. Derm. Syph. (Chlcagol, 75. 1957, s P.: Schwerste akute Chlorakne durch Tri743--748. -- 5. Breun, W.: Chlorakne. Mo chlorphenol-Zersetzungxprodukte. Arbeits nographien zur Zeitschr. nnnifsdvrmatosen. medizin (ASA), 7. 1972. S' 12--18. -- 12. Band !.. Aulendorf 1, IVOrtu Edition Cantor Goldmans, P. j.: Schwerste akute Chlor 1955. s 73. -- 8. Braun, W.: Klinische Ileo- akne. eine Mnsscninfoxlkatlon durch 2, X hachtungen zur Entstehung der Chloracne. H, 7-Tetrachlordihcnzodlosln. Hautarzt, 24. Hautarzt, 10, 1959. s 123--129. -- 7. Brink- 1973. s 149-152. -- 13. Combot, B , Po* 316 /, i ; v G6TOTMOCJ VBf. spp kJ iaaagfju mi ? J j , i THIS IS A SINGLE PHQTQCQFY MADE BY THE NATIQNA1 LIBRARY QF MED ^ O 4 1 * A 7093 was caused by some lead compound that formed on the cham!-- - > lead walls as a result of a reaction with chlorine. Renon {35] also connected chloracnc with lung tuberculosis in one of his patients and ascribed both diseases to gaseous chlorine. However, Hirsch [20] concluded in that same year that the cause must be a solid substance which formed on the anodes during electrolysis because exposure to chlorine gas alone did not cause the disease. This finding was supported by Bettmann 12] who ob served 21 patients who were engaged in the production of hydro- . chloric acid and who were in contact -with chlorinated tar com pounds but not with chlorine gas. Lehmann [26], in 1903, concluded that the actual causes of th disease were chlorinated products such as hexachlorobenzene, pentachlorobenzoic acid and hexachloroethane which formed on the car- ' bon anode during electrolysis. Because similar cases of acne chlorina were also observed in the production and processing of pnitrochlorobenzene, W. Lehmann [27] concluded that acne chlorina was caused primarily by chlorobenzenes. As soon as carbon anodes were replaced by magnetite anodes, the incidence of acne during electrolysis ceased. Before World War I, Wahle [46] observed acne chlorina in two chemists which was probably caused by a chlorine compound - phenyl dioxide - not identified or known in more detail at that time. The next .wave of the disease's incidence occurred at the end of World War I during the production of chlorinated naphthalenes or perchloronaphthalenes. In this period the disease was given an additional new name - Pernakrankheit (Wauer [42]). Chlorinated . naphthalenes and diphenyls with a waxy consistency, also known as halowaxcs, have some very valuable properties such as waterproof ness, flame resistance and acid resistance. These properties earmarked these materials for the production of protective gas masks and other military equipment. A new increase in the incidence of the disease occurred in the nineteen-twenties during the use of chlorinated naphthalenes 0001118 co ,, P 709 5 as an insulating material in the mining industry (Tcleky [42]). Acne chlorina also appeared in the electrolytic production of chlorine when insulating coatings of tar were used. This fact brought about numerous disagreements and questions as to whether th disease in those production processes v/as not actually acne picea. On the other hand, Teleky supported the contention that the tar acne could be caused by chlorinated hydrocarbons contam inating the tar. .- . ' . The excellent dielectric properties of the chlorinated naphthalenes ("haftax", "halowax", "nibrene") led to their wide-' utilization in the production of cables and condensers and anothe: mass occurrence of the disease was produced in the nineteen-thirt: when the U.S.A. and other countries recorded hundreds of stricken individuals. Holtzmann [22] also noted the occurrence of acne .among the family members of workers employed in the production of. condensers. These family members handled and washed work clothes contaminated with the chlorinated naphthalenes. Based on these observations, Holtzmann corrected his previous original'conclusior and stated that the chloracne was actually tar acne and judged the effect of halowaxes to be that of an external achegenic cause. Ormsby [31] observed acne chlorina with the use of a fungicic used in the impregnation of lumber (sodium tetrachloro-o-phenyl- phenolate). Jones and Alden [24], Schwartz [39] and later many others also observed total disorders in their patients and'Flinn and Jarvik [8] noted a death due to yellow liver atrophy. In 193 Mayers and his coworkers [28] were unsuccessful in clarifying the mechanism of occurrence of dermatitides and- acne chlorina in thei: patients with the aid of epicutaneous tests - the tests were nega tive. - .. . During World War II, chlorinated' naphthalenes were used for the impregnation of ships' hulls when it was discovered that these substances could protect ships from damage of magnetic mines. The use of halowaxes increased significantly, especially in-the elect: cal-industry. American authors gave acne chlorina another.-name, : 'o o o i l id . ..m -o o OD 7 0 9 0 O P .y c- .) "cable rash", because of the occupation (cable production) of the patients (Good and coworkers {14), Schwort?. [40], Morris 129]). At that time, massive outbriteaks of acne and several deaths were recorded. After World War II, the incidence of acne did not sub side because the undesirable experiences with the chlorinated naphthalenes did not prevent their use in the electrical industry for some time. An interesting observation was recorded by Herzber [19] in 1947. He observed seven cases of acne chlorina in patient who had ingested chlorinated paraffins in the form of a food shortening. This prompted Herzberg to conclude that acne chlorina must occur after the internal ingestion of chlorinated hydro carbons. Similarly, Fuchs (9) also noted the affliction.of three children and their mothers after they had eaten potatoes fried in the "paraffin". Winkler [48] recorded 10 cases of dermatitis and 13 cases of acne chlorina among 23 workers in a condenser factory, that used nibrene in the production sequence. From these cases Winkler concluded that "chlorinated hydrocarbons cause the forma tion of reagents that in turn cause an increased secretion from lubricating glands". In 1949 Gavrilova' [10] warned of the photo sensitization effect of polychloronaphthalenes.' In recent years, plastics have replaced chlorinated naphthalenes in industrial use. Even today the production of sodium hydroxide may be a source of acne chlorina. We have observed more cases between 1950 and 1965. The main causes of acne chlorina in these cases have been the combination of chlorine gas with the hydrocarbons in the tar gaskets and fittings on the electrolytic containers and tubs and with the mineral oils which were used to protect the electrode contacts. The authors noted several cases of acne chlorina among laboratory workers after the use of nibrene in the electrical industry in the 1950's. Furthermore, the acnegenic effects of the mineral oil based coolant mixtures'used in metal working increase v/hen chlorinated . hydrocarbons are added. Therefore; some cases of acne oleosa were observed which exhibited a partial acne chlorina character (more . . "s . . : ` . '- J COG 1 1 0 0 ' o b* on follicular hyperkeratosis, loss inflammation, cyst form, tion and atypical localization). The last wave of the disease's occurrence was recorded in ti 1950's. This outbreak was noted among persons engaged in the production of chlorinated phenol type insecticides and herbicidei From 1950 through 1951, Baader and Bauer [1] and Brinkmnnn [7]' published information on the occurrence of acne chlorina which involved the complete internal, nervous and psychic symptoms amor 17 workers engaged in the production of pcntachlorcphonol. Sever years later, Hergt [17], Oettel 130] and Hoffmann [21] observed a total of .80 afflicted workers who were employed in the production of 2,4,5-trichlorophenol by the alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene. From 1954 through'1956, Kimmig and Schultz [25] recorded 31 cases of acne chlorina in one Hamburg (Germany) plant that produced 2,4,5-trichlorophenoxyacetic acid and its esters. By very detailed research and tests, these autho. .have shown that'acne chlorina and total intoxication with damage the internal organs, mainly to the liver, with psychovcgctative d: orders were net caused by the basic chlorinated hydrocarbons, i.e. tctrachlorobenzene, trichlorophenol and trichlorophenoxyacetic acid, but rather were caused by a ballast substance concentrated in these compounds in small quantities, namely, 2,3,6,7-tetrachlorodibenzodioxine. These authors contributed significantly with their other experimental works to the question of what sub stances cause acne chlorina and how these substances act. .In 1964, Bleiberg and coworkers [3] observed 29 cases of acne chlorina among persons engaged in the production of 2,4,5-trichlorophenol and 2,4-dichlorophenol. In eleven of these cases they also diagnosed symptoms of porphyria cutanea tarda. In 1957 Bowen and coworkers [4] observed acne chlorina among workers em ployed in the production of DDT from trichlorobcnzene and hexachlorofcenzene. The fates of 53 patients who in 1953 had worked In the alkaline hydrolysis of tetrachlorobenzene to trichloropheno were described only last year by Goldmann [11]. In addition to acne,. Goldmann also diagnosed in his patients bronchitides and \ * . Q... .-r:iA : ,O GO 7098 d7 disorders of the liver, kidney, myocardium and nervous system. T h e a u t h o r d e s c r i b e d the death -of a patient f r o m n e c r o s i s of the pancreas as a special case. Furthermore, he also observed dermatitis in a nurse who'had had contact only with experimental ,animals. F i n a l l y he d i a g n o s e d acne chlorina i n the f o u r t e e n year old son of an employee whose disease was contracted only from the household environment - by wearing his father's con taminated scarf and by using his father's towel. . . * '. Actual observations The process called PCP (pentachlorophenol) which caused mass damage in Czechoslovakia was, as far as production is con cerned, identical to the process from which Kimmig and Schulz drew their patients for observation. The final products of this process were sodium pentachlorophenolate, 2,4,5-tricholorphenoxy- acetic acid and the latter's sodium salt and butyl ester. The primary raw material was technical grade trichlorobenzene which was produced in a neighboring building for.the production' of hexachlorocyclohexane (KCH) and lindane. .The table shows the entire production scheme (Table I). The chlorination of tri chlorobenzene produced tetrachlorobenzene and hexachlorobenzene. Sodium pentachlorophenolate was formed by the alkaline hydrolysis of hexachlorobenzene. Sodium trichloropher.olate was produced by the alkaline hydrolysis of tetrachlorobenzene. The hydrolysis of tetrachlorobenzene with sodium hydroxide in the presence o f . methanol took place in an autoclave at 190C and at 45 atm of pressure for.one hour. After cooling, the methanol was distilled from the hydrolysate, the hydrolysate was diluted with water to a 25% concentration and it was then syphoned into a storage tank. The condensation of sodium trichlorophenolate with monochloroaceti acid produced sodium trichlorophenoxyacetate. Following cooling, i. centrifugation and flushing with water, the dried sodium trichlorc phenoxyacetate was picked out by hand and placed in small barrels. Some of this product was used to produce the butyl e s t ^ of ' . ' o . $. 00011:2 CD O Cl 7099 fd y o $ trichlQrophonoxyacetic acid. This production took place in . another building. The production of both the primary products took place in a four story building with grated floors (WEMA grates) which was one reason why the entire..building was contaminated with the high risk substances. . The building was not sufficiently airtight, the local exhausts were in most cases improperly installed and h e r m e t i z a t i o n and m e c h a n i z a t i o n were insufficient. Initi a l l y , th; s o d i u m te t r a c h l o r o p h e n o l a t e [sic] was escaping f r o m the c e n t r i fuge into the work space in the form of a mist. A series of tasks such as the removal of solid substances and the pumping of liquids were performed by hand. The workers were dressed in linen work clothes. The thorough cleaning and frequent changing of the work clothing was impossible to guarantee over the.entire interval of production. Rubber gloves and respirators were used at some work sites. The conditions surrounding the production of the butyl ester of trichlorophenoxyacetic acid were the same as those described above. Kimmig and Schulz [25] found that the main source of acnegenic hydrocarbons was that segment of the production process in which sodium trichlorophenolate is formed by the alkaline hydro lysis of tetrachlorobenzene in the presence of methanol. A co worker of these authors, Doctor (of Chemistry) Sorge, had de duced on theoretical grounds that if this reaction were conducted at 190C and 45 atm of pressure, chlorinated hydrocarbon by-pro-, ducts such as polychlorodibenzo-p-dioxines, polychlorodibenzo-. furans, polychlorodiphenyl ethers and polychlorodiphenyl oxides may be formed in small amounts (Schulz [36]). Sorge also pre pared some of these substances by his synthetic process. Animal tests carried out by the authors (Schulz and Kimmig). with tetrachlorodibenzofuran and 2,3,6,7-tetrachlorodibenzodioxine I .. have proved an extraordinarily high toxicity for these substances. A simple smear of a 0.1% solution of tri- and tetrachlorodibenzo furan on a small area of a rabbit's auricle caused heavy J,iver :. : o s 0 0 0 -11 2 3 ' -.-. .o CO O 7.100 ... f damage and death of the test animal within 2 to 3 weeks (Cchulz i36)). 2,3,6,7-Tctrachlorodibenzodioxine, which also has very strong acnegenic effects, was proved to be far more toxic. Only then was Dr. Sorgo successful in the identification of this enormously toxic substance-in the production process and in the final product. He proved that this toxic substance is formed only at certain temperatures and pressures. If during the alkaline hydrolysis of tetrachlorobenzene to sodium trichlorophenolate the temperature is reduced below 153C, the toxic substances do not form. However, at this low temperature, the reaction tine is extended from one to five hours and the productivity is ob viously lower. Furthermore, it was proved that the condensation of two molecules of trichlorophenolate to give tetrachlorodibenzo- dioxine is an exothermic reaction. Several explosions of auto claves with injuries and the lethal intoxication of workers were reported to be the result of this reaction in the German Demo cratic Republic (East Germany) (Schulz 138], Goldmann [11,12]). . .Tetrachlorodibenzodioxine was also identified in the Czechoslo vakian production process and in the products as well. v Th first two cases of acne chlorin were noted in 1965 in two technicians who operated the production machinery. It was as sumed at that time that these two cases were caused by careless work and an undeveloped production technology under semiproductio: conditions. After one year without a reported case and during which production was temporarily halted, a mass outbreak of 78 cases was recorded within the following three years. Finally, after a detailed investigation and study of the entire problem, the process was terminated and production ceased in 1968. Worker: from all stages of production v/ere stricken. At that time, it' was impossible to determine which production stage was the most risky because most of the workers were involved in. several stages at various locations in the plant within a relatively short time. Workers directly involved in production as well, as maintenance pe sonncl and workers digging sewage ditches were stricken. Later, a research scientist working to isolate 'the acnegcnic b^last -.0 00 o u i 4 7101 CD C ty y e . 10 substances from the products of the individual production phases .was even stricken. His research work was carried out out&idc the production plant in a research institute's laboratory. la addi tion to the 73 patients'with dangerous symptoms of acne chlorina or porphyria, other workers in this plant had symptoms of acne 'which were, however, symptoms of juvenile acne or of a similar character and could not therefore be counted as an occupational disease. . " With the exception of two female laboratory technicians, the patients consisted of males from 18 to 57 years of age (4 persons below 20, 4G persons from 21 to 30, 11 persons from 31'to 40, . 10 persons from 41-50 and 7 persons'from 51 to 57). Therefore, the acne chlorina patients were mostly young people. On the other hand, more than half the patients with porphyria cutarieai tarda were older than 40 years. Generally, the disease followed a normal course as described by other authors. In only six patients did the disease begin sud denly as acute solar dermatitis, i.e., by erythrema and edema in regions exposed to solar radiation and continued by acne sym ptoms (Photographs 1 and 2), as observed "by Goldmann 112], Grimmer [15], Braun [6] and before these authors, also by Touraine and co workers [45]. Therefore, chlorinated phenols in some cases have similar photodynamic effects as the chloronaphthalenes described by Gavrilova [10].' Erythema and edema in these patients were obvious mainly on their auricles.. Sometimes, small blisters .in "thick groupings were.observed there. In all the other patients the acne developed slowly. Among the less severe cases the disease was limited to single or densely grouped comedones, conditoned by follicular hyperkeratosis, which were so small and sometimes so numerous that they appeared to be a slate grayish-brown color, like dirty spots on the skin. These symptoms were noted mainly on the face, with maximum occurrence above the cheek bones (Photograph 3). In the more severe cases, . the comedones gradually developed into small white cysts^inilar Q. ' 00011-5 & - ' 'o 7 1 0 2 00 iU ljC U tj nili/ a l t h o u g h w i th black centers,, a n d i n t o largo c y s t s r a ng ing f r o m the size of a pea to that of a nut (Photographs 4, 5 and 6). As opposed to other acne forming dermatoses, the described symptoms and manifestations mainly lacked signs of an infection. Only in the more severe cases and rather in the vici nity of large cysts were some reactive inflammations, infections and abscesses noted. The emptied cysts and abscesses left behind large atrophied scars with raised edges and with bridges of intact skin under which a probe could be inserted (Photograph 7). This basic clinical picture was supplemented by diffused hyperpig mentation in the face' and by papulopustules on the torso and ex tremities. Patients who were found to suffer from a disorder in porphyrin metabolism had symptoms of porphyria cutanea tarda in regions exposed to the sun (hyperpigmentation and hypertrichosis, blisters,- excoriation and small scars). However,' in this group, there were two patients with prophyria but almost without any acne at all. . Histological investigation is not much help in diagnosis.. The symptoms begin with hyperkeratosis at the mouth, of the follicl After one to three weeks, the follicle changes into a cystic for mation filled w'ith a horny substance [.keratin?] (Photograph 11) and to a small extent with sebum. However, the epidermal changes are small. After the rupture of the full follicle, the contents of the cyst penetrate into the surrounding tissue and a granuloma of foreign bodies forms around the area (Photograph 12)... . The disease normally starts on the face'above the cheek bones However, in 17 patients the disease started in an unconventional manner by papulopustular eruptions on the extremities, primarily on the lower extremities. This characteristic, resembling'.more acne oleosa, remained in some of the patients for the entire dura tion of their illness. The most common localizations of cne chlorina were the cheek temples arid to a lesser extent, the entire face." Eyebrows, eye lids, the perioral region and the scalp and hair area jmained fre o nO L* 00 o 7103 I Other predilcctcd areas were the auricles, the back of the neck, the back and the chest in a seborrheic localization and the genitals. The auricles and genitals and especially the scrotum were stricken by the formation of largo cysts (Photographs 8 and 9 Among the five most seriously ill patients,' the disease spread all. over the body and had a discouraging effect that was heightone by the.disease's usual persistence, the long term healing and by medical helplessness (Photographs 10 and 7).` The contents of the cysts produced a repulsive and rancid odor. . For some of the patients the disease disrupted their social and family relations and caused severe depressions. Some authors, such as Braun [6], emphasize itching as the initial symptom, but we noted this symptom in only three cases. .In 20 patients with extensive symptoms, the occurrence of pustules, cysts and abscesses was also noted in axillae although this location was not mentioned by other authors. Because of the great number of follicles in the axillae and the similarity of the axillary skin to that of the genitals and for several other reasons, this finding appears to be just as valid as localization on the genitals and the genital area. On the other hand, it is surprising that acne chlorina does not strike at the hairline or scalp. In the case of one patient, the acne symptoms disappeared in the facial areas when he grew a ruxi beard while in other area: the symptoms continued without a reduction in intensity. So far there is.no explanation for this phenomenon. Hypertrichosis or hyperpigmentation or both were determined in the faces of 19 patients without a laboratory diagnosis of a deficiency in porphyrin metabolism. Bleiberg [3] made similar ob servations.' These symptoms receded simultaneously with the heali: of acne chlorina. .' 41orO" younger-porsons suffered from acne e)\log-ina-than older- porsons.* This study group contained 50 persons (851) below 30 ' *T-rai>6'iefcor'o-note-;-- --Beginning-- of this sontonce-illog.ih.le^. Many authors believe that younger persons have a greater tendency to develop acnc chlorina than older individuals. (Correction by KIH Translation Un ... o Page 13 years of age. However, this fact cannot io used -as -a hard and fast rule or to support the findings because the production plant > under study employed mostly young workers. The preillness medi cal history among the patients did not indicate any significant predisposing factors and no incidence of serious illnesses, intoxi cation or e tiolation*.. Furthermore, no relation to hair color, eye or complexion color, to the degree of.body hair, to the per spiration of skir. oils or to hereditary follicular hyperkeratcrin and other skin diseases was noted. Only one patient reported a more severe prior case of acne juvenilis, 12 patients reported mil cases during puberty and 14 patients reported only insignificant acne juvenilis symptoms. 18 patients also had some infrequent con tact with mineral oils either during the appearance of the symptom or at some time in the past. ` These patients were mostly involved in maintenance work as mechanics. However none of them had ever had an oil acne in the past. Their illnesses following'contact with tetrachlorodibenzodioxine were of the same character as the illnesses of the other patients. Their diseases were not similar . to acne oleosa either in appearance or in localization. The latent period from the initial contact with the risk substance to the appearnace of symptoms was quite varied. Some workers became ill after exposure of from several months to severa. years and the disease in their cases.was rather mild although the indications pointed to a massive exposure. In other cases, severe damage occurred after a very short time of several weeks and after an apparently small exposure. For example, one of the most seriou; ill patients worked under the semiproduction conditions for only two and a half weeks before the outbreak of the disease (acne and porphyria cutanea tarda). After that he had no contact with the risk materials. it is not clear whether individual predispbsition or the leve* of personal and work hygiene is more responsible for the intensity *E d i t o r To -n o te;-- T h 'ro- m a y -- b e a-dubious c h o i c e . tgnnoli-feoffxrfceo to "etiliom" alcoholism (Correction by Kill Translation Unit) . : . oooii:" The Czech- "etyliami .c I--* o 00 x ' u y u j l - of damage or latency of the disease. Most authors discount individual predispositon. However it is believed that the deciding factor must be the degree of actual exposure which is undoubtedly dependent on a scries of other factors such as work discipline, personal discipline, adherence to preventive measures, frequency of changing work clothes and undergarments, the degree and regularity of hygienic decontamination, work site order and cleanliness, dust in the environment, etc. . These patients also had a series of subjective difficulties: tiredness, weakness in the lower extremities, muscle pains, sleepi ness and insomnia, increased perspiration,, lack of apetite, head aches and other disorders in the mental and sexual spheres. These complaints were more recurrent and more intense in patients with the more extensive skin symptoms. In more serious acne cases, a. significant weight loss was also noted. It is our opinion that al' these difficulties and symptoms are manifestations of a rather overall intoxication than of only the acne chlorina. All the patients were admitted to a dispensary. Regular examinations of their dermatological, internal and neurological conditons were arranged. The patients who were still working were transferred to prevent further contact with any acnegenic factor. All contact with chlorinated hydrocarbons of any type was prevente: even with those substances whose acengenic effects have not been thoroughly proved. Furthermore, all contact with mineral oils and tar and its products were prevented. The application of mineral based ointments was discounted for external therapy. The plant's medical doctors were cautioned against prescription of any interna.' medication with an acnegenic effect (iodine and bromium preparatio: corticoids and others): The patients were given detailed medical directions concerning their daily regimen, personal 'and work hy giene and secondary disease prevention and treatment. All available medications for acne were utilized in the treat ment. ' The expressions of comedones and the pressure relief of the contents of cysts and abscesses necessitated many periods of . O. ' a 0001129 0 6*47 J1. ` hospitalization and special ambulatory treatment hours attended by a specially trained nurse. No special results were observed as a result' of repeated hospitalization. The best results were noted after the expression of.comedones by the use of a sauna constructed for the patients by the factory on the author's re commendations. It was not possible to test the effects of vitamin A acid (retinoic acid) which has been praised by some authors in the medical literature. However/ in agreement with the findings of other authors/ it was determined that no local or internal medication exists which has an effective and permanent healing .effect on acne chlorina. Some of the patients have been under . observation for eight years and their .disease still persists. Dr. Schulz's (38] patients have manifested continuous symptoms for more than ten years. Goldmann [11] has now been treating one patient with comedones and cysts on his penis and scrotum for 18 years! In most of the patients discussed by these authors, the symptoms and manifestations of the disease progressed, and reached a maximum after about one year, even after transfer of the patient from the high risk work area. . A regression takes place after two or three years at the earliest. In some isolated cases, the pro gress of a mild course of the disease was observed even after, five years. However, the regression of the disease in all the patients was very slow and, it may be said, even lasts a number ,of years. Of the 78 patients treated by the authors, 76 suffered from acne chlorina. Of these,.11 simultaneously had hepatic lesions with a deficiency in porphyrin metabolism. Two patients did not have acne chlorina. One of these two had porphyria and the second one died in 1966 from acute intoxication. Pentachlorophenol was-, considered to be the cause of death in that instance. When the toxicological analysis was carried out at that time, the investigators were unaware of tetrachlorodibcnzodioxine and, therefore,. it is our contention now that in that case, this highly toxic substance was primarily responsible for the acute intoxication. ' ; Ct ? -*.' 0 0 0 1130 I IU Combos [13] m e n t i o n e d one patient w ith acne c h l o r i n a and p o r p h y r i a in connection w i t h this pr o d u c t i o n process.. The authors analyzed 55 patients internally and ncurologi- cally in detail. About half of these patients had a deficiency in lipid metabolism and in more than one-third, some minor biochemi cal deviations and mild hepatic lesions were noted in the first stages of the disease. In 17 patients, the authors found symptoms of central nervous system disorder, the majority with lesions of the peripheral neurons of the lower extremities (verified by EMG examination). In most of the patients, physical disorders, pri marily an acute neurasthenic syndrome, were diagnosed. In addi tion to the death due to acute intoxication (pcntachlorophenol? tetrachlorodibenzodioxine?), three more patients died. In one patient, the porphyria developed'unusually .rapidly into an. arteriosclerosis of the brain with an atypical morphological diag nosis and demention. Two patients died from bronchogenic carcinom All these patients with their complete symptoms are discussed in other papers [32,23]. ; .Discussion ' ' .i In agreement with Kimmig and Schulz, we cite the tetrachloro dibenzodioxine which is formed during the production process as the main reason for the skin symptoms as well as for all the other deficiencies mentioned and diagnosed in the authors' patients. This toxic compound was confirmed in considerable quantities in the final product (Arboricide E50), in the butyl trichlorophenoxyacetate and also in the mortar and wall paint of the factory building. 'Even adjacent rooms such as worker dressing and locker rooms were contaminated with this compound. Test rabbits placed into these rooms soon died. In his monograph, Braun [5] cited the following chlorinated hydrocarbons as the then known causes of acne chlorina: '. chloro benzenes with various degrees of chlorination, chlorinated phenols (sodium totrachlorophenolatc, pcntachlorophenol), para-nitrochloro benzene, sodium tetrachloro-c-phcnylphenolate, chlorinated ; \ ' i 000V13'1 -O o 7108 i-- i 00 naphthalenes, various chlorinated diphenyls,, trichloroxydiphcnyl, chlorinated diphenyl oxides, sodium o-{2-chlorophcnyl)phenolate and others. However, Kimmit and Schulz [25] ascertained that pure pcntachlorophenol and pure 2,4,5-trichlorophonoxyacctic acid do not have any acnegenic effects. Therefore, they checked the presumed effects of the other substances that were blamed for causing acne and found that neither pure nor chlorinated benzenes '.(from 1 to 8 chlorinated positions) nor chlorinated diphenyl- ethers have any acnegenic effects. A definite acnegenic effect was proved only for the following substances: chlorinated naph thalenes (5 and 6 chlorinated positions) in agreement with Shelley and Klingman [41], higher chlorinated.dibenzofurans (diphenyl oxidt and 2,3,6,7-tetrachlorodibenzodioxine. Therefore, it is possible and even probable that some chlor inated hydrocarbons that have been previously considered to have an acnegenic effect do not have this effect and the actual causes were other ballast chlorinated hydrocarbons contained in .those hydrocarbons. . /: Tetrachlorodiphenylene oxide (tetrachlorodibenzofuran). 2,3,6,7-tetrachlorodiLir.zodioxine (tetrachlorodipher.v dioxide) 'In an animal experiment, Kimmig and Schulz found an ex ceptionally high toxicity for tetrachlorodibenzodioxine. In a local application on rabbits' auricles (keratosis test), this com pound produced a strong reaction even at a concentration of 0.001%. Also, a skinapplication of this substance at the same concentra tion produced acne chlorina (Schulz [38]). Tetrachlorodibenzofura: was proved to be 10 to 20 times weaker. Chlorinated naphthalenes had a thousand times weaker effect. Generally, acne chlorina is caused by the direct contact of tctrachlorobenzodioxine with the skin. Absorption through the skin is not ruled out and ah effect :D 0001139. c 5 ; w 7109 J- through the ingestive or respiration tracts is also conceded. However# in a pure ingestive experiment with a test animal# acne chlorina did not occur. However, the cases of acne chlorina con tracted after the ingestion of chlorinated naphthalenes as food fat substitutes should not be forgotten [19#9], Tetrachlorodibenzodioxine used internally has a strong he- patotoxic effect. A dose of 20 to 50 mg per kg of rabbit's body weight causes death in all the test animals by yellow liver atroph; Because of this enormous toxicity# all the preventive measure taken at the high risk processes in East Germany and Czechoslo vakia were proved to be completely ineffective. The only de pendable solution to that problem was the reduction of the re action temperature and pressure during the alkaline hydrolysis of tetrachlorophenolate to sodium trichlorophenolate. In East Germany# the decontamination cost of the high risk areas and buildings was extremely high. One chemical plant attempted to use for other purposes a contaminated building which' had not been decontaminated after the building had remained unused for two years, but new cases of acne chlorina were shortly reported. With normal decontamination procedures new outbreaks of the diseas occurred and therefore, decontamination had to be carried out usin special pressurized chemical protective suits with pressurized breathing apparatus because gas.masks were not sufficient. Doors, windows, the wooden trim and other wooden building components had to be burned; the steel construction materials in the building had to be sandblasted; the plaster, facade and floors had to be torn out and cast into cement blocks together with the tubing, plumbing and other equipment. These blocks were t h e n .transported to the Atlantic Ocean and sunk. The disposition of those non flammable materials by burying would have run the danger of con taminating the subterranean water supply. Only the most expensive components, autoclaves and some valves, were successfully decon taminated by repeated washing with special detergents. The Czechoslovakian plants are still closed and their fate is still undecided. The patients are still under a continuous ob servation. r> 0001123 o t-* 00 7110 ; , . .References *. 1. Enadcr, C. V/., lla s a r . II. J.: la d u sir ie l . In lc u c alio n duu Io ]>i:niaclilor|HirroL Ind. Med. S u r * . 2d. 1951. s. 253. -- 2. D d t u s i o : Clilt.f-Al.iic. i-iiiu besondere r e n n vca pro- . lo iin n c llc r llju tciM jnliunr.. Duett. ined. W lir , 27. f J J t . s. 437. -- 3. tllcibarr. J . U'olinn, M., E.-odkln, 8.: lm luslutilly ncrjulr- 7u0d3 |iuri>liyriu. -- 7 3 7 . -- 4. Arcli. Durm, 93, ISSt. Honen. S.S..Miiuui.nd. z. M. IV Clilorccno (a thu nsnulnccuto ol DOT. . Arch. Der. Syph. (C'j !c j0 ]. 73. 1237. *. 7 C l- 7 iJ. -- 5. Uroao, IW ^klarakr.a. Mo, no^rjplilia :u.- Zosahr. nirufsderr.iaiessn. 73.: . Band 1 , Aulundorf I., v.'Urit. JMl'ion Cantor-, ,.'35S5. s. -- C. Braun, V/.: Klinische BaO- *' 'li.iClitar.c;:! sur Entstehung eis? Chlaiccao.': vJIauM itt, 10. lOs'J. s. IM --1 2?.-- sasr.::. Un Tcntaclilsrphanel -- Veredlung. Vortraf. Ceivurb.lritl. T a jj. ln Hanna L W. 1909 -- d l. Bauer -- {9. -- 3. H ina. F. 3t, ' .. Jaxilk. D. H aphlnlcnns : Act'.oa on Ute lolvtccr.crVtatolne. chlo.-lnstcd Soc. nxjicr. : fliol. .Med, 33. 1230.' sl 310--120. -- 9. ' Fuchs, Fa d t B.-aua -- 5. -- 13. C an ilcra, - V. Ma O paiogcncze loiodcraatliov ot chlo- rlrovanoych naltalinov. V ista. D er. Vo- ` J ^ b e r , 1919. . 3, 5. 32. -- 33. ColdtatiES, J. J F a Dcliwcrsle akute Chlcratna durch Tri- / tlitOiiiListSi-lenfwisss^spfuliiaie. Artslt** V xsoJUln [ASA], 7. 1972, S. 32--10. -- l t * . eM m oa, 1`. j.t Schwerste akuto Chlor-! 'Kilo, olna Massenlntoxlleatlca durch 2, -3, * V0. 7 -Tlr3 ch!s:dU,2n:cdIo.tJa. .il.iuurr.f, ^.1'J7.V:J J . s2--3 ii- - r `-73:'Ci.vhaV.'.r.*T.w^ : `eh;.oA,',': KoVi-.v. v,* '-!i.-::ssi:3 v A .' i.< ' M 'orpiiyita cutanua tarda aeno cMe.-uia' / n r l virobo cykllchich ehlurixvanfsli ulilo- * a-adlhov. llr a t lOJs. Idsty, 53, 3993. *. 010-- - i i -- i<L Dual, Cli. 1h, rausky, Iks HMu seal aeno {.eublo rash 'J. Arch. * Darts. Syptt (Chicago], 45. 1313. J . 251-257. -- -- 35. Crltitmor, Ha Ucntnich bitdlui'ie Akne durch clilorlcrlc eromallscliu Kohlca- leJSicrMoife. Zbl. Arbcllsntcd, S, 1955, s. 70. -- 10. Haltopeaa. II.: Acn clilcri<|ue. Seat. icd. 1503. s. 231 -- r t t Crauo 5. -- 1 7 . B arst, UV Diskussion. V/crklrztc-lCua- .4le3r.e--n z.13U. nldl oDa hilcrkl clitcclra,t, 193S -- Km Uber eil. Dauer Chloraknc. ' Jlaclu leeit Witi'.r., Id. 3039, s. 270. -- 19. Jlerzhurg. J. J a Chlorakcc nach denuss vca ..1c9b1l7c,ilie.r<tt2a5j. JVrafln. Dem -- 20. lilreeli: . V.'sch r. 130. HauterkrafAuae ,1a chicr FafciIV zur clcktrclytuahen CUsr- . crzcurur.s. 2tscli. d. Zentralstelle 1. WLaJ- Icr-V.,u!illjl:rf-Clurlal::uuj;un. 7, 1503, a. 01 -- CIL Eren 3. -- 21. Httl.-aaua. H. Tlt: Vorlr. V.'crkDratc-iCaziarr:J7.Iln:tcr-rKhen, . 3935 -- d t Bauer 53. -- 22. Hu'.tzzszoit, IV Clt'.oracno U. rernakrankketL Artlv. Hy5, 117. 3933. 1, z. Rcu-.-edena -- d t l'-iaun u . -- 73. Jl.iitA-, I.,JlstauskJ. J, Itckac. It, T i A l t r , lV zlBK tiJ. J. ot Jda I'B.v.c'.ty fa.otu littoiikucu u '.tu cltUvrtua pH vj- uti licrbtdl-- )i;<i).u-.e.-3toda ll-.ku.-- Page 19 2 L Jouer, J. V/^ T-Hux, IX. J t : Tin uci-xtorm deraiatL-rfosIs. Arett B rir.t Syjih. . CJ. 33. 13J3. s.3032--ldJJL -- 23. KiuuiU. J , Sclutl;. K. It: Bcrudichc Aknc tsbf. ' Chloralmu] dutch chlurlarte aronatisebc lyhllfchr Atlicr. Demistj:;i:;!aa. 115. 1317, .3.510-- 513. -- 20.Lokuiann, K. B.: Cxjiurl* .aicntctlo Sludlen Ober den ELnlluss tech* - . nlsch u.hyslenlscb wtchllser Car? u.Ca:a- Jc out den Orsatihsous. X L Eiudiou ilber .Chloratr.c*. Arch, llyj, 1G. 1903. s.322-- 333. -- ell. lireun 5. -- 27. ttbinoaa, VjV * Ober Chlorakac. Arch. Darns, 77, 1905. s. 703-233. Ibldcn 323-- 3ii. -- 20. Nayerr. I t J-t. Stlrcrlicr^, i i . a 5kIn ccndltious ...'7Tl2ec01ds.ourh1li9uyi3nsd,3"r`,oGIcsr.a.or1n2bC1o,e1nx--7sp.2ob]5s.c3ucb.raietdbiutatoMirc,Bre.rrtaUt.auyinnsR-jc5-Tl.iol.o--Cxriaicnb2ual2l*e,.'- . . xasli* -- A noto on a new c lc sn sto j mix- i 3tore. J. Aracr. m id. A ss. 121,19i3, s. 192-- 33. -- -Clt Braun -- 33. Onltul. 11s X7II- r Zilscka i t /JcnrxpcrJiacntc'lo ilrluktxiusua **o lt hochtoxlschcn Chlarkoldcnwasscrstor* *ilun; ola Ilellraszitnrarna-Prahluia.Vrcticr nJSrsio Kenierr-tz, Bad DCrklicIm, 1955, c lt * .Bauer <3.---- pr.zuliuka ' It t3l1ln.`kUuiuiiJolrny-,*:D.7t1V, Dh--kus..2u2t. "/, rard o iu v i, J , t n t l l , D , S jilliiu r l, I I , J|. ' rds-jlc, L . K idauit/, J . riA iti.1, J . Jlristk . * A . KimeutA I t , Juluv, J-- !'<<>t.ezuul zilrasl- lvp ric o u ilk i pH v / r c t i 7,{.5-lt!c`>il6r!enox)-> octnau vcdnClio. Trac.LV. IX ':, IIsLuJ. -- 33. ric u U . Cm Zur Klitcltk ilk! Csuednacn.* - Bllduuf bul Chlorakiis (linlow asaacej. Arch. klht. exp. Uerzn. 233. I97i). s. 223-- 241. -- 31. Pljsvij. C.t LokalLid.as-.dlm.s dtr CliloraCi'.c (Ilaluwaxaeu.i] n:ir Vliarcla A- BOurc. Haulurzl, 21, I'JVO, s. -1C3-- 170. -- ' 3S..3tiu:a, Ms tntcixlceilua vruies::isf.a!)e par lux vapours de chlorc; cblerlr,iu) et tuberculosa pultuunulra. Eerj. titdcL, 1933, *..123. -- ell Braun 5L -- 3. SrUuU. I t Hz Xllnlsche u. ci'perl::ic:iteJIi: UnlLisushun* pen zur Aticlo^ia dor Chloraeuc. A rcit ' kiln. exp. Dcra:. 203. 1957. s. 5 0 3 - 0 3 . -- 37. Svhulz, I t I t : Zur Kik:lk x:. Atlalopls der Culoraknc. ArbeUsnikdizbs |A 5A !, 3. 1933. s . 2 5 -2 9 . -- 33. S-abulz, X. lta OsoVd sd iV aJ. -- 39. 5 cln varlt IV D urasilU s Irons tynitztic resins and v.-.-.rcs. Amur. J. otjiuliL llUh. 211, 1030. s . 533--502. -- -i3. SeUwartr. t x As aiiltvcak llalov-.-az aeuc (.cable rash*] r.ate::p AShnteel.l-c, yi,n1r7d.. A ss. 122. JU It. tiliuta-au, i3. A. crl.cu25'.r1l.sa--r.s.i lJ.. J Ja Ilia t-ipu- " rln e n u l jirri'ncti&z ot cc:i>: .'> pu-;:a- and huxaiJilorjiupitialenes. Arch. Us-rus. C-yjilL a(Clticapu). 73. 1357. *. Ci23. -- <7- Talufy. Xm Di lvriLak.unk.kci: (C tlera'sne]. Kiul V.'se b r. 15. 1327. S. CIS--CC4. JtlilcM U9/-- 90L -- 43. Tb'iilervn. C.: ileni "c'oisddoa pGsurnlisle due 5 rbihalailo.i pratcsslcacl* le de vape-uts ehlociquis. 5i:sn. vx.i.d, 1039, s. 24. Cit. Ilrau 5. -- 0001134 PQW'ioi817 7.111 44. TI|.:I| t i C , Vdi- K i c t , 'I n . : L 'ac-iS tli'.j.-ii.ui'. U c ru u 5s,,.*,. rjtni. n r a / i , ^ 4 i ; - : a . -- o \ jl.-j-a . -- 45. Tnjral.'c. A., So'.aute. 0 , ' B.. A ctrya: S! c ai i!e evuatltcs par i;:iiiluruaji:iidlt::c. Uull. i'oe. <lu;iu. n,n.1, 41. J'JH. *. : n s - :v,1. -- .vj. V/aUla. ~ j\ : L'a.-r : `.Me voa ,,CUi.tfacni:*. luauj;. t j I Wvi. u i t . 1014. -- C. Jiraur. 5. -- 47. V/auer: lcw .- lu h c S i K rai:!-o n * liurcJi KK!i'.u.:vassiTSM: ll'cinaitrs.ik- hell I. 7.W. Cf.wrteijyx^ 3. l'Jl. *. 1W1-- jCl.-- v4.Vlu.tir,K.:Zur CfclorcXnc fPer- n--'.:ar.!.;.c:;J. ?_ Haul. 11. J i i J J . a . < 2 'J -- t i t 4 5 . H a u n t . >1.. S e r i a l * . K . u , i,i\( yllti/Z. V j l'.cru:i:t: y.;n l-.;I er ItyrMcUun* ves Culurpiiaiicl--* . V.Miiiimrcn. A :c)t .CowuK-r.v.riOl. C a*T*- v v rc iM ly fr, 10. 1531. 5 . 5 il - 5 i5 . ` . * . .' ; ' ' MO] P a t h o g e n e s i s of p h o t o d e r m a t i d e s f r o m c h l o r i n a t e d n a p h t h a l e n e s M3] Gombos, et al., Porphyris cutanea tarda and acne chlorina .occurring during the production of cyclic chlorinated hydro carbons. 123] Symptoms of total intoxication in acne chlorina during the p r o d u c t i o n of h e r b i c i d e s - in f i n a l p r e p a r a t i o n f o r .printing. 131] Discussion on paper by Jones [24] * 132] Health hazards to workers in production of sodium 2,4,5-trichlorophenoxyace tate.. O. o O* * I. 0002135 7112 i re i *1? i 909866 ^ a DRAFT 6 APR 1*377 Dioxin: Position Document Dioxin Working Croup U.S. Environmental Protection Agency 115' 000257n DR!; Dioxin: Position Document Page I. B a c k g r o u n d .............................................. 2 A. C h e m i c a l and P h y s i c a l Prope.rtles of T C DD . .. 2 1. S y n t h e s i s ..................................... 2 2. E n v i r o n m e n t a l C h a r a c t e r i s t i c s ............ 2 B. 2 , 4 , 5 - T L i t i g a t i o n H i s t o r y .....................3 II. D i o x i n I m p l e m e n t a t i o n P l a n .......................... 7 A. I n t r o d u c t i o n ...... .............................. 7 B. A n a l y t i c a l ............... t . . . ................... 8 1. P h a s e I Beef Fat and L i v e r ................8 2. S u r v e y of T r i c h l o r o p h e n o l P e s t i c i d e s for T C D D R e s i d u e s ........................... 8 C. T o x i c o l o g i c a l E f f e c t L e v e l ...................... 9 III. F u t u r e A n a l y t i c a l S t u d i e s ....................... 9 IV . S u m m a r y ................................................. .11 V. A p p e n d i x .............................. .....12 A. I n t e r p r e t a t i o n of P h a s e I Data by A n a l y t i c a l C o l l a b o r a t o r s ............. ... ....................12 B. P h a s e II E x p e r i m e n t a l D e s i g n . . . . ............... 13 s i * .. CO ' CO 00 1 t 711T- 0002571 I . Background - 3 HP/* Air i Z G A P ft h v A Chemical and Physical Properties 1. S y n t h e s i s T C D D is not f ou nd n a t u r a l l y in the e n v i r on me nt , its s o u r c e cones as a b y - p r o d u c t in the m a n u f a c t u r i n g p r o c e s s of several chenical products. 398638 Although there are several synthetic routes for the f o r n a t i o n of TCDD, the m o r e c o m m o n r e a c t i o n oc cu rs u n de r c o nditions which are used to p r e p a r e 2 , 4 , 5 - t richlorophenol, tetrachlorobenzene being the precursor compound. Under extreme sa p o n i f i c a t i o n reactions, 2 . 4 . 5 - t r i c h l o r o p h e n o l is formed. This reaction was first (stablishe) by Schwetz, et al. (1). T h i s m e a n s that T C D D 'or T H e r c h l o r o d i b e n z o - p dioxins will always be present in trichlorophenol products a n d / o r t h ei r de ri va t iv e s , and that any use of these p-roducts will result in environmental contamination by dioxins. 2. E n v i r o n m e n t a l C h a r a c t e r i s t i c s Basically there are three ways in which TCD D may be i n t r o d u c e d into the e n v i r o n m e n t : (1) it may be p r e s e n t as a c o n t aminant in c h l o r o phenols or their deri vatives, (2) it m ay be f o r m e d f r o m c h l o r o p h e n o l s or t h e i r m o n o m e r i c c h l o r o p h e n o x y d e r i v a t i v e s u n der c o n d i t i o n s of u s e or s t o r a g e , or (3) it m a y be f o r m e d f r o m p h e n o x y p h e n o l s or o t h e r p o l y meric derivatives under similar conditions. In those cases where TCDD residues have been found in environmental samples, it is not k n o wn at p r e s e n t w h i c h of these ro ute s is r e s p o n si b le . K e a r n e y ' s , et al. (2) s t u d y of the f a t e of T C D D in the environment, however, has resulted in the following observations: 1). T C D D does not l e a c h v e r t i c a l l y in so il s; 2). S i g n i f i c a n t a m o u n t s of T C D D are not ta ken up by plants and none could be h a r vested in grain or soybeans; 3) T C D D d i s a p p e a r s s l o w l y f r o m s o i l s an d a b o u t h a l f is lost, a f t e r one y e a r. It is less persistent than most chlorinated hydrocarbon insecticides, but more persistent than 2,4,5-T; j 4) T C D D is not t r a n s l o c a t e d f r o m the p o i n t of application on the leaf surface to o ther parts of the plant. S o m e of it is w a s h e d of f w i t h rain water; 71 .7 U K A J " fi:! i. 5) . TCDD destruction may be caused by sunlight In!/ ^ water, but not on so l surfaces; -x ' 6 ) . T C D D is not made froii b r e a k d o w n p r o d u c t s of 2 , 4 , 5 - T in soils or in su nl ig h t; /> T /.* .. 7) . A l t h o u g h there are some r e s i d u e s in the liver, l a rg e a m o u n t s of T C D D fed in an a n i m a l ' s diet can be eliminated in the urine and feces; * 8 ) T. C D D was a c c u m u l a t e d f r o m w a t e r by fish in )^ t i laboratory studies. ^ .V ^\ 68986 S M O a f urther I n f o r m a t i o n on the p e r s i s t e n c e and m o b i l i t y of TCDD in the environment will be available in late 1977 as a result of an interagency research agreement between EPA and USDA. The research being conducted at the USDA-ARS D e g r a d a t i o n L a b o r a t o r y is d e s i g n e d to p r o v i d e da t a on the fate of TCDD when Silvex is applied in s imulated home and r e c r e a t io na l turf plots. Dr. K e a r n e y is the Laboratory, le ad er for the investigation. S in ce TC D D is l i ke l y to be fo rm ed in the p r e p a r a t i o n of 2,4 , 5 - t r l c h l o r o p h e n o l and s in ce this c o m p o u n d is c o m m o n to the m a n u f a c t u r i n g p r o c e s s of the p e s t i c i d e s 2,4,5-T , S i l v ex , Erbon, Ronnel and 2,4 , 5 - t r i c h l o r o p h e n o l , all four compounds were included in the dioxin monitoring program as possible s o u rc es of T C D D c o n t a m i n a t i o n . B. 2 , 4 , 5 - T L i t i g a t i o n H i s t o r y On October 29, 1969, the President's Science Advisor a n n o u n c e d that a s e ri e s of c o o r d i n a t e d a c t i o n s was being taken by several g o v e r n m e n t a l a gencies to restrict the use of the h e r b i c i d e 2 ,4 , 5 - t r i c h l o r o p h e n o x y a c e t i c acid ( 2 , 4 , 5 - T ) . This was precip i t a t e d by the release of a screening study c o n d u c t e d by the B i o n c t i c s R e s e a r c h L a b o r a t o r i e s in w h i c h it was found that mice and rats treated during early pregnancy with l a rge doses of 2 , 4 , 5 - T ga v e b i r t h to d e f e c t i v e o f f s p r i n g T he a n n o u n c e m e n t , t o g e t h e r w l t h A r e p o r t s of an i n c r e a s e d o c c u r r e n c e of b i r t h d e f e c t s by Vietnamese newspapers (June and July 1969), elicited Immediate reactions from g o v e r n m e n t a l a g e n c i e s , s e g m e n t s of the s c i e n t i f i c c o m m u n i t y , various lay groups concerned with environmental problems, and from public communications media. Government-sponsored panels of experts^ special c o m m i s s i o n s set up by s c i e n t i f i c o r g a n i z a t i o n s , h e a r i n g s b e f o r e s u b c o m m i t t e e s of the U.S. Senate, and conferences attended by representatives from Industry, government, and academia examined available data and h e a r d expert opin ions. No ne of these g roups, h o w e v e r , were able to c o n c l u s i v e l y resolve the c e n t r a l q u e s t i o n of whether 2 ,4,5-T^con s t i t u ted a risk for human fetuses during p r e g n a n cy as currently produced and used. At least one r e a s o n for fa i l u r e to reach a s a t i s f a c t o r y r e s o l u t i o n of the I s s u e was the p a u c i t y of r e l i a b l e , s c i e n t i f i c ev id enc e. I 0002573 2 A d d i t i o n a l a n i m a l e x p e r i m e n t s p e r f o r m e d early in 1970 c o n f i r m e d that the purest ava il a bl e sa mpl e of 2 , 4 , 5- T g i v e n in large do ses to pr e g n a n t mice, did indeed result in the birth* of m a l f o r m e d o f f s p r i n g (3,4). It was l a t e r reported (5,6) that the 2,4,5-T formulation employed in these studies contained 30ug/g of 2 , 3 , 7 ,8-tetrachloro- d i b e n z o - p - d i o x i n ( T C D D ) , one of the most toxic and potent terat o g e n i c substances known (1,7). On A pril 14, 1970, p r e c a u t i o n a r y actions were taken by the Secretary of Health Edu c a t ion and Welfare, who advised the Secretary of A g r i c u l ture that: "In s p it e of the u n c e r t a i n t i e s of the e x p e r i m e n tal data related to 2,4,5-T, the Surgeon General feels that a p r u d e n t course of ac t i o n mus t be based on the d e c i s i o n t ha t e x p o s u r e to this h e r b i c i d e m,ay p r e s e n t an i m m i n e n t h a z a r d to wo men of child b e a r i n g age." A c c o r d i n g l y , on the f o l l o w i n g day the S e c e t a r l e s of A g r i c u l t u r e , of Hea lth , Education, and Welfare and of the Interior jointly announced the s u s p e n s i o n of the r e g i s t r a t i o n s of 2 , 4 , 5 - T for: "I. In all us es in lakes, ponds or on ditch banks. II. Li q u i d formulations for use around the home, recreation areas and s i milar sites" (USDA-PRD PR 70-1, 20 April, 1970). A notice for c a n c e l l a t i o n of r e g i s t r a t i o n was is su ed on M a y 1, 1970 for: "I. All g r a n u l a r 2,4 , 5 - T f o r m u l a t i o n s for use ar ound the home, recreation areas and similar sites. II. All 2,4, 5 - T uses on crops intended for human consumption." ( U S D A - P R D PR 7 0 -1 3, M a y 1, 1970) All r e g i s t r a n t s were a d v i s e d of these a c t ions, and two of the r e g i s t r a n t s , Do w Chemical and Hercules Incorporated, exercised their right u n d e r S ection 4.e of the Fe d e r a l I n s e c t i c i d e , Fu ngicide, and R o d e n t i c i d e Act (FIF RA) [7 U . S . C . 135 t_ s e q . ] to p e t i t i o n for r.eferral of the m a t t e r (for the c a n c e l l a t i o n 'of r i ce only) to an A d v i s o r y Committee. As provided by that statute (8), the National Academy of Sciences supplied a list from which was selected a n i n e - m e m b e r A d v i s o r y C o m m i t t e e of s c i e n t i s t s with a p p r o priate qualifications from universities and research insti tutes throughout the county. The charge g iven to the C o m m i t t e e w a s to: (a) c o n s i d e r all r e l e v a n t facts, (b) submit a report and recommendations regarding registration for c e r t a i n u s e s of 2 , 4 , 5 - T , and (c) s t a t e the r e a s o n s or bases for these recommendations. The Report from the C o m m i t t e e was s u b m i t t e d to the A d m i n i s t r a t o r of the E n v i r o n m e n t a l P r o t e c t i o n A g e n c y on M a y 7, 1971 (9). The report, r e c o m m e n d e d that 2 , 4 ,5 -T m a y be p e r m i t t e d u n d e r c e r t a in c`ondit*r3'rrsr~nTr u s e s in f o r e s t r y , r a n ge and r i g h t s - o f - w a y p r o v i d i n g : -' ' ... .... pOV/ 398640 >* I i 000257-1 I UKAFT 1. T h a t the limit of 0.1 p p m of c o n t a m i n a t i o n with T C D D be set for all f u t u r e p r o d u c t i o n of 2 ,A ,5-T . 2. T h a t 2 , 4 , 5 - T be a p p l i e d no m o r e than once a year at any one site. - I-".- DOW398641 3. T h a t 2 , 4 , 5 - T be a p p l i e d w i t h p r o p e r c a u t i o n so that it w i l l not c o n t a m i n a t e o t h e r a r e a s w h e r e it may c o m e into h u m a n c o n t a c t . T h e C o m m i t t e e also recommended that this action be reviewed a g ai n w h e n the e x is tin g d e f i c i e n c i e s in i n f o r m a t i o n r e l a t i v e to p o s s i b l e m a g n i f i c a t i o n in the food c ha i n of T C D D have been r e c t i f i e d by specific research directed toward that end. I In July 1972, Dow Chemical obtained an injunction against EPA enjoining further administrative action against 2,4,5-T. In 1973, the U n i t e d S t a t e s C o u r t of A p p e a l s for the Eighth Circuit overturned the injunction and a d m i n i - ' strative proceedings were allowed to go forward (10). A c c o r d i n g l y , a n o t i c e of intent to h ol d P u b l i c H e a r i n g s on all uses of 2 , 4 , 5 - T b r u s h - w e e d ki l l e r was filed wit h the EPA Hearing Clerk on J uly 20, 1973 , under Section 6(b)(2) of FIFRA. All f e d e r a l l y - a p p r o v e d uses of the c o n t r o v e r s i a l brush-and-weed killer, 2,4,5-T, alleged to have caused adverse effects on human and animal health, were to be explored in a public hearing scheduled for April 1974, following com pletion of an Intensive m onitoring p r ogram for detecting dioxin in the parts per trillion (ppt) range (38 FR 19869, July 24 , 1 9 7 3 ) . Th e hearing was to a f f o r d -tB-all c o n c e r n e d parties-- manufacturers and formulators, the U.S. Department of A g r i c u l t u r e (USDA) , the E n v i r o n m e n t a l D e f e n s e Fund ( E D F ) , and other interested groups as well as the EPA - - t h e o p p o r tunity to present ^^r^iji^nt scientific, economic, and o t h e r r e l e v a n t da t"a_ n e e d e d by E P A to w e i g h the b e n e f i t s of the herbicide against the risks involved. The basic question to be resolved by EPA through the hearing process was whether the remaining Federal registra tions of 2 , 4 , 5 - T shou ld be c a n c e l l e d or the c l a s s i f i c a t i o n changed. D u r i n g the p r e p a r a t i o n of the h e a r i n g s , it was e v i d e n t that the p e s t i c i d e 2 , 4 , 5 - T , p e r - s e , p o s e d no' s p e c i a l cause for concern, when used as d i rected.* * Note: 2,4,5-T, *2,4,5-TCP, Silvex, Erbon, and Ronnel are n o w u n d e r r e v i e w in the O f f i c e of S p e c i a l P e s t i c i d e Reviews ( O S P R ) , EPA, as candidates for Rebut t a b l e P r e s u m p tion A g a i n s t R e r e g i s t r a t i o n ( R P A R ) . The toxicity of these pesticides and/or the associated dioxin contaminant are c onsidered in this review process. 712l_ 0002575 I -7- DRAFT ' O1' }([ ' Residue monitoring analyses for 2,4,5-T in environmental samples, including food, have for the most part led to negative findings. In those cases where positive results were obtained, levels were low (normally less than 0.1 ppm)_. Thi-s^Fo^clusion was based primarily on the results of EPA forced feeding study of the levels of 2,4,5-T in c(ows-nri'lk and calf tissue, following exposure. These observat-ions, coupled to the low order of terategenicity for 2,4,5-T when tested at the highest purity available, lead to the conclusion that human exposure to 2,4,5-T from normal usage posed no discernible problem. On M a y 10, 1974, the information hearing was expanded to Include all insecticides and herbicides having in their manufacturing process 2,4 ,5-trichlorophenol (TCP). These include the pesticides Silvex, Erbon, Ronnel and 2,4,5TCP all of which have the potential of containing TCDD. On July 24, 1974, the Agency withdrew cancellation and information gathering proceedings Initiated against the herbicide 2,4,5-T and related compounds. The proceeding was w i t h d r a w n because of the inability of the Agency to monitor food for residues of 2,4,5-T's. highly toxic contaminant TCDD with the necessary analytical precision. The lack of evidence that 2,4,5-T use results in exposure of man to TCDD made a final determination on the "unreasonable adverse effects" caused by the use of 2,4,5-T extremely difficult, if not impossible. Accordingly, while the 2,4,5-T notice of hearing was withdrawn, the Agency stated that it: "will continue its TCDD residue monitoring program and will take such further action as it deems a p p r o pr i a t e once the results of the monitoring project are a v a i l a b l e . " 39 FR 24050 June 28, 1974. On July 25-26, 1974 the Agency held a Dioxin Planning C o n f e r e n c e in W a s h i n g t o n ,.D .C . The public meeting was held primarily for those parties having an Interest in the w i t h d r a w n 2 ,4 ,5-T/dioxin hearings. The nature of the m e e t i n g was to address data analysis and retreival (in the areas of anal y t i c a l methodology, toxicology and monitoring) with empha s i s on analytical m e t h o d o l o g y for TCDD at the parts per trillion level (ppt). As a result, the Ag'ency promulgated the Dioxin Implementation Plan which is designed to develop the required analytical methods for the det e r m i na t i o n of dioxin residues in e n v i r o n m e n t a l samples. DOW 398642 i 000257G DRAFT II. Dioxin Implementation Plan A. Introduction / I-' In September 1974 , the Agency sent out a Preliminary Dioxin Implementation Plan (DIP) to all attendees of the Planning Conference requesting that it be reviewed and that they " provide input on what changes or additions would be a d v i s a ble in the overall plan. After these responses were received a final plan was prepared in February 1975. It consists of two parts, a short term monitoring program and a broad research plan which would take four to five years to complete. The EDF, USDA and D o w Chemical agreed to participate in a monitoring program established by EPA, which would utilize improved analytical m ethodology capable of TCDD d e t e c t i o n at O O_ the ppt level. ~ The short term beef fat monitoring plan was a joint plan (representatives from EPA, USDA, EDF and Dow Chemical) in which beef fat and liver samples were to be collected at six month intervals to permit timely rvaluation of any available evidence for TCDD residues. Such rvaluation could lead to (1) m o d i f i c a t i o n s in plans for future research and monitoring and/or (2) r e c o n s i deration of the d e s i r a b i l ity of initiating some form of regulatory action on 2,4, 5 - T or other pesticides contaminated by TCDD. At each point all of the Information then available should be considered even though the specific experiment producing it might not be completed. The guiding principles for the sampling plan were: CO oq OD ' CO a. The samples should be representative of beef actually being consumed by some segment of the population. b. The sample should represent cattle likely to be marketed for human consumption and grazed on lands treated with 2,4,5-T and thus likely to maximize the probability of containing 2,4,5-T (or TCDD). Between February and March, 1975, the first 85 beef fat and 43 liver samples wete collected. At the outset it was decided that emphasis would be placed on analytical m e t h o dology. Due to the complexity of the analytical tec h n i q u e to determine TCDD at the ppt level, analysis for these first 128 samples were delayed and not completed until M a y 1976. A p p r o x i m a t e l y twenty-five percent of these samples was taken from non-treated areas, i.e., where 2 , 4 , 5 - T or TCDD is not likely to be found. One laboratory prepared all sample extracts, and identical aliquots were sent to all participating analytical laboratories. The purp o s e of having one laboratory perform all cleanup of samples was to minimize possible errors in the procedure. 7122 0002577 i B. Analytical -9- h in &$:? :L. _____ 2 5 Ahi 1977 1. Phase I Beef Fat and Beef Liver A n aylyses The primary analytical achievements of 1976 have been the; 1) completion of phase I beef fat and liver analyses using combined gas c h r o m a t ographic (GC) and using high resolution, mass spect rome tric (!IS) techniques; 2) analysis of technical grade pesticides for TCDO residues; 3) further refinement of cleanup method o l o g y for e n v i ronmental samples; and 4) the development of a preliminary method for the extraction analysis of human tissues and milk for TCDD residues. The progress made in the area of TCDD anal y t i c a l m e t h o dology is noteworthy. By far the most significant data out of the above is the phase I beef and liver analyses. B ased on the repeated ability of more than 1 laboratory to detect TCDD in identical sample aliquots, it is clear that dioxin can be detected at the ppt level. Th e data as report- ed by the collaborators at the June 15 , 1976 m e e t i n g in i . - *. / Washi n g t o n (see section V A p p endix part A) are sugges tive of >- TCDD residues in beef fat ranging from 20-60 ppt. Phase 1 of the prog r a m was primarily intended to identify' a cceptable methods for extraction and analysis of TCDD in environmental samples. This phase of the p r o g r a m is essentially complete. A short term study (90 days) will be conducted, prior to initiation of phase 11, to determine the optimal combination of extraction and a n a l y t i c a l methods. Once this combination has been determined, the Agency will immediately proceed with the second p hase of the program. Phase 11 will proceed under an experimental design intended to insure the accuracy and legal su f f i c i e n c y of the a n a l y tical data being developed (see section 111 Future AnalyticalStudies). y? 2. Survey of T r i c h l o r o p h e n o l P e s t i c i d e s for TCDD Residues. Samples of tric h l o r o p h e n o l materials wer e received from the basic m a n u f a c t u r ers of pesticides for a n a l y s i s of TCDD content by EPA. The limit of detection in the analysis was 0 ppm, corresponding to the 0.1 ppm limit for TCDD conta m i n a t i o n of 2,4,5-T, as set by the 2,4,5-T Advis o r y C o m m i t tee (NAS) in 1970. S e v e n t y - t h r e e out of the 75 samples col l e c t e d did not contain TCDD at. the above limit of_dotec- tion. The remaining 2 samples, tonneT^and N a - 2 ,4,5-TCphenate are believed to contain 0.107 and 0T312 ppm of TCDD r e s p e c t i v e l y . These samples will be reanalyzed for confirmation. If the residue level continues to exceed 0.1 ppm, the Agency w ill take regulatory action to insure that T C D D is reduced to an acceptable level. 0002 i.78 -.1 0 - DRAFT A second study of the TCDD residues In 'technical pesticides will begin in mid-1977. The limit of detection in this study will be lOppb. Samples for this study will be taken by representatives from Pesticide Toxic Substances ^ Enforcement Division, EPA. The data will provide EPA with a more compren h e n s i v e analysis of the intrinsic level of TCDD residues in the pesticides under c o n s i d e r a t i o n , prior to use. C. T o x i c o l o g i c a l Effect Level The TCDD toxicology data is well documented in the EPA conference report of January, 1976 and in the Nay, 1976 addendum and therefore does not need to be reviewed extensively herein. Additional data however have been g e n erated which expand our knowledge of the risks associated with human and nontarget exposure to TCDD. Dow Chemical Company has recently completed a two-year feeding study with 2,3,7,8-TCDD in male and female rats. Preliminary data indicate that at a level of 0.001 ug/ k g / d a y of TCDD (which approximates 20 ppt TCDD in the rat's daily diet) the effect was similar to controls in all parameters measured. At the dose of 0.01 ug/kg/day however increased urinary exc r e t i o n of p o rphyrin and Increased liver weights were observed (personal communication, Dr. Koyiba, Dow Chemical Co.). Toxicologists from the Criteria and Evaluation Divi sion (CED) , O P P , EPA have reviewed this data and established a range of 80 to 200 ppt as the effect level (EL) for human e x p o s u r e to beef fat containing TCDD. To determine the EL it was assumed that the amount of TCDD in the beef was constant and that f h e amount of beef fat ingested is from 1 to 2.SZ of a humanV total diet. The data generated thus far for the 85 beef fat and 43 liver samples analyzed in phase I of the monitoring program do not Indicate that the EL for TCDD exposure through^1 beef fat ingestion has been exceeded or reached. It is e m p h a sized howe v e r that exposure to TCDD through Ingestion of beef fat is only one possible source of exposure. The e n v i r o n m e n t a l samples scheduled for analysis in phase II of the Dioxin Implementation Plan range from soli to human types. The d i v e r s i t y of these samples is Intended tp provide EPA with a better understanding of the potential for TCDD to b i o a c c u m u l a t e once it is released into the e n v i r o n ment and thus present multiple sources of exposure to humans or other non-target organisms. III. Future Analytical Studies Phase II of the Dioxin I m p l e m e n t a t i o n Plan is designed to provide the Agency with precise analytical data on the residues of TCDD in e n v i ronmental samples. -n - Thc purpose of the design is to establish a practicable experimental procedure that (a) will permit development of a "best fit" recovery curve (or curves) for quantification of .35 C^-TCDD spiked in beef fat and liver at levels ranging from 0 to 100 ppt, and (b) will allow statistically reliable m e a s u r e m e n t s of the following parameters: 1. The accuracy and p r e cision with which ppt levels of 35 C ^ - T C D D radded to beef fat and liver can be extracted at OPP's Pesticide Monitoring Laboratory and quantified at the collaborative analytical laboratories; DOW 3186+6 2. The accuracy and pre c i s i o n with which these a n a l y tical laboratories can quantify ppt levels of 35 CJ^-TCDD providedasstandards; 1 3. The relationships between measurements of TCDD in standards and measurements of TCDD extracted from beef fat and liver spiked at the same levels as the standards; 4. The consistency of laboratory performance throughout the experiments, as measured by the relative magnitude of statistical interactions; 5* The precision of PML's extraction procedure relative to the precision of the GC-MS quantitation. Based on the ability of the collaborator to detect TCDD in spiked samples, an eval u a t i o n can be made of the v a l i d i t y of the TCDD levels reported in blind environmental samples. It is anticipated that the above procedure can be applied to all environmental samples scheduled for analysis on phase II. Highest priority however will be given to the analysis of phase II beef (fat and liver) and human (fat, liver, and milk) samples. The Agency presently has a total of 167 human samples (milk, liver, adipose) in storage at our Pesticide Monitoring Facility, Bay St. Louis, Miss. These samples were collected in M i s s i s s i p p i and Arkansas from persons who might have been exposed to TCDD through the use of 2 , 4 , 5-T on rice. A d d i t i o n a l l y , EPA is establishing a v o l u n t a r y bio p s y and mother's milk monitoring program in Oregon, to further i n v e s t i g a t e the signific ance of direct and indirect human e x p o s u r e to TCDD through the use of 2 ,4 ,5-T. This program is intended to provide EPA with information on the s i g n i f i cance of indirect and direct human exposure to TCDD through the comp a r i s o n of samples taken from persons living in urban versus forested sites, respectively. 7125 For specific details on the phase II analytical p r o c e dures refer to Section V, A p p endix, part B. 0002580 IV. Summary UKAi' i / /, EPA is investigating the frequency and level of TCDD residues in the environment as basis for determining the toxicological risk associated with human and other non target exposure to TCDD. - OOV/ 398647 A. Phas*e I of the Dioxin Impleme n t a t i o n Plan was primarily designed to identify acceptable research approaches e xtraction and analysis of TCDD residues in environmental samples. Existing data indicate that the methods are satisfactory for analysis of beef samples (fat and liver) and that TCDD is present In a small percentage of the beef fat samples taken from cattle with a known exposure to 2 , 4 , 5-T. All of the b e ef liver samples a n a l y z e d _were negative. As a part of phase I ,i the Agency is conducting "further research to determine the a c c e ptability of these methods for analysis of other sample types (humans, small mammals, birds, soil, etc.) and the optimal combination of extraction and analytical methods. The latter study will be short term (90 days) in order that the Agency might rapidly proceed with phase II analysis. for B. The Agency has analyzed 75 samples of pesticides which, due to their manufacturing process, could contain TCDD. Seventy three of the 75 samples analyzed, did not contain TCDD at or above the 0.1 ppm tolerance level set by the Science Advisory Committe|c^n 1970. The 2 remaining samples r o n n e l . and Na-2.4.5-T phenate are believed to contain .107 ppm and .312 ppm of TCDD respectiv e l y . These samples will be reanalyzed for confirmation. If confirmed EPA will take regulatory action to Insure that the-level of TCDD in the affected p e st icides is reduced to the 0.1 ppm 1 ev e 1. An a dditional analytical study for T CDD residues in technical grade pesticides will be conducted in 1977. This study will analyze a greater number of samples at a lower limit of det e c t i o n (10 ppb) than the preceeding analyses. EPA is presently selecting an analytical laboratory for this research. C. Phase II of the Dioxin I m p l e m e n t a t i o n Plan is intended to provide the Agency with increased information on the range and possible tendency of TCDD to b i o a c c u m u l a t e in the environment. All analyses in phase II will be based on a s t a t i s t i c a l approach designed to improve the precision of the q u a n t i f i c a t i o n of TCDD residues reported. I A n a l y s e s of the human (fat, liver and milk) and beef (fat and liver) samples now in storage will receive the highest priority when the Agency proceeds with phase II of the plan. 0002581 I U'flW'-u a t D. Existing beef fat and liver analytical data from phase I of the Dioxin Implementation Plan do not indicate that the toxicological effect level for exposure to TCDD through ingestion of contaminated beef, has been exceeded. This is only one source of exposure however. Additional analytical* -- - data are necessary to a^fTess the overall toxicological risk associated with accumulative exposure from different sources of TCDD. In addition to the human samples now in ^tirage, EPA is establishing an elective biopsy and mother^ milk program in Oregon to further assess the potential for human exposure to TCDD through the use of 2,4,5-T. V. A p p e n d i x ' , A. I n t erpretation of Phase I Data by A n alytical Collaborators A meeting of the analytical collaborators (Dow Chemical, Harvard University, and Wright State University) was held on June 15 . 1976 . to discuss the results obtained to date. As a r e s u l t ot this meeting the following statement was Issued by the analytical collaborators. 1. Of the beef fat famples (85) analyzed, one shows positive TCDD level at 60 ppt; two samples appear to have TCDD levels at 20 ppt; five may have TCDD levels which range from 5-10 ppt. While several laboratories detected levels (5-10 ppt) in this range, the values reported were very near the sample limits of detection. T here exist a great deal of uncert a i n t y of the procedure below 10 ppt. 2. The a n alytical method is not v a l i d b e l o w 10 ppt O j M*~*s**~* 3. An I n t r o duction to a neutral 'extraction technique shows promise of the capability of detecting levels below 10 ppt. This was d e monstrated by comparative data at the lower parts per trillion range (5-10 ppt). However, this method has been demonstrated by only one laboratory at this time and has. not been validated below 10 ppt by another competent analyti cal facility. A. The samples analyzed were peritoneal fat and kidney fat taken from cattle which had grazed on rangelands of_known treatment with 2 ,4 , 5 ,-T. Controls were "the same s a m p l e " T y p e -TiTken from cattle from non treated areas within the same state. DOW398648 7'i1jZ*94,7( 0002582 i 1\ -in- UKAt* I' 5. Of Che liver samples (43) analyzed, only one sample sugge,sf\any TCDD residue, but the residue observed was toOclose to the sensitivity of the sample detecVfon limits for quantitation. The fat sample analyzed from the same animal showed no TCDD * residue. Three liver samples (for which fat samples were analyzed and showed positive data) showed no TCDD residues. 6. None of the collaborators reported TCDD in samples of beef fat taken from cattle in non-treated areas (at the sensitivity of the analytical method). Three of the laboratories receiving liver samples from cattle in non-treated areas observed no TCDD in the samples. 1 ( e-* Due to the improvement and agreement among the anlytical collaborators with the analytical data, the above data supercedes the EPA beef fat monitoring data released in m e m o r a n d a dated August 5, 1975 and December 19, 1975. 8. Phase II Experimental Design The design phase II calls for preparation of two "pools" of control beef fat (say. Pool F and Pool G) and two pools of control beef liver (Pool L and Pool M) from which all spiked samples are to be constructed. The fat pools are c o n s t r u c t e d from equal amounts of fat from each control animal selected, using a separate set of animals for each pool. Liver pools are constructed in a similar manner. (Necessary pool sizes are to be determined by Dr. Aubry D u p u y after study of the design). 0_ 1 . CO CD 00 CD \ >u CO , Eleven samples each are prepared from fat pool F and liver pool L. T h e samples from each pool are spiked.* i n d i v idually at levels between 0 and 100 ppt of 35 QjL-TCDD. Samples are then extracted, and the extract is divided into three equal aliquots for shipment to the analytical labs, (the spiking levels are systematically spaced). The spiking system allows close spacing at lower levls and moderate increases in spacing at higher levels). Next, five samples each are prepared from fat {fool G and liver pool M. The samples from each of these pools are spiked individually at levels between 0 and 100 ppt of TCDD. These samples are extracted and divided into three equal aliquots for shipment as above. } , | It is necessary that each laboratory receive enough extract from sets G and M and from the comparably-spiked samples of sets F and L to allow dup l i c a t e m e a s u r e m e n t s of each sample. For other samples, labs need receive only enough extract for a single measurement. 0002583 O O W 398650 -11)- A single s e r i e s ^ s t a n d a r d s , prepared as sec S and set will serve for comparison both with fat and liver samples Sec S will c o m p r i s e eleven a l i q u o C s spiked aC levels, c o m p a r a b l e Co faC sec F and l i v e r sec L. T, Scandard seC T will contain 5 aliquoCs spiked aC levels comparable Co faC set G and liver set M. Again labs must receive enough standard from set T and corresponding levels of set S to permit duplicate me a s u r e m e n t of each standard. Thus, a total of A8 samples -- 16 standards, 16 fat and 16 liver -- are needed; these will require a total of 78 measurements by each laboratory, pounting duplicate measure ments of specified samples. A diagram of the design is ' attached. All samples -- fat, liver and standards -- are to be prepared and shipped in random order,except that aliquots from a given sample wil be shipped to the three labs slmultaneously. Laboratories are to analyze the samples in t h A ^ order in which they are received. Labs are to p e r f o r m blind analyses, i.e., they are not to know either the origin of the material or the level of TCDD in any sample. A sample numbering system should be used that provides no clue to sample identity. All data will be used to develop recovery curves. Data from duplicate analyses of replicated samples will be used to measure extraction and GC-MS precision. Data evaluation will be by analysis of v a r i a n c e and regression methods. i t 0002  '..I'1 ' ' ' Ji*-. DRAFT TCDD Standard ..' .*1*.1*. *, : , Beef Fat oof Llvor ^ ** ^Pii . Lovcl innt) Measurements Pool Codo tiCibt A L&bi B Lftb G Measurements Pool Codo Lab. A Lab. B Lab. G Moaouremonts Pool Code lab. A lab. B .lab. G .* A !. 3 A 'T 5 : s Cs D' E .. s F. T F is 0s r, ii." ip H s i s i- : V-' T j- s K; s K .= T ?V 'r 2 ' 2 1 ' 1 ! 2 2 1 '2 2 1 2 2 *1 2 .2 ' ' 2,: 2 ... ' ` 2 ' :*2` 1 .1. . 1 ; - 1 1 i 22 22 11 . 2 / 2.' 22 1 .. 1 22 2 .2 11 2 2 . '2 ` - 2 : ' 1 Tot. Poolo 16. Tot. M . \ .>M.`;>>' < if:f*.V. '/ \ v*` . v.V. '*i' '.iV., :\V." /u;> . .V o' o ro cn GO S--V* .j 1 ' ' Ti 2 2 2 H2 2 "2 L1 11 L 1 * 1. 1 I. 17 .1 1 I. 2 2 ' 2 M2 22 L1 11 L2 .2 2 M2 22 L1 11 L2 22 M2 2 2 ' L ' 1 " . 1 . 1 .L 2 ; 2 2 M 2 2 2 26 26 j ,..T998&e,i|'00 . * l *. t #. I < DRAFT l-: !);/ REFERENCES 1. B.A. Scliwetz, J.M. Norris, G.L. Sparshu, V.K. Rowe, P.J. Gehering, J.L. Emerson, and C.C. Gerbig in Chlorodioxins-- -Origin and Fate American Chemical Society Advances in Chemistry Series, 1 2 0 , (E.H. Blair, ed) Washington, D.C., American Chemical Society, 1974. p5 5. 2. P.C. Kearney, C.S., Helling,A.R. Isensee, E.A. Woolson, P.O.J. Ensor, G.E. Jones, J.Ri. Plimraer, Chlorodioxins In Pesticides Soils & Plants, J. E n v . Qual 2:171-7 1973. 3 . - K.D. Courtney,D.W. Gaylor, M.D. Hogan, H.F. Falk, R . R . . Bates and I. Mitchell, S c i e n c e , 168 , 864(1970). 4. K.D. Courtney and J.A. Moore, T o xicology and Applied Pharamacology, 2 0 , 396(1971). 5. I.L. Emerson, D.J. Thompson, C.G. Gerbig and V.B. Robinson, Toxicology and Applied Pharmacology, 1 7 , 317(1970). 6. G.L. Sparshu, F.L. Dunn and V.K. Rowe, Food Cosmetic T o x i c o l o g y , 9, 405(1971). 7. G.W. Cribble, C h e m i s t r y , 47 15(1974). 8 . FIFRA, Section 6(d), as amended in 1972. 9. Report on the Advisory Committee on 2,4,5-T to the A d m i n i s t r a t o r of the Environmental Protection Agency, . M a y 7, 1971. 10. Dow Chemical Company vs. Ruckelshaus, 477 F. 2nd 1317 (8CRI 1973). 11. 6 (b) 2 FIFRA as amended in 1972, information gathering procedure to determine whether or not a pesticide should be cancelled or its registration changed. Changes in the Act in 1972 added 6(b) (2) to supplement 6(b) (1), c a n c e l l a t i o n -of a pesticide or to change its classification. 12. Toxicology Meeting Summary, February 21, 1976. Office of Pesticide Program, U.S. Environmental ^Protection Agency, Washington, D.C. D O W 398652 i 0002556 U a. IAA. .\A 82 THE LANCET, JULY 9,1977 * ' THE LA Letters to the Editor EFFECTS OF DIOXIN EXPOSURE Sir,--Dr Lapone (May 14, p. 1049) continue! the inter minable argument--often largely political--in which the allqtcd-long-term effect of 2,4,5-T/2,4-D herbicide praying in Vietnam are compared with industrial accidents over the 28 years or so during which factory workers have been exposed to tetrachlordibenzo-P-dioxin (dioxin, t.cjld.). Dr Lapone cites extensively from Prof. Ton That Tung and similar Viet namese sources; having several times visited Italy to assist the Italian Government with the Scveso problem, 1 fed I must try to put some oftheseassertions into perspective. inteI rsphraeltledexawmitihnesoimneilaqruoctaautitoionni.nTdheetaijlo; uthrneaol thSeorpsemra,ythbee source of almost all Dr Lapone's data, slates that from 1961 to 1969, 500 kg of dioxin were sprayed over Vietnam. During these 9 years, the total US. production of 2,4,5-T for every purpose was 97*5 million lb of 2,4,5-T add. This contained an average of 1-91 pan per million o f 2^,7,8-t.c.d.d. (i.e., 2 p.p.m. approximately). Thus the total t.c j>.d. content of the entire U.S. output for this period was only 195 lb (82-5 kg)', and only pan ofthis ever reachedVietnam. Dr Lapone, by referring only iO the explosions, also attempts to suggest that the numerous factory workers exposed to T .C JJJ). over a period of nearly 30 years, have not been chronically as well as acutely exposed. However, the explo sions, dramatic though they have been, have poisoned far less workers than has poor plant hygiene with iu chronic taxidty. The imponant Czech experience,* for example, is essentially one of chronic poisoning yet Dr Lapone does not mention it, and when we compare the relatively enormous concentrations of T.C.D.D. in such industrial exposures with an average con centration of T.CJLD. in the Vietnam defoliant of only^-O mg/kg we can readily appredate that those industrially exposed should by now be manifesting a high rate of liver cancer, chromosome damage, and so on if Prof. Ton That Tung's assertions are correct. So far there is no published evi dence to confirm this among those who have been poisoned in industry in yean gone by. 2,4,5-T and 2,4-D are not "both synthesised from trichlorophenol". 2,4-D is made from 2,4-dichlorophenal which con tains no toxic chlorinated dioxin at an and made up 50% of the agent-orange herbiddeused in Vietnam. The statement that chloracne is a -consequence of hepatic damage revealsa profound ignoranceo fthesubject. There is no foundation for the statement that T.C.P.D. is bring disseminated either superficially or deep in the soil at Scveso.4 Finally, the spraying of a 37 hectare (ha) plot of lakdand soil in Florida from 1962 to 1964 with the Vietnam defoliant (507 2,4,5-T and 2,4-D), at a rate of 4950 kg/ha, is informa tive. The TX.P.P. contamination o f the 2,4,5-T averaged 2-0 p.p.m. (i.e_, 0-5 kg/km2). Since the total 2,4,5-T'produced in the U.S. between 1961 and 1969 contained only 82-5 kg T.C.D.D. it follows that had the above concentration of herbi cide been sprayed on to Vietnamese soil the total area covered each year would have been approximately 18 km2, which we know to be absurdly low. Therefore the Vietnam concentration per km2 must have been less than the Florida concentration. The significance of the Florida experiment lies in the fact that post-mortem and ultramicroscopic examination of beach mice, fish, and reptiles in the sprayed area revealed no significant al- teration from controls some years later, although tissue levels Respecte ofT .cn.D . were still appreciable.2 1 rate of - All such facts, should bekept in the forefront ofone's mind ' when evaluating such data about Vietnam and Italy as ^ k I doub: Z mouth, died by Dr Lapone. Only by so doing will the truth finally impassi emerge and Scveso cease to be both a medical and polit y ,abartio: battleground. .lattea 1 UP enough Depennimi o f Dermatology, Piinetti Margsm Hotptial, Swindon5N14JU K.D.CROW , Vietnar :in my I Ithan ine VThis letter has been shown to Dr Lapone whose reply foL Q rinogeni lows.--Ed.L . cutaneo damage Sir,--I thank Dr Crow for-his interesting letter on dioxin and for his corrections to someofmy statements. As the accompanying figure shows, the total U.S. produc tion of 2,4,5-T between 1961 and 1969 was 97-5 million lb. In 1969 2,4,5-T production dropped back to 5 million lb, m it may be concluded that about 52 million lb was used in Via. nam (i.e., about 44 kg of t.c jl d . and not 500 kg, the figuic I died1). However, in -Vietnamspraying may not have been the only source of dioxin. 2,4,5-T heated to 500-600C will yiey 50 000 p.pjn. T.C.D.D.,2 and such temperatures may have H--n * achieved on the battlegroundsofVictnam. 2,4-D gisefor of2,4-D Odor; I stated is pania, to th U oeous sec ceousdai Dr O ment ths deepmti fini lette places at 20 an an n in fa li.1' Severo, a: we bave profondi I too w happa if dai intere HeepiialdeS tinte I. SAC The commission of the American Assodathm for the Advan cement of Sdences, which went to Vietnam in 1972, took sa pies of edible fish from Vietnamese rivers. These were analysed at Harvard University.2 A specially designed spearopbote . metric method revealed mean dioxin concentrations of 540, yg/kg, the l.d.m for guineapig being 600 yg T.cn.DVkg bed^ _wdghi.J In 1974 the U.S. National Academy of Sdences C o "mince on the Effects of Herbiddes in Vietnam was not aUew collect animals with a view to investigating the Vietnsfflwe food chain more fully, but the Committee did say of diaxie that "it is a proven teratogen in mice and rats" and it strongly recommended " repeated sampling of the Vietnamese food chain, where low levels of dioxin have already been found".4 The Czech experience mentioned by Dr Crow could beoat of chronic poisoning, though very different from the shuatica in Vietnam or Scveso. A high rate of liver cancer would 1* Si,--F dene in p: bas b ea ; subdinical fisiNing w k r lie m velocities ir 27 patic ere indie the rem: a more gei *udy 17 ps {ability of < the extraoc tUe 1. Kenner. P. C . Uanhon. E. A. Henne. A. R, Hcihng. C S. Bw m . H ltk. P crtpett. I973.no. 5,p.273. 2. Jimrfc,1. Kokmkv.K . Kabcr.K.C ai.D m . 1973,43,306. 3. Henne.A. K. Kraknct greento (he Eneironmenl Contente a t theOrel SmlnhAcademeofSciences,Keb,7-9,1977. 4. Rice. A. Cretner It Vainer, csnwhaai eciniiu to ibe Lombardy Region. Pcnonalcnmnwiucaiian. 1. Young. A. U Hwikcn. C L . Arnold, E. U CupcOo, j. AL, C fk^ L G . depanotem of dnaotinr and biologirl testato, U.SAJ. 4* deny,ColoradoS0S40.0ewko.1976. ' fr 1. Rignaaa.G.,Frenuli,N-,Zito,R.opera,1976,796,67. 2. Jaubcn.A.Xckerrkc. 1974,7,161. ". 3. Shaphv.D.Sctmce. 1973.ISO,2S3. 4. Shipley.D.M l. 1974,113,1179. 0006704 : S ta ; V n-Coh, 1Shu 7133? 153 133 WORLD HEALTH ORGANIZATION O M INTERNATIONAL AGENCY FOR RESEARCH ON C A N C E R S co IARC MONOGRAPHS en CO w ON THE EVALUATION OF THE CARCINOGENIC RISK OF CHEMICALS TO MAN Some Fumigants, the Herbicides 2,4-D and 2,4,5-T, Chlorinated Dibenzodioxins and Miscellaneous Industrial Chemicals V O L U M E 15 CG067SS INTERNATIONAL AGENCY FOR RESEARCH ON CANCER LYON AUGUST 1977 7135 USZ3ls. Preliminary, 3-Tu.cal.q /erment 2,4-0 AND ESTERS 1- Chemical and Physical Data ^ 1-1 Synonyms.and tr*d< names diem- Abstr. Services Reg. No.: 94-75-7 ~ Ctem. Abstr- Name: (2,4-Dichlorouhenaxy)acetic acid DichlorophencBcyacetic acid; 2,4-dichlcrophenOKyacetic acid For a representative list of otter synonyms ana trade names of products containing 2,4-0, its salts or esters, either as die sole active ingredient or as mixtures with other compounds, see Appendix A. 1.2 Chemical formula and molecular- weiaht ci OH Mol. wt: 221.0 1.3 Chem ical and physical properties of the pure substance Fran Weed Science Society of America (1974), unless otherwise specified (a) Description: Odourless .white crysta ls (b) Boiling-point: 160C at 0.4 mn (c) Melting-point: 140-141C (d) Spectroscopy data: Infra-red and ultra-violet spectra are given by Gore e t a l . (1971). (e) Solubility: Soluble in 95% ethanol and in acetone, dioxane and isopropyl alcohol (f) Stability; Stable up to and including its melting-point (q) Reactivity: Forms--^its that are soluble in water G006786 7136 HI DOW 1 37*5306  O W J375307 1.4 Technical products and impurities Technical grade 2,4-D is available in the OS as the free acid (98% purity), as salts (dimetbylamine, mixed ethanolamine and iscprcpanalamine, lithium and sodium) and as esters of the following alcohols; isopropyl, -butyl, sec-butyl, iso-octyl, 2-butaxyethyl and butnxypolyprcpylene glycol (CS International Trade Cancissian, 1976a). Of 28 samples of 2,4-0 tested for content of chlorodifcen2o-pcrc-dixBdns, one was found to contain <10 mg/kg bexachlorodibenzo-ptzra-diaxin (Woolscn a lat ., 1972). Bis(2,4-dichlarophenaxy)methane has been identified as the major contaminant of 2,4-D, and bis(2,6-dichlarophenoocy)methane and 2,2' ,4,6'-tetrar.hlorndiphenaxynethane as minor contaminants- (Huston, 1972). ff-Nitrosodioethylamine1 has been detected at a level of 300 u g A in dimethylamine salt of 2,4-D which was stored in metal containers the interiors of which had been presprayed with sodium nitrite as an antioxi dant (Fine a t a l . , 1977). Technical 2,4-D produced in Japan is more than 99% pure. 2. Production, Use, Occurrence and Analysis Far background information an this section, see preamble, p. 17. 2.1 Production and use (a) Production 2,4-D was prepared in 1941 by the interaction of 2,4-dichlorophenol, mcoochloracetic acid and sodium hydroxide (Pokomy, 1941), and a similar process is believed to be used in its connercial production . Production vos first reported in the US in 1944 (US Tariff Comission, 1946). The quantity of 2,4-D produced,increased steadily between 1963 and 1968, when it reached a maxim m of 36 million kg; production decreased to about 20 million kg in 1970 (US Department of Agriculture, 1973) and See IABC, 1972 `1 1 2 000(3767 ire, l, toxins, sen the 972) . Clf ar sien, and -3 i DOW 1 375308 gradually increased again, to an estimated 27 million kg in 1974. In 1975, three US companies reported production of 2,4-0 acid; three others SSSmreported the production of esters or salts of 2,4-0, presumably purchased acid. Separate production data far 1975 are availablexrdy for the dimethylamine salt of 2,4-D, 11.6 million kg of which were produced, ' and for the iso-octyl ester, 4.5 minion kg of which were produced (US International Trade Connissicn, 1976a). In 1973, 115 thousand kg of 2,4-0 acid and 57 thousand kg of mixed butyl esters were imported through the principal US customs districts (US Tariff Carraission, 1974). In 1974, 365 kg of the mixed butyl esters were imported (US International Trade Cannissian, 1976b). Garibined U5 exports of 2,4-0 and 2,4,5-T amounted to 5.7 million kg in 1975 (US Department of Connerce, 1975). The Federal Republic of Germany and the UK are the major producing countries in 'western Europe, where annual production is estimated to be 3-30 million kg; in eastern Europe it is estimated to be less than 10 million kg. 2.4- 0 was first produced commercially in Japan before 1945 by a process similar to that used in'the US. Production in 1975 by two pro ducers amounted to 511 thousand kg, and 176 thousand kg were exported. About 90,000 kg were imported into Australia in 1975-76. (b) Use 2.4- 0 is a systemic herbicide widely used for control of broadleaf weeds in cereal crops and sugar cane and on turf, pastures and non-crop land (Weed Science Society of America, 1974). It is also used to control the ripening of bananas and citrus fruits, to delay preharvest dropping of seme fruits and in sane countries as a fungicide for the control of A lts r r a r ia rots when lemons are to be held for storage (WHO, 1975). An estimated 27 million kg or 2,4-D acid equivalent, largely in the form of esters and salts, were used in the US in 1975, as follows: wheat and ocher small grains, 31%; c o m and grain sorghum, 26%; pasture and rangeland, 25%; industrial and carmercial uses, 9%; lawns and turf, 5%; aquatic weed control, 3%; rice and fruit, 1%. 0006788 <*71JLOo'Hs 113 DOW 1375309 2.4- 0 was used to defoliate jungle areas in south Vietnam, where it was a component of 'Agent Orange' (a 50:50 mixture of the -butyl.esters of 2,4-0 and 2,4,5-T, containing up to 30 mg/kg or more TCTD) (Davis, 1974). About 40 million litres of 'Agent Orange' were sprayed in south Vietnam between 1965-1971 for defoliation or crop destruction (Cccmittee on the Effects of Herbicides in Vietnam, 1974). National tolerances are in effect in several countries. Examples were reported to the Joint Meeting of the EAO Working Party of EScperts cn Pesticide Residues and the WHO Expert Canoittee on Pesticide Residues in 1975. The previously established *<-rapt-ahia daily intake for man of 0-0.3 mg/kg bw was considered and confirmed at this meeting (WHO, 1977). The OS Occnraticnal Safety and Health Administration health standards far exposure to air contaminants require that an employee's exposure to 2.4- 0 does not exceed an eight-hour time-weighted average of 10 mg/m3 in the working atmosphere during any eight-hour work shift of a forty-hour work week. The corresponding standard in the Federal Republic of Germany is also 10 mg/ta3, and the acceptable ceiling concentration in the USSR is 1 mg/in3 (Winell, 1975). 2.2 Occurxe ce 2.4- 0 is not known to occur as a natural product. It is broken down by soil microorganisns, and there is reportedly no -accumulation in the soil as a result of normal agricultural use (('feed 'Science Society of America, 1974). In a continuing program involving the monitoring of pesticide residues in food, the U5 Department of Health, Education and Welfare found a decreasing level of 2,4-0 in food samples collected at retail outlets during the period 1965-1973. Between June 1964 and April 1965, 2.4- 0 was found in 10 of 216 ocnyosite food samples examined, including leafy vegetables, oils, fat and shortening, sugar and adjuvants ..(Duggan a t a t . , 1966). In 1973, an insignificant amount of 2,4-0 was fcuhd during this program*: a trace was *rar*in cne of 360 carposite potato sanples (US Bureau of Foods, 1975). 114 0G06783 ~ T l0 ,-1 A 2 it .ars vrh :tee :s .uas of '7) . tds to in .-or ;R is / no an DOW 1375310 Air samples were collected near wheat-growing areas around-^pUman and Kennewick Highlands, Washington, USA between April and AuguiT!~l964 after applications of 2,4-0. Although data were given for several products containing 2,4-D, those far the isopropyl ester were the highest:- an average concentration of 0.116 ug/in3 and a maximum concentration of 1.96 _ Ug/ta3 of an aerosol farm of the ester and an average concentration of 0.007 ug/m3 and a maximum concentration of 0.69 wg/fti3 of the vaporized ester were found in 24-hour samples near Pullman (Finkelstein, 1969). Residues of 2,4-0 in pond waters declined fran maximums of 0.345 and 0.692 a q /1 in Florida and Georgia, respectively, to less than 0.005 ng/1 28 days after treatment and from 0.630 ng/l in Missouri pond waters to less than 0.005 mg/1 56 days after treatment. Residues in nud fron the Florida and Georgia ponds never exceeded 0.05 irq/kg and had declined to trace or detectable levels 56 days after treatment. The highest residue fcxind was 0.170 ng/kg in samples of mud taken an die first and third days in the most heavily treated Missouri pend. In nud fran one Missouri pond, residues were detected as late as 28 days after treatment; no residues occurred in any ponds after that time (Schultz s Hannan, 1974). In September 1971, soil samples were obtained fran an area in Thailand that had been used far calibrating aeriaI. herbicide spray equipment a..a that had received about 940 kg/ha 2,4-0 and large amounts of other herbi o fcides in 1964-65. TV*} 6 samples contained 0.18 and 0.21 kg/ha 2,4-0 (Oannittee on the Effects of Herbicides in Vietnam, 1974). 2.3 Analysis *7-; ' The Association of Official Analytical Chemists (AQAC) has published _ three Official Final Actions for the determination of 2,4-0 in femulations: /;;* (1) titration, far femulations of the free acid; (2) determination of :-/ total chlorine after calorimetric destruction for femulations of 2,4-0 caipounds^ and (3) infra-red spectroscopy for 3,6-diehloro-2-methoxy- benzoic acid (Dicamba)/2,4-0 formulations. Official First Actions have been puhl ishert for determination of the free acid in formulations of 1-0 esters by titration to pH 7, tor determination in formulations with 4-amino-3,5,6-trichloropicmIonic acid (picloram) by high-pressure liquid 0006y Su t 7133 115 DOW 1 375311 (Beroza, 1976? Horwitz, 1975). Results of collaborative studies of several of these methods have been published (Haimcnd, 1973; Malina, 1971; Skelly e t a l . , 1976). Another method for determining 2,4-0 in formulations involves gas chromatography of its trimethylsilyl derivative (Zweig, 1972). With a similar method, recoveries of 100.5 s 1% compare favourably with those obtained using the AQAC infra-red spectroscopy method (Collier & Grimes, 1974). Methods of analysis far 2,4-0 residues ir. various ocnurdities using gas chromatography have been sumnarized and tahii in a recent review (WHO, 1975). In a more recent publication (WHO, 1977) attention was drawn to the Low recoveries frequently encountered due to conjugation of' 2.4- 0 with plant constituents. Hie Pesticide Analytical Manual (US Food & Drug administration, 1975a,b) surrmarizes several methods far determining 2.4- 0 residues, using gas, thin-layer and paper chromatography; a general method, far extraction and clean-up of chlorophenoxy acids in a variety of foods provides more, than- 80% recovery. Limits of detection for gas chromatography are in the order of 10 ug/kg when nrierocoulcm eeric detec tion is used and 0.2 ug/kg with electron capture. A gas chromatographic procedure has been used to separate mixtures of herbitidal acids as their pentafluorobenzy1 derivatives, which generally provide better separation and better responses in the electron capture detector than do the methyl or silyl derivatives; the method has a sensi tivity of 0..4 yg/kg for 2,4-0 (Chau & Terry, 1976). An automated, gel- permeation chromatographic procedure permits clean-up of pesticide residues in lipid-containing plant and animal extracts prior to their determination by gas ehronatography with 1 detection, and provides recovery of more than 80% 2,4-D esters (Johnson e t a l ., 1976). A low-taiperature extraction and clean-up method is used to separate multiple residues of 2,4-0 and other for eventual gas >~hrcmato- gratiiic analysis with electron capture and flame photometric detectors ; 82--108% of added 4 mg/kg 2,4-0 wni-' be recovered (McLeod & Wales, 1972). OGOfoWI 7 1 3Q 116 DOW 1 375312 Gas chromatography cortoined with electron-capture detection has been used to determine 0.5 trg/kg 2,4-0 residues in oysters (Duffy & Shelfoon, 1967) and 0.05 ng/kg in soil, wheat and barley (Khan, 1975). 3. Biological Data Pelevant to the Evaluation of Carcinogenic Risk to Van 3.1 Carcinogenicity and related s^vHes in animal; (a) Oral administration -Mo-u-s-e: Grouos of 18 mal-e-and 18 fsnale (C57BL/6xdH/Anf)Fi mice and 18 male and. 18 female (C57EL/6xAKR)F mice received c m raercial 2,4-0 (90%, nup. 136-140oC) according to the fo1llowing dose schedule: 46.4 mg/kg bw in 0.5% gelatine by stanach tube at 7 days of age and the same amount -"-g (not adjusted for increasing body weight) daily up to 28 days of age; subsequently, the mice were given 149 mg/kg of diet. A further group of ai 18 male and 18 female (C57BL/6xAKR)F mice were given oral doses of 100 mg/kg bw/day froa 7-28 days of age and subsequently fed 323 mg/kg of diet. The experiment was terminated 'when the mice were about 78 weeks of age, at which time 15, 16, 16 and 16 given the lower dose level and 11 and 13 given the higher dose level were still alive in the respective of groups. Tumour incidences were compared with those observed among groups of 79, 87, 90 and 82 control mice, which had either been untreated or had received gelatine only: the incidences were not significantly greater li (P>0.05) when any group or combination of groups was considered. Similar results were obtained in groups of mice given 2,4-0 isopropyl, butyl or isooctyl esters (99%, 99% and 97% pure) at doses of 46.4 mg/kg fcw from on 7-28 days of age and, subsequently, 111, 149 and 130 mg/kg of diet, respectively, up to 78 weeks of age (limes e t a l . t 1969; NTIS, 1968a). Rat: Groups of 2S male and 25 female 3-week old Qsbome-Mendel rats were fed for two years on diets containing 0, 5, 25, 125, 625 or 1250 mg/kg of diet 2,4-0. The 2,4-0 was 96.7% pure and contained no detectable levels of 2,7-dichloro- or 2,3,7,8-tetrachlorodihem o-parg-dioxin; the limit of sensitivity of the method of analysis was 1 mg/kg . The numbers of male and fanale rats with malignant tumours were 6 in controls and 3, 7, 7, 8 0006792 v. 117 DOW 1 375313 and 14 in the treated groups, respectively. Tunours were randomly distri buted and were also found in ageing rats of this strain. According to the authors, a statistical increase (P<0.05) in the number of treated rats with malignant tumours over controls was found only in males receiving the highest dose level (Hansen et a l . , 1971). Groups of 120 male and 45 female randan-bred rats, weighing 80-100 g at the start of the test, were given 2,4-0 as the amine salt (amount not specified) mimed in the food at a concentration which to a daily intake of cne-tenth of the ID . (not specified, but see Table 1, s section 3.2). Two treated rats developed tumours (a mammary fibroadenoma and a haemangioma of the mesenterium) afteir 23 months, and one untreated rat had a mammary fibroadenoma after 27 months (Arkhipov & Koslcva, 1974) . (b) Subcutaneous and/or intramuscular administration Mouse? Groups of 18 male and 18 female (C57EL/6xC3E/P>n) F mice and 18 male and 18 female (C57HL/6xAKR)F mice were given single s.c. injections of 215 mg/kg fcw 2,4-0 (90% pure, m.p. 136-140C) in dimethyl sulphaxide on the 28th day of life and observed up to 78 weeks of age, at which time 16, 17, 18 and 18 mice in the four groups, respectively, were still alive. Tumour incidences were compared with those in groups of 141, 154, 161 and 157 controls that were either untreated or were injected with dimethyl sulphaxide, 0.5% aqueous gelatin or c o m oil. The tumour incidence in any group or ccnfainatieh of groups was not significantly different from that in controls (P>0.05). NO increase in the incidence of tumours was observed in similar groups of mice treated with single s.c. injections of 21.5 mg/kg fcw butyl or 100 mg/kg bw isopropyl esters of 2,4-0 (both 99% pure). Of mice treated with 21.5 mg/kg bw isooctyl ester of 2,4-0 (97% pure), 5/17 females of the second strain developed reticulum-cell sarcomas (P=0.01) (OTIS, 1968a). 3.2 Other relevant biolocical data 0006793 (a) Experimental systems The toxicity of 2,4-0 was reviewed by Dalgaard-Mikkelsen & Poulsen (1962) 71 U iesiElIWW Acute and short-term toxicitv Ciere are species differences in the acute oral toxicity of'J?T4-0 and of its derivatives (Table 1). The symptoms of acute toxicity'in mice, rabbits, guinea-pigs and rats are essentially similar (Bucher, 19'46; Hill & Carlisle, 1947). Seme animals died suddenly, apparently iron ventricular fibrillation; those that did not die iamediately developed stiffness of the extraaities, incoordination, lethargy, stacr and prior to death (Hill & Carlisle, 1947). Symptoms of nyotonia were evident in mice after the parenteral administration of 150-200 my/kg bw; and in mice acutely intoxicated with 2,4-D, dilatation of the blood vessels of lungs, liver and kidneys was observed (Bucher, 1946). Bats and guinea-pigs administered lethal doses of 2,4-0 exhibited congestion of the viscera and enlarged, swollen kidneys; microscopically, there was massive cloudy swelling of the proximal, convoluted tubules with cast formation (Hill & Carlisle, 1947). S.c. injections of 100 rrry/kg bw 2,4-0 resulted in a decrease of both thyroid and body weights in male rats (Florsheim & Velcoff, 1962). Oral administration of 625 mg/kg bw to rats resulted in changes in the levels of several serum enzymes and globulins (Szdcs s t a t,., 1970). 2,4-0 was found to be a potent uncoupling agent for oxidative phosphoryl ation and to stisulate the respiration of rat liver mitochondria in a phosphate-deficient medium (Brody, 1952). In dogs, toxic synptons were often not present until 6 hours after oral administration of a lethal dose of 2,4-D; the animals became ataxic, with progressive increase in spaan. Death appeared to be due in most cases to hepatic congestion or to' pneumonia. Pathological changes, limited to the gastrointestinal tract, lung and liver, followed the development of anorexia, weight loss and myotonia (Drill 6 Hiratzka, 1953). Dogs exhibited evidence of liver damage more frequently than other animals studied (Bucher, 1946; Drill & Hiratzka, 1953). (to toxic symptoms were noted in monkeys given 214 mg/kg bw orally, or 428 mg/kg bw intraperitcmeally; hewever, oral plus i.p. injection of a total of 500 mg/kg bw 2,4-0 caused nausea, voniting, lethargy, muscular incoordination and head drop (Hill & Carlisle, 1947). 0006794 Acute toxic doses *87X1 Ao 'X us TRB1E 1 Canpound of 2,4-D Acute oral toxicity of 2,4-0 and esters Species Sex ID 5 0 (ng/kg bw) Beference Acid ax Mice Mice Bats Bats Guinea-pigs Guinea-pio3 Babbits Dogs Chicks M M M M&F M&F 375 368 375 666 469 1000 800 100 541 Hill & Carlisle (1947) Bowe & Hymas (1954) h n it Hill & Caflisle (1947) Bcwe & Hvmas (1954) Hill & Carlisle (1947) it it Drill & Hlratzka. (1953) Bowe & Hymas (1954) Butyl ester Mice Bats Bats Cats 380 1500 920 820 Konstantinova (1970) Schillinger (1960) Konstantinova (1970) If II Esters of mmo-, di- and tripro pylene glycol butyl ethers Bats F 570 Bowe & Hymas (1954) Isopropyl ester Mice Bats Guinea-pigs Chicks M M&F M M&F 541 700 550 1420 Bowe & Hymas (1954) ft ft tl it m ft Mixed butyl esters Mice Bats Guinea-pigs Babbits Chicks F F F M&F M&F 713 620 848 1420 2000 Bowe & Hymas (1954) ft ft ft It ft It ft M It Sodim salt Mice Bats Bats Guinea-pigs Babbits F M 375 805 2000 551 800 Bowe & Hymas (1954) ft ft Schillinger (1960) Bowe & Hymas (1954) f t f t It Alkanolamine salt Chicks 380-765 Howe & Hymas (1954) 000G735 ( 7143 120 a (1947) '.354) 3 (1947) 1954) 3 (1947) II -J3. (1953) 1954) 0) iou) (1970) 1954) L954) N n u 1i9f54) if tl 1n954) '50) .954) -954) of 2,4-0 (765 mg/kg bw) produce fatty degeneration of the liver, spleen, kidneys and heart and haemorrhagic gastrcenteritis in chickens (Bjorn & Narthen, 1948), and acute toxic doses have similar effects in shdei? and cattle (Palmer & Badeleff, 1964). Subacute and chronic toxicit1/ 2,4-0 administered subcutaneously to mice at doses of 50-90 mg/kg 2w once or twice daily for 3 weeks to 3 months produced no clear-cut.chronic symptans. levels of 70 mg/kg bw and more retarded growth, probably by reducing food intake (Bucher, 1946). Bats fed 1000 ng/kg of diet 2,4-0 far cne month showed no signs of toxic effects (Hill & Carlisle, 1947) . No adverse effects were noted in young female rats fed 100 and 300 mg/kg of diet 2,4-0 (purity unspecified) far periods of up to 113 days, while those given 1000 mg/kg of diet over the same period had increased mortality, depressed grcwth rate, slightly increased liver weight and slight cloudy swelling of the liver. Animals fed 3000 or 10,000 mg/kg of diet were sacrificed after 12 days because of food refusal and rapid weight loss. Increased liver and kidney weights and unstated slight pathological changes were noted in these organs (Bcwe & Hymas, 1954). No significant adverse effects were noted when 15 ml of each of 3 ccamercially available formulations of 2,4-0 (the dimethylamine salt and the isooctyl and butyl esters) were administered 5 times weekly for 3 weeks to the intact and abraded skin of rabbits at two concentrations, 0.626% and 3.13% (the dimethylamine salt was diluted in water and the ester in either oil or water) (Kay e t - a l ., 1965). Groups of 3 male and 3 female beagle dogs were fed 10, 50, 100 or 500 mg/kg of diet 2,4-D (96.7% pure, with no detectable chlorodibento-rait- dicxin content) for 2 years, starting at 6-8 months of age. Twenty-eight dogs that survived the 2-year period were clinically normal. No adverse e t a t . ,effects related to 2,4-0 aininistration were, observed (Hansen 1971). Dogs of both sexes *re given 2, 5 or 10 rrg/kg fcw cocnercial 2,4-0 (98.5% pure) orally by capsule for 5 days per week for 13 weeks; no signs 0006736 *jy-* a A 121 9TQld MOO ; *=i'-T T'" fi? HOW 137531? of taxicity were observed. Dogs given 20 mg/kg bw survived far periods ranging f r m 18-49 days, with loss of weight occurring after 7-12 days and with ataxia, increased nuscle tonus and a terminal fall in lymphocyte count prior to death (Drill & Hiratzka, 1353). Young pigs treated at varying intervals up to 103 days with 50, 100 or 300 mg/kg fcw of the ccmercial triethanolamine salt or butyl ester of 2,4-0 exhibited svrotars of intoxication and pathology analagous to those seen in laboratory animals. Clinical signs of anorexia and retarded growth were found in one animal given 51 doses of 50 mg/kg bw triethanolamine salt over 103 days. Pigs fed 500 mg/kg of diet triethanolamine salt of 2,4-0 for up to 12 months developed locateter disturbances of increasing severity after about one month. Animals sacrificed after 2-12 months had normal organ weights and no gross pathological changes. nirvirry<ThwTriral obser vations included lowered haemoglobin and hematocrit values, elevation of glutamic-oxaloacetic transaminase and reduced albumin and albumin: globulin ratios in the treated animals (Bjflrklund & Zme, 1366). Absorption, distribution and excretion In rats, pigs, calves and chickens, 2,4-D administered in doses of 50-100 mg/kg bw orally as salts was readily absorbed and eliminated, mainly in the urine, with plasma half-lives varying fron 3-12 hours (Erne, 1966a,b). The rate of 2,4-D elimination in rats was dosage dependent. Following administration of ll*C-2,4-D, .radioactivity was found in all organs and tissues examined (Kharaia s Fang, 1966). 2,4-D esters are hydrolysed in animals. The phenoxy acids are excre ted predominantly as such in the urine of rats after their oral admini stration, although a.minor portion is conjugated with the amino acids glycine and taurine and with glucuronic acid (Grunow & Bfime, 1974). No 2,4-dichlorophenol was, however, detected in the urine or C57BL/6 mice treated subcutaneously with 2,4-0 or its butyl or isooctyl esters. The _ rates of disappearance from the plaana of 2,4-0 and its butyl and isooctyl esters following single s.c. injections of 100 mg/kg bw of the cancounds to renale C57BL/6 mice were: & Fishbein. 1967). 122 butyl ester>isooctyl estar>2,4-Q (Zielinski ' 7H1Jl 0006737 Is r/te .00 or 2,4-0 .-en. in nre : over >r Y J. f me. :re- 'o m /1 i3 After oral administration of 0.05 mg/kg bw 2,4-0 to rats, traces were detected in the milk of lactating animals for 6 days. Within hoars after administration of 2,4-0 to *orea~nant rats, 16.8% of the dose 533 detected in the uterus, placenta, foetus and aimiotic fluid (FecarSfe s Belova, 1974). 2,4-D was also found to pass the placental barriervuv pigs (Bjrklund & Erne, 1966). Eafarvotcrcicitv and taratocenicitr 03 '"<F CJI ca V-- k 00 Results of teratologica1 studies with 2,4-D were variable; terato genic effects are observed with doses close to those which cause maternal toxicity. Administration of 2,4-D or its isopropyl, butyl or isooctyl esters orally or subcutaneously during days 6-14 of pregnancy increased the incidence of foetal ananalies among BL6, AKR and/or C3H strains of mice but not among B6AK and A/Ha strains. Hie purity of the compounds was: 2,4-D, 90%, m.p. 136-140C; isopropyl and butyl esters, 99%; isooctyl ester, 97% {OTIS, 1968a,b). >. Sprague-Dawley rats were given 1000 mg/1 2,4-0 in the drinking-water during pregnancy and for a further 10 months, and 2,4-D was administered to the second generation for up to 2 years. Pregnancy and parturition were normal; litter size was not significantly reduced, and no malfor mations were noted in the young. Except for retarded growth and increased mortality in the second generation, no unequivocal clinical or morpho logical changes were seen. Vfcen 2,4-D was aninistered at a concentration o> 500 mg/kg of diet during die entire pregnancy of a sew, anorexia was noted; the newborn piglets were underdeveloped and apathetic, and 10/15 died within 24 hours. Continued feeding of 500 mg/kg of diet to the survivors until they were 8 months of age marked growth depression, persistent anaemia and moderate degenerative changes of liver and kidneys (Bjflrklund & Erne, 1966). Female rats (10 per group) were fed 2,4-0 (purity unspecified)' at levels of 0, 1000 and 2000 mg/kg of diet for 95 days and then mated with ' -'ntreated males and continued on their respective diets through gestation and lactation. Pups bo m to females fed the highest level were small at ?.. .. : i. : -W. 0006798 123 DOW 1 375319 birth; 94% HipH before weaning; smw deaths also occurred in pups of rats e t a lfed the Lower Level (T.B. Gaines & R.D. Kimbrough, cited by Hansen ., 1971). The maximum tolerated oral dose of 2,4-0 (98.7% purity, no chlorinated dibenzo-pura-dioxins found with the limit of detection of 0.2 wq/kq) ar an equimolar dose of 2,4-D propylene glycol butyl ether ester or of the iso octyl ester of 2,4-D haul embryolethal and growth retarding effects but no teratogenicity when given to pregnant Spracue--Dawlev (Spartan strain) rats on days 6-15 of gestation. Other signs of foetotoxicity were s.c. recema, delayed ossification and wavy ribs. 2,4-0 did not affect fertility, gestation, lactation or viability of the ne wborn; the propylene glycol butyl ether and isooctyl esters dpcrpasorf viability of the newborn and lactation indices1 (Schwetz e t a t . , 1971).. s^nriia-r effects were observed in Wistar rats given single daily oral doses of 100-150 mg/kg bw 2,4-0 or the butyl, isooctyl, butoxyethanol dimethylamine derivatives of 2,4-0 on days 6-15 of pregnancy (Khera & McKinley, 1972). No consistent embryotoxic effects were noted when 2,4-0 was admini stered orally to hamsters at doses its to 100 mg/kg cn days 6-10 of gestation (Collins & Williams, 1971). Ictz-Ostertag & Lutz (1970) tried to simulate field conditions for spraying with 2,4-0 and to evaluate the effect cn pheasant eggs and develop ment of chicks. A high mortality rate aryi morphological alterations were observed, in the embryos and chicks, the toxicity apparently being higher than for mammalian species. Tbtal or pTfiai paralysis was observed in most of ie surviving embryos, and 50% of the surviving chides were sterile. In a 3-generation reproduction study, Osbome-ttendel rats were fed 100 or 500 mg/kg of diet 2,4-0 that was 96.7% pure, with no detectable (<1 mg/kg) 2,7-dichloro- or 2,3,7,8-tetrach]ornditenzo-pero-dioxin. No adverse effects were observed. Diets containing 1500 mg/kg 2,4-D, while `lactation index: (pics weaned/pups alive cn day 4) x 100 124 0006799 of rats ~.t a l., orinated j or an a isoout no n) rats oedema. /col and rved 4-0 or -D on .nini- gestaticn for develcps ware igher i in sterile. fed jle No iiile apparently affecting neither the fertility of either sex nor litter size, sharply reduced the percentage of pups surviving to weaning and tftweights of the weanlings (Hansen e t c l . , 1971). Mutacenicitv ' 2,4-0 was not mutagenic on Escherichia con WP2 her or her -or.in Salmonella typhvnurium strains 7A1535, 73U536, 170537 or TA1533 (Aadersen e t c l . , 1972; Nagy e t a l . , 1975; Shixasu, 1975; Shirasu e t a l.^^tS p S ; Zetterberg e t a l . , _ 1977). -j i i . 1 In the rec assay, which is believed to give an indication of reparable EiCV damage, 2,4-0 was not mere toxic to B acillus s u b tilis M45 (rec~) than to H17 (rec4'), suggesting that this canpourri does not damage ENA. (Shirasu, 1975). In Saccharamyces cereo isia e 04, gene conversion was increased by concentrations of 2,4-0 above 400 ug/W.. Mitotic recombination in 5. c e r e v is ia e 05 was also increased by 2,4-0 (300 ug/tal) (Siebert & Ierperle, 1974; -Zetterberg e t a l . , 1977). S. cerevisia e HAD 18 (a histidine- dependent haploid strain) was reverted to histidine independence by 250 ug/tal 2,4-0. `Baxic and mutagenic effects in 5. c e re v isia e were dependent cn lew pH (4.3) (Zetterberg, 1977) [Metabolic activation systans were not included in any of these tests]. ,, S .In host-mediated assays with typhim nium strains TA1530 or TA1531, or with 5. c e re v isia e D4, no nutagenic effects were observed when adult male mice were given 6 mg 2,4-D (200 mg/Tcg) by gavage (Zetterberg e t a l . , 1977). S.Serum frcm orally dosed rats was not nutagenic to tyvhimuriifa (Styles, 1973). The effect of 2,4-0 cn chroiosa ne aberrations in cultured plant is carpiex because 2,4-0 is required as a plant growth regulator in tissue culture. Singh & Harvey (1975a,b) found an inverse correlation between 2,4-0 concentration and chromosome aberrations in '.'i d s ra jesten a and Haplopappu3 g r a c ilis . Horever, with a strain of .Via o tia ra that does not require 2,4-0 it increased chromosome breakage (Ranchi e t a l . , 1976,. 1 ?148 0006800 125 DOW 1 375320 zes^ l MOO 2,4-0 induced chrcmoscme aberrations in a nimber of cultivated plants and weeds. The cytological abnormalities included chrcmoscme bridges, frag ments, lagging chromosomes, C-mitoses and chronatin bodies (Mohandas & Grant, 1972). No increase in the number of recessive lethals was observed when 2-day- old adult male DroaovkiZa melanogaster flies were fed 4.5 or 9.0 rrM 2,4-0 in sucrose (Vogel & Chandler, 1974). In v itr o exposure of embryonic bovine kidney nns and of bovine peripheral blood cells to concentrations of 1-1000 ug/ral 2,4-0 for 6-96 hours resulted in stimulation.of mitosis. Qircmosanal aberrations were not detected in the peripheral blood cells, but nucleolar irregularities and polyploid mitotic stages were observed in the kidney cells (Bcngso & Basrur, 1973). Treatment of cultured human lymphocytes with 2.5 x 10~r.M (0.02 ug/ml) 2.4- 0 increased the number of chranatid aberrations (single acentric fragments) and, to a lesser extent, of chromoscmal aberrations (paired acentric fragments). In mice, toxic nrrpn n H w w (100-300 mg/kg bw) of 2.4- 0 administered as a single oral dose significantly increased the fre quency of aberrant metaphases (2-4-fold); single fragments were the primary aberration (Pilinskaya, 1974). 2,4-D hai no effect on cultured cells nor cn bone marrow after its oral administration to rats (Styles, 1973). There was no detectable increase of micronuclei in the erythrocytes of mouse bone marrow after i.p. administration of 100 mg/kg bw 2,4-D (Jenssen & Renberg, 1976). 2,4-0 did not increase dominant lethal nutations in mice when given as a single i.p. injection of 125 mg/kg bw, or when given orally' on 5 successive days for a total dose of 75 mg/kg lx/ (Epstein e t a i . , 1972). (b) Man In a case of suicide of a 23-year old farming student, the total' amount of 2,4-0 in the body was estimated to be no less than 6 g, corres ponding to a dose of about 30 mg/kg bw. All organs shewed marked acute congestion. Severe, degenerative changes of the ganglion cells were found 126 O G O G s m rsgrant. ^y3 -ary in the central nervous systan (Nielsen a t a l . , 1965). A man vio accidentally ingested about 30 ml of concentrated weeckillr containing 50% of a thiolcarbamate and 36% 2,4-D isooctyl ester in aqueous-- solution exhibited fibrillary twitching and paralysis of the intercostal * v muscles. There was evidence of generalized skeletal itosele damage, as indicated by marked elevation of the levels of several imscle enzymes, as r> well as haemcglcbinuria and nyoglcbinuria. Recovery was ccmplete after several months (Berwick, 1970). 03 a 03 ro to No adverse effects were reported in a man who took 500 mg 2,4-0 orally daily for 3 weeks (approximately 8 mg/kg bw/day) (E.J. Krauss, cited in Mitchell e t a l . , 1946). Wien 2,4-0 was used as a treatment far a patient in the terminal stages of disseminated coccidiodrnycosis, ns side-effects were observed following 18 i.v. doses over 33 days: in the 7th to the 17th injection the dose of 2,4-0 was 800-960 ng (about 15 mg/kg bw), and the 18th dose was 2000 mg (about 37 mg/kg bw). A 19th and final dose of 3600 ng (67 mg/kg bw) produced sysptoms of toxicity comprising a semi-stuporous status, fibrillar novroents, hyporeflexia and urinary incontinence, which persisted far 24 hours. Seventeen days after the last administration, the patient died (Seabury, 1963). Three cases of peripheral neuropathy have been reported following spraying of 2,4-0. Initial symptans were, nausea, vomiting, muscular weakness, diarrhoea and swelling or aching of the feet and legs, with malaise and headache, which persisted for 10-20 days. In one case, pares thesia in the extremities and pain in the legs appeared within 4-5 days, followed by twitching of the muscles in the calves and arms; fasciculaticns became generalized, without neurological or electronyographical changes. In the second case, cne week after a second exposure to 2,4-0, numbness and aching of the fingers and toes occurred, followed 6 weeks later by a welldeveloped neuropathy, in the third case, after a second exposure, severe pains occurred in the legs, with swelling of the metacarpal joints of f-^th hands; 5 months later flaccid paraparesis was seen (Goldstein e t a l . , 1959). c * .7 * i S & ? TV $ I fi r, Ik. ff V! 0006S02 7150 127 D O W J 375323 Similar recocts of poisoning in agricultural workers have been r^porT*^ following spraying of 2,4-0 (Monarca 4 Di Vito, 1961; Paggiaro at a t . , 1974; Todd, 1962). The main initial synptems were, noscular weakness, vomiting, diarrhoea, fever, hyperthermia and tachycardia. In two cases, neurological synptems occurred, which continued for 40 days "2to years after exposure and included loss of deep-tendon reflexes and paralysis of thigh and. leg nuscles. Assauly (1951) reported that workers enployed in the fabrication of 2,4-0 developed synptems of somelance, anorexia and gastralgia, increased salivation, a sweet taste in the mouth, a sensation of drunkenness, heavi ness of the legs and hyperacusia. Subjective in-irai synptems reported among workers using various esters and salts of 2,4-0 included rapid fatigue, headache, loss of appetite and pains in the region of the liver and stomach. Sensitivity to taste and snell was lowered (Fetisov, 1966). Bashirov (1969) examined 292 persons (248 men and 44 woman) engaged in the manufacture of the amine salt and butyl ester of 2,4-0, with expo sure ranging from under 5 years to 6-10 years (for 194 and 98 persons, respectively); 63% of these workers frequently of weakness, rapid fatigue, headache or vertigo. About 20% had disturbances of the cardiovascular system (mainly hypotension and bradycardia) and of the digestive organs (dyspeptic synptems and gastritis). The various liver dysfunctions that were found were more pronounced in workers with longer exposures to die herbicides. Changes in metabolic processes were observed in workers engaged in the production of 2,4-0, in particular, increases in blood cholesterol caxtent, with no change in the lecithin:cholesterol ratio. Decreases in serun albumins, increases in globulins, decreases in KinrH sugar levels c tand altered responses to sugar loads were also noted (Lukoshkina cl., 1970). In a report on 220 workers exposed in a manufacturing plant to 30-40 mg/day 2,4-0 for periods ranging from 0.5 to 22 years, Johnson (1971) stated [without providing any supporting evidence] that no 'meaningful' 0006S03 CT >: differences were observed in [unspecified] clinical assessment, in cotnan- san to a control group of 4600 men, and that no chromosomal effects were observed in 10 workers whose chromosomes were karyotyped. Feldmann & Maibach (1974) studied the absorption through the skin of 4 ug/ca2 1'`C-labelled 2,4-0 dissolved in a small amount of acetone. lkC activity was measured in urine over a 5-day period and compared with that in urine after i.v. administration of the compound: urinary excretion of CO : . cn -v c o 1 to 2,4-0 after i.v. administration was 100% of the dose in 120 hours, while excretion after topical administration was 5.8% of the dose. e t a t.Kchli (1974) administered 5 m g /k g bw pure 2,4-0 in a gelatin capsule with water to 6 health^ male volunteers, aged 22-20 years. None of the subjects onmplained of any ill-effects; no charges in blocd pressure, pulse rate, haemoglobin content or total or differential white cell counts were observed. 2,4-0 was absorbed fairly rapidly; the highest concentration in bleed was reached in 7-24 hours. In urine, 2,4-0 was present as early as 2 hours after ingestion, and more than 75% was excreted in 96 hours without undergoing transformation in the body. In 5 male volunteers given a single oral dose of 5 mgAg tw 2,4-0, Vthe ha 'ife in the plasma was 11.7 hours, and elimination in the urine occurred with a half-life of 17.7 hours. About 82% was excreted as such and 12.8% as a conjugate (Sanerboff i t a t . , 1976). Fas: details of adverse troricnlogi.na-1 effects resulting from the spraying of 'Agent Orange1 (a 50:50 mixture of the -butyl esters of 2,4-0 and 2,4,5-T, contaminated with up to 30 mg/kg or more TQ3D) and other herbicides in Vietnam, see monograph on chlorinated dibenzodioxins, p. 3.3 Case reports and epidemiological studies Axelson & Sunde11 (1974) reported that in a cohort study of Swedish railway workers exposed to a variety of herbicides a significant, two-fold excess of all cancers was observed in exposed workers, as compared with the national average. The situation was difficult to evaluate because of the xinliined exposure of many workers to more than one herbicu-.-.. Mast of the 2excess, however, seemed to be due to exposure to 3-arnino-l, ,4-triazole hi Ir t.; * 2 . fc-l 4(5 V: ft I I1 i tin e % 0006804 7152 129 DOW 1 375325 (amitrole);' within the subgroups that had been exposed to phenaxyacids (2,4-0 and/or 2,4,5-T), only a small difference was detected; 5 cancers at all sites observed versus 2.8 expected. The authors stated, however, that use of 2,4-0 and 2,4,5-T had probably been higher than that they oould trace. See also monograph on chlorinated dibenzodioxins, p. 41. , 4. Comments on Data Reported and Evaluation 4.1 Animal data 2,4-0 and several of its esters were tested in rats and mice by oral adninistratlon and in mice by subcutaneous minit-ra-Hnn. All of these studies had limitations, due either to inadequate reporting or to the small number of animals used. Therefore, although increased incidences of t m c urs were observed in one study in which rats received 2,4-0 orally and in another in which mice received its isooctyl ester by subcutaneous injection, no evaluation of the carcinogenicity of this oanpound could be made. 4.2 Human data The results of the single cohort study of a snail number of workers exposed to various herbicides, including 2,4-0, 2,4,5-^T and 3-amino-1,2,4- i Jtriazole (amitrole) are not sufficient to evaluate the carcinogenicity of 2,4-0 to man [Because 2,4-0 may be used with 2,4,5-T, which is contami nated with 2,3,7,8-tetrachlarodibenzo-pgra-dioxin, see also monograph on chlorinated dibenzodioxins, p. 41]. ISee also XABC, 1974. 130 0006805 S lu O 5. References &Andersen, K.J,, Leighty, E.G. Takahashi, M.T. (1972) Evaluation of herbicides far passible mutagenic properties. J. acrric. Fd Chem., 20, 649-656 Arkhipov, G.N. & Roslova, I.N. (1974) The study of the carcinogenic properties of the amine salt of 2,4-0. Vc p . Pitan., 5_, 83-84 Assculy, M. (1951) Dsherbants slectifs et substances de croissance. Aperu technique. Effet pathologique sur l'hottne au cours de la fabrication de l'ester du 2,4-0. Arch. Mal, nrof., 12, 26-30 L.Axelson, O. & Sundell, (1974) Herbicide exposure, mortality and t m r incidence. An epideminlogica1 investigation on Swedish railroad workers. Work-environm.-hlth, 11, 21-28 Bashirov, A.A. (1969) Health condition of workers producing herbicides of amine salt and butyl ether of 2,4-0 acid. Vrach. De3o, 10,-92-95 Beroza, M., ed. (1976) Changes in official methods of analysis made at the eichty-ninth annual meeting, October 13-16, 1975. J. Ass, off, analvt. C a m . , 59, 456-457 Berwick, P. (1970) 2,4-OirhLnrophenoxyacetic acid poisoning in man. Sane interesting clinical and laboratory findings. J. Amer. med. Ass., 214, 1114-1117 Bjrklund, N.-E. & Erne, K. (1966) Toxicological studies of phenoxyacetic herbicides in animals. Acta vet, scand., 7_, 364-390 Bjorn, M.K. & ttorthen, H.T. (1948) Effects of 2,4-dichiorophenoxyacetic acid on chicks. Science, 108, 479-480 In v i t r o----------------------- ---------- Bongso, T.A. & Basrur, P.K. (1973) response of bovine cells to 2,4-dichlorophenaxy acetic acid. In Vitro, 8_, 416-417 Brody, T.M . (1952) Effect of certain plant growth substances an oxidative phosphorylation in rat liver mitochondria. Proc. Soc, exo. Biol. (N.Y.), 80, 533-536 Bucher, N.L.R. (1946) Effects of 2,4-dichlorophenoxyacetic acid on experi mental animals. Proc, Soc. exp. Biol. (N.Y.), 63, 204-205 Chau, A.S.Y. & Terry, K. (1976) Analysis of pesticides by chemical .derivatizaticn. IH. Gas chromatographic characteristics and conditions far the formation of pentafluorbenzyl derivatives of ten herbicidal acids. J. Ass, off, analvt. Chem., 59, 633-ojo 000680G 7154 UI DOW 1 37532G j ; j ]\ f 375327 &Collier, R.H. Grimes, G.S. (1974) Determination of chlorophenoxy acids in formulations by gas-liquid chrcm atography of their trimethylsilyl derivatives. J. Ass, off, analvt.- Chem.,. 57, 781-734 Collins, T.F.X. & Williams, C.H. (19711 Teratogenic studies with 2,4,5-T and 2,4-0 in the hamster. Bull, environm. Contam. Toxicol., 6_, 559-567 Carmittee an the Effects of Herbicides in Vietnam (19741 The Effects of Herbicides in South Vietnam, Part A, Surnnarv ani Conclusions, wasnington DC, National Academy of Sciences Dalgaard-Miktelsen, S. & Poulsen, E. (1962) Toxicology of herbicides. Pharmacol. Rev., 14, 225-250 Davis, D.E. (1974) The 2,4,5-T story. Weeds Today, spring, pp. 12-13, 13 Drill, V.A. & Hiratzka, T. (1953) Toxicity of 2,4-diehlorophencxyacetic acid and 2,4,5-trichlorophenoxyacetic acid: a report on their acute and chronic toxicity in dogs. Arch, icdustr. Hyer. occuo. Med., 7, 61-67 Diffy, J.R. & Shelfccn, P. (1967) Determination of 2,4-0 and its butcxyethanol ester in oysters by gas chranatography. J. Ass. off. analvt. Chen., 50, 1098-1102 Duggan, R.E., Barry, H.C. & Johnson, L.Y. (1966) Pesticide residues in total-diet samples. Science, 151, 101-104 Epstein, S.S., Arnold, E., Andrea, J., Bass, w. & Bishop, Y. (1972) Detection of chemical nutagens by the dominant lethal assay in the mouse. Tcxicol. apol. Pharmacol., 23, 288-325 1_,Erne, K. (1966a) Distribution and elimination of chlorinated phenoxyacetic acids in animals. Acta vet, scand., 240-256 Erne, K. (1966b) Studies on the animal metabolism of phenoxyacetic herbi cides. Acta vet, scand., 7, 264-271 Fedorova, L.M. & Belova, R.S. (1974) Incorporation of 2,4-dichlnropbenoxy- 2,acetic acid into the organs of animals: paths and dynamics of its excretion. Gig, i Sanit., 105-107 Feldmann, R.J. & Maibach, H.I. (1974) Percutaneous penetration of sane pesticides and.herbicides in man. Tacicol. anol. Pharmacol., 28, 126-132 Fetisov, M.I. (1966) Problems of occupational hygiene in work with herbicides of 2,4-0 group. Gig, i Sanit., 31, 28-30 Fine, D.H., Ross, R., Rounbehler, D.P. & Fan, S. (1977) .V-Nitroso compound impurities in herbicide formulations. J. aerie. Rd Chan, (in press) 0006807 132 t 7 1 5 5 .ZS vl -T 9-567 18 -C 61-67 ,v t. :etic :bi- -.o xy- 126-132 bicides curd s) Finkelstein, H. (1969) Preliminary Air Pollution Survey of Pesticides, Raleigh, North Carolina, US Department of Health, Education and Welfare, pp. 52, 136 =2 & Florsheim, W.H. & Velcoff, S.M. (1962) Sane effects of 2,4-dichlorcphenoxy- acetic acid on thyroid function in the rati effects on iodine accirai-'T"" latian. Endccrinolcav, 71, 1-6 V> CO Goldstein, N.P., Jones, P.H. Brown, J.R. (1959) Peripheral neuropathy after exocsure to an ester of dichlarophenoxyacetic acid. J. Amer. L C JT _ med. Ass~., 171, 1306-1309 328 Gore, R.C., Hannah, R.W., Pattaciai, S.C. Poixo, T.J. (1971) Infrared and ultraviolet, spectra of seventy-six pesticides. J. Ass, off, analvt. Chen., 54, 1040-1082 Gruncw, W. Bhme, C- (1974) Cher den Stoffwechsel von 2.4.5-T und 2.4-0 bei Ratten und Musen. Arch. Hsxicol., 32, 217-225 Hngend, H. (1973) Free acid in esters of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and their femulations. J. Ass, off, analvt. Chan., 56, 596-597 Hansen, W.H., Quaife, M.L., Habermam, R.T. Fitzhugh, O.G. (1971) Ouronic toxicity of 2,4-dichlnrophenoxyacetic acid in rats and dogs. TPxicol. aopl. Pharnacol., 20, 122-129 Hill, E.V. Carlisle, H. (1947) Ibxicity of 2,4-dichlorophenoxyacetic acid for experimental animals. J. industr. Hyg. TQxiool., 29, 85-95 Harwitz, W., ed. (1975) Official Methods of Analysis of the Association of Official Analytical Chemists, 12th ed., Washington DC, Association of Official Analytical Chemists, pp. 110-111 Hustan, B.L. (1972) Identification of three neutral contaminants in production grade 2,4-0. J. aerie. Fd Chan., 20, 724-727 IARC (1972) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, 1, Lyon, pp. 95-106 '~ IARC (1974) IARC Monocrraohs on the Evaluation of Carcinogenic Risk of Chemicals to Man, 7, Sere Anti-thyroid and Related Substances, Nitrofurans and Industrial Chanicals, to. 31-43 Innes, J.R.H., Ulland, B.M., Valerio, M.G., Petrucelii', L., Fishbein, L.,. Hart, E.R., Pallotta, A.J., Bates, R.R., FaDc, H.l., Gart, J.J., Klein, M., Mitchell, I. Peters, J. (1969) Bioossay of pesticides and industrial chemicals for tunerigenicity in mice: a preliminary note. J. nat. Cancer Inst., 42, 1101-1114 C006808 133 DOW 1375329 Jenssen, D. & Renberg, L. (1976) Distribution and cytogenetic test of 2,4-0 and 2,4,5-T phenaxyacetic acids in mouse blood tissues, Chem.-biol. Interact., 14, 291-299 Johnson, J.E. (1971) The public health implications of widespread use of the phenoxy herbicides and picloram. Bioscience, 21, 899-905 Johnson, L.D., Waltz, R.H., Ussary, J.P. & Kaiser, F.E. (1976) Automated gel permeation chranatocraphic cleanup of animal and plant extracts for pesticide residue determination. J. Ass. off. analvt. Chem., 59, 174-187 Kay, J.H., Palazzolo, R.J. & Calandra, J.C. (1965) Subacute dermal toxicity of 2,4-D. Arch, environm. Hlth, 11, 648-651 Khan, S.U. (1975) 'Electron capture gas-liquid chromatographic method for the simultaneous analysis of 2,4-D, dicamba, and mecoprop residues in soil, wheat, and barley. J. Ass, off, analvt. Q oem., 58, 1027-1031 Khanna, S. & Fang, S.C. (1966) Metabolism of C 11*-labeled 2,4-dichlorophenoxyacetic acid in rats. J. aoric. Fd Chem., 14, 500-503 Khera, K.S. & McKinley, W.P. (1972) Pre- and postnatal studies on 2,4,5trichlorcphencocyacetic acid, 2,4-dichlorophencxyacetic acid and their derivatives in rats. Toxicol. appl. Pharmacol., 22, 14-28 Kohli, J.D., Khanna, R.N., Gupta, B.N., Dhar, M.M., Tandon, J.5. & Sircar, K.P. (1974) Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man. Xenobiotica, , 97-100 Konstantinova, T.K. (1970) TOxicolooy and sane problems of the embrvotrooic action of the butvl ester of 2,4-0. In: Vygodchikov, G.V., ed., Proceedings of a Conference on Problems on Hygiene and Ttoxicolcoy of Pesticides, USSR, 1967, Moscow; Meditsina, pp. 177-179 Lukoshkina, L.P., Guseinova, R.S. & Melik-Zade, T.M. (1970) Lipid-proteincarbohydrate metabolism in workers during 2,4-dichlorophenoxyacetic acid production. Proc. Res. Inst. Occup. Hlth, Azerbaijan, 5_, 146-149 Lutz^Ostertag, Y. & Lutz, H. (1970) Action nfaste de l'herbicide 2,4-0 sur le dveloppement embryonnaire et la fcondit du gibier d plumes. C.R. Acad. Sci. (Paris), Ser. D , 271, 2418-2421 Malina, M. (1971) Collaborative study of infrared analysis of dicamba-2methyl-4-chloraphenoj^acetic acid and dicamba-2,4-dichlorophencxyacetic acid fannulations. J. Ass, off, analvt. Chem., 54, 706-710 McLeod, H.A. & Wales, P.J. (1972) A low temperature cleanup procedure for pesticides and their metabolites in biological.samples. J. aoric. Fd Chem., 20, 624-627 0006S09 7157 134 ,4-0 1 =ity m joxy- LT r, K.P. in man. II in149 :tic :r Mitchell, J.W., Hadgscn, R.E. & Gaetjens, C.F. (1946) Tolerance of farm ~ T Zanima 1s to feed, containing 2,4-dichlarophenoxyacetic acid. Set., 5_, 226-232 J. tAnimal Mohandas, T. & Grant, W.F. (1972) Cytogenetic effects of 2,4-0 and^aftiitrole in relation to nuclear volume and ENA. oantent in sane higher plants. Canad. J. Garlet. Cvtol., 14, 773-783 ' Msnarca, G. Di Vito, G. (1961) Sull'intossicazione acuta da diserbante (acido 2-4 dichlnrofencssiacetico): contribute clinico. Folia med., ,, 44, 480-485 Nagy,oZn.E,sMcihleer,ic'In.ia&sAonlit.onWiP,2 Ft.r y .(19A75c)ta The mutagenic effect of pesticides micsobiol. acad. sci. hunc., 22, ' 309-314 ;~ KNielsen, ., Kaenpe, B. & Jensen-flola, J. (1965) Fatal poisoning in man by 2,4-dichInrophenaxyecetic. acid (2,4-0): determination of the agent in forensic materials. Acta charmacol. tnxicol., 22, 224-234 OTIS (National Technical Information Service) (1968a) Evaluation of Carcino genic, Teratocenic and Mutagenic Activities of Selected Pesticides and Industrial Chemicals, Vol. 1,. Carcinogenic Study, Washington DC, US Department of Cacmerce OTIS (National. Technical Information Service) (1968b) Evaluation of Carcino genic, Teratocenic and Mutaoenic Activities of Selected Pesticides and Industrial Chemicals, Vol. 2, Teratocenic Study in Mice and Rats, Washington DC, US Department oFconferce Paggiaco, P.L., Martino, E. Mariotti, S. (1974) Su un caso di intossicazione ' da acido 2,4-diclorofenossiacRtico. Med. Lavoro, 65, 128-135 Palmer, J.S. & Radeleff, R.D. (1964) U>e toxicologic effects of certain fungicides and herbicides an sheep and cattle. Ann. N.Y. Acad. Sci., ill, 729-736 Pilinskaya, M.A. (1974) Cytogenetic effect of the herbicide 2,4-0 on hunan and animal chranosanes. Tsitol. Genet., 8, 202-206 Pokomy, R. (1941) Sane chlorophenoxyacetic acids. J. Amer. chem. Soc., 63, 1768 Ronchi, V.N., Martini, G. Buiatti, M. (1976) Genotype-hormone interaction ::ic o ti^rain the induction of chromosome aberrations; effect of 2,4-dichloro-- phenoxyacetic acid (2,4-D) and kinetin on tissue cultures fran spp. Mutation Res,, 36, 67-72 Bomb, V.K. Hymas, T.A. (1954) Smmary of. toxicological information on 2,4-0 and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Amer. J. vet. Res., 15, 622-629 0006810 7.158 135 DOW 1375330 f  of - -S i, .-cnal, \ and R e s ., m on 'as., .ties itians :1 . i ii i i US Bureau of Poods (1975) Caroljanee Program Evaluation. Total Diet Studies: FY 1973 (7320.08) Washington CC. US Government Printing Office, p. 19 US Department: of Agriculture (1973) Stabilization and Conservation sTehrev irP*e*sf ttioHsn^ioneRfetveinewn,cr1u9s72, AgSSfeultural Printing Office, p. 44 US Department of Carneree (1975) US Escorts, Schedule B, Ccinm d itrr.t? Country, ^Danestie Merchandise, FT 410/December 1975, Washington DC, US Government Printing Office, p . 2-74 US Food and Drug Administration (1975a) Pearirifir* Analytical ManualT'Vol. I, Methods Which Detect Multiple Residues, Rockville,~Md, US Dtartrent of Health, Education and welfare, sections 221.12a, 221.12b, 22.14, 221.15, 221.13, 222.12a, 222.12b, 222.18 US Pood and Drug Adninistration (1975b) P e s H ^ e Analytical Manual, Vol, U. Methods far Individual Pesticide Herod, Rn^-u-i i i ^ r Mdr t:s D e p a r r a ta it of Health, Education. and Welfare, section 120.142 US International Trade Caanission (1976a) Synthetic Organic Chemicals. US Production and Sales, 1974, ITC Ptihi "5, wsnington DC, US Government Printing Office, p. 189 US International Trade Comission (1976b) Iteorts of Benzenoid Chemicals and Products, 1974, ITC Publication 762, Washington DC, US Government Printing Office, p. 96 US Tariff Commission (1946) Synthetic Organic Chemicals, US Production and Sales, 1944, Report No- 5, Second Series, Washington DC, US Govemmait Printing Office, p. 116 US Tariff Comission (1974) Imports of Benzenoid Chemicals and Products, 1973, 7C Publication 688, Washington DC, US Government Printing Office, p. 92 Vogel, E. sate S Chandler, pesticides iJn.LD.rRo.so(p1h9i7l4a)n eMlauntaoggeanaitceirt. y testing of cyclomate and Doerientio, 30, 621-623 Weed Science Society of America (1974) Herbicide Handbook, 3rd ed., Champaign, Illinois, pp. 116-122 ' WHO (1975) 1974 Evaluations of sate pesticide residues in food. Wld Hlth Ora. Pest. Res. Ser...No. 4, pp. 159-183 WHO (1977) 1975 Evaluations of same Pest. Res. Ser., No. 5 (in press) residues in food. Wld Hlth Ora. Winell, M.A. (197b, n international comparison of hygienic standards for chemicals in the work environment. Anbio, 4, 34-36 CG0661S 71 59 137 DOW 1375332 UH IM 7160 I ;) r. V* /- / / It'- ' : / / DEPARTMENT 01: H E A L T H , EDUCATION AND WELFARE Public Health Service . . National Institutes of Health National Cancer Institute Division of Cancer Cause and Prevention Bethcsda, Maryland ` * #< I M IN U T E S * o Ni V*l ro oo o Yi D O W 765340 Fifth Meeting of the Data Evaluation/Risk Assessment Subgroup of the Clearinghouse on Environmental Carcinogens September 26, 1977 Building 31 Conference Room 10 NIH, Bethcsda, MD OOOSiSi * The.^e m i n u t e s have not y e t been a c c e p t e d by t h e Da t a E v a 1. j a t i o n / Ui k As s e s s m e n t S u b g r o u p o f t h e Cl e a r i n g h o on E n v i r o n m e n t a l Ca r c i n o g e n s . ?161 1he occurrence of r a n tumor.'. Dr. Grie >:;:er n* tha , whenever significant, historical control data are included and analyzed in bioassay report:;. O W 765341 Review of Pi pass ay Reports* 1) P i l a v t r Dr. Brown served as tlie p r ima ry rev iewe r of the report describing the bioassay of !'ic 1o r a m . In summarizing the findings, l)r. Brown said that hepatic neo- plastic nodules were found in both inn le and female treated rats. In the females the incidence w as significant in the test for a positive J o s e -related tren d and rhe incidence in the.high-dose group was statistically significant when compared to pooled controls. The bio assay reports concluded, he said, that "the findings are sugge st ive of the a h i 1 it" o f the compound to inJucc benign tumors in tlie livers of fcm a 1e Osborne-Meiule 1 rats." Dr. Brown was particularly critical of the small number of matched control animals used, that the ten percent impurities in the Picloram were not analyzed, and that the diagnosis of "neoplastic nodules" had not been confirmed by additional pathologists. He felt that the conclusion drawn in the report on the carcinogenicity of Picloram was inordinately conservative. Hi? thought that the findings lent enough support to the hypothesis that Picloram may he a carcinogen, lie added, that consideration should be given to repeating, the bioassay using a more appropriate design. A lengthy discussion ensued on the biological nature and s ign if i.cance of the neoplastic liver lesions found in. the treated rats. hr . Brown recommended that the lesions be reviewed by additional pathologist's. Mr. Samuels said that P ic 1o ram siioulJ. be. regarded as a care ino gen since the li v e r feslor.s', i f not malignant, arc a"t lea st a- step in the p a t h o genesis of cancer. In regard to the ten percent impurities in the tested material, Dr. Griescraer noted that the Picloram was the -techa ica'l grade product to which humans arc exposed. Dr. Brown moved that, the Subgroup accept his critique of the biuass.ay of Picloram. Dr. Woisburgcr seconded the motion 'and it was accepted unanimously. 2 ) ('!;! ) rrP, >.. Dr. Wolfe served as the primary r e v i e w e r for the- report describing the hioassay of. Ghlorambcn. * S i n g l e c o p i e s - o f t lie i e p o r t s may he r e q u e s t ed from the 7162 Oi f i v e . o f Ga n c e r Gomr.ua i c a t i o n s , N a t i o n a l (lancer Inst itut B u i l d i n g 31, K->om 1(1.\ 2I , N a t i o n a l I n s t i t ut of H e a l tii B e t i i v s d a , Ma r y l a n d J 00 14. / - 1 'ii/e 4^ 4 J6 9 . HI O. * > ; . P s, V>'A. . . A. U lt Ki. . I CVlf. f>N I h < tri- r.iT., i-vt , 3 ^ - T H . t '*> *-*' ? * v W ? TT--n-f 3 a-! f i * - * : jc :d rd;PJ^:u'. 4 -< n i" -:z.iT.- r .' 2 31*jlr:rhi r 1 2 .) .i - in A ... i i. f-i f r i v? il .* ' < ti7n> j i T ` Mtdrasea. ,-ri: iie jtee* :*1- r ? - im; fie ld In- as**avs `if fieiJ t-J<".* ; J'T-j'-f-' iJ- v-j. c^r. !.. 4*e - -* **> r -.t % iL.* M'-r? ! *1C| efJfi M -*:- a r e /< *** - n ^ '**- le n r r . 2 . 3 . 6 - T H A 'j ? inf* 1-i.ate. affi itrj.r r . lMj ir.*.- i* j *t ' r- i>t**r.pp a c r ***s N rt-rjv*. T r- r.rta a a l pr.-. i fi.rttv -i p.-t r - a > :! a a * n-.< r.-ee.4*ir.lv : *iirit :7*-'r. ra ;r Jjl. t - * ,:re--t* -f r i;w u n j r ^ vi.I a;th Mener -ireanrf u t t e r r i r p * . - 4 3 7 a bCIFBKS. C .1. f t p . Rance fc i.. Tee A r:r !! .. ! > : t P C iliece it o li. . T *.. VSA. . R P. HAHN", and SI..U. V F PHI. I - - -:nal;i>n { r : * l .r.irr fr-'n: . tjSj >n f rrm : 1r !: r J 1J "Ii - . "AFTT': f-'K :*A S #r pa- Umc ae;*i pici r a n * r ver* Srtfriw f p r.r.a r. * InifJlocrJM- d jr t v i--teff Sett.* Er^elm. .v*! ni** e r i r / a __'i- tri u g racili Wil ex h1kT J .* e s Or fU h *.. i3iir.ed.jtfIt fr*r 'plication uf 0 .S k*z ha p id ruts 0 2 * * c fu 2,4.S-tr.Tr.; c n "-*" * ' " acetic acid I2.4.3-T- in *:irtbvtt fe x a *. Xe--- tfun 1 npir. f p r i t i n U5ualiv wan defected in ra se t;.v<ue 30 tv 0 liv > -ter tre.trv *t At 1 lociti-jn. detectaMe p;*rl r m . r r m s H r. c r a v i : ^ : # fr -m J I t dam after application. IncreiM** f p.cit ram :c *er;a* !.*;* were attributed tu r**i4 upt-** dar-u: a fl*a> f Tei;M4t.ie ^r-.mtr.. P;* ciuram dissipation from was n* affected ; r r . m . e .t j ran- off at 10. 20. t 30 dar? after applicati-or etectitrfr pset cam wa- reduced by 93** in bertuce'ais. hraadleaf Spec.-** tn JO i a f t e r ap plication. 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' r.A j _ w rt- i . :.;. t -/ i i3 5 T " f --:.--;r V .* * : ir-.. : .*;. : * *. **-ji . g" *?* i f * * - : - i ' .* - . t i . ' ar t . ***..' .v.v. j ^ -artrr n-j * * - tv ." \ . " - * r . 0005120 63 \ US' 7164 D.G, ED. MI. 4-4-73; DCW,. EPA AGREEMENT 9-/9 TOXICOLOGY RESEARCH LABORATORY HEALTH AND ENVIRONMENTAL "RESEARCH DOW CHEMICAL U.S.A. o O LOCATER SHEET FILE NO. H g T DEAD STORAGE NO. l-lg oe co CO CD R e s u i T lu e-Y eA RT I T L E O F R E P O R T ! ' - T 3 o ^-a / o w /c T i c (r y a a / * / t 4 F ; / a j T y S r u t y o r 1 ' 3,7,r-TeTR*cUL0KoOitU!Hjeo - P - i m v ( . T C W n RATS s e />+-** ber-D A T E R E P O R T I S S U E D ! 1 T (^ 7 7 AUTHOR (S ) i. fCOc'ii* , U . <i. ( C c y ^ /?.>?. fG e.V.ftrfc , /t.W u w // ici 'Notebook( s) - (Number and Pa g e (s ): 1 ) TB k V J i - S . 6 l + C i ______ 4 ) B k N f l T 7 - 6 U^i , D Z > , p * y 5 ~ - v < . i ` r - ? . K. K*h*i*% -73- 7 y S /B T n -x i -ji tiL -/a2 3 6 k i - 6 , 100 30 5 ) ____________ .__________________________ 3 ) S x V 8 T - 7 . r ^ 3 f ^ 3 f c _________ 6 ) _______________________________________ SPECIMENS: 1 ) U/ed T is s u e s PATHOLOGY NUMBERS: - H I-SO tZ 7 V - V 7 f / __________ 2 ) T 'i s s u e B l o e k s (g r- . 4. H 7 - V l o g 7 V - V 7 .r< __________ 3 ^ _ T l 5 ^ c i C ? "//< /* s _________________ - f V - V I S ~ST 7 V - V ? r / _________ s e e P & L o g )i f ) ___________ athqlo v _______ I________________________ _________ AD D ITION AL DOCUMENTS: LOCATION: PofVPH y R i M A A / A t y ^ f a ( B ie s c e H c e ^ --V e ^ J f-t-er-iq* F i l ___________ # S G T A n a ly s e s C Q 'tiienee) De^J F / ' / e ___________ 30 P/'/y jSax 3 A A -B D _ E l e c + r o * M ,r-o-C O fiV 5/i dg A>'/<; rrt-ifl /" //<_______ Fi'i.-g-- Slid* Cast* 7 HbevAxwrii^S'-i/-i3it-tT lr D r>u>or / RESTRICTED F O R U S E W I T H I N T H E DOW C H E M I C A L C O M P A N Y O N L Y . THIS PAGE S H O U L D F O L L O W T H E DOW S U M M A R Y P A G E A N D B E A T T A C H E D T O C O P M A I N T A I N E D ONLY IN THE T O X IC O L O G Y R E SE A R C H A R C H I V E S AND ES7165 QUALITY ASSURANCE UNIT F IL E S . 0004433 RESULTS OF A TWO YEAR CHRONIC TOXICITY AND ONCOGENICITY STUDY OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) IN RATS CO on co R. J. Kociba, D. G. Keyes, R. M. Carreon, C. E. Wade, D. Dittenber, R. Kalnins, L. Frauson, C. N. Park, R. Hummel and C. G. Humiston Checked by: B. A. Schwetz Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan September 28, 1977 0004433 7186 -2- ' SUMM ARY AND C O N C L U SIO N S Male and female rats were maintained continuously on diets supplying 0, 0.1, 0.01 or 0.001 yg TCDD/kg body weight/day for 2 years. These dose levels were equivalent to dietary levels of ~2,200, 210 and 22 parts per trillion (ppt) of TCDD. Ingestion of the high dose level of 0.1 yg vO- C cr # c TCDD/kg/day caused multiple toxicologic effects, including increased mortality, decreased body weight gain, slight depression of certain hematologic parameters, increased urinary excretion of porphyrins and delta-aminolevulinic acid, increased serum levels of alkaline phos phatase, gamma-glutamyl transferase, glutamic pyruvic transaminase and morphologic changes primarily of the hepatic, lymphoid, respiratory, oral and vascular tissues of the body. These morphologic changes included an increase in the incidence of hepatocellular carcinomas of the liver (females only) and squamous cell carcinomas of the lung, hard palate/nasal turbinates or tongue. Also seen at the high dose level was a significant decrease in the incidence of numerous age-related lesions, including a decrease in tumor incidence of the pituitary, uterus, mam mary gland, pancreas and adrenal gland. The incidence and severity of chronic renal disease was also decreased. Terminal content of TCDD in female rats given this high dose level averaged 24,000 ppt in the liver and 8,100 ppt in the fat. Ingestion of the intermediate dose level of 0.01 yg TCDD/kg/day caused lesser degrees of toxicity, including primarily an increased urinary excretion of porphyrins (females only), liver alterations, Including an increased Incidence of hepatocellular nodules 7167 OGtK-iSQ -3 - and lung alterations in the form of an Increased incidence of focal alveolar hyperplasia. Females given this dose level had a decrease in the incidence of tumors of the uterus. Terminal liver and fat content of TCDD averaged 5,100 and 1,700 ppt, respectively. Tf 00 CD **3 O Q Ingestion of 0.001 yg/kg/day caused no discernible effects in male rats and a statistical increase above the background incidence of swollen hepatocytes in female rats that was judged to be reversible in nature and likely associated with the hepatic detoxification process for TCDD. At this dose level, both liver and fat of female rats contained approxi mately 540 ppt TCDD. Evaluation of the data collected in this study indicates that doses of TCDD sufficient to induce severe manifestations of toxicity increase the incidence of some types of tumors in rats, while reducing the inci dence of other types. No increase in tumors occurred in rats receiving sufficient TCDD during the two-year study to induce mild manifestations of exposure. 0004461 I*<0/ ?189 I .. 4 Stnciiy oinicnu c o :.T T n -\7 r.\L ii.jd accident at a chemical factory in the Netherlands Ac cH i'hilips-Dvphar 2,4,5-T factory, sodium trichlorophenate -'.is -ynthetized by the hydrolysis of 1,2,4,5-tetrac'nlorobenzene using sodiumhydroxide and methanol at a temperature of approx. 170C and a pressure of approx. 22 atm. Temperature and pressure within the autoclave (capacity 900 litres) were registered autoraatically, but the machinery was handoperated. DOW 372641 UM05J305 Description of events c On March 6, 1963, during the start of the synthesis, the reaction became uncontrollable. Because of the "explosion" the safety-valve blew as well as the lid of the autoclave, and the contents of the vessel con taminated the inside of the A,COO m^ building, that became covered with soot. Nobody was injured by the.explosion and there was no fire. It vas ' estimated that the final temperature in the autoclave was 400-450C -:nd the'pressure > 80 atm. Concerning the cause of the accident, no mechanical failure could be found, but a mistake of the operators could not be excluded. . Immediately after the accident, the building was closed. Animal ex periments were started since the management knew of problems encountered with 2,4,5-T production in the U.S.A. and in the Federal Republic of Germany. Animal experiments On March 7, 1963, the first animal experiment was started. Three groups of 10 rats were used. The first group was placed in cages inside the building (exposure by inhalation). The second and third group was treated respectively by the oral and dermal route with 1 g of soot per kg body weight, rive animals i i 0002326 ^1170 DO W 372642 --n- of the firsc group vere killed after 3 days. Ac macroscopic examination, no lesions were observed. Microscopically, lipid accumulation was present in the liver. Of the remaining animals of this group, one died after 11 days. Of the dermally exposed rats, 2 out of 10 died within 11 days. All remaining animals were killed after II days. Microscopically, there was lipid accumulation in the liver. In a second dermal toxicity study, started on March IS, 10 rats were exposed to 1 g of soot/.kg body weight. Four rats died after 6, 8, 13 and 14 days. Again at microscopic examination, there, was lipid accumulation in Che liver. At the end of March the efficacy of the cleaning of the building was studied; 75 rats were placed inside the building (exposure by inhalation). Croups of 25 rats were killed after 1, 2 and 3 weeks. There was no mor tality; microscopically there was lipid accumulation in the liver. In ad dition, 6 rabbits were placed in the building. After 3 weeks they were re moved from the building. One animal died one week later. From these results it was concluded that the building was still contaminated despite thorough cleaning. In June, another experiment was done. At that time, the activities in the building included removal of paint from machinery, walls etc. A to tal of 48 rats were kept in open cages. In this manner, dust, peeled-off paint etc. contaminated the inside of Che cages. Six of these animals died. At pathological examination, thymus atrophy, enlarged tastes and lipid accumulation in the liver was observed. At the end f 1963, after the reconstruction activities, a final toxicity study was carried out in the building, using rats and rabbits. From the results it appeared chat the factory-building was still contam inated. Therefore, the final decision was reached to close the building. 000327 DOW372643 Analyses In March 1963, the compound TCDD was unknown to the plant management. At speccrcphotometric analysis, a peak occurred in the toxic material that was present in the building. Using this peak, the effect of the different cleanir.g-phases was studied. From more recent data it is known that this peak did not represent TCDO, but possibly a phenolic compound. In 1966, the CLC method for TCOD analysis, as developed by Dow Chemical Co., was introduced. TCDO levels in Che soot that was formed during the explosion were 1,000 ppm ^ 20Z (samples obtained from the autoclave as well as from the building). Ba-sed on these levels it is estimated that 200-500 g TCDD were released in the building. Samples obtained after the cleaning and reconstruction of the building were also analysed. TCDO levels in samples of floor scrapings ranged from 0 to 3 ppm; in paint from columns 6 to 25 ppm, and in paint from machinery 10 to 30 ppm. On the basis of these data it is estimated that between 20 and 200 g TCDD was still present in the building after Che thorough cleaning and reconstruction activities. Cleaning and reconstruction activities As Che results of the first toxicity study were not alarming, dust and soot in the building was removed and Che building was cleaned by Philips- Duphar employees, with water and a surface-active compound. Since animal experiments and analyses (measurements at 290 nm) showed that the building vt * as still contaminated, it was cried again to clean Che building. During the months April and May, factory employees sprayed Che building (for a total of II times) with 1,000 - 1,500 litres of 2Z sodium hydroxide, followed by spraying with water and a surface-active compound. As a result of this, ^ analyses at 290 nm showed chat the building was practically clean. Mext, 0002328the building was ventilated with fans (capacity of A0,COO a^/hr) . I c ( During the months May until July, reconstruction activities began. Paine vas removed from machinery, walls etc., as was insulating material. Next, the building was painted and damaged machinery was replaced. During this period, also non-factory personnel was hired: a clean-up crew for removal of paint (this group made use of organic solvents) as well as personnel for plumbing, painting and insulating. Protective clothing for all workers in the building included a mask (depending on the nature of the activities; ranging from a dust mask till a fresh-air helmet), overall, boots and gloves. In particular the hygienic measures (shower, changing of clothes) were very strict, but difficult to enforce for the non-factory employees. Demolition of the building o O $ CO -a to <T. At the end of 1963, the building was-closed, as decontamination was impossible. In 1973 the fa'ctory-building was dismantled, embedded in concrete and*dumped in the Atlantic Ocean. Working with fully protective clothing (air-supplied suit with integral helmet) proved a suitable method to prevent TCDD exposure. Details of the safety measures are published by Dalderup (T.soc.Geneesk. 52 (1974) 532 and 616). Medical data A total of 106 men are likely exposed to TCDD as they were in the building during the period March-July 1963. These workers can be divided into three groups: * A. Factory .employees (exposed during all phases of the cleaning and reconstruction activities). B. Clean-up crew (non-facto-y personnel; exposed during the months May-July). 71 7 3 00&2329 - 5- C. Personnel for plumbing, painting end insulating (non-factory workers; exposed during the months May-July). During the explosion, 2 operators were present in the building. With the exception of these men, who were likely exposed by the inhalatcry route, all other workers are probably exposed by dermal contact, because of the use of masks. This is possibly the reason that chloracne was the prominent feature of TCDD exposure. Liver function tests (thymol turbidity, serum glutamic-pyruvic and glutamic-oxaloacetic transaminases) did not indicate DOW372645 liver damage. A few men complained of fatigue. The latent period between exposure and the appearance oi chloracne, characterized by comedones, pustules and cysts on the face and sometimes on other parts of the body, was approximately 4 to 6 weeks. Sometimes, erythematous and oedematous skin lesions, possibly due to phenolic compounds, were noted one day after exposure. No skin lesions (chloracne) were reported in family members of the employees. Group A. A total of 44 factory employees had worked in the buildir.g. All were medically examined (during a period until March 1964; and further on at least once a year); 26 had chloracne. In 1977, t( of the 27 men who were still employed, 20 still had acne. Of the remaining 17 men, 3 died and 14 men went elsewhere or are retired. One man died in 1964, at the age of 54, from pancreas carcinoma with extensive metaStases in the liver. He died 14 months after slight exposure (cleaning of slightly contaminated room, next to the autoclave room). Already before the accident he complained of abdominal pain; he had no chloracne. One died in 1977, at the age of 69, from a second myocardial infarction and one man died in 1972 from a traffic accident, at the age of 76. . 717 4 00^2330 II DOW3 7 2 b`4 6 6 Group 3. Of a total of 13 men of the clean-up' crew, 16 were examined in 1963. Ten of ciiese 16 had chloracne. In 1977, 13 out of 17 men were alive, but no medical data are available (one man could not be traced). Four died: two of them in 1966 (sudden death, prob ably from myocardial infarction, at ages of 41 and 53 years); one died in 1967 at the age of 44 (progressive shortness of breath, not known whether of pulmonary or cardiac origin); one died in 1976 at the age of 65 (sudden death, probably myocardial infarction). Group C. Of a total of 44 men1 in this group of non-factory workers, 16 were examined .in 1963, eight had chloracne. In 1977, 31 out of 32 were alive; one died in 1969 (sudden death, probably myocardial infarction, at the age of 50). From a total number of 93 men (of the remaining 13, no data are avail able), 7 died a natural death, in the period March 1963 till October 1977, between the age of 40 and 69 years. This does not seem to indicate an ex cess mortality, in comparison with mortality data of men of similar age group in Che community of Amsterdam although,this is of course not a matched control group. However, caking group B (clean-up crew) separate, of 17 persons, 4 died which possibly indicates excess mortality. Moreover, three of these four persons died a cardio-vascular death. Deaths from malignancies were not observed, and morbidity data are not available. The causes of death of the / /y group as a whole is unexpectedly drawn towards cardio-vascular death. In the male Amsterdam population, deaths in the same age groups occur more from malignancies chan from cardio-vascular origin. The relative high in cidence of chloracne and mortality in group B versus groups C and A, is 0002331 OOW372B47 7 ro-crk.'bli Especially, since roup E was in die building at a time that the building was certainly less contaminated then early after the accidenc when factory employees removed contaminated dust and soot. Possibly, group B had increased exposition to TCDD because of the use of organic solvents to remove paint. According to their profession, they had in any case multiple exposures to more or less toxic substances both before and after the exposure to TCDO. _Proposed future medical studies 1 During 1973, the exposed factory employees (of whom 27 are still employed), will be medically exanined. Laboratory tests will include haema- tological examination, liver function tests (serum glutamic-pyruvic and i glutamic-oxaloacetic transaminases, lactic dehydrogenase, alkaline phospha tase), serum cholesterol levels, serum immunoglobulin levels, and possibly the in vicro responsiveness of lymphocytes to mitogens, and levels of porphyrins or porphyrin precursors in the urine. It will be tried to com pare these data with a matched control group (non-exposed factory employees of similar age). Of all men traced, mortality data will be collected in future years. It will also be tried to obtain morbidity data, but medical examination of the non-factory employees is not possible. The present report presents preliminary data, detailed information ,/ - 8- 0002332 -8- as well as Che results of che proposed study will be reported in the near future. November, 1977 J.G.Vos National Institute of Public Health, Bilthoven Tj.Sterringa (plant manager) D.Zellenrath (industrial medical officer) L.M.Dalderup (medical advisor) Factory Inspectorate, Amsterdam ( H.J.Docter (formerly industrial medical ^officer) Philips-Duphar B.V., Keesp DOW 372648 Vi ;/ 0002333 I2*1 1a i Cheoosphire No. 8 , pp 461 463, 1977* Pergamon Pt c s e . P rin te d in G reat B r it a in . FORMATION OF POLYCHLORINATED DIBENZO-P-DIGXINS AND DIBENZDFURANS DURING COMBUSTION OF A 2 A 5 - T FORMULATION B. Ahling, A. Lindskog Swedish Hater and Air Pollution Research Laboratory Box 21060, S-100 31 Stockholm, Sweden and B. Jansaon, G. SundstrSm National Swedish Environment Protection Board Special Analytical Laboratory Wallenberg Laboratory S-106 91 Stockholm, Sweden (Received in The Netherlands 1 July 1977; accepted for publication 4 July 1977) The use.in forestry and agriculture of products derived from 2,4,5-trichlorophenoxy qpids (2,4,5-T), e.g. I, has given rise to much concern during a number of years. The major cause of this concern rose from the findings of the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (II) as a contaminant in many of these preparations. 1 '2 Toxicity tests with II have given LD5Q values of 2 ug/kg in guinea pigs, the most sensitive species known, and around 80pg/kg in monkeys.^ The compound also causes irreversible liver damage and is a potent inducer of enzyme systems. In man exposed to II one symptom is chloracne, a severe skin disease. 4'5 Cl OCH2 COOCH2 CH2 OCH2 CH2 CH2 CH3 Cl I CL IICl' O' 461 462 No. 8 The presence of II In the chlorophenoxy acids is due:to its formation as a byproduct in-the-preparation of 2,4,5-trichlorophenol used as a starting material for the manufacture of 2,4,5-T products. 5' It might also be formed during the coupling reaction between the sodium salt of 2,4,5-trichloro phenol and the appropriate Qt-chloroacid ester. Although the content of II in 2,4,5-T preparations is nowaday under strict regulations and control, there is still some uncertainty about the possible formation of II from 2,4,5-T products during fires or by photochemical trans formations. Laboratory experiments have shown that large amounts of II are formed when the sodium salt of 2,4,5-trichlorophenoxyacetic acid or of the 2-(2,4,5-trichlorophenoxy)propionic acid is heated in a melt at 500-600o .7'8 The present investigation was performed in order to establish whether any formation of II might occur during burning of 2,4,5-T products, i.e. under simulated environmental conditions. These experiments were carried out in a combustion installation for pilot scale tests with possibilities for varia- 9 tion of conditions such as temperature, transit time and available oxygen. In this study account was also taken of the possible formation of other' polychlorinated dibenzo-p-dioxins (PCDD) than II and also formation of poly chlorinated dibenzcfurans (PCDF). Although II seems to be the most toxic isomer of the chlorinated dibenzo-p-dioxins, many pentachloro-(5CDD) and hexachlorodibenzo-p-dioxins (6CDD) show similarly low LD^g values. ^ It was recently shown that some preparations of 2,4,5-T in addition to II also contain pentachlorodibenzo-p-dioxins and tetrachloro- (4CDF) and pentachlorodibenzofurans (5CDF). 10 Chlorinated dibenzofurans have been reported to show almost the same high degree of toxicity as the PCDD. EXPERIMENTAL Analysis of the herbicide formulation. The 2,4,5-T product used was speci fied by the manufacturer to contain 500 g/1 of 2-butoxyethyl 2 ,4,5-trichlorophenoxyacetate (I) in an inert hydrocarbon solvent. The concentration was determined by GLC using a synthetic standard. Free 2,4,5-trichlorophenoxy acetic acid in`the formulation was determined by GLC as the methyl ester after appropriate dilution of the product with hexane-diethyl ether (1:1) followed by treatment with diazomethane. 2,4,5-Trichloro- and pentachlorophenol were determined as their methyl ethers after extraction from a dichlaromethane solution of the formulation (0.1 g in 3 ml CHjClg) with 1 M potassium hydroxide solution (3 x 3 ml). After acidification and re-extraction with diethyl ether ( 3 x 3 ml) the extract was treated with diazomethane and after appropriate dilution used for quantitation by GLC. Analyses for chlorinated dibenzo-p-dioxins and dibenzofurans were performed by GC-MS after clean-up of the 2,4,5-T preparation by the use of a charcoalpolyurethane adsorbent according to Huckins et all** with the exception that toluene (100 ml) was used to elute PCDD and PCDF. Burning experiments. The pilot scale plant has been described earlier. 9 In a first series of nine experiments the possibility to destruct Z was studied. The temperature was varied between 300 ("open fire") and 950 with transit times of 2-3 seconds. The formulation was?in all experiments except under "open fire" conditions, dissolved in isopropanol and injected through an atomizing nozzle into the oven, charged with sawmill wood chips. In the open fire experiment the formulation was mixed with sawdust and wood chips '30 kg)- and introduced into the oven. In a second series of experiments the formation of chlorinated dibenzo-pdioxins and chlorinated dibenzofurans from I was studied. Three experiments (1-3 in Table 2) were carried out with a mixture of 30-75 kg sawmill wood chips and 1 kg 2,4,5-T formulation which was burned at temperatures bet ween 500 and 850 with transit times of about 0.7 seconds in the burning zone. In experiments 4-6 a mixture of 50-100 kg wood chips, 8-10 kg dry--- leaves and r~kg~"bf the formulation was burned. One experiment (7) was carried out under pyrolytic conditions, i.e. in an oxygen-deficient atmosphere and one (8) as a simulation of an open fire. In the latter experiment sawmill wood chips (30 kg) and leaves (8 kg) together with 1 kg of the formulation were allowed to burn without any attempts to raise the temperature. Sampling and clean-up of PCDD and PCDF. Equipment made entirely of glass O were used for Isokinetic sampling of emissions from the oven. Particulate matter was collected on filters of glass wool which were eluted with toluene for analysis. Gaseous components were collected on concentration columns filled with Chromosorb W coated with 30% Apiezon M.13 The columns were elut ed with hexane and toluene. From each experiment the three extracts were combined and the solvent eva porated. The residue was dissolved in 1 ml hexane and chromatographed on 1g basic alumina (Aluminiumoxid 90, aktiv, basisch, Merck, activated at 250 rfor 2 h). The column was eluted with hexane (10 ml, fraction A) to remove non-polar co-extractives followed by ethyl acetate (10 ml, fraction B) to elute PCDD and PCDF . Recovery experiments showed that PCDD added to the extracts were recovered to about 70% in fraction B. 464 No. 8 Analyses of PCDD and PCDF. The dioxins and furans were analysed by mass fragmentography on a UP 5930 A <30--MS with a "HP 5933 A computer system. In each run five chlorination degrees of PCDD or PCDF with two ions each and one internal standard were analysed. As internal standard was used a 2,3,7,8tetrachlorodibenzo-p-dioxin with four 37Cl and four deuterium atoms (M=332).13 The reference compounds available for the quantitations were IX, a mixture of two 6CDD (unknown isomers), 8CDD, 4CDF (2,3,7,8-) and 8CDF. Since these do not cover all the chlorination degrees determined, Interpolations had to be made. Thus the area measured for one m/e value was corrected to a total area which correspond to all m/e values in the chlorine isotope cluster of that ion. The amount injected divided by this total area give fairly constant response factors for the references available. RESULTS AND DISCUSSION Impurities in the herbicide formulation. The results of the analyses of the formulation are given in table 1. A notably high concentration of the free 2,4,5-trichlorophenoxyacetic acid was found. In addition to the listed results, the specified content of the active substance (I) (500 g per 1 formulation) was verified. Table 1. Impurities in .'the herbicide formulation used for combustion experiments. Compound 1^ g per g formulation 2,4,5-Trichlorophenoxyacetic acid 2,4,5-Trichlorophenol Pentachlorophenol Dioxins 4CDD 5CDU 6CDD . 7CDD8CDD Furans 4CDF 5CDF 6CDF 7CDF 8CDF 4.7 10"2 2 . 3 1(T4 1 .2 -e; 10 3 1.8 10-8 .Q 7 10 * -Q <5 10 * <5 10 * .5 I Q " 9 1 . 3 10-7 8 10-9 <5 i o ' 9 <5 10-9 <5 10-9 1) 4CDD, 5CDD etc. comprise all isomers containing 4^5 etc. chlorine atoms, respectively. No. 8 465 The content of 4CDD is well below concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin accepted by the Swedish authorities. The level of 4CDF is much higher than that of 5CDF and also that of 4CD0. In an earlier investigation 10 the same dimers were found, but no quantita tive data were given. Destruction of 2-butoxyethyl 2,4,5-trichlorophenoxyacetate. The experiments performed to study the destruction of I showed that, with one exception,---99,995% of I is destructed at 400-500C and a transit time of 2 sec. However, when "wood chip's with a very high water content was burned under "open fire" con ditions, the temperature in the flue gas duct decreased temporarily to around 100C and only 92% of I was destructed. Emission of PCDD. The conditions in the burning experiments and the emitted amounts of PCDD and PCDF with 4 to 8 chlorine atoms are given in table 2. Table-2.~ Emitted"amounts-of PCDD and'PCDF from combustion of a herbicide formulation containing 2-butoxyethyl 2,4,5-trichlorophenoxyacetate Experiment no Temperature (C) Transit time (s) Oj In flue gas (%) (>2 in flue gas (%) 00 in flue gas (%) Emitted amounts (mg/kg formulation) Dioxins 4CH> 5CJD 6CDD 7CED 8CDD Furans 4CDF 5CDF 6CDF 7CDF 8GDF 1 500 0.7 15.0 2.6 0.0 23 675 0.6 12.0 4.0 0.0 850 0.7 10.5 6.1 0.0 45 500 0.8 13.0 4.1 0.0 625 0.8 12.0 7.2 0.0 678 850 0.9 7.5 7.7 0.0 750 3.7 0.0 9.0 5.5 100 1.0 17.5 0.9 0.0 3.0 <0.4 2.0 <0.4 <0.4 2.3 <0.5 <0.5 <0.5 0.5 <0.1 <0.2 <0.2 0.2 0.2 1.3 <0.6 <0.6 0.6 0.6 0.4 <0.3 <0.3 0.3 0.3 0.2 <0.3 <0.3 0.3 1.4 1.4 1.2 0.8 0.3 0.05 2.5 <0.5 <0.5 0.5 0.5 <0.2 1.8 1.3 <0.7 <0.7 8.0 7.1 2.8 <0.7 <0.7 1.3 1.9 1.8 1.4 0.6 0.6 1.3 0.6 0.6 0.6 0.2 0.3 0.3 0.3 0.3 2.9 7.6 1.0 4.2 5.7 0.5 4.9 1.7 0.4| 3.4 0.2 2.3 3.9 0.1 0.4 466 No. 8 Background values were obtained after combustions without the herbicide. About 0.03 mg 4CDD were emitted during half an hour, which corresponds to the period required to burn 1 kg of the formulation in the experiments. Higher chlorinated dioxins could not be detected. The results show that, with a few exceptions, only 4CDD was detected. This emission decreases with increased temperature and transit time. The very low temperature under "open fire" conditions (experiment 8) does not give extremely large 4CDD amounts. The explanation would be that the formation of dioxins is inefficient at this low temperature or that the formed dioxins are effectively destructed during the relatively long transit time. The formation of higher homologues of PCDD in some cases, expecially under the oxygen-deficient conditions in experiment 7, can not be explained at present. The content of PCDD in the formulation can not explain the amounts of these compounds present in the flue gases. Furthermore, combustion experiments with chlorophenol formulations carried out by us 1 4 indica*te that the con tent of chlorophenols in the 2,4,5-T formulation can not give the PCDD amounts found in the present study. The 2,4,5-trichlorophenoxyacetic acid in the formulation could, however, possibly be the precursor if extrapola tions are made to earlier studies which showed that II was formed from the free chlorophenoxy acid in about 1% yield when heated as a melt to 500-600C. 7,8 Emission of PCDF. The results of the PCDF analyses are presented in table 2. The background levels of PCDF were unexpectedly high and emitted amounts up to 0.5 mg during a normal experiment run time have been detected. .The results given in table 2 are not corrected for these background values and at least the low values must be regarded as unreliable. In most experiments the emissions of PCDF are higher than that of the PCDD. These amounts of furans can not be explained by the content in the formula tion. It is unlikely that the chlorophenols present in the formulation have any significance for these emissions. 14 Under the experimental conditions used the emission of PCDF can not be corre lated to either temperature or transit time. CONCLUSIONS t Under normal combustion temperatures (>500C) the investigated 2,4,5-T ester was destructed to 99.995%. * It is evident that under certain conditions, the combustion of a 2,4,5-T formulation may lead to a formation of small amounts of PCDD and PCDF. The role of the free 2,4,5-trichlorophenoxyacetic acid in this context is not clear but should be further Investigated. The emission of PCDD decreases with increased temperature and transit time. The highest emitted amount was 3 mg 4CDD per kg formulation at a temperature of 500C. For the PCDF emissions up to 8 mg per kg formulation have been measured (4CDF at 675C) but no correlation to either temperature or transit time can be found. The concentration of the 2,4,5-T ester in the burnt material in the present _2 study (about 10 g/g wood chips) is higher than the concentrations found after normal use in the environment (10-4 -10-3 g/g dry leaves 13'1). A high concentration will favour the formation of dimers and smaller amounts are to^be expected from e.g. a forest fire. The largest formation of 4CDD found in this work should correspond to a formation of about 1 ug 4CDD per 2 m in a forest fire directly after application of the investigated herbicide formulation. Even if the formation in the environment is probably smaller than this amount, extreme caution is necessary not to disseminate these very toxic xenobiotics. ACKNOWLEDGEMENT We are indebted to Dr. S.Jensen for valuable discussions and critical advise. The skilful technical assistance by Miss L.'Fahlberg, Mr L.Johansson, Mr-A.LindstrSm, Mrs K.Nylund and Miss K.Wiberger is greatfully acknowledged. REFERENCES 201. Woolson, E.A., Thomas, R.F. and Ensor, P.D.J., J.Agric.Food Chem. (1972) 351. 2. Ramstad, T., Mahle, N.H. and Matalon, R., Anal.Chem. 9 (1977) 386. 3. McConnell, E.E., personal communication. 4. Kimbrough, R.D., Arch.Environ.Health 25 (1972) 125. 5. Schulz, K.H., Arch.Klin.Exp.Derm. 206 (1957) 589. 6. Kimmig, J. and Schulz, K.H., Naturwissenschaften 4j4 (1957) 337. 7. Buu-Hoi, N.P., Saint-Ruf, G., Bigot, P. and Mangane, M., Compt.Rend. 1971 708. 8. Saint-Ruf, G., Naturwissenschaften 59 (1972) 648. 468 No. 8 Chemoephere No. 9. Ahling, B. and Johansson, L., Chemosphere, in press. 10. Huckins, J.N., Stalling, D.L. and Smith, A.L. Presented at the 90th Annual Meeting of the Association of Official Analytical Chemists, Oct. 18-21, 1976, Washington D.C. 11. McKinney, J.D., Chae, K., Gupta, B.N., Moore, J.A. and Goldstein, J.A., Toxicol.Appl.Pharmacol. 36 (1976) 65. 12. Laveskog, A. and Lindskog, A., Chem.Ing.Tech. 8 (1976) 65. 13. GOthe, R. and Leander, K., to be published. 14. Jansson, B., Sundstrfim, G. and Ahling, B., to be published. 15. Plumb, T.R., Norris, L.A. and Montgomery, M.L., Bull.Environ.Contam. Toxicol. 22 (1977) 1. 16. Sundstrdm, G., Jansson, B. and Jensen, S., to be published.. STP POL'. (Receive Polychloi . food poisoninc -,vJti'i western Japan of gas chroma 'pi Yusho were di ''t' observed in t) I ' after the inci & .tfv:.of these PCBs m - Jensen ar grti-: in PCB product 'fS^E'? Apiezon L and \ as !8 \ IS ..sIt --hl owingpouipsoinn m--odthioexrisn' miik By Um Washington Post " We do not know w hether 10 o r 40 p p t o f dioxin in W ASHINGTON -- H arvard U niversity research ers .- m o th e r s ' m ilk f a t w ill b e in ju rio u s to h u m an b a b i e s , " rep o rt th at four sam p les of m others' m ilk tested in tw o sta te s contain traces of a super-toxic industrial chetnical -- a poison the Environm ental Protection A gency describes a s "p erh ap s the m o st toxic sm all m olecu le known to m a n ." -r sa id D r. Ja m e s R . Allen, a research er a t the Univer city of W isconsin M edical School, who is studying the ' effects of dioxin on m onkeys. B ut he said the d ata h as ; '-i 's o m e v e r y se r io u s im p lic a t io n s ." c ; : . " T h om as H ollaw ay, an official in the E nvironm ental o O ''.` T h e c h e m ic a l is c a lle d d ioxin . It's a m a n u fa c tu r in g * '^ P r o t e c t io n A g e n cy 's O ffice o f P e s tic id e s , s a y s th e im p u r ity th a t often c o n ta m in a te s c e r ta in in d u stria l V.-.-t " a g e n c y w ill " r e s e r v e opinion" on H a r v a r d 's fin d in g s O 3 COp r o d u c t s , in c lu d in g th e w id ely u s e d h e r b ic id e 2,4.5-T. .. j. u n til th ey h a v e b e e n " c o n fir m e d b y a n o u tsid e lab o - VJ1 oT h e h e r b ic id e h a s b e en u s e d in th is c o u n try s in c e 1948 . - .r a t o r y ." VM on p a stu re s and in national fo rests, along h ighw ays an d pow er line rights-of-w ay, and on rice crop s. *. Scien tists have w orried about the potential th reat o.. > i ' - W illiam B . S e w a r d , a s p o k e sm a n f o r D ow , s a i d , - - " We don't believe H arvard h as a reliab le m ethod (fo r .. . / d e t e c t i n g d io x in ) w hen d e a lin g w ith le v e ls b elo w 10 V A 03 to o f d io x in s in c e th e e a r ly 1970's , w h e n la b o ra to ry . y . p p t. We'v e an aly z ed m o th ers' m ilk , a n d w e'v e n e v e r . stu d ies show ed a sin gle dose of the ch em ical k ills ani ' found an y ." m a ls in the sev eral hundred p arts p er trillion ran ge, low er than any other chem ical ever tested. T he H arvard m others' m ilk findings open the latest round in th e -right-year battle betw een environm en- . * D r . M atth ew M eselson, ch airm an o f th e d e p a r t < --.ta lists and the fed eral governm ent over the herbicide m e n t o f b io c h e m istr y an d m o le c u la r b io lo g y a t H a r- j ; : , .'.2 ,4 ,5 -T . T h e he rb ic id e f i r s t b e c a m e th e t a r g e t o f w id e- v a rd , sa id that if confirm ed by furth er testin g, the . ... sp read public protests when U .S . w arp lan es sp ray ed n ew fin d in g s w o u ld p ro v id e th e f ir s t c o n c re te - ` it in m a ssiv e quan tities to defoliate V ietnam fo re sts ev id en ce th a t dioxin from 2,4,5-T an d p o ssib ly other i. ..d u r in g th e In d o c h in a w a r . - so u rce s m ay be accu m u latin g in hum an tissu e s. I f / : r e se a r c h e r s r e p e a t H a rv a rd 's fin din gs in a la rg e r ' ` When a go /em m en t-spo n sored stu d y in dicated in sam p le of women across the country. D r. M eselson . ' ^ 1969 th a t 2 ,4 , >-T c a u s e d b irth d e f e c t s in la b o r a t o r y s a id " th at would be cau se fo r co n cern ." m ice, the Pentagon bowed to public p ressu re an d 'D r . M eselson and oth er H a rv a rd re se a rc h e rs banned the he rbicide from its V ietnam arsen al. te ste d the b re ast m ilk from 18 w om en livin g in M is L a te r stud es showed the chief culprit in cau sin g so u r i, T e x a s and O regon, n e a r a r e a s w here 2,4,5-T is the birth deft rm ities and other toxic effects w as the routinely sprayed. - d ioxin in 2,4,,` -T. Soon a fte r E P A w a s fo rm e d in 1971, A ccording to a new chem ical an alytic technique de \ - th e agen cy a inounced it w ould hold h earin gs to con veloped at H arv ard , the b re a st m ilk fro m four sid e r banning 2,4,5-T from the m a rk e t. w o m e n iA T e x a s a n d O rego n a p p e a r e d to c o n ta in . But those 1earin gs w ere canceled and have never d io x in a t le v e ls f r o m 0.6-1.6 p a r t s p e r trillio n ( p p t ) . been held. E l 'A officials contend they cannot ban the T h e levels tran slate to 1040 ppt of dioxin in the m ilk- . ; h erbicide u w il, an d un less, they gath er conclusive fat. the research ers said. ' - evidence th a t dioxin actually threatens th e public T h ree of the b reast m ilk sam p les, fro m T e x a s, - health by " b oaccum uiating in m an and the hum an w ere collected by L a Leche L eagu e, a national group food ch ain ." F o r the past four y ears, research ers at th at ad vocates b reast feeding. T h e O regon sam p le . the E nviron n en tal Protection Agency (E P A ), H ar- w as provided by a m em ber of G tizen s A gain st Toxic Z: v a rd , Dow a id oth er re se arc h institutions h ave con- S p ra y s. It is one of num erous environm ental groups . ; . ducted a spec ial " dioxin m onitoring p ro g ra m " to fin d . a c r o ss the nation that h ave su ed the U .S. F o re st Serv- * w hether diox n con tam in ates food and hum an tissu es. ic e in a n effort to h alt 2,4,5-T sp ra y in g in n ation al fo re sts. *: B u t th eir s- a r c h h a s b o gged down in a co m p lex an d F iv e m ore w om en w ere tested in Boston, w here the ' '* fru stra tin g tec h n o lo g ic al b a ttle . F o r ev e n a s re- h erbicide is not w idely used. They did not h ave dioxin search ers lock for dioxin in the environm ent, they are '' <ln th e ir b re a st m ilk . D r. M eselson sa id the H a rv a rd ' still trying to perfect the an alytic techniques cap ab le - fin d in g s h ave riot been con firm ed b y o th er lab o rato o f m easuring: dioxin in the p a r ts p er trillion ra n g e -- rie s -- research ers used all the m ilk sam p les -- and . a " pioneering e ffo rt," accordin g to E P A docum ents. h e ch aracterized them a s "p re lim in a ry ." Sin ce 1974. re se arch e rs h av e reportedly d etected But the H arvard findings m ay ad d w eight to previ -dioxin r e s id ie s in b ird s an d fish in O regon 's S iu slaw o u s re se a rc h su gg estin g th a t dioxin from 2,4,5-T m a y N ational F o tc st, in beef fat an d now m oth ers' m ilk. con tam in ate p art of the nation's food supply. : B ut scientists at E P A , Dow and H arvard have con R esearch ers a t H arvard and Dow C hem ical Co. of sisten tly disagreed ju st how m an y p a rts p er trillion of - M id la n d , the m a jo r m a n u fa c tu r e r o f 2,4.5-T , r e p o r te d dioxin they're finding in ju s t how m a n y sam p les. CD GO ro O* J 5 o la st y e a r they detected dioxin a t le v e ls up to 69 ppt in b eef fa t sliced from cattle which h ad g raz ed on p as- H ollaw ay said the d ata " certain ly indicate from a q u a litativ e stan dpoin t that th ere is d io x in " in beef * tu re s sp ray e d with 2,4,5-T. " I f th ere w ere le v e ls o f dioxin o f even 1 p p t in th e fat. And he sa y s " I think D r. M eselson's stu d ies are .. in d icativ e o f dioxin re sid u e s in h u m a n s." B u t until h u m an diet, a n d if th e m a te ria l w ere highly H a r v a r d 's m e th o d s a r e " reco g n ized b y sc ie n tists in accum ulative, a s m onkey tests in d icate," D r. M esel so n sa id in a recen t telephone in tereview , "th e n th at w ould put u s o v er a period of y e a r s in the region of -' the field a s being a positive and valid m eth od," the E P A w on't know th e p re cise lev els of dioxin, Holl aw ay said. 7188 h avin g a body burden th at would be lethal for a gu i W ithout that inform ation, the E P A won't be ab le to nea p ig." determ ine for su re w hether dioxin in the environm ent th reaten s the p ublic's health, he said . H ollaw ay said from the d a ta h e's seen so fa r , " w e don't feel th ere is a significant r is k of e x p o su re to h u m a n s" fro m - -dioxin. ' <:''bt C Som e scien tists and en viron m en talists re je c t E P A 's view . F o r one thing, a s E nvironm ental D e fense Fun d gen eral counsel W illiam B utler sa y s, "N o scien tist h a s ev e r been ab le to define a 'no-effect* le v e l l o r a c h e m ic a l w h ic h c a u s e s b ir th d e f e c t s .1' - r ' * Scientists such a s D r^M eselson w arn that stu d ies a t the U niversity of W isconsin su gg est p rim ates " a r e rath er surprisingly sensitive (to dioxin) an d th at it is highly accum u lative in their b o d ies." . .. W hile the dioxin d eb ate h a s focu sed on the herbi- ` cid e 2,4,5-T, an E P A rep o rt w arn s th at dioxin m a y con tam in ate other, h erbicides an d num erous chem i'c a ls u sed in the p a p e r an d pulp, leath er tanning, tex tile and other industries. L a st su m m er, hundreds o f people in S ev eso , Italy , w ere evacu ated -- and m uch of the town w as se ale d o ff by barbed w ire -- afte r a nearby factory th at m ak es chem icals for herbicides, m edicines an d cos- m etics leak ed a cloud o f dioxin into the sk y . Thousands of an im als died, pregnant women w ere u rged to h av e abortion s, an d the once-lush cro p lan d s .w ere contam inated, possibly for y e a rs. .. . < :v *. Researchers see C3US6 for COnCem * O iTl n e w I c S l S in areas Spraying f herbicide . 907625 b Q t CGC5&39 (19?o~5o); ace-Scie-ce iltursv'"*the CoiTk_.*ttee lobiological .roleofthe :hthe chroiiswork es)me sizeor diosensitivfali into 3 srchromo>fthestud* iedata on rreased by th a basic -st,a very edon this :ousphysnetic test chemical indetect* iavenNa'logyDe- r. xO l M utation Research, 4 2 (1 9 7 7 ) 3--18 3 Elsevier/NorCh'HoIland Biom edical Pres 3 THE 1NFLUENCE OF pH ON THE EFFECTS OF 2,4-D (n 4-D1CHLOROPHENOXYACETIC ACID, Na SALT) ON S A C C H A R O M Y C E S C E R E V I S I A E AND S A L M O N E L L A T Y P H I M U R 1U M HS T ' L B U S K ' * * E L O V SO N . * STA R EC -N O R D EN H A M M A R and Department o f Genetics and Plant Breeding, University o f Uppsala, Uppsala (Sweden) (Received March 15th, 1976) (Revision received August 2 5 th , 1976) (Accepted Septem ber 9th, 1976) Summary The genetic effectsof 2,4*D (2,4-dichlorophenoxyacetic acid,Na salt)have been investigated in cells of the yeast Saccharomyces cerevisiae and of the bacterium Salmonella typhimurium in experiments in vitro and in vivo. Experiments in vitro showed that the killing of both yeast and bacteria is dependent on the pH in the treatment solution of 2,4*D. A dose-dependent increase of the frequency of mitotic geneconversion and mitoticrecombina tion in yeastwas observed atpH 4.50 and 4.30.Inexperimentsinvitrowith two strainsof Salmonellano significantincreaseofthenumberofrevertantsto prototrophy was obtained.The positive correlation between survival of cells and dissociation of 2,4-D in the pH region2.8--5.0indicatesthatthecellsare unable to take pH *hen most up dissociated 2,4-D. Therefore the survival ishigh 2,4-D isin dissociatedform, and thesurvivalislow at at aa high rela tivelylow pH when more of the 2,4-D isinitsundissociatedform.No genetic effectswere induced by oraladministrationoftolerabledosesof2,4-Dinhost- mediated assaysusingmice ashosts and yeast orSalmonellaasindicatorcells. Introduction The intensive use of chlorinated phenoxyalkanoic acids as herbicides in agriculture and forestry and alsoasdefoliantsduringthew?* inVietnam has initiated a largenumber of investigatorsinto theiracute toxicityinmammals Abbreviations: 2,4-D, 2,4-diehlorophnoxyac*tie add; 2,4,5-T, 2,4,S-(ilchlorophmoayacatic acid; MCPA. 2-mathyl-4hlorophanoxyac*tic acid; Macoprop, 2K2-rathyl-4-cbloroph*noxy)-propionic add: EMS, ethyl methanasutfonata. * 0005630 % o O o vn V*4 O CCOo - V* hu i 4 includingman. The geneticeffectsseem tobemuch lessinvestigated. With mammals astestsubjectstheresultsarecontradictory.Buselmaieretal. [3] observed no increase of dominant lethals in mice treated with 2,4,5-T. Cytogenetic testsonratcellstreatedinvivoand invitrowith2,4,5-Tand 2,4-D gavenegativeresults[16].Inmice [5] andMongoliangerbils[11]chromosome aberrationshave been induced with highdosesof2,4,5-T. In higherplantsmany investigatorshave found cytologicaleffectsofchlori nated phenoxyalkanoic acids but their relevance isconsidered questionable because inplantsthesecompound haveahormonal effefct.However,Dark [4] and Kabiesch [8] have induced mutants inthe offspringoftomatoes treated with2,4-Dand ofpotatoestreatedwithMecoprop,respectively. In experiments with Drosophilalow concentrations of2,4,5-Thad effects on oogenesis and fertility [6,7],andhighconcentrationsincreasedthenumber ofX-bound recessivelethals[12]. Results from experiments with microorganisms areatvariance.Andersenet al. [2] found no increase of the frequency of revertants inSalmonellaafter treatment with ninedifferentchlorinatedphenoxyalkanoicacids.Two ofthem were also tested in experiments with bacteriophage T4 and were found nega tive. Buselmaier et al. [3] reported negative results with 2,4,5-T, 2,4,5-T-nbutyl esterand MCPA tested inSalmonella (invitroand inthehost-mediated assay with mice) and Serratia(invitro).Negativeresultswerealsoreportedby Styles [16] from treatments of Salmonella in serum of ratsgiven2,4-D and 2,4,5-T.The lattercompound was alsowithout effecton mitoticgeneconver sion inSaccharomyces cerevisiae.[15].Positiveeffectshavealsobeenreported: mutationshavingeffectson sporulationandproductionofantibioticshavebeen induced with 2,4-D inStreptomyces [9];mitotic geneconversioninSaccharo myceshasbeeninducedwithMecoprop [13] and 2,4-D [15]. We reportinthispaper the resultsfrom treatmentsinvitroandinvivowith 2,4-D on Saccharomyces cerevisiae and Salmonella typhimurium. Forthetreat mentsinvivowe usedmiceinthehost-mediatedassay. Materialsandmethods - >phy by o*. Division rn o Q ed in the bought CD CD CT ents in vi< O jene c o m 'me method desc were grown in-ye source to eliminat when thisstrainis tureswere incubate forspontaneous co "convertants were 1 Before the treatme sion was standard: spectrophotometer solutions of 2,4-D buffer(pH 7.0).W inMcllvaine'scitn on yeast extract-j: - white on this m (petites)were recc after5-days*incub Mitotic recombi The method de grown overnightc glucose. The treat werecountedaftei Test agents 2,4-Dichlorophenoxyacetic acid, Na salt, preparation from Monsanto Chemical Co.,St.Louis,Mo.,U.S.A.,wasagiftfromDr.A.Aamisepp,Depart ment of Plant Cultivation, Royal Agricultural College, Uppsala, Sweden. Freshlypreparedsolutionswereusedinallexperiments. Ethyl methanesulfonate,usedasapositivecontrol,wasobtainedfromSigma Chemical Co.,St.Louis,Mo., U.S.A. Back-mutation The methods c were grown overr. ments were mad: effectof 2,4-D w which coloniesw revertants was de after3 days*incu Biological test material Saccharomyces cerevisiae,strainD4, used fordetectionofmitoticgenecon version intwo lod, trp5 and ade2, andstrainD5, usedfordetectionofmitotic recombination, were obtained from Dr. F.K. Zimmerman, Technische Hoch schule,Darmstadt,BRD. Salmonella typhimurium, strains TA 1530, TA 1535 (his", reverting to prototrophy by base-pairsubstitutions),TA 1531 andTA 1538 (his-,reverting Experiments in vi The methods Legator and Mai food were remov a suspension of was injected int CG05631 'd. iolmaieretal. with % 4,5-T. >'T 2,4-D ch.^mosome ctsofchloriquestionable 'er,Dark [4] itoes treated 'had effects thenumber Andersen et onellaafter wo ofthem 'ound nega,2,4,5-T-nst-mediated eported by 2,4-D and -*neconverireported: shavebeen Saccharo- vivowith ti "it- VTonsanto ',DepartSweden. m Sigma *neconmitotic 1Hoch- ing to verting to prototrophy by frame-shift mutation) were obtained from theWallenberg Laboratory, Division of Genetics,Stockholm, Sweden. Mice, used in the host-mediated assay,were males weighing about 30 g,of strain CBA, bought from the Instituteof Genetics,University ofStockholm, Sweden. j Experiments in vitro Mitotic gene conversion in Saccharomyces cerevisiae,strain D 4 The method described by Zimmermann [17] was followed ingeneral. Cells were grown in yeast-extract-peptone medium, with 2% glycerol as carbon source to eliminate respiration-deficient cells that occur in high proportion when this strain isgrown in medium with glucose ascarbon source.The cul tureswere incubated on ashakerat18--20C tilllatelogarithmicphase,tested forspontaneous convertants, and cultureswith low spontaneous frequency of convertants were kept at 4C tillthey were used in the test(max. 3 weeks). _ Before the treatment thecellswere washed and the densityofthecellsuspen- "* sion was standardized to E t*," 0.25 (about 1 X107cells/ml) with a Linson spectrophotometer. The treatments were made inthe dark at37C withfresh solutions of 2,4-D in buffers and stopped by repeated washing inphosphate buffer (pH 7.0).When the effectsoflow pH were studied2,4-Dwasdissolved inMcllvaine'scitrate-phosphatebuffer.CellsofstrainD4 formed redcolonies on yeast extract-peptone agar. However, respiration-deficient colonies were white on this medium. The frequencies of respiration-deficient colonies (petites)were recorded insome oftheexperiments.The colonieswerecounted after5-days'incubationat30"C. Mitotic recombination in Saccharomyces cerevisiae, strain D 5 The method described by Zimmermann [17] was followed. The cellswere grown overnight on ashakerat30C inyeast-extract-peptonemedium with 2% glucose. The treatments with 2,4-D were made as described above. Colonies werecountedafter7 days'incubationat30C. Bad-mutation test in Salmonella typhimurium The methods described by Ames et al.[1] were followed ingeneral.Cells were grown overnighton a shakerat37C inDifcoPenassayBroth.The treat ments were made as in the experiments with yeast (see above). The killing effectof 2,4-D was determined by platingthecellsinOxoid NutrientAgaron which colonies were counted after2 days'incubation at37"C.The number of revertants was determined on minimal medium where colonieswere counted after3 days'incubation. Experiments in vivo The methods used in the host-mediated assay have been described by Legator and Mailing [10] and were followed in general. Drinking water and food were removed from themice 3h beforethestartofthetreatments.From a suspension of the indicator organisms (cellsofyeastorSalmonella) 1.0 ml was injected into the peritoneum of each mouse. The yeast cells were sus- 0005632 DQW908952 6 pended inyeast-extract-peptone medium, thebacteriainphosphatebuffer(pH 6.8).Immediatelyaftertheinjectionoftheindicatorcells,0.25ml ofasolution of 2,4-D (24 mg per ml buffer, * i.e.6 mg permouse) wasadministeredorally with the aid ofa ball-tippedsyringeinserted about 20 m m intotheesophagus of the mouse. After 3 h the mice were killedby cervicaldislocation,and the indicator cells were recovered and washed twice.Plating,incubation ofplates and counting colonies were done asdescribedabove.The dose of2,4-D used was close to the maximal tolerabledose forthesemice. During theperiod of treatment a singlemouse died and wasthereforeeliminatedfromtheprotocol. As a negative control the mice were given 0.25 ml bufferorallybut inother respectsweretreatedidentically. Results Experiments in vitro Mitotic gene conversion in Saccharomyces cerevisiae,strain D 4 Siebertand Lempede [15] reported that 2,4-D gaveanincreasedfrequency ofmitoticgene conversion inthestrainofSaccharomyces used inourexperi ments. Siebertand Lemperle usedacommercialpreparation,U46 D-Fluid,and made thetreatmentsatalow pH,4.6.Theydidnotgiveany reasonfortreating the cellsunder these conditions.With 1.0 mg 2,4-D/ml they got 22% survival after 16 h treatment at25C. In aseriesof experiments we have investigated the pH dependenceforinductionwith2,4-Dofkilling,mitoticgeneconversion and petitecharacterinstrainD4 ofSaccharomyces cerevisiae. Fig.1 shows that the killingeffectisstronglydependent on thepH during thetreatment. Owing to difficultiesindetermining and maintainingthepH at a desiredvalue with high precision (tothesecond decimal) the resultsmay be expected to vary from one experiment to another. This isalso true for the resultsshown in Table I,asummary oftheresultsfromexperimentswherethe pH was varied but the concentration of 2,4-D (0.6 mg/ml) and duration of treatment^ h) were keptconstant.Undertheseconditionstherewasacritical pH forsurvivalaround 4.5. . Table IIshows the resultsfrom aseriesofexperimentswherethetreatments were made atpH 4.50.Therewasadose-dependenteffecton thekillingand on thefrequency of convertantsand petites.Also,when thedurationofthetreat ment was varieda dose-dependence forthese effectswas obtained (TableIII). The increaseofthe frequency ofgene conversion after3 h treatmentwith0.6 mg 2,4-D/ml inbufferatpH 4.50 (TablesI--III)wastestedstatisticallyby the t test. The increase was significanton the 5% level(t12/0.053 1.78)forthe ade2 locus (f3 1.96)butnotquitesignificantforthetrpS locus(f3 1.36).The increaseofthefrequencyofpetiteswashighlysignificant(t3 3.00). The physiological condition ofthe cellswas important forthesensitivityto 2,4-D. Fig.2 shows thatcellsinthelogarithmicphaseofgrowth aremoresensi tivethan cellsin the stationaryphase. Cellsgrown inmedium with glucoseas carbon source were more resistant than cells grown on glycerol as carbon source. ,/ ~-s=r-=i-i.v..-:-.. CGG5633 K- o O 3 ? |*11 a fi ? ? ^ r J ki ti - s , B a * g - S r is hj*f EC f tSr O. -i np> o" 3 n =| a nH- a "r/ 8 <aiq *0 * . *0 f S* tt a # 4 C 5 Ho ) ?m 5 at ^r fg ' " WJ % *% Z * i t^l'O 3 2* &JT`O ' ? of Hi ^ f s, a -- P- t o r s' l S ^ s | l (O M= i o-- <3D S ? a g & - i 3 iC* * to 3 3 UE "* `t*` .**" r ~ - r i f r r r i nn w mi n j TABLE I T H E EPFECTS O N T H E F R E Q U E N C Y O F MITOTIC Q E N E CONVERSION, SURV I V AL A N D F R E Q U E N C Y O F PETITE MU T A T I O N IN STRAIN D4 OF SACCHAHOUYCBS CBHBVISIAB A F T E R T R E A T M E N T F O R S h A T 37C WITH 0.6 mg 2,4-D PER ml BUFFER AT DIFFERENT pHi Number within parentheaea tie the totalsof convartanta cored. Tha rarulti are from 4 Independent experiment. pH of buffer . 4.0 4.2 4.3 4.4 4.6 4.6 4.7 6.0 6.1 6.6 6.0 0.6 mg 2,4*D Convertante per ml buffer X 10"* trpS edti Survival (ft) Petltes (ft) <0.1 io -J 12 0.11 61 7.3 83 1.72 (82) 1.27 (24) 28 48 1.28 (338) 0.70 (209) 36 40 0.71 (291) 0.67 (287) 61 11 0.96 (241) 0.69 (147) 98 6.6 0.63 (286) 0.44 (263) 96 2.8 . 0.96 (35) 1.08 (40) 90 6.6 0.93 (40) 0.98 (42) 106 6.8 Negative control Convertanta X X0-* frpff od2 Survival (ft) Petltea (ft) 1.26 (44) 1.2S (42) 98 7.0 0.64 (237) 0.34 (163) 94 2.6 0.86 (176) 0.72 (148) 04 4.4 ; 0.89 (190) 0.60 (107) 100 2.6 0.39 (241) 0.30 (187) 100 3.4 0.90 (37) 0.78 (32) 100 2.4 G00CS3 esG806M O a `3 8 ie '% -4 i2/y G / o O CD O 00 CO Ul 7TS ON TH IF PETITES ITH 0.6 mg irnbsroi cor trC oaverter ; X IO-* ! an Survival ( Petits*(% Converts X 10-* t' Oi Survival Paths* (' 4,0 4,1 44 4.4 4,4 S,4 . . . pH M eone. 2 .4 - 44 0, m gtml 4.4 4.4 Fig. 1.The killing effect of 3 h treatment at 37*C fatbuffer of differentpH containing 0.6 rag 2,4-D/raL Strain 04 of ScccAoromyccs arrotila*. Fig. 2. The killing affect of 3 h treatment at 37*C with different concentrations of 2,4-0 in buffer at pH 4.S on stationary phase cells(O) and cellsinlogarithmic phase (X).Strain 04 of Sscchsromyces c r r w v iiia t. T A B L E II T H E EFFECTS O N THE F R E Q U E N C Y OF MITOTIC GENE CONVERSION, SURVIVAL A N D FRE SACCHAROMYCESCEREVISIAEQUENCY OF PETITES IN STRAIN 04 OF AFTER TREATMENT F O R 3 h A T 37*C WITH D IF FERENT C O N C E N T R A T I O N S O F 2,4-D (SODIUM SALT) IN B U F F E R A T PH4.S 'V The total number of convertants scored are within Parenth priment*. The results are from 7 independent ex- 2.4-D (mg/ml) Duration (h) Survivors. - -, Petits* (%> Convcrtsnt* X 10''* survivor* trpS od*2 0 0.10 0.25 0.40 0.50 0.60 0.75 0 Positive control E M S (1%) 0 3 3 3 3 3 3 3 3 -- - - 100 78 64 37 33 23 4.0 95 88 . >' 1.5 i-a ^ 1.7 7.2 12- <1 95 2.7 0.56 ( 616) 0.66 ( 154) 0.53 ( 94) 0.96 ( 173) 1.26 ( 368) 1.52 ( 248) 1.42 ( 36) 0.63 (1049) 36 (8000) 0.25 (263) 0.28 ( 66) 0.32 ( 57) 1.06 (192) 0.66 (194) 0.92 (160) 0.35 ( 10) 0.42 (673) 46 <10000) t * O So^ ---- - ... 4.- . -- Reconstructic parent cellswa effectbetween the ade-2 locus trp+,were some sensitivethan g for the smallei 2,4-D. The rec quency of gen induction by 1 existinginthe Experiments effect on surv dose depende; dependencew EMS was u: centration (15 shows thatwl were compare 2,4-Datamo The effect during'T.5h v cellswereme atpH 7.00.T owing tothe The effect .* *$ H - p>. ti 3 0 TAbLE III t h e e f f e c t s o n t h e F R E Q U E N C Y o f MITOTIC G E N E CONVERSION c , Q U E N C Y or PETITES IN STRAIN D4 O F S A C C H A R O V Y C E S C E R E V , * ^ * TM ^ ANO FRE- A T 37C w i t h 0.6 mi 2,4-D PER ml B U F F E R A T pH 4.5 RFV/S/AF AFTER TREATMENT f mTeh.nuto.taln u m b of convrrunU scored ar. within parentheses.'TThh.* "*."i,tu. t,rTM 2 Independent exp.* 2.4-n (m|/wD 0.6 Convertants X 10_i trpS ad*2 Survival (%) PetK (%) 0 Convertants X 10-* trpS adc2 Survival (%) Petit <) Duration of treatment (h) 0.6 1JO 1.67 ( 845) 1.92 ( 620) 0.61 ( 293) 112 14 0.72 ( 233) 72 13 1.96 (1037) 2.07(1093) 0.68 ( 313) 100 . 7.0 0.69 ( 329) 100 4.0 2.0 2.21 ( 921) 1.46 ( 606) 41 16 1.88 (1009) 0.69 ( 341) 100 8.7 3D 3.48 ( 886) 2.03 ( 520) 28 17 2.12(1004) 0.82 ( 382) 100 5.4 * 2.4-D/ml. ai/er at pH tcrrroio*. \SD FRE:a t m e n t :f f e r a t rodent ex- Reconstruction experiments inwhichthesensitivityofconvertantsand their parent cells was compared showed no difference insensitivityforthekilling effectbetween the parent cells,ade~ trp~, on the one hand andconvertantsin the ade-2 locus, ade* trp~,on the other. Convertants in the trp5 locus,ade~ trp*, were somewhat more sensitivethanade~ trp~ cells.Alsopetitesweremore sensitivethan grandes forthe killingeffectof 2,4-D,which may bethereason for the smaller effect on petite induction obtained with thehighestdoses of 2.4- D. The reconstruction experiments showed that the increase of the fre quency of gene conversion aswellasthefrequency ofpetiteswas a resultof induction by 2,4-D and not dependent on selectionofconvertantsorpetites existinginthesuspensionsbeforethetreatments. Experiments in which the treatment was made at pH 4.30 gave a higher effect on survivaland gene conversion than thatatpH 4.50. Fig.3 shows the. dose dependence at concentrations up to 0.3 mg/ml. Fig.4 shows the dose dependencewhen thedurationofthetreatmentwasvaried. EMS was used asapositivecontrol. EMS isrelativelynon-toxicathighcon centration (1%) and gave a strongincreaseofgeneconversion(TableII).Fig.5 shows thatwhen the effectsonsurvivalandgeneconversionof2,4-Dand EMS were compared on a molar basis there was a significant effecton both with 2.4- D atamolaritywhereEMS wasalmostwithouteffect. The effect of the low pH was reversible. Table IV shows how the killing during1.5h with0.3mg 3,4-D/mlbufferatpH 4.30couldbeinterruptedifthe cellswere moved to a medium containingthesameconcentrationof2,4-Dbut atpH 7.00.The effecton geneconversionwasnotsoevidentinthisexperiment owingtothelow frequencyobtainedafter3 h atpH 4.30. The effects of longer treatments with 2,4-D at pH 4.30 werealsoinvesti- CG03S3S r,'r -4^-S*s f sA&J\- r 10 oo ^ o 00 CD C J1 05 I. I. ! Fit. 3.The i!(k U of 3 h tnatmcnt at 37*C with diffannt concantntioiu of 2,4-D in bufferat pH 4.30 oa nixvival (X) and the frequency of mitotic gene conversion in trpS <0)and ad2 (*) lociof strain D4 of Saccharomycn ctmitiat. Filled symbols indicate the frequency of conversion in the control (buffer at pH 4.30). Fig. 4.The effects of different lengths of treatment at 37*C with 0J O m g 2,4-D/ml buffer atpH 4.30 on survival (X), and the frequency of mitotic gene conversion in trpS (O) and ad*2 (t) locus of strain 0 4 of Saccharomyen ccreeistae. Filled symbols indicate the frequency of conversion in the control (buffer at pH 4JO). i Fig. S . T h c e f i asd of 1J4 X sion in trpS (~ frequency of c Fig. 6. The ki T A 1530 of S gated, but under these conditions the cellsinthe negativecontrolsshowed a lowsurvival:thereforeno dataarereportedfrom theseexperiments. Mitotic recombination in Saccharomyces cerevisioe,strain D S t a b l e rv f -'Si Table V shows theresultsfrom three differentexperimentsmade underthe T H E EFFE STRAIN D following conditions: pH 4.30, temp. 37C, 3 h treatment, 0.20 and 0.30 mg A N D / O R Pi *: 2,4-D perml. Aftertreatmentwith 0.30 mg 2,4-D/ml the percentageof aber N um boi w; rantcolonieswas aboutthesameinallthreeexperimentsandclearlyabovethe control value. Evidently 2,4-D increases the frequency ofmitoticrecombina tionundertheseconditions. . 2,4-D (mg/ml> Back-mutation test in Salmonella typhimurium A strongdependence on the pH in the treatmentsolution of2,4-D was obobtained when the killingeffecton Salmonellawas studied (Fig.6).The con centration of 2,4-D used in the experiment in Fig/6 (0.5 mg/ml) was com pletelywithout effectwhen thetreatmentwas made atpH 6.3(Fig.7).Treat ments with 2,4-Dvat pH 4.30 did not increase thenumber ofprototrophs in - %. o o 0 0.6 0.6 0.6 0.6 11 e 0> ?0* 2 1 4 c .e2 ? at pH 4.30 train D 4 of (buffar at >H 4.30 on '.rain0 4 of (buffar at a u Cone.2,4-O.MxIff* M 44 PH 4P 4,* FsK* 5.The cffccU o( 3 h tmtmtnt i 37 C with diffrat eoncntx&tions o( 2,4-D inbuffe* it pH 4,30 and of U 4 X IO"3 M E MS atpH 6.80 and 4.30 on nuvival (X) and tha frequency of mitotictana conver* xion in trpS (O) and ad2 (t) loci of itrain D4 of Soccharomyca* caraaioa. Filiad lymbob indicata tha Izequaney of eonvanion in tha control (buffer at p H 430). Fi*. 6. Tha killln* affact of 3 h treatment at 37*C vrith 0.5 m g 2,4-D/ml buffer at different pH. Strain T A 1530 of SalmontUa typhimurium. owed a lerthe 30 mg fabervethe nbina- 'jsobconcomreat- 'is n T A B L E IV T H E EFFECTS O N T H E F R E Q U E N C Y O F MITOTIC G E N E CONVERSION A N D SURVIVAL IN STRAIN D4 O F S A C C H A R O M Y C E S CEREVISIAE A F T E R T R E A T M E N T IN B U F F E R A T p H 4.30 A N D / O R pH 7.00 WITH 0.3 m* 2.4-D/mL Numbers within parenthesesare tha totals of convertants scored. 2.4-D (mg/ml) H treatment at pH 4JO p H 7.00 Survivors Convertants X 10*5 survivors trpS adaS 0 0 0 loo 0.54 (106) 0.50 ( 98) 0 3 0 100 0.74 (233) 1.06 (334) 0 0 3 100 0.33 (124) 0.37 (140) 0.6 1.5 0 70 3.60 (494) 2.26 (310) 0.6 1.5 a- 1.5 80 3.46 (539) 2.03 (316) 0.6 3 0 11 2.02 ( 43) 2.49 ( 53) 0.6 0 S 100 0.33 (132) 0.28(113) 1 ..... / ** . . . ... ------ -- --- CG0S633 r ii 12 TABLE V T H E EFFECTS O N SU R V I V A L A N D T H E F R E Q U E N C Y O F A B E R R A N T COLONIES IN SACCIIAHO. MYCES CEHEVISIAE STRAIN DS A FTER T R E A T M E N T F O R 3 h AT 37C WITH DIFFERENT CON CENTRATIONS O F 2.4-D IN B U F F E R AT pH 4.30 The result*are from three independent experiments. 2.4-D . Survival (mg/ml) (*> Number Red/ Pink/ Red/ Pink of colo pink white white nics scored Red Hair Total Percent line aber aberrant rant 0.20 0.30 0 53 11 100 510 1. 1 0.2 1630 13 1 3 11 6 4 38 2.3 4557 11 11 2 6 0.1 1 ,D n c c CD <3 o 00 ^ _ cocn o o ' i -I o'". T A B L E VIII 1NDUCTIOI ode2 LOCL O F 2.4-0, 6 Duration of Expt. No. I I 34 1 Cooc-2.4-D.m g/im * Fie. 7.Tbs killing effect of 3 h treatment at 37C with differentconcentrations of 2,4-D in buffer at pH 6.8.Strain T A 1530 of Salmonalla typhimurium. T A B L E VI T H E EFFECTS O N S UR V I V A L A N D T H E T O T A L N U M B E R O F hit* R E V E R TA N T S A F T E R 3 h T R E A T M E N T IN VITRO A T 37C WITH DIFFERENT C O NC E N TRATIONS O F 2.4-D O R E M S IN B U F F E R A T pH 4.30 Strain T A 1335 of Salmonella typhimurium. The result*are from taro independent experiments. Treatment with pH Survival (*) Total number of reversantsscored 2.4-D (0.08 ms/ml) 2,4-D (0.07 ms/ml) 2.4-D (0.05 mt/ml) 2,4-D (0.03 mg/ml) Buffer only Buffer only 4M 4M 4.30 4.30 4.30 7.00 30 57 67 108 65 100 72 51 75 63 2 70 , EMS. 0.5* EMS. 0J * 4.30 4.30 10 59 1400 730 4 ,/ ".* . ' ' . CGQ5S33 7ri 20 I SACCIIAKOF E ' ~ CON- Pereant aberrant 0.2 2.3 0.1 13 T A BLE VII tT Rh Ee AETFMFEENCTTSINOvNi SUR tro VIVAL AT 31 AND C WIT T H H D E I TOTAL NUMBER FFERENT CONCEN OF TRA hit* R E V E TIONS OF RT ANTS 2.4-D IN AFTER BUFFER 3 A H T pSltira4in3 0 T A 1536 o fSalmonella lyphlmurtum. The results at. from 4 independent e.perimeniv pH Survival (%) Total number of revertantiscored 0 .0 * 0.07 0.05 0.03 0 0 4.30 4.30 4.30 4.30 4.30 7.00 27 42 73 93 72 100 122 179 227 194 157 154 d u lle r ' pH AFTER 3 h OR E M S TS -s. -cored TABLE V m INDUCTION IN VIVO (HOST-MEDIATED ASSAY) O F MITOTIC G E N E CONVERSION IN trpS A N D atleS L O C U S O F STRAIN D4 O F SACCUAROMYCES CEREVISiAE BY O R A L ADMINISTRATION O F 2.4-D. 6 mg PER MOUSE. Duration of treatment, 3 h. Number within parentheses are the totalsof conversant -- -- H ExPL No. Mouse No. Number of cellstested X106 % of contr. Convertants per 10s edc2 dpi i1 5.0 199 - 2.24 (112) 330 (195) 2 7.8 1.59(121) ' 3.82(291) 3 Contr. U 2.05 ( 84) 435 (179) . 4 2 3 100 1.47 ( 41) 234 ( 69) 21 2 3 4 ' Contr. 5 6 7 31 2 3 4 Contr. 5 6 7 41 2 3 4 Contr. 5 6 7 23 4JO 1.2 . 53 5.8 6.7 8.9 40.0 3sa 253 33.8 26.9 31.3 19.0 28.0 15.6 3.4 41.4 24J. 23.4 83 47 100 134 100 118 100 2.73 ( 77) 2.90 (117) 3.62 ( 42) 2.18(115) - 238 ( 84) 336 (148) 239 ( 30) 3.15 (166) 1.96 (114) 2.70 (181) 1.38 (123) 2.62 (153) 334 (224) 239 (213) 0.35 (143) 037 (140) 0.48 (124) 0.30 ( 81) 0.52 (208) 032 (273) 0.72 (186) 0.57 (192) 0.48 (130) 0.41 (128) 0.51 ( 96) 0.78 (211) 0.59(184) 0.75 (142) .46 (130) 0.58 ( 91) 0.71 ( 24) 0.44 (180) 135(321) 0.77 (120) 037 ( 33) 0.72 (296) 0.69 (166) 0.68 (159) 0.56 ( 46) 0.92 (222) 0.86 (202) 0.81 ( 66) OLiOStdQ -- " - r.. - 7 ~ ` . U wtiwtohesxtrpaeirniTmeAnt1s53wi8t(hTastbrlaeinVITI)A.1535 (TableVI)orinany offourexperiments Host-mediated assay Indicator organism: Saccharomyces cereuisiae,strain D 4 Table VIII shows the results from four different experiments where we TABLEix MIUNDRUICUTMISOTNRIANIVNITVAO1(S3H0OSWTI-TMHE2D.I4A-TDED ASSAY) O F hu* R E V E R T A N T S IN SALMONELLA TYPlIlOn) d/ninixtntioB,5 rat pr noun; duration ottraatmnt,3 b. " Mouw No. vj 1 2 3 4 Control 5 6 7 8 1* 2 3 4 ' Control 63 7 8 1 2 3 4 3.9 3.6 4.1 4.8 6.6 10.4 6.2 6J 6.5 2.7 10.4 8.2 0.9 8.3 8.4 11.0 9.6 6.3 16.6 11.4 4.8 7.6 4.3 3.6 100 104 100 ' 66 100 338 1.2 100 2.1 9 8 8 14 12 21 16 14 13 4 12 12 0 1 1 2 0 13 6 8 90 81 91 83 76 1.60 1.46 1.16 1.46 0.1 0.1 0.2 2.1 0.3 0.7 18.7 10.6 21.2 23.1 CGQST1 o O oc c 00 CD 05 O O N VIVO JM STRA1 rationS mi : Mt>uu : No. I1 !1 i. 4 Control 'S 6 7 8 1 2 8 Control 4 65 7 tested whether c increase the frequ 3 h exposurein 1 no significantinci Indicator organ Infourdiffrer ent.experiments i revertantsto prot same conditionsa Discussion Of theresultsj pH dependent is was more active form andt>h.atcel thesurvivalofye; Like the kiUin; dependent on the with thatobtaine without effect a gene conversion. 7202- offour experiments * c riments where we S A L M O N E L LA TYPltt. hi* X 10~* 2 .4 7 2 .9 2 .8 2 2-35 2.33 2.49 1.97 2.92 1.82 2.03 2.59 1.85 i.e r - \ i 1 .- ,, 1.46 0.1 0.1 0.2 2.1 0.3 0.7 3.7 XS 31..12 : .3s .9 s SOOCTION O N VIVO (HOST-MEDIATED ASSAY) O F HU* REVERTANTS IN SALMONELLA lTYf> m M U H W M STRAIN TA1531 WITH 2.4-D Oral administration 6 mg pr mous: duration of treatment. 3 h. ExP*> No- Mous* No. Number of cellstested X10* % of contr. Total number of his scored his* X 10-* 1 1.3 219 2 2.9 3 2.7 4 3.4 0 1 0 13 0.3 3.8 1 Control 6 0J 100 6 1J. 7 1.0 8 1.7 0 5 3 6 4.8 3.0 3.6 1 41 2 23 8 r 2.6 290 1 7 9 0.2 2.5 cv 3.6 2 Control 4 0.6 100 6 0.8 6 0.6 7 2.5 7 14 0 10 12 19 4.1 oo CD "o Co CD Ob tested whether oral administration of 2,4-D (6 mg/mouse) to mice could increase the frequency of mitotic geneconversion incellsofstrainD4 during 3 h exposure intheperitoneum. Compared with thecontrolvalues therewas no significantincrease. Indicator organisms: Salmonella typhimurium, strains T A 1530 and T A 1531 InfourdifferentexperimentswithstrainTA 1530 (TableIX)and twodiffer* ent experiments withstrainTA 1531 (Table X) no increaseinthe number of revertantsto prototrophy was obtained.The treatmentswere made under the same conditionsasthosewith yeast(seeabove). Discussion Of the resultspresentedabovetheobservationthattheeffectsof2,4-Dwere pwaHsdmeopreendaecnttiviesatthea most interesting.We proposethatthereason why 2,4-D low pH isbecause more 2,4-D was in itsundissociated form and thatcellscan only takeup theundissociatedform.Fig.8 showshow thesurvivalofyeastcells,was correlatedwiththedissociationof2,4-D. Like the killingeffect,the increase ofmitotic gene conversion in yeastwas dependent on the pH and the dose of2,4-D.The increasewassmallcompared wwiitthhotuhtateofbfetcatinaetdtwhiethlEoMwSm.olHaorwietvyerw,heitreisw2,o4r-tDhniontdiuncgedthaatcEleMaSr wasalmost increase of gene conversion. The two compounds behaved very differently as to dose CG057Q2 16 io -I o--O io 'H io H o2 9 10 1.sO9 io 1 pia h IO PH Fif. B,Th* avivai of catta of Saecharornyc** trtoUo train D& aitar 3 h treatment at 37*C with 0.05 mi 3.4*0 par ml buffar at dlffarant pH (X). Tha nacativa control (O) was buffer. Tha dotted curva raprasanta tha diaaociation of 2,4*0 at dlffarantpH. response: increaseddose of2,4-D gave mainly an increaseofthetoxic effect, whereas EMS with increasing dose became only slightlymore toxicbut very mutagenic. At low pH,cellsofSalmonellawerekilledby amuch lowerconcentrationof 2.4-D than were yeastcells.Itmay bethatisSalmonellano mutagenicchanges can occurattolerabledosesof2,4-0. The resultsfrom thehost*mediatedassayswerenegativeforbothSalmonella and yeast.Itispossiblethatthe conditions were such thatthe compound did not reach the indicatorcellsinthe peritoneumataconcentrationhighenough to cause an effect [14]. Furthermore, the pH in the peritoneum iscircumneutral,i.e.farfrom the optimalvaluefortheeffectsof2,4-D. In situationswhere 2,4-D occurs ata lowpH therearepossibilitiesfortoxic and mut&genic effects to occur.Thisindicatesthateatingfood contaminated with 2,4-D would be ariskforanimals with low pH intheiralimentarycanal, e.g.man. The high toxicityof2,4-D onmicroorganismsmay beofimportance also for ruminant animals that are dependent on microbial processesintheir rumen for the decomposition of food. The observation that the effects of 2.4-D were stopped by increasing the pH indicatesthatthecompound when administered orally would have smallchance tocauseeffectson cellssuch as gametes. The exposure ofthegametes would have to be precededbydistribu tion in the blood plasma where the pH isabout 7.4,i.e.much above thepH atwhich 2,4-Dcanenterthecells. ,frp n c e s o CO0 .MeCn iTdin^ 1 L fc.G. At*. Foc a 00^* CD CD to 1 A ** i,. 1 1 1 1 9 - cV w *n . E nl i Effect i 7 mDetivanroin<.*-...L..<r,anHedraMd.i and early cmbtyocer B Kabietch. W.. Carry- krankb. POaiuaapati a t Kiasne, M. aad N. S t r a p U ju jy e , Ain: 10 lo cato r. M .S. aad 1 tial nuuienic agen Y o rk. 1971, pp. 56 1 1 M ajum du, SJC. ao< lian fertile, i . Herat 12 Majumdar, SJC . ar mrlanotailtr, Can. 13 Fairy.J.M.. The >r. (1073) 83-91. 14 Ryxtmaa. H. aad < ocuaaidiaa CMNft the hoit*mdiatcd 15 Siabert, D. and Saccharom yctt ce 16 Style*.J.A..Cyto: 17 Zimmermann. F.! yea*tSaceheramy Acknowledgement Partof theinvestigationwas supported by Grant B-2468 from the Swedish Natural ScienceResearchCouncil. ./ . - ; 005703 iJT'C with *5rdetud effect, h;iveiy roneJJa -*ddid tro u g h j * ~ .Tum- - toxic References A m *. B.N-. J- McCann and K. Yam asaki, Method for detecting carcinogen and m utagen with tha * SabnoneUa/mammaHan-micro*on muunnielly test.Mutation Reg^ 31 (1975) 347-- 364. a d en n , K J . . E .G . L tllh ty and M.T. Vakahajhi, Evaluation o f herbicides for possible mutagenic properties'-J. A ir. Food Cham. 20 (1972) 649. 3 Busclmaicr. W ,, G. Rhrbom and P. Propping. Comparative investigation on the mutagenicity of p esticid es in mammalian testsystams. Mutation Re.. 21 (1973) 25. 4 Pirk. S.O.S.. A genetic effect on the progeny of tomato plants sprayed with 2.4-D, J. Natl. Inst. Asrie* Botany. 11 (1967) 199-204. j Davring. 1-- Cytoacnetiska effektar av 2,4.5-TriklorfenoaySttikayn pa Drosophila melanoeaster och .tfus musculus. En frberedande iamfrande studic. Inatitute of Genetics. University of Lund. Lund. Sweden. 6 Pvrinc. L*. Effect of a 2.4.5-T "ester on early oogenesis, fertility and development in Drosophila mrlanogaster. Hereditas. 80 (1975) 255*262. 7 Davring. L. and M. Sunner, Cytogenetic effects of 2.4.5-trichiorophenoxyacetic acid on oogenesis and early embryogenesis in Drosophila mrlanogaster. Hereditas. 68 (1971) 115-122. 8 Kabiesch. W ,, Carry-over effects of mecoprop in potatoes in the 1stand 2nd generation. 7. POanaea- krankh. PflansenpathoL Pllanzenschutt. 4 (1968) 137. /9 Kisane. M. and N. Kiss, Investigating the genotypic effects of herbicides on individual specie* of Streptomyces. Agrartud. Egyet. KSzlemen. (Gddld). (1966) 100-114. **. 10 Legator, R 3. and H.V. Mailing. The host-mediated assay, a practical procedure for evaluatingpoten tial mutagenic agents in mammals, in A. Hollaender (ad.). Chemical Mutagens, VoL 2, Plenum. New York. 1971. pp. 569-589. 11.Maiumdar. SX. and R.C. Hall, Cytogenetic effectsof 2,4.5-T on invivobona marrow cellsoi mongo- liangcrbUs. J. Herod. 64 (1973) 213-- 216. 12 Maiumdar. SJC. and JX. Golia. Mutation test of 2.4.5-trichlorophenozyacetic acid on Drosophila melanogasltr. Can. J. Genet. CytoL. 16 (1974) 48S-- 466. 13 Parry, JJi. The induction of gene conversion in yeast by herbicide preparations. Mutation Res. 21 (1973) 83-91. 14 Ryttman.H. and G. Zetterberg. Induction of mitotic recombination with N-methyl-N'-nitro-N-nitro- soguanidine (MNNG) in Saccharomyces cerevtsiae. A comparison between treatment in vitro ^nd in the host-mediated assay. Mutation Res., 34 (1976) 201-- 216. 15 .Sichert. . and E. Lemperle, Genetic effects of herbicides: induction of mitotic gene conversion in Saccharomyces cereuislae.Mutation Res.. 22 (1974) 111-- 120. 16 Styles.J.A.. Cytotoxic effectsof various pesticidesin vivo and in vitro. Mutation Res., 21 (1973) 50. 17 Zimmermann, FJC. Procedures used in the induction of mitotic recombination and mutation inthe yeast Saccharomyces cerevisiae, Mutation Res.,31 (1975) 71-- 86. M o -coO oo^* CO 03 c o -r as CG0'0704 .$ 13 d l3o . t" r* ` . > * *} S V* .* V .4 1: A. Jr\ :- The Problem of Mutagen Effects on the nd 2 G eneration a fte r ' Exposure to herbicides /. Ton-That-Tung Ton-Duc-Lang Do-Duc-Van S'9.29Z6OM00 The aberrations of chromosomes, in both their structure and their number have been pointed out in 1970 by Ton- ^ .That-Tung and his collaborator^// on Vietnamese patients '' who have been exposed to the herbicide sprays,especially } the later orange agent. This work has been confirmed by Fujita, Fujita and Funazaki^ Yefimenko/and by Hev#fol- lowing Seveso. The mutagenesis of the orange agent has b been affirmed for the Salmonella Bacteria b y Hussain*^ with the flies Drosophila by Majumdar and Hallwith Rodents, In 1977, Murray and his collaborator^fHhink that the TCDD can act like a dominant lethal agent, whereas Seilei^proves reduction of DNA production by experiment with testis'rats' with TCDD(1977). ^ -- ? : S * ,* * ' t _ S Through the newspapers, one knows that in man, the former American veterans of Vietnam back in the U.S.A. complain about birth defects in their descent and about the abortion frequency among their wives, iAt the time of a visit in the D.S., consequent to a private interview with American Veterans Administration in Washington in May 1979, it has been suggested tpdus $o study the remote effects .of herbicides on the 2 Q generation among the former vietnamesesoldiers who fight in the South and who come back in the North to found a family, that is to say in the same conditions of the American veterans, ,, Here are the first results of our investigations: * % ' ` l--r. / ` -,,v Methodology - Our investigations first proceed on statistics in obstetrics services. Unfortunately in most of the^ cases, the father's -' military service has not been mentionSed in the observations " (besides, this has always been considered as a secret), By chance, we ran adc^ross the town of Yen-Bai; being coupled with a Southern town,ftreceives a certain number of Southern veterans (and usually native from the N o r t h ) ,Yen-Bai belongs } '; to the province of Hoang-lien-Son, whose sanitary service .. boss, Doctor Mai became interested in our work. The town of Yen-Bai counts 10,000 inhabitants and about 700- .'j former soldiers coming from the South. The former soldiers ..)* gather in agricultural and handicraft cooperatives in the town and its vicinity. t*.* * . Vr Ar. \ >.,** i ?:..r 1J .\ .>f V# nA . + U *Y > - V 'j*' , - -lv > ;A S t # # D0WO978266 After Yen-Bai, we appealed to former southern soldiers groups in 5 big provinces of the Delta of the Red River: ' Hanoi, Hunghoa, Namdinh, Haiphong, and Thai Binh. There, we sent teams in order to personally interview each indivi dual. Localities in which our investigations have been taking 1 place, have never received herbicide sprays.. *\ % t ,V . .* 7 RESULTS * ` 1-i . ".v; ' A) Hospital investigations aJMaternity of ^en-Bai Material : 3058 births from 1975 to 1978 (4 years) Number of birth defects: 30 Number of birth defects depending on the couple: father, southern veteran, mother, native from the North, not having left the country: 15 1 . i *t . ''' Out of these defects there were 22 due date deliveries, 8 non due date deliveries. Weight of these anomalous children: Above 2.5 Kg (about 5.5 pounds) 20 Between 2,5 and 2 (between 5.5 and 4.4) 5 2 kg ( 4.4 pounds) 4 Non registered 1 I. Birth defects coming from father, former southern soldier, and mother, living in Yen-Bai: / Pure Anencephaly 2 Anencephaly with absence of nose and left ear hare lip short neck elevation of navel against sternum * 1 Anencephaly with protuberant eyes short neck shoulder retraction facial cleft 1 Anencephaly with batrachian abdomen shortening of limbs (upper limbs 8 centimeters lower limbs 11 centimeters) 1 Anencephaly with absence of nose and eyes 1 7208 C 005725 0OWO9Z8267 T Ii ' , i I Hydro cephal3r with angulation of upper limbs endowed of 3 fingers on the right hand, and four fingers on the left hand. absence of anus 1 Absence of nose with auricles jointed to the ear feet jointed to the legs hands jointed to the arms bilateral syndactyly 1 Abnormal location of the eyes with auricles jointed to the ear varus equine . ' Batrachian abdomen with agenesis of the upper lip Absence of forearm 1 Polydactyly 1 Syndactyly of fingers and toes 1 Hare lip and cleft palate ' Absence of the upper abdominal wall II. Birth defects observed on the civil population: 1 1 15 Hare lip Cleft palate and hare lip 2 -j Club-foot Batrachian abdomen Batrachian abdomen +Ballot Anal imperforation -j 4 1 1 Batrachian abdomen +hermaphroditism Hydrocephaly + batrachian abdomen Hydrocephaly + batrachian abdomen + shortening of the limbs -j 1 1 Hydrocephaly + absence of arms and forearms, replaced by 3 fingers on each side ` Hydrocephaly -j 1 15 coos1; * U f j **u D0NCI978268 On the whole, the number, of anencephalies obviously predprni-' nates on the group of children born of southern fig'nters (6/15) relative to the civil population (0/15)- b ) Maternity of Quv mong* district Material : 233 births for a civil population of 4500 inhabitants with 30 families whose father has been a southern fighter during the years 1976, 1977, 1978 ( 3 years). Number of birth defects 9 Number of birth defects among the civil population 0/9 Number of birth defects coming from father, southern veteran/ mother living in Yen-Bai 9/9 These birth defects are dependent on only 6 couples: Anencephaly with absence of upper maxilla shortening of limbs and batrachian abdomen Pure anencephaly Anencephaly with cleft palate batrachian abdomen absence of penis shortening of limbs (These 3 anencephalies come from the same couple) *" Cleft palate, deceased on the 6 day (cardiac disease?) Hydrocephaly Hare lip , atrophx^of uvula Deceased on the 3 day by cyanosis (cardiac disease?) Hare lip, shorten^gg of limbs Deceased on the 6 day One single lower limb set up in a mass of shapeless flesh t Shortening of limbs, big ears ,batrachian abdomen , .. 1 .. 1 ^ 1 1 1 ^ 1 . . --4 0no t i-. X. C&05727 In this little group predominate : The anencephaly (3/9) The hare lip with or without cleft palate (3/9) I 4\ The possibility of cardiac diseases (3/9) 1 1 The shortening of limbs (5/9) o O B ) Investigations in the cities of southern veterans, in the delta of the Red River : tOo Our teams went to: I. Gia lam (neighborhood of Hanoi) II. Tu ky (Hai hung) III. Hung ha (Thai binh) V.Chuong Ky (Ha dong) 09 to 09 \D VI: Haiphong VII. Ba dinh (Hanoi) VIII. iiam dinh IX. Ninh binh X. Phu ly XI. Thanh hoa Ij' On the whole our teams have questionned 1102 veterans, of whom 836 coming back from the southern sprayed zones(Group A), and 266 not having been in the South (Group B). Out of 1102 soldiers from group A, 670 couples sire composed of a southern veteran husband and a wife having always lived in the North. The group witness is composed of 116 couples. The Jesuits are registered on the board I BOARD I Individual investigation on 1102 former soldiers (836 coming back from the southern sprayed zones) in the communities of North Vietnam. Birth Defects Abortions and Sterility V premature deliveries Number % Number % Number % Group A with 43 *670 couples (out of ( former sol 1187 births diers from the on due time South married 3.6$ 214 (out of 1401 pregnancies) to a northern woman i Group B {2bb) 0 with 116 couu (0ut of 309 ples(former births on oe/ U/0 36 (out of345 pregnancies) soldiers not due time) aav -* . the South,mar ried to a nor thern woman p 0.001 GG05723 15,39 t 10.4? p^e,03 23 (out of 670 cou- pies) -z 2(11 3,5/6 2 (out of ' 116 cou- 1.91$ pies) 7211 V D0WCI978270 Here the nature of defects is: Anencephaly Microcephalia dementia Anophtalmy Hydrocephaly Genu valgum Absence of left forearm Congenital bilateral cataract Hare lip and cleft palate Polydactyly Retraction of the right upper limb in flexion Hermaphroditism Absence of the right auricular auricle Congenital diseases of the heart Paralysis of the external ocular motor mongolism In this group predominate: 1. Cardiac diseases 15/43 2. Defects of the neural tube 9/43 Dicussion I. The clinical relationships These first results seem to justify the complaints of the'American VN veterans who have resided in the sprayed regions (Tay ninh, Da nang, etc*..) and to suggest the spread through the father to the second generation. V/e have been able to study 70 of these files whose rate of the birth defects and abortions on 70 couples is as follows; Birth defects Abortions 70 couples (VN veterans + 1/10 8/70 women in the U.S) Here is the nature of these defects: 2 cleft palates 1 spina bifida 1 absence of anus with spina bifida 1 absence of genilo-urinary system 2 congenital cardiac diseases * 0G05723 7212 . The former Vietnamese soldiers coming back from the southern sprayed zones present suggestive signs of a mutagen ao* transmitted by the father. There is a significative difference for the defects between the group of Vietnamese soldiers fighting in the South (group A), and the others (group B). In the first group, the ; congenital anomalies occur 43 times out of the 1 1 8 7 births, ;' i.e 3.656 (the normal number of defects seems to be about 1 . 5 to 2 50 whereas on the witness group , they are not any out of -.s 309 births (p./o.ool). Abortions and premature deliveries represent 15.35 in the group A and 10.45$ in the group B ( p ^ a o l ) About the characteristic of these defects, one must insist on the excessively high frequency of brain defects ( anencepha- ly, microcephaly, anophtalmy) in Vietnam on the second generatic We should normally have: ' - One anenceph?ly out of 2777 births, whereas we have 1 anencephaly out of 197,8 births among the southern veterans.. - The microcephalic dementia exists once out of 2 5 ,000 birtl and it represents among the southern soldiers once out o f - 1 1 8 7 births. :|v - Anophtalmie should normally be 1 out of 10,000 births, whereas it is here 1 out of 1 1 8 7 births. On the other hand , the important place of the cardiac defects has to be emphasized: 34.856 of the defects.i.e. 1 5 cases out of 43 defects. The elective harm of the neural tube on the second generatic seems to be in accordance with Barbara F i e l d 's works, who - ' proves that in Australia, a linear relationship exists between the increasing rate of spina bifida on the newborns of the . first generation and the rate of 2,4,5T used every year. : The number of molar pregancies is abnormally high in Hanoi: many of them stem from women whose husband has been a soldier in South Vietnam. For instance, at the time we are writing this, one can count, in the obstetrical Service of the Mother and the Child Institute,,19 hospitalized cases by mole or by chorioepi- thelioma: out of this number 1 9 , there are 9 cases coming from southern veterans ' wives and 1 0 belonging to the civil popula tion.* II. Experimental tests (1 1 ) Jackson' was the first one to note that the dioxin inhibi mitoses of,Haemanthus katharinae Baker( 1372^ Davring and Summer * This will be the object of another work C G 0S730 D 0 1 N 0 9 7 8 2 7 1 ....... ; . \ ... t v v D0W0978272. reported that the commercial form of 2,4,51 with less than,.o .1p p m ' of dioxin, affects theoogenesis and causes sterility on the-fly . Drosophila melanogaster: for the first time, sterility with a genetic origin of the 2,4,51 is then evoked. Many authors, after a dioxin treatment.,have observed multinucleate cells.(Greig and his collaborators' ^'*.Buu Hoi and his collaborators'TM , Kimbroug and his c o l l a b o r a t o r s ^ ) on experimental animals. . Green Moreland and Sheu^1^ , in 1975, have been able to demonstrate that the dioxin could increase the number of the chro mosomal aberrations in the r a t s ' b o n e marrow in "1977, after Green and M o r e l a n d '/s,,failure ' '' 2 years before. Czeizel and Kiraly . i n 1976, i.e..6 years after the work of Ton-That-Tung and his collaborators^*', studying Hungarian workers' blood working in a Budapest plant stained with dioxin, have found a high percentage.of chromosomal aberrations . . Fujita and his collaborators^2 ' have also observed chromosomal anc lies among the Japanese workers in contact with the 2,4,5T as well as Yefimenko'0 ' among rats in the cells of the bone marrow. After Seveso, a Hew seminary has.allowed to report on the Italian population, from 2 to 25 years old, a high number of chromosomal anomalies (gaps, b r e a k s .....). The mutagenesis of the dioxin has been the first assertion by Hussain and his c o l l a b o r a t o r s w i t h ESCHERICHIA COLI Sd-4 Salmonella typhirmurium TA-1532. With Hussain,dioxin seems to be mutagenic by intercalation with DHA. Kondorosi and his collaborators^"" ' have found that the dioxin is a-powerful mutager. of Salmonella typhimurium TA-1532: according to them, the dioxin seems to cause an intercalation with DMA rather than to chemically react with the nucleic acid. Khera and Ruddick' s wprks have shown that on the YTistar ''rats, the fepididymises^male ratg^, treated by the dioxin, present some granulomatous forms ~similar to the auto-immunologic reactions observed after a bacterial infection or a flow of foreign bodies. Van Hiller and Allen' ' have proved that the Sprague Dawley male rats, after a treatment by oral route, by daily doses . T. of 0.05, 0.5, and 1,0 ppm of TCDD present on.the fourth week, a decrease of the spermatogenesis, whereas Seiler'" ', points out that 0.4mg/lcg of dioxin given by intraperitoneal route to. the male mouse, provokes a 50% reduction of testicular DNA. ' Besides, one knows that dioxin causes adverse effects in the teste of mice, chickens, guinea-pigs, and monkeys. The double assertion that, first the dioxin reaches the testes, and secondly, provokes anomalies, implies the idea that the chromosomal dioxin can act as a dominant lethal agent, like Murray and his collaborators. ' wo seem to s u g g e s t ' c o v e r i n g three generations of Sprague-Dawley rats. < It seems that the immediate |*roblem is the cytologic and biologic paternal sperm in animals as well as in nan, and moreover the paternal sterility can already appear like a problem according to the animal experimentation.  Z Q 6Q M G Q J Furthermore, the study of herbicides and of dioxin seems to bring an Unitarian proof to the actual conception according to which the transformations caused to DMA by environmental mutagens can be ',,the main cause of cancer, birth defects, cardiac diseases and senility. Work done by the surgical clinic of the University of Medicine in Hanoi. v CG05732 ^o 9,C tS .cri " 1'-* . ;V%^ >j :. ".**: " 'iWfv? - *k ' :r Tj.,*on D0WO97827 4/"^. 1 Ton-l'hat-Tung, Trinh Kim Anli, Bach q u o c 'Tuyen, Bao xaan' T r a `,:i Nguven xu-^n Huven, Clinical effects of massive and -continuous * i - a * *% utilization of defoliants. f 1971, (N?:.,9),.*: $1 .'.iiv-.^r-vx; . W * 'y--r " 2 . Fujita K, Fujita K, F u n a s a k i :Z, Chrnmospheric.;.ahnomali.tift's:@ - * ' ,V.i * 'J *# brought about by the use of 2,4,51', J ..`J a p V :Ass o c .*<Rural" Ked-. 1975, 24 : 77 - 7 9 . 5 .* ; ,-v `; \ ^ . ' f '' '. V ' M i. - \ 5 \* : - ; &- *S#v- ili !'Jfr ` V * *' ;*J.*\; * Ii88 T 'tr y . .M & . V ^ . bv. , && !'^ i .,*%. *fi IW : ''V l,;4 V .\: * *L fc/*7; J* . f efif*i 7 V *if 5 . Hussain- f fp c t 6 . Majundar SK G0 iia JK, Mutation test of> 2 ,4 .,5T on1Brosohila? ' :y , * , ... #. .. /.! melapogcisrer. Coil. u . wenec. -Cytol. I^V'i,>1 o .: 4b 5 .-^466^X**^Tv' '. V `- ' y ! - ' 7 . Majumdar K Hall K.C., Cytogenetic ^effects- df'-B,4 ,5T *on^iir^; vivo bone, marrow cells of;.Mongoli^;5 erbiis,''-.i.^ered.^il9/7^i\: - 6 4 : 213 - 216. ,, 8 . Murray JP, Smith PA, Nitschke CG, Humi'ston.-.CG, *Ko ciba- RJ-V*^v ' .;' \ ;' Schwetz BA, Three generation reproduction study...ofrats?rin-v^' . .;.* f* ' gesting TCBD, Toxicol. Appi.;?^armacol, '1 ^7-7 , ; 2 0 0 <-201j1 9 . Sei]er JP, Inhibition of testicular ENA synthesis.bycherni-iS. I cal carcinogens ana niuoageus, Mutation Res. 1 9 7 7 , 46 : .5 0 5 -3 1 ' ' '. ' 1 0 . Beu*bai'a Field, Charles K e r r , Herbicide use and incidence ofi> neural tube defects, Lancet 1 9 7 9 , i : 1541 - -1 J4 2 . * . _.. . .*- * y - y - : A V Y* J *-C o T 1 6003733 7 2 1 6 /;^ i - D0 W 0 .9 7 8 2 7 5 1 2 . Dyvri.nrr L, Summer M. Cytogenetic effects cf 2 ,4 ,5T on oogc--'% liesis and early embryogene sis in Droscnhila melanoeaster. Hereaitas, 1 9 7 1 , 68 : 1 1 5 _ 122 3 . ',Grcig JB, Jones TCDD Pood Cosmet 4 . Buu Hoi, Pham huu Chanh, Sesque G, Azum Gelada MC, Saint Ruf Organs as targets TCDD intoxication, Naturwissenscgaften, . 1972, 59 : 174 175. 15- Kimbrought RD, Carter CD Epidemiology and pathology of`.TCDD'poisoning ^episode -'Archiy*. Environ. Health 1 9 7 7 , 52 s 7 7 '- . 8 6 .. V ' -4- * ' 1 7 . G re e n S , M o r e la n d 'P S , C y r o g e n e t ic 'ie y ^ u a t io n 'o f . s e y e r ^ l+ ^ ip ir ^ x i n c o n t h e ' - r a t , T o x i c o l . ' A p p l - , . - P h a r m a c o l . *'1 .9 7 5 ,'- * 5 3 1 8 . C z e is e l E , K ir a ly J . , ' . * < '1 ^ 1 C h r o m o s o m e e x a m i n a t i o n s i n w o r k e r s ';: p r o r * t 9 . K o n d o r o s i A , P e d o r c s a I , S o l m q s y : P , . ? E h r e n b e r g . / L , ; rO s t e r m a n - i ^ G o l k a r S , I n a c t i v a t i o n o f ;-2"-- ; R E A b y . i e l e c . t r o p h i l e s , - ' ^ ^ q t f o n . 20. R e s. 1973, 17 : 149 E h e ra K S , R u d d ic k JA , 1 6 1 :''' Y - : :'; , r ` * ' v ; .V '-*ri*-. * 11 P o l y c h l o r o --d i b e n z o --p --d i o x i n s / . : ' p e n i n a r * . t o l e f f e c t s a n d t h e d o m i n a n t l e t h a l t e s t i n W i s t a r E a t s ,-'r- i n !: E .H . B l a i r (E d ) C h lo r o - d io x in s O r ig in s a n d P a t e , A d v a n c e s in C h e m is tr y s e r ie s n 1 2 0 , A m e ric a n C h e m ic a l s o c ie t y , W a s h in g tc DC, 1973 pp : 70 - 8 4 YY . 1 2 1 . Var, M i l l e r J P , A l l e n J R , G h r o n ic t o x i c i t y o f T C D D i n R a t s , : ; ] . ; P c u . Am . S e c . E x p . B i o l . 1 9 7 7 , 3 6 ' : '3 9 6 ' * . t" -. - ? 7> . Norhack DH, Allen J P , biological reponses of the nonhuman primate, chicken and rat to TCDD, Environ. Health Percpect. 1975, 5 : 235 - 240 `V t , .,.v r 24, Bruce N, Ames, Ident ifymAg^environmental^Che'mihais ^causing? Mutations and Cancer, S c i e n c e '1979, '204 ; `587. - '59" ^ \: *\ *.**;~ ' J p 1*: t . . ` [rt4 V* ` 1*\ -1 -`- . A j ;*-ir * y O !?** V jV ,.. ., ; .i.` a *-'*. . 1 " ^f V - *., . -, - . i i * _ .? - *-rr.>.....v.1-/- * * y . *-r *. } v ;:> , . i > ' . .* y V . t. 'fy //; 0005735. 7218- Ii i f ' \3\ 7219 FJ C'Kwt. VH.15rp PcrnmonPrr*1477PrimaliaGreatBrium DOW280834 MN 022777 J.R. Allen*+. D. A. Barsotti*+.J.P. Van M iller'+. L J.Abrahamson*+ and J. J.Lauch* D epartm ent o f P a th o log y *. L 'n icersity o f W isconsin M ed icu l S J io n l. anil R eg io n a l Prim ate R esea rch C e n te r* . L'n icers ity o f W isconsin. M a d iso n . Wisconsin J 7II6 . C S A (R eceived I A pril 1977) Abstract-- Femalerhesusmonkeysgivenadietcontaining500ppt(t- 10*-)2J.7.3-tetrachlorodibenzop-dio.xiniTCDD) for9 months became anaemic within 6 months and pancytopnie alter9 months of exposure. The marked thrombocytopenia was associated with widespread haemorrhage. Death occurred infiveoftheeight animals between months 7and 12 ofthe experiment at total exposure levelsof2 -3 u g TCDD kg body weight. At autopsy, inaddition to theextensive haemorrhage, there was a distincthypoceilulantyofthe bone marrow and lymph nodes. Hypertrophy, hyperplasia and metaplasia oftheepithelium in the bronchial tree,bileducts, pancreatic ducts, salivary-gland ducts and palpebralconjunctivaewereobserved. Squamous metaplasia and keratinizaiion ofthe sebaceous glands and hair follicleswere present in the skin. Death was attributed tocomplications from the severepancytopenia. INTRODUCTION 2J.7.S-Tetrachlorodibizo-/>-dioxin (TCDD) is produced in small amounts during the production of trichlorophenois. Uncontrolled chemical reactions in the presence of heat have led to the development of considerable amounts- of TCDD in the commercial preparations. Explosions resulting from overheated chemical reactions have been responsible for human exposure to TCDD in Europe and the United States. One of the serious complications of this exposure has been persistent chloracnc. Tlie health implications of TCDD. other than those related to industrial exposure, were brought to the attention of the general public during the Vietnam conflict. Defoliants widely used in the combat zones contained varying concentrations of TCDD. which has been held responsible for physical abnormalities that have developed in the human population (Rose & Rose. 1972). In addition to chloracnc. exposed indi &viduals have experienced fatigue, penplier.il numbness IRose Rose. 19721 increased abortions and infant deaths, and liver cancer (Tung. 1973). More recently, serious physical problems, which included severe weight loss. acne, alopecia, haematurea and abortions, have appeared in horses that inhabited arenas sprayed with residues of tridiloropheiiol production containing TCDD (Carter. Kimbrough. Liddlc. Cline. Zack & BarthcL 1975). The detection of minute amounts of dioxins in human milk IM. Mesclson. per sonal communication 1977) and beef fat iR. T. Ross, personal communication 19761 have caused concern about the exposure of man and animals to TCDD. In the Mudy now reported, cmpiiusis is placed on determining the pathological alterations that occur in primates after low-leveL long-term exposure to TCDD in food. EXPERIMENTAL Eight young healthy female rhesus monkeys weigh ing approximately 5-6 kg were placed on a diet of pelleted monkey food to which 500 ppt; TCDD tDowChemical Company. Midland. Mich.) had been added. The experimental diet was prepared in ICO-ib lots by suspending TCDD 122-7 ,ug) in approximately 1 mi of acetone and adding this to 250 ml of coni oil: the eom oil was pre-mixed in 251b of finely ground monkey food (Purina Monkey Chow. Raiston-Purina Co- S l Louis. Mo.), and this mixture was then added to an additional 75 lb of food and mixed thoroughly for 10 min. The food was then pelleted and stored at --5JC until used. The monkeys remained on the experimental diet for 9 months. Gen eral health was monitored by daily observation, and in addition complete blood counts were made and scrum protein, serum lipid, serum glutamic -pyruvic transaminase (SGPT) and blood-urea nitrogen were determined on a monthly basis. The food intake for each animal was monitored daily throughout the ex periment. A complete autopsy was performed on the monkeys that died. Sections of the tissues were fixed in lb'*,, neutral buffered formalin, dehydrated, embedded in parailin and sectioned at 5 pm. The sections were stained with haematoxyiin and cosin and examined using a light microscope. RESULTS C lin ica l oh scria thn ts 3 !Uu througnout thispaper. Follow ing 3 months of TCDD feeding i5-5 ; (>*> ug toul intake per ammail the monkeys developed peri orbital oedema, loss of facial hair and eyelashes. 401 0002029 1 G80RZ'MOa 4 0 : J. R. Allen. D. A. Barwittl J. P. Van Milllr. L. J. Abrahamson and J. J. L alicii Tabic I. 9tta e m n q ru m s o f m o n k e y s fe d M l ppt T C D D fo r m o n th s ' White bloodcelli Ixll)JmmJl Animal 1 -- -- no. Initial Terminal Blood platelets Ixio-1 mmJl - - - . Initial Terminal Haemoglobin Hucmaiocm (gm';,) r'..J 1 ---------- Initial Terminal Initial Terminai 7t 1 1 0 2-3 240 23 13-5 40 41-0 12 0 9+ 7-9 2-4' 360 44 141 6-9 43-0 215 23 10-3 3-8 380 23 146 126 45-5 -UHI 31 10-6 10-4 400 480 14-1 10-7 45-0 322 s-s 4-1 300 50 141 6-0 43-0 9-5 33t 9-5 3-3 330 34 13-6 6-6 43-0 2 2 0 41 7-8 3-0 350 340 13-1 115 400 44-5 49$ 8-6 8-7 260 54 (3-7 8-5 43-0 29-5 'Valuesobtained a months alter the end of treatment unless otherwise stated. tOied. during wit 44 ofexperimenL Died during u-k 23of penmenl iOicd during wit 36 of expenmenl accentuated hair follicles and dry scaly skin. These pchanges became more prominent in six of the eight animals by month 6 of exposure 111-3 1-7 g total intake per animal). The majority of the animals showed a decrease in haemoglobin and haemutocrit by month 6. The haematologicul changes became more accentuated in six of the seven animals that survived 9 months of TCD D exposure (Table 1 k However, blood urea nitrogen, total' serum lipid, serum cholesterol. SGPT. total serum protein and albumin, globulin ratios were not altered appreciably during the experiment except in terminal animals where there was a slight decrease in serum albumin and an increase in SGPT. The monkeys also lost 21weight throughout the experiment although their food intake was unaltered (Table Animal no. 32 became severely anaemic, thrombo cytopenic and Ieucopcnic in month 7 of exposure after a total intake of 11-2 ^ig (Table Ik Prior to death the peripheral blood smears were practically devoid of immature red and white blood cells. During the sub sequent 4 months four additional animals (nos 7. 9. 33 and 49) showed similar clinical changes and died. Their peripheral leucocytes and platelets decreased to levels as low as 2300 and 24.000 mmJ from normal values of 7-I0.IXX) and 200--HX).00U mmJ. respect ively. The total TCDD intake of these animals at (he time of death was IS-6 I S /ig. animal Although all animals were removed from the TCDD diet after 9 Table 2 B o d y H eig h ts o f 'm o n k e y s le d :<*> r y t T C D D lo r Vim m illi Body weight1Lei) Animal no. Imtul Terminal' 1 6-14 5-S6 9 6-74 5-74 -- ' 23 Ji 6 : 6-02 5-03 <.35 511) 4-12 38 i*f* 41 5-73 5-44 49 5-39 4-46 ' Weight at autopsy or alter 12 months on inai months, the three animals (nos 23. 31 and 41) that survived for 12 months experienced a continuing loss of hair and periorbital oedema. One of the surviving monkeys (no. 23) developed a severe leucopenia and thrombocytopenia by month 12 (Table Ik Cross observations at necropsy The animals that died experienced hair loss from all parts of the body. The skin was dry and rtakey. while the hair follicles about the lace became accen tuated. Loss of eyelashes and swelling of the upper eyelids along with facial oedema and peteehiae were present in all animals. There were also large haemorr hagic areas around the nares. on the gums and on the surfaces of the buccal cavity (Fig. II In addition, there were petechial haemorrhages over the entire surface of the body. Subcutaneous oedema was par ticularly prominent in the lower abdominal region and inner surfaces of the thighs. Marked irregularities in the growth of the toenails and lingemails iFig. 2) and gangrenous necrosis of the distal plialanges were also recorded. Ascites was prominent in all of the animals. There was marked distension of the inimhcpatic biliary- sys tem. with oedema and dilatation (up to I cm in dia meter) of the common bile duct (Fig. 3l Patency of bile ducts was demonstrated by the tlow of bile through the ampulla of Vater and by careful dissec tion of the intrnhcpaiic duets. The lymph nodes throughout the abdominal cavity had undergone atrophy. In addition to (he pale uppeaiai-.ee of all abdominal organs, there were foci of haemorrhage in (he adrenals, pancreas, liver, endometrium, serosal and mucosal surfaces of the gastro-intestmai tract and the urinary bladder. In (he thoracic cavity, the lungs exhibited \vu l haemorrhage in all lobes iFig. 41. There was bilateral ventricular dilatation, cardiac enlargement and oedema. Focal areas of haemorrhage were prc'eni in the epiearditim. myocardium and endocardium. The skeletal musculature, particularly in the extremities, was oedematuus. pale and discoloured by haemorrha gic loci. Extensive haemorrhage was found in the meninges, and there were isolated haemorrhagic areas in the brain parenchyma. The bone marrow ifrom the femur, vertebrae and sternum) had the apfvu ranee 7221 0002030 r. 1 t. '.i crminal 1:0 :i-5 -UK) 35-5 19-5 "0 U-5 29-5 41) that ling loss urviving mia and ss from ;; flakey. accen- i upper ae were lemorr- and on :ideitnitoinre. as par- region jianties Fig. 2) is were There ry sys- in diancy of )l' bile Jissccnodos .Tgone of all age in erosal n and focal ateral and .tu in . The nines, rrhai the areas from ranee * UU Fie. I.Facialviewofanimal no..'X which diedalteru monthson a dietcontaining 500 ppt TCDO. showingpetechialhaemorrhagesaboutdienose,absenceofeyelashesand oedema of(heuppereyelids and lips. Fig.2.Toesofanimal no.7.whichdied afterhmonths on adietcontaining500 pptTCDO. showingmarked thickening and irregular growth ofthe toenails and swelling ih (lielermuial phalanges. In themoreseverelyalfeetedanimals,therewasgangrenousnecrosisoftlieterminalphalangesisecinsetl. ZZZl 00020 I 9RRnQ?M O c J D O W 280837 DOW 280838 i Fig. 3. In animal no. 9. which died after receiving TCDD over 9month*, aprobe(arrowed) was i introduced through the ampulla of Vater to demonstrateoedema anddilatation inthe common bile t ducts. I | Fig. 4. Lungs and heart of animal no. 49. which died after 35 wlc on a diet containing 500 ppt I TCDD, showing multiple haematomas in the lobes of the lung adjacent to apparently normal paren1 chyma. and an enlarged heart of globoid appearance. | Fig. 5. Sample of femoral bone marrow from animal no. 49. which died I wlc after receiving a diet containing 500 ppt TCDD for 9 months, showing the fatty appearance of the marrow and focal haemor rhages in the darker areasi 0002033 A l l c m . D. A. B.x k s o t t i. J. P. V a n M i l l i .k . L. J. A o u a iia x is o s and J. ]. L.xurn 0002034 l'ig A Hone marrow from animal no. .13, inch died will) severe pancylo|>cnia after ingesting 112 /rg I t I X ) . showing small foci nl d.nker cells dispel sed among llie Incenl lal cells ami consisting |inscl| nl small lymphoid like cells with iiinnd l>asi>|iliilic nuclei and scant cytoplasm. Ilacinuloxyliit and eosm : 12 (inset MMt l'ig 7. Section of a mesenteric lymph mule from animal no. <W, which tlieil after receiving a diet containing fttlp p t I t ' l f l f for til VIk. showing the liypnccHnlaiily of the nicdullaiy colds ami, in the sinuses, uidely dis|>crxcd cells which are mainly small lymphocytes and huge vacuolated cells with eeceiilncally located nuclei ll.icinaloxylm and eosin MM. Ii g H. Section of cerchrum from animal no. IV which, died after !> months on diet coninining 500 ppl T l 'O I) . showing in the cortex extensive haemonhage limited lo the Virchoxv-Hohin space around the lilood vessels. I laein.iliisyliil and eosin a IIKi. CJ1 6C 808Z M O O 0002035 co CV c\ r- < V IT I/ TCDD-induccd changes in monkeys 40V Fig.9. Mucus-secreting cells(arrowedIin the ductal epithelium of the salivary glands from animal no.9.Haematoxylin and cosin x 104. Fig. 10. Hypertrophy, hyperplasia and metaplasia ofthe mucus-sccretin; cellsin the gastric mucosa ofanimalno.J2.typicalofallmonkeysexposed toTCDO. withpenetrationofthemuscularismucosae by the epithelium {arrowed) and the development of large areas of mucus-secreting elands in the submucosa. Haematosyiinand cosin x11 Fig.1 1.FeminizationintheMeibomian elandsoftheeyelidsIK)occurringasanaftermathofTCDD exposure inthemonkeys,with squamous metaplasiaintheepithelialcellssurvivingaround theperi pheryofthegland.Hacmatoxylin and cosin x11 DOW280842 of fatty tissue in which there were focal areas of haem orrhage (Fig. 5). Microscopic observations The major microscopic changes may be separated into three categories: degeneration or atrophy of the bone marrow and lymphopoietic tissues, haemor rhage. and changes related to cellular hypertrophy, hyperplasia and metaplasia. The bone marrow displayed a decided hypoceilularitv. The haematopoietic ceils of the marrow were replaced primarily by fat ceils with focal areas in \which ceils that were lymphoid in appearance predo minated (Fig. 6 There was also a conspicuous reduc tion of discernible erythroid or myeloid stem cells as well as of megakaryocytes in the marrow. The lymph nodes throughout thg body were hypocellular. In addition to the absence of any distinct lymphoid ger minal centres, the cortical lymphocytes were sparse and the medullary cords were narrow or inconspi cuous. Cellular debris and large vacuolated cells pre dominated in the sinuses (Fig. 7V The spleen, also, was devoid of any distinct lymphoid germinal centres and the small lymphocytes were widely dispersed. The second m ajor category o f m icroscopic changes w as related to the h aem orrh age that occurred in tis sues throughout the body. W eil-circum scribed focal are as o f h aem orrh age in the h eart, lungs, liver, adrenals, pancreas and skeletal m uscle consisted of collections o f intact red blood cells that partially dis rupted the architectural pattern o f the alie n e d tissue. The acute nature o f the haem orrhage w as shown by the lack o f tissue necrosis an d an absen ce o f reactive cells. Petechial a re a s w ere prom inent in the derm is o f the skin, su b m u cosa o f the urinary bladder an d epithelium o f the alim entary trun. H aem orrhages of the en d om etriu m a n d m eninges w ere m ore dilfuse in their distribution. T h ose in the bruin w ere lim ited to the V irchow -Robin spaces surrounding the blood ves sels IFig. U T h e haem orrhage in the bone m arrow varied con sid erab ly , being ditliisc in som e are as an d focal in others. Considerable modification in the morphological features of epithelium occurred in the TCDD-treated animals. Mciuplastic changes characterized by numerous mucus-secreting cells were present tn the ductal epithelium of the salivary glands tFig. 9). bile ducts and pancreatic ducts. Similar changes also occurred jn the bronchial epithelium and in the palpe bral eonjunctivae. In addition to the metaplasia, the epithelium of the bile ducts and palpebral eoniuneiivac developed considerable hypertrophy and hyper plasia. Squamous metaplasia of the sebaceous glands occurred more extensively in the skin of the lace and to a lesser extent in other areas. Hypertrophy, hyperplasia and metaplasia were observed in the gastric epithelium where the parietal and zymogenic ceils were replaced by those that se creted mucus. In addition to the'thickened mucosal lining, many of the epithelial cells invaded the submu cosa through interruptions in the muscularis mucosa (Fig. 10k Such ectopic epithelium assumed many dif ferent patterns. Some isolated cells encompassed large mucinous cysts, while others were arranged in sheets or formed acinar structures. Considerable oedematous fluid usually surrounded the epithelium. Inflam matory cells with a predominance of neutrophils were seen in close proximity to ruptured submucosal mucinous cysts. Rupture of such cysts produced the ulcers that were found in the gastric mucosa. Hyper trophy and hyperplasia were also apparent in the transitional epithelium throughout the urinary tract. In addition to squamous metaplasia in the epithe lium of the sebaceous glands, there was hyperkera tosis of the skin with keratintzation of the hair folli cles and the adjacent sebaceous glands. The latter alterations were particularly prominent in the Meibo mian glands of the eyelids (Fig. Ilk The pronounced thickening of the fingernails and toenails was con sidered to be secondary to excessive keratin produc tion. In the heart, petechial haemorrhages were promi nent in the atria and in the tips of the papillary muscles. Additionally, foci of subendotheiiai and peri cardial haemorriiage were frequently found in the ventricles. In two of the live hearts the ery throcytes were haemoiysed. so that haem pigments and haemosiderin could be identified by Peris' stain in the intra cellular spaces. Intra- and intercellular oedema led to separation of the muscle tibres and myoiibnls. DISCUSSION The data presented in this report indicate that pro found cellular alterations are induced in many tissues following the ingesuon of minute concentrations of TCDO by primates for 9 mouths. Most notable are the ellects of TCDD on the haematopoietic system. As exposure time lengthens, the seventy of the cellular deterioration in bone marrow and lymphoid tissue becomes widespread. Terminally the animals develop severe pancytopenia. The haematologieal data pres ented here agree quite well with the previous studies conducted in this laboratory1, where mixtures of dioxins, including TCDD. were fed to nonhuman pri mates (Allen & Carsicns. 1967k In the previous inves tigation the monkeys also developed anaemia and^J O O O leueopenia prior to death. Similar haematologieal ' O changes have been recorded in rats given a smgle dose of 50pg TCDD kg body weight |Van Miller & Allen. 0002037 410 J. R. Allen. D. A. Barsotti. J. P. Van Mil u .*. L J. Arrahamsos and J. J. Lalicm 1977). In addition to the depletion of circulating lym phocytes. atrophy of the thymus, spleen and lymph nodes has been observed in several species (Vos. Moore & Zinkl. 197?). Diese changes may be related to the immune suppression which has been recorded in TCDD-exposed animals. Subletha! doses of TCDD have been shown humoral immunity to in suppress guinea-pigs e(cVlio-ms cedtiaatle.d19a7n3d). There are other complications that arise as a result of the decrease in haematopoiesis. D ie widespread haemorrhage that was observed in the monkeys prior to death corresponded to the decrease in blood platelets. It is logical to assume that the haemorrhage was associated at least in parr with the altered clot ting capability of the blood. The ventricular dilatation and myocardial hypertrophy in the TCDD-exposed monkeys may be related in part to the reduction of circulating erythrocytes and subsequent increase in the cardiac workload. mIn this experiment it was shown that ingestion of 2-3 TCDD kg body weight over a 9-month period was capable of producing in several organs cellular destruction or alteration sufficiently great to cause death in over 50*; of the non-human primates. It is of interest that the L D . 0 of monkeys exposed to a single oral dose of TCDD is 50-70 /<g/kg body weight (McConnell. Moore Dalgard. 1977) with death occurring within 45 days. There are some major dif ferences in the changes that occur in monkeys acutely and chronically exposed to TCDD. While death was associated with severe pancytopenia in the chronically exposed animals, haematological changes were not a constant feature of those acutely exposed. In addition, widespread haemorrhage occurred only in the chroni cally exposed animals. Changes that followed both acute and chronic exposure were loss of hair and nails, keratinization of the Meibomian glands and hair follicles, hyperplasia of the transitional epithe lium in the renal pelvis and of the mucosal epithelium of the stomach and bile duct, a decrease in scrum albumin and an increase in SCPT. The morphological changes that have been recorded in monkeys indicate that TCDD is capable of suppressing haematopoiesis and inducing cellular alterations in epidermal appendages and mucosal epithelium of the biliary, pancreatic, intestinal and urinary tran. After exposure to TCDD. the cell population in the bone marrow and lymphoid ele ments underwent degeneration and necrosis. Ollier less specialized cells such as the bile-duct epithelium and mucous cells of the stomach were injured less severely. However, as a result of injury the latter cells showed a tendency to compensate by undergoing hypertrophy and or hyperplasia. Some of the injured ceils also reverted to more primitive forms that were capable of surviving in a less desirable environment. This was seen in the sebaceous glands where the sec retory and ductal epithelium reverted to squamous cells. D ie observations made on the monkeys used in this experiment indicate that there are areas that need special attention in animals, including man. exposed to TCDD. Perhaps of foremost importance are the alterations that occur in the haematopoietic (issue. Anaemia, thrombocytopenia, and leucopenia were the most debilitating changes in the primates. The altered lymphopoiesis could be associated with immune sup pression. The possibility of reproductive abnormali ties also exists. Altered levels of serum progesterone and oestradiol associated with difficulties in concep tion and early abortions have been observed in female Stmonkeys exposed to low levels of TCDD tAllen. Bar sotti Van Miller. 1977). and testicular atrophy has Stdeveloped in male monkeys given small amounts of dioxins (Allen Carstens. 1967). In addition, the widespread hypertrophy, hyperplasia and metaplasia that occurred in the epithelium of monkeys exposed Stto TCDD. and a greater frequency of tumours in rats (Van Miller Allen. 1977) suggests (hat the com pound has carcinogenic potential. AcknowInH/tm --ienis This research, published as Primate Center Publication No. 16-049.was supportedinpart by theUniversityofWisconsin Sea Grant Program and US Public Health Service grants ESQ0472. GM00IJ0 and RR00167fromtheNationalInstitutesofHealth.A portion ofthisresearchwas conducted inthe UniversityofWis consin Biotron.a controlledenvironmental research lacilitysupported by the National Science Foundation and theUniversityofWisconsin.Die TCDD wassupplied by Dow Chemical Company. We thank Mr. R. Murlar and Mrs. L Carstensfortheirtechnicalassistance. REFERENCES Allen.J.R.St Carstens. L A. 11967) Light and electron microscopic observanons in Mueucu muiatta monkeys fed toxic faL 4l J.ret. Rex 23. Ii I3. Allen.J. R. Barsotti. D. A. & Van Miller. J. P. 119771. Pathologicalalterations inprimates resultingfrom lowlevel (ctrachlorodibenzo-f>-dio.un exposure. Proc. Inti Symp. Environ. Pollutants.Storrs.CT. 1-3 June 1977. Inpress. Carter.G D- Kimbrough.R.D_ Liddlc.J.A..Gine.R.E_ Zack. M. M. & BarthcL W. F. (I97ji. Tcirachlorodibenzodioviu:an accidental poisoning episode inliorse arenas. Science. .S'.)'.133. 733. McConnell E.E_ Moore.J.A.JfcDalgard. D. W. 11** 1. Toxtcityof2J.7.3-tctruchlorodibcnzo-p-dio\in iTODDl inrhesusmonkeys:a singleoraldose. T o x ic ..ippi. P 'tar m ac. Inpress. Rose. H. R. & Rose. S. P. R. (19771.Gicmical spraying as reported hy refugees from South S'ietiuni. Science. ,V.Y. 177.710.' Tunc.T.T.(1973VLecancerprinuircdufoieau Viei-ium. CUtruro. 99.427. Van Miller.J.P.& Allen.J. R. 119771 Gtrome toxicity of2J.7.S-ieirachlorodibcnzo-p-diovinm rats.FcJn. P roc. F eih i. . In.Sorte x p . B io l. 36.396. Vos.J.G.. Moore. J.A. & Zinkl J.G. <1973i Filceiof TJ.T.S-ictrachlorodihcnzo-p-dio.Mn on*the immune sys tem oflaboratory animals E m ir. U tili R i-n p cet. no. 5. 149. 7229 OOOtOr'S 13. ' 7230 \3 A DOW 089489 78-2108. Miller. C-: Sonlum. S.: Crosby. D. C . (Dep. En-v* , 0 iron. Toxicol.. Univ. California. Davis. CA 05616) Elec* tBruoll-.aEccnewpirtoonr. pCroonptaerinti.eTs ooxficcohl.lo1r8i(n5a)t:e6d11d*i6b1e6n;z1o9-7^7.d(i2o3xirnesf*. ) ' j*. erences) Molecubr orbital calculations were performed to * \H,PL / analyze the electron distribution in both the unchlorinaled r f c ' * ' dibcnzo-/> dinsin and TCDD. particularly their first unoc cupied orbitals. It was hoped that this would allow a demon stration of the theory that neutral TCDD can'be viewed as an electron acceptor, rationalize the necessity of haiogenaiion for toxicity, and explain the chlorination pattern required for toxicity. The calculations made indication that haiogenaiion o f dibenro-p- dioxins at certain positions offers considerable structural stabilization to added charge. Greater chlorination at these positions yields stronger charge- transfer complexes. Chlorination at other positions provides poorer acceptors, precisely the observed chlorination requirement for dioxin toxicity. These cakubl inns may help to explain the unusually firm binding ofTCD D to soil and protein, both of which lend to be electron-rich or to contain electron-donors. TC D D may be solubilized by the presence of suitable electron- donors, displaced from its complexes by stronger electron- acceptors, or bound prcfercntblly by a specific electronegative protein in target organs such as the liver. PESTICIDES ABSTRACTS 7231 0000901 7232 fiGLTE AND CHRONIC TOXICITY OF 75 PESTICIDES TO VARIOUS ANIMAL SPECIES Lugcnc K. Kcnaga. Dow Chemical I '.S.A .. Midland. Michigan INTRODUCTION Dcish(mwlvIcoeteieionaiasrbtaptyiosainhleiieftcnlucrhunsrimreoctdilaIahnrslIcneieoce.mnAtdennillIinclaapo`re)!tileul7rnhneof(tI>vegmlt'hIirienrpoSuoepsotdrsitntrhreinoeoiitenAetacvmcogyttaiIeiidefIootsIonbeitafsnnolofdxtn.aniAoclc*eirshf)hegfe4efcfe-qS!4eomfnfeuSucl>cI'ictVbi(yc'ir.t'hsvAaSsoet<lue.na.lmnS-pmst.(Ilocuur*aa)eenbxAccxns---r Considering the m im herorchem ic.il compounds which may need quick en vironmental evaluation, the costs in volved. and (lie shortage o f toxicity testing facilities, the important ques lion which remains to he answered is: " W h a t arc the best and m inim um number <// in tin a tar test methnds am i '/**/I'.x J t i r a.s .sex ain i? In s ir it y In ti i'rnatl \/>t i tram nj anim at tanani.sms 1 1 ertebrate and invertebrate, ter restrial and aquatic f " I here is a need for making a critical selection o f good toxicity indicator organisms lor evaluating hazards of chemicals. Data from a large base o f acute toxicity for ten species o f animal organisms and 75 chemicals was as sembled from the literature lor use foi correlative purposes. I hese data are tabulated here ;md serve as the basis for another paper (X). The table has not been ptihl.shcd prev io its lv . due to space limitations. Hecause the tabul ated d.ita are so difficult lo obtain in any one place and includes so manv c o m p o u n d s, it a p p e a ls v a lu a b le lo make this database available. In d icato r organism s and test met!) oils are n eed ed loi both acute and ch to m c toxicity studies | xtensive el.ita lot c lito m c lo x ic itv is not a v a i l able on many species olh ei than lisli and i.its . ( hum ic to x icity d a ia on lisli is discus-scd m a se p al a le p .ip e i In the .1111In *t i ~ i I lie p ie s e iil p .ip ei n u lu d es 1 1.11.1 Oil I lie I III Mile I. >Xli Its o l ~2 t o l l I o VaI ' ro 00 o pounds to rats and a summary o f the daplinids i2. 4. In 14. lf>-2>.2bi. I he a cu te to x icity data the a u th o r ha-, lai test melhods alni Illuse lui w ild- given pieviously (,X). Iile species are essenlially illuse de si lib e ri in th IT 'A p ro p o s e il t k s t )r <;a n i s m s a n d (iu id e lin e s lu i R e g is ti a lim i ot' l 'estieides 1241. Iloncv bee contact MI. EATcuHteOTDoxSicity test m elhod s are d esei ihed by Atkins r i a l il). I he originai leler- Certain organisms have been used ence-- may be examined loi details sufficiently in acute loxicitv tests io Idi v.uiables in test methods lo i support a common data base Idi 75 mugli loxicitv scieeiiing puiposes. dillcrcnl pesticides, these oiga- siici) vaiiables aie consideied Ilei e nisius, listed in 'Iable I. include the lo r is iili in v a iia tio tis tu w itliin rat {Manns nnrveyit ns ) ami mallatd abolii otte onici ni magnitude lui (Anas /dalsrlivnehns ) toi acute oral one compound oli otte species. Il 1.1) 50 studies: the bobvvlute (( nl- pivciscly si.uidardi/eil test methods inns vityinianns I (or Japanese quail weie requiied Idi every test lesili! iCntarni.s t. itqnmicti) lor diclaiv bere, little computativi- data vvoiild I.C50 feeding studies: the i.iinbovv be avatlable Idi ibis papei. I bus. trout {.Salm a y a ir d n e r i t. h lu e g ill sudi a data tcqim cm ciit is noi a {.epntnis mat hmt hints u. fathead piuctical solulioii loi cnnipniison 2. Chronic Invilii'minnow {/`imcfditdes firnmeltis i. the striped mullet {.Munti < 'Indus i puiposes al th pi escili ime. lor spot {l.einstumns stallina ns )). I b i l e is no p u b lislie d l a i c i d a ta Pa film ia manna (or D. fades i. and base Idi co i relation o f c h io m e tox- shrimp (any o f the lin ee 1`enaeus icity tests vv11h n n iiie ro u s C h e m ic a ls species;/', aziet as. /'. dnm arnm . /'. o l l u i t Itati lliat a s s e m b le i! by th .teliferns ) for aquatic I .( '50 studies: I \<) \\ I H ) e x p e il c o im m tte e s o li and the honey bee t.l/uv an Ihlt ra i. p e s tic id e le s id n e s in lo o il i h . I he a terrestrial arthropod lot contact uiily sp ecies oli w h ic ll acu te alivi 1.1) 50 studies. A nunibei o f these c h in ili,. l2 v i a l i o rai to x icily itala species w eie chosen because thev an: coiisisleutly available Idi com- were umlei pi eliminai y cousiileia p a iis o n is th ta l. I I i i i s . th la t is lion for scieeiting use bv the I S< A piesenlly the oliiy c h o u e o l species Interagency Advisoiy Committee, a va ila b le Ini c o n c ia tim i ut acu te anil all are piesenllv used loi and ch io m e loxicitv Scven tv-tw o .5. IJasis olClassilkatimi of ( hi mii alspesticide registialiou purposes In I I 'A l24). Ille se species lepresent Chem icals a ie lluis lo m p .u e d oiganisins which it is known horn In t.'rmips experience can be leaied read il v I he d ive tsilv ol ib i s tiu ilm e s ol anil tested in the labniaioiv .uni th ' i Ile im , al ' 'Mini in i u i.ik cs ile whii.li ale sensitive lepiesentalives la lle il .lle llM .a l u - ip u iC ' .*1 a n-i ol then I .mill y ni c lassi it oi g.iiusms. lui , o liv i.:!iv e si/c u n ii.ili.c c .iH c M o st o f the acute loxicitv test I II si*, 11, |.1e . . a l i li. n i l *ei he nielli.>ds used lot data bases .ue g im ip id aito Ioni e.iunps. , !i|,u i those ot the l S I)e p .u lin eili ol iia e d h .d lo ,,|lb .l||- piu ph> : i.- In tel loi 1 Is|i and U ild lile S i l v i , e lo i b u d s I S I S | I I . j, s 111III l|1 a ln i 0004333 .o lii.n u m e d ic m i.u !- . . uih.m -.itc' all, I II il -, i l ai li . ai - II,-. In ! l't. ;. !i - .r. j . i n I / . M l - I I I ' 1Im - j I- ' l \ \ * M M l > V U l l ) V\ . I N 44 * I ! * "*. 4 !l! 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( h e e n i i e l . i l m i l ' w e t e e'.il e i i l . i t e i l l i v i n g .i i m 1111pie l e i M e ' M n n '.mpiitiT p r.'C i.iin I he \ .iliiliti nl [III- c m ie l.il intis .u c p re s e n te il m lei ms ii| the i i m im iv i ni ni ilei n ni '.11U *' *1 <; K vj it . , !; i;- i u n 1 : 1 k 1! \ . .li: * . ! : !tf ! t it '.v III 1' 1i '. * n j.! !'i \ 11 * v i i ' i . 1ll >11' ` \ |N | 1 1> v i ' f u l . r i i ' i i . i 11k u n i * .tini N V , v 1' I I I llll lle Itili-:'' .ill- Mie pi ni e iple n u l l e . l i n i ' nt lile tIf'JU V " I v 1le e e " I n i p i l lile INI'.: .1 I n e ' I m i : ; ei|ii:ilin!i' RI SI ITS 1. \ e iiU - Inviliti \ v i i l e I n v i e i l v il.il.i I m |n . p e . ie- m ill i . n n i p m i n i U , n e : : i \ e n in l.i I'll" I l i l i s Is lile I'. I ' l l In i t h e .nl ! \ . . :i ); 1* 11 1 'M - ' v : | , l i; . 1 ! !* \ . t!! :| '11 * i M u i ii.ii . . : -.-1.:. 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( iiiiiiiln i: ir iitlu -r V lllH - fnr IV * t ii-id i'4 IN SM TK IIHiS f ( liriiim Inviliti I )il;i Km N i>-i-lli-it m i* k u d u i in ilH-t K :il 1 1150 m i: kt; / .// \ i i i t i - " K l 1 11 N ll . il. i M .ill.in t/ 1.1)50 / nm ku lt lllllllilt1 < 50 P im i I ll l ll`\ f*l 1 ) 1)50 .<1! I n i IIIlH U ill R jiiitm ii 1 50 Irn llt ppm ^ 1 (5 0 |l|llll K iltlt.iO M im im i M io |>|)IM DOW763444 S iili V 'iiii r I i'll 1( 5o ppnt Piifihnnt I t 50 ppm M in iti 1 t 0 ppm a. ( M o n n u u d llvilrmarhini ln\n tn i<h \ H iilrin .ili.5 52 r i liJurcUii- IH ! *% liM 'lilriM l.tl im i 25 2M - x w XT-500 40- M H I CIM I 224 5NI i MI r rm litM ilM li. ju d riti htjiliM lilnr liniM m m i j h t i i i hfcr .u n it \ l i b t n * |||m (Hll 1*1U l i J i N | i . \ j | i!m ih 11.05 25 1.25 IH 11.25 Mi i: tK -llll .45 M M X IN N I. 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I C ontinu vd V IN S U I H ll>l S O O W, 763446 ( brun*luxiiiU Data <'.mimmi iir ullirr Nmiiii- for hxlk'iilf` Uni NiH'ITn'l Hat 1.1)50 ni| kgriat mit*kj; indiri d. Mi\rtUanruu\ ln\rrtitidi4i firlhnn ^ *-?C 7 7 - ohriitni-fl*.}*V ! 25 .1.0 ptft'lhrtrm-/v*0 rulritonr IU.U 1.25 H t*i ?.? > Onu-looo IKM-N20 200.2600 60.1500 Mallard 1.1)50 KkK _ - 10.000 '2000 Atult- I ONICI I I lala Hub*hitr 1.(51 iliinn lire 1.1)50 lllurgill 1(50 KaiiiIii*" 1mut ppm HKIl*-r ppin I.C50 ppm kalbi-ad Minim* I.CStl ppm 2000 1*5 soooijyi iwuijgi 0.51 .M.2 0 .6 .) 2.42 O.IWN 0.070 0.022 0 .01* 0.0012 0.054 0.026 Salt Ualrr Daphniu filli 1 .(5 0 1 .(5 0 ppm ppm -- -- -- 0.0571 Mi 0.021ill.p.i 0.002* OMUO.p., 0 .1lit pi Shrin H '50 ppm _ ornar _ _ U. 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M i.ili.liti K 1 i . u i i K ' vs j ( N -p ..t W vk 1.it. 'h i t i r . f 7235 S l i m i v i ' i d i v ii e n t i e s e s n i l i t e t t e . J m .i l e .ii - . m i l . 1 . . . 11tv i \ U l e i i v c t*m . i <t .L it .i I U I . '. . .1 .l l ll i J U u j j i Ic u ftv c i n . i ' t N ^ . i . , i.: -1 i. c - ii i >.i G0043421 h i x i 'i l i i 'H n . i i t i e * o 1 ili 1hi* 1 I'm' .if ' i i'i i . - l . i . IV. e s ' .iSu? ' - , e ' ! ! M ie liti. '..i? i / 1 ( lUlllllIHli \ . i n s i ( 11< i m s DOW763445 ( liriMiH jnxitil lllu \inii- io\ k in ifcii.i ( iminiiHi r iilln-r Naror fur Kul Hui N i n l l n l 1115 ini* k c tlu* m u kl! in ftt-t M ullunl 11150 ll)(! k|> lluliu hilf . I l fll IM'*' 1lullt llfc 1 05)1 iili In t- Kltifttill 1(511 IM'* Kuiiili'iu 1n u l l l.< 5 IM*" f;il lu-;it1 Miiiiinti 1(5 IM*" li. I`knsphnru\ Cunlainiitg / ir r /in i/n a/Mn*n<mihl u.125 V** / rtotfpvnM . J _ imiJ l-IO *7.27 no 70.0)1 400 2*<J(2> (1.42 114 .52 ii.ioi.M 11.1114 U.IMI (i. 2.45 hfcnfi/v A,| *41-21411 2MN) 5)00) U..0* .1114 1.22 HiniMpIkn^ ' -V- Vl.25 dttnritiN p ~'H V* -I dOMWtiifii 5 duumiy .I A iM irim .*-li H, O.O.I.I dkrunphi u.lPf t 1Kii H.2tnuni dkMfiOit rllwHlK . , ,,,r 0.115 0. finit rtNlutm .25 frnwttfiHhiiiii .25 fc-Mlikin K * 1'i ' .15 1*4. ' 12 141*.'nn OM 25-17 22-75 250-5) 212 27-11* 25)147 21 255-2* 2*.0 7.2 -- .*.5 7.0 42 42 0.5 -- ll*u 0.7 5.* 12 5*0 5.* 245 2*0 .12 .u o ijg i 715 5100) 157 .*5 M nwkri*hiiHiir u 5.0 0520)1)1 1405 .4V7 nu-llnl |Mr. iM.ni ' * (i UNOM u |iltn ,, nofcd b- i / . 0. 25 .nnini 02 0.0|5 .,17 *42 .1.7 21 4M 1 4.0 4. 52 <NI 2oo.igi 2.4 2117 luittlhNtn . .Ii5miani lilurali K . j t / , r . -- |lll<JtM1 2-0. irtknuhMKtniik/inym* 0 .1 nmnrl 9 ?V B.i Mirofti* -- Irmrpfei* (l. t irfthfcirCin 2.5 1-5 147-2** 15-*.* DO-.M25 4MM5 luui. 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II.JO 1.4 24 51 Sll N tiltr li-J 1 1) 5 IM" Ihlfihriii 1. 5 IM M iriitip l.( 5 IM" 11.101551M. ll7tSi lliait. kt I5 im tt.20t('i 0.55i Si l.utSi 11.25tMi 0.5.5t M ' kl kt U.74tl't ll.OOltl 1 ( 1 n.ll55tki l.5*i Mi l.22iM 11.57t Mi 0.55tSi li tt.Mitelip i II.IH444iHi 0.10117 II.INI| 11.1*01*1Hi II.IHI24t l*l 11.10017t Hl <I.IHOI7l|-| II.IHII -- Itlll U.IOI.IOt|>l U.UOll>l 1.10 Hl II.INU 11.044t Hl O.IMOOIOO1)p.t (11144t l*t ii.o/i p i n.25iHi 2.5 Hl U.IIINHII ll.H2.5iHi ' ll.ll.50t 1*1 - 11.10125t Hi - II.INOW t/l /i l ti.iMai'i .1 U.IOOOUi ll*l ll..4i l't (1.1014* II imi55i Hi 11. W i kl .<5iMi II.44i Si II 005.I r II.10014t k > ll.ll<5iM.ki liM .si l l . l ' iSl II.IHailo/i p , ll.25i 1 - ll.llhi Hi II.100115 Iip . II.INI! 1*1 II.INI* .0125 U.MI57 II.IOI Hi II l*HI?l il t 1110145t Hl 11.44t l't ii.ihi52<Hi l.liaSl -- 11.20t l*t I lOSt - ii.ihi2Ki Hi 4 t .2t'>t INIII2 tl Wtl-I II IHN-5,1-, i , (arbatnuf* * i*muhHtrt * III .*) %ailllall Vlr. . . . ,m |II.U tmlhuHrffh .5 itr* 1 M-l.5 iii<ul<dli 5*M* 1*0 |2 5 *5-IU4 ;i 2iai - .*4 12 2)o*i i n . 5)MII 1 4 I42'jytat. | tf, ii in i i | 2it*> 1 * _ i i 2 l.u 14 lt < i in (4 lo * |7 II | *: 0004341 lt i 1 *'.M. li 5. k . lt " il.oiir4/i / , n ui h / . II <.#.. II IlllJ. II II ll< Iii iiiW'Mn|l r 7?36 I.*viv*[\ valili s I nuli U n s e il.il.i t C l l a l l l UC.IIIIsllls il'-' m o l e ' C l l ' l U v e .ili*l s h o w II.- c i c a l e s l tir. ci sil i *1 i<\i*.M> l i t u i ) v . m o i i s * henne.iK. I l I>111 a l i a l i SIN *| | i l l es i * i t a l a ili*. k d milicalm oruanisiiis ot loxicilv lo iti.ui .iihI o l h c i am m .il m u a m s m s m die c n i i i o n m c n l are lite lai. a s p c c ic s ol listi aliti tlaplmia. 2. ( 'limine loxicitv I he chiome no-clicct tlala on rais s i m u n in lahle I are illuse Irom iliclaiv leciliiit: Nliitlie.s over a i no - vear perititi ol 'lime . Ih en o-elteel level is nevei Iclhalilv a* il is in llie nenie loxicilv I.DMt ami I.C'ti le*!*. I lie p i i n e i p a l u s e l'or Mie eluonie no-eleel ilain liere is loi eompaiiMiii u i i h acute m a l I . D 50 ilal.i idilli: IIle mi. 3. ('iMiipnrismi of dirmi ic invil iti No-I .ffeel Ia*u*Is uith Acuii Orni 1.1)50IhiluinKnls Hv use ol Mie Mulislical mclhtuls slaleil previoiislv lite clironic no- e lic c i levelstin rais vi ere correlateli vi il h lite I D 'O mi rais ami \v il li all ol lite o llie r acute Io m c iI v lesis. I he Ih's | slalislical coiielalions liom illese pairinps are sliovvn in lahle ( o iie la lio n eoeliicieiils l'or some specilic Chemical proups aie Iv lle r thaii Illuse troni all couil'iucil Chem ical pimips as show li m lahle V W hilc llie coiielalions eoeliicieiils in veita le sipniilcanl al th I ' level. lite *'* coniiilence limils lum i c a l cili.ileil values are piite prcal ini veiv accurate pieilielions. A iiieasuivol cliionicilv tur eacli Mi l l - - .................. . I-. - III * licmn .|| I- lli'1 11iv l.i! *I:- 1.11V 11I t 11* . I 1N I i level -.. i111 ! Il i a 1 .unit- >i .i I i \ i 11 *l>>>.i:i- \ N| \( I loxit ilv l.ilio via-- c.il* a laleil lot e.nll l ompotunl liolii la Iile I. Due 1*> lack ol p.Kt.. di rs e iiuliv ulii.il i.ilios .iic no! riven lie:*1. I oi a il.ila base. Ilie exlicme laiiees ill loxicilv lalio v*iItit`s w i l e lakeu liom each chemical aiul mil mai i/eil In chemical pinups in la hie -4. I he non-umloimils ol die NI-.:AO loxicilv lalio v allies lot each chemical pump is shown. A c c o n lin e lo w ulcsprcun cm lent opinion am i scientific m in im aiio n . I he "p e is is ie n l chlm inaletl h vtlio cn ilm il iiis e c lic u le s " slittulil have a hipli ch io m citv value, am i uuleetl m a in ilo. In lo x icilv tests w ith c h io lin aleil hviliocarh on iiiseclicules. ml m a in s p e c ific e llc c ls p r c c c ilc le th a l e llc c ls . I l o w e v e i . I he i e is still vviile v a r ia tio n in the N| : \<) r a t i o s vv l i i c l i i l l u s i i a l e s d ie v n lc va ria tio n in sp eeil am i ilc u ic c ol m elaholism ot ch lm m a ieil liiilio - ca ih o n iiiseclicu les. I he speeil ol m e la h o lis m is otic o i l l i e im p o it.m i im lica im s o rc lu o n ic lo x icilv loi all chem icals since m eaiiisiiis w liisli rapnllv m clah o li/e cm iipm iinls m Itie e i|iiiv a le u l o l s e v e ia l Imi-s the a c u te m a l I D m ' in th eii ihet il.uiv torn uiim hci ol *lavsh elm e iatism e eip iivaleu l nioitalilv do A c io h Iiiii! In w n le s p ie .n l cm tent o p in io n p h o sp h o iu s c o iila in iiiu n ise i - ticiiles. caih am ale in seiliciile s. ho la n ica l iiiseclicu le s. .uni h eih icu les MO I OO I I I ... I I ................... . tli I ' t.l l 11' I* . i. < . * 11: 1. 1 1 . ( 1 ' |-|m. 1 , %1 *:111111!'11 .Il'.t v I !* 11 it .:I:I .1 *1 i 11 I f' K 11 .V n .ip p r.ii I * It : . r h 1 11 V ill* I I IV. 1I ' its lllllNll.lt. *1 I n use ol 1he N I :A< t lain* A losl lie ih n nli s .u noi \ e iv lo x ii. eilhei ihiotiii.illv m acnlelv. hnl ; he N I : \<) 1 alios lui suine Ilei ' h iiales aie I.nee. itnlie.ilmc i In o li li 1 1 v . Ilie h lv w ale 1 so lu b le heihicules ili. not accumulale um ili in animal tissues such as lai ami muscle aIvove cm iceiilialim ix m the am om uline walei m llie animals tliel ami aie olieii elim n ialeii lapiillv m I lie mine. H ow ever, it h c ih ic h lc s a le not la p iillv melaI'oli/eil hv animal muumsnis oi are eonslaiilli presetil in the viale! or looil ol the oiuanisiii thev mav e - luhii chiome loxicilv e llccls. ( callo phospluu us contamini! insect n iiles mav have a la ice N| : \l> i a ' 10 not onlv he c au se ol d e a l iliilereiices in melaholism hut a l s o h e c a u s e c o m p u i i s n i i *>l the NI : M ) 1alio inv olv es c In>1m e s t e i a sc Nl 'letali conceiilialioiis in 11eaIeif mis-. I liolinesieiase meaMiieinents mav he seiisiiivc lo 2-' oi ilei s o l n i a e i u l mie lowei cmi cetili.iiimis than ihose de c e s s a l i lot let ha Iit v. Ihe ciileiia use*l tor \< ) lesis I he li' e c a i h a ma l e insci denies have small Nl :.\< > ratios, piohahlv h ecause ol llie lapiil melaholism know n to occur w ilh these c h e m i c a l s in anim al oi c a u i s i n s . <hie heihiciile cmnpmiml know u lo ........... TT IIVVIMtII IH.VfM't W'lhmuo-fi Mifl<t<Wlf**flWitb liinMMphthvrtWtRtn^IKilOSiiSni'lQ^T) 'fidtib0 ( 'o i i i |i a n i t i v i O r c a i i i M m a n d l i s t M h Ih h Is M a t a i 'u i i * r a l I .D 5 I I i s M a l l a r d 1 .1 *5 0 i s Ito li Itili-1 1 M l M a l l a r d I I IM I i s I to li** liil* 1 ( M l K a ia lM iw I r o n ! I .C f t l * s S a lt u a li- r lis ti s|i*- *i - s I t it ) i s Itlu ix ill 1 < M l i s i a l l u a d M i n i i o u 1 ( .M l i s M ir in i|i *sI t l i H x i l l 1 ( .M l t a ll i * a d M in ilo t .l M l ?(l \sV i t l u ^ u r 11 s i i v j i t t i t x | < sh u n t) | < C iir r ila liiu i ( in -flH ic n l ill 1 1 .7 7 u .5 *i (U i7 II.M l O 'K i II.`1 | I t i. II.M l II I t " CG04343 II 7J 71 Ml 55 41 :n. <1 (T 2 t i* V 'J tO l tS : a -i :i r 7237 i DOW 763448 . j;< i i i M i l"ii|Tr rii I;.' 15*: * li ' h ( tinnirai ( >ri<ii|. I i ' l 1Ircaiiisnix. ami Ivxl M illiu ilx Ih-iii: 4'nrrciali'it ' 4 ni ii l.iliiiii 4 i i i H h i u I II 5 4 .1.. lair lin k lit \alue l 'ri'ilii I n i 1" 44 illiin l \ 14ll'lllT i III Matiiliiili\ = ; vali' liflntt 1. ( Itlmiit.ilt'il lltilrmarlHin tin n inilrx KjI iVkiilirliir! filli: 1. Kat I I 511 ;. iiniiHiiiii-1< in 71 . II.' II. l'hiii|ilNiriix 1 anitainiiii* liist'ilkiilix Hai 'V I i ili'lan utili 1. vlall.ird 1.1)50 2. Ili nifi lirv 1.4'50 II.7X .(: III. VII 1 Itriiiiial Itriuiiix kal M'. uliflari i uilli: 1. Kiiliuliili' ilii lart 14 5 .iti :. Kal 1.115' ..<7 .4. >liriiii| 1.4 50 11.55 4. Iliiin t Iter l i l i l 1J.ll.l l.lM tl Itl'IM l.llOl* 1 * .5. \n ri|ii.ilinii ik'iiwil 1**f iiiiiu tini.* i.il 1 |)*u It* i.jf NI VvcK i> .p |mIU*v\ 1 i.: M ' : \:i i*| fi*.* im u i.itiitiN .ir M^iplie.mi .il llw I*. Uvei l|| '* i.1. . r 1 1r 1 1.5' I.J 0.`K l. '4 I.X4 l.l I.'* 1.'IV - _1 I ll'-.Milll! ;| il (iimi!!n.ii'ii b lltni Cl liVi.'nii'i '-iit' li fucili l'i l! il/.'.v(:|L't:'.lHu V>i'.'oTni1li-`Xoii! i'iO lp / fl/ai'iJti I____________________ ______ ____ __________________________ Clii-niHiil(rniip Nu. il ( '|I I I I |I I I I I I I ( I n Kan^r i'lKal Ni'.:M lKuln l'unii lalili-I --u ( liliil in,i(rd litaliiHarlNiii HM 'l'l H lIt'N l'lllll|ll|lll X l'lllllIIII|! MI tl lll'S ( ill ll.ltlin li' M M 'llH Ht'1 . MmiII.iiiiiiiiiiiim-iliiiilex llirhii'ialei i l l XII I l i l i lilil- . H l i l l l i i l i u l i t i ll t l . l ' .1 l .l ll l i ' l 11 IK IM KII l.< :x i 4 !<* II.I.W IIM IM II 11.11.1-M.IIIHNI.4 i i ..V'H.ii.<;5 ll.llf-llminx . ll.ll-U.IHMI.n It.n e .i l;n)i- N i :.\ i) i.iliu ih .nuil i n lv vv lie i e llic N I le v e l i> l'.netl imi i!iiileii>ueiiK le x p o o 'e ' U lieji mui.leeme. i.iiiiiineenie. U'i.ilneeme .iiw!niliei lepnuliiilive nuleii/.vni.'iivi'Ilei.Ih <im.tili.t>iii pimulh .ile l.ikeii inti>.lumini. Ilie NI leve! m.iv be emixiilei.ibl'. tel .iih!limn 1lie NI \<>i.itinx Um . li.iiiei wis.jilei.il'lv Ihn, il ix11|'\iini ilui! n,i ijii'iiiil.il'K- V.tl U ..in be ir. i if i i .1 if. in .K u le i'i.il i'VI.I'. ll.ll.l .llnllf l.'l ll'l tu pii' . ' u H. .' N I I. i' 11 11; : *l i i .il I : I e . i I. .1 J . ;' I . I. ' . ! I I. I I:. I l .. I l 'i e i l i i liv e a b i l i l i e x li*i N I I r i e l m l . l l i n i till IC I n i ' J . l l l l .' l l l ' I . l l l b t1' lie Veli ' ( ' i l l liitii k in ti |i *il ee ! |'ti'.xi i . l l .illil i Ik 'IHIi .i I |U n p e 1 I ie; !n| pei Hinleiii e .nul m nbiliiv ni ihe V,l 'li M I '. k l V i I I I ' M I U .il* 111 >1'J.|i j ,.| I)n .1 " i l m I l l e e i n il > > n ii: e ii! I l u x l.in'V. I i 'i l e e p i l i ' .i k i m v . li-ile i .n i h e ili.-! e il'iliv e lui elle. I nie.i-iile m e n ! .il ul n u u l e n i . u ! n >n . >| \ .u m . i x I k ' 111u .i I X 11 V .i I n -1 i'ie.iii.ixir'- V- ili i v i i ' i Tul .ix p i f i lit !i*. r - m i ' . . i,. i i l l ,l|li t . i U i l l ' . 1III I IliL'I. i.lll|>.ill 'i..>1!h-,m |'i.elIit- .::I .>1im. ..'u:i;. COM 1.1 SIONS lv i i i . i ki ne I.i l' ii l.i : eil In Vie il t tl.il.i .i .iil.il'li1. beili, i il.-, ixi *i X nu tlii' pte il U 1111' i i'.i! mi ixliipx ni ti ll !el i'iil i li' n ' u . i l ' . U ' ! il tel In >tl X .nul li il n '.:.iiiixi:ix .u: be m. ule li.:xeJ mi tl.il.i piexeiil-e.! \' i 'I e . i i i i ' i i i x .t u l " f i e n i l i .ii~ ' ite li .i 111e i n 11 e I.i 1: 11X *.*. i i I. r i *. * :. *.* lii.illv 'leniile.ini .iui i:-,l.illi vi. il i . : i i n :i;.' ' 't i I m . : . :. i In mi. .il I \l ili i: : .. -I .il I I >' i . il' 0G04244 7238 2 N.ili water li'li spceivs I l ' O n v .hi l v picdiclcd I i i >111 i.uiihovv lim il >. BI (iuv'(gI isll. . rainbow flout. ninDatlivad minnow I.C5<fs can lv picdiclcd limn each others I.C5<'s. 4. ( u n cia lio n s between Ilie above oinanisms are improvcil when de rived lim n chemical groups closely icla lcd b y struct me. Olhesi cm ivlatiotis may be useiul depending on the sensitivity needed lor decision making. 5. Ih e most useful predictive indica tor organism s anil methods lo r acute toxicity from among the ten organisms tested would appear to l v the rat 1.1)50. one species o ffish (rainbow troiitl I.C50. audom- spe cies o f aquatic arthropod (dapDnia) l .('50. C o r r e la tio n o l\ hrm i 'n and n/< oral !.1)50 toxicity data lor rats indi cates that ihe predictive usefulness o f equations are limited in sensitivity to generalizations with a few exceptions for results from related chemical using the identical test methods. This lim ita tion is due principally to the use o f ' I lili. I I I Ic.iili l< <i sp.iini I W U llll.illls. I |) pi '< | j-iti.d dlt i.u \ loxicilies ol emiioiimelil.il I'ollnlants lobiitls. I S D | I S | isli.n.tl W ild lile Se t v ice. S p e c lal V t-nl it it. K e p o t i W i l d l i f e N o . 1**1. W a s h i n g t o n . I M hi P ti. K e naga. 1-..I-.. I at tois to be co n sidered in the evaluation ol the tovii.ni ol pesticides to butls in then e n iu .a i inenl. l-.nv. Dual. Sale J:l>h M. 7. Kenaga. I-..I-.. I*~X. Aquatic oiganisms and methods useiul I'm assessinent ol chronic lo vicib. o f chem icals. (In piess. Am et . Soc. le s t .M a i i 2' p. X. Kenaga. I..P.. PCX. lest oigamsms ami methoils useful for early assessment ot environm ental loxicitv ol chemicals. In press, l-.nvironmeiitul Science ami T e c h n o lo g y . N o v e m b e r . 2X p .. 1)22-2*. 1 *. Kenaga. P..P!.. A lo ig an . K W l,'X Cm um ercial and expcim iciiial igame insecticides. (M ay. I^ 'X K evisio u Special Publication ~X |. I iiiomologi eal Society o f A iueiica. ( ollege Pa ik . M l). 7*p. | In. M a c c k . K . J . . Hiitchiiison. t .. C o p .. O .M . I**h**. J he effects o l lem peralm e oil the susceptibility til blm-gills ami rainbow trout to selected pesticides Mull. I.n v iio il. Contam in. lo v 4 il i: 174-S.V many non-lethal. no-effect indicators for chronic toxicity from chem icals having different modes of toxicity compared to use o f lethal effects as an indicator o f acute toxicity, and to d if ferences in the rate o f metabolism o f the va rio u s ch em icals effecting chronicily. II M acek. K .J.. M cA lh siei. W .A I'Cii Insecticide susceptibiliii ol some com mon tisli family ic p ic 'c n l.iliv c s l.nui aiy lia iis a c tio iis ot the I.Am eiiean fish e n e s S o c. `***<1 i:2tr~ | 12. M e m a . .1AN.. I pele. P J . I`* "V I he e ffe c ts o f m eth o x ych lo i tin a q u atic biota. C .S . lin v uoiiuient.il Piotcction Agency. I-.PA -KV"* U4h Sept. ' ,v RKFKRKNCKS 1. Atkins. |-..l...'(ireyW (Kid. I-..A.. M a c d o n a ld . K . I . . l `*" .v T o v ic it v o f pesticides and other agricultural Chem ieals to honey bees. C iipiihlishcd le- poit no. Agricultiii.il l.xtension. ( Diversity ol ('niilorma. Kiver-ide. ( 'A . Sept. V i p. 2. D o w C h e m i c a l C S A . 1**77. l i t - published data Midland. M l V I \ M \ N | | t ). |'i.s.||-s I v a l i i . i l i o i i s o l som e pesticide lesulues. I ood and \gneiiltiire < ig.iiii/ation ol I lie l nitevl V i i i o i i s a m l the W o iid I(c.dtii ( h gam /a I i o n s M oil o n i a ('ll s . (telle v a. Sw il/eiland 4 I i e i i d e i s o n . ( .. P i c k c i i n u . (, I I . I . u / w ell. ( A I I**'*< K e l a l r e lo x ic it v o l ten . Id o l m a t e d hv tli Ov.il b o n i i i ' i k t i c i . l c ' i n Itiui s j K k i e s o l tisli 11.m s \ i n c i I i s o St>t .' v .2 > ' 2 I) . Paik s. S .M .. Wo/Ih. H.M.:i*M5. Ih e etfects o f ti lm ahn to fish.' P io c . So. W e e iK 'o n f. Dallas. I X . :4i.l-"4. 14. Sanders. 11.0. I*" , lo\ivi:,es o f some lleibicitles to six species ol 're-'.iwalet crustaceans. August. J. W ater Po.lul. C o n i i o l I e tie i at io n P a it I . V2(XI:I544-'U. ' I V I ticker. K . K . . C rabtiee. I i . ( i . |*>_0. ilam lbtiok o f toxicity ol pesticides to w ih llile l .S I) I I ish and \\ ildlilc Setvice. Minean ol Spoil l isheiies ami W lidate. D eiivei W ildlife K csc.iieh ( e n le i. Kestuuee Piib lk . N o X4. IM p (A lso lettei iiili'i malimi Iio iii liu k e i ami llm lstini | Ih. I S I ep.iiIment ol It Inteiim I :s|i ami W ild lilv Set ; .e >*h2 I fleets ol pesticides on t ' an.I ivildhtc. A tv- vie w til inte ..gal lolls tint me* I'llrf) t ll t. n l .ii 14 1 1 I1 l X l)e| 1- . -11 1 Ihe* I n i e i u m I is h alitl W il. ' Se :! ' U v S| .. tlie ' 1' t * . ' I 'f s l l e lll(' W ildlil. N* ' \ i et lew . H list, .h u ! at lld lllc - i'l ' M ' l i n e * J*y'.i! it i i' ..a .: ii 1 all.! 1..lit 1 a , ai. i a n dI - I N I : ii-i -ii : ; I .. W .Mii[> s- i ... I tt. 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I nviioum eul.il PitUe^ lioa \ u ckcv l'i"4. lie ib k it lv le p o it. e l k ' i m s l i v .trul a nal s i s . e n v i i . u i m e n tal ettecis. agiie u llu i.il .itid t Uh ei a p plietl uses. Kep.il P P A S A M - "4 uo|. Ila / .iit lt u is M a l e n -.1' A d v i s o i v l o m MiiMee a m i t iisuli.iiils. M a v !*( p 24. I S 1 nv ii.M im e iil.il Pi let, t ion A g e n c v . ,;-s P e s tic id e P io g ia m ( uiidc'liiic's toi Kegistem ig Pesluuk-s in l lin e d S l a t e s f e d . K e g . 4 / / i | 2 . M : 2 h X i i 2 - `* ! 2 2 v W eed Science Society ot Am cnc.i. I*"4. Ile ib ie it l e H a n d b o o k o l the W eed Science Society of America. I Intel I d i l i o ! ' . C h a m p a ig n . I I . 4 h i p . 2h. W i l l l o r d . W . A . !(. , 'l o x i c i t v o f 22 I h e i a p e u i i c v o m p o i i . i H l s it S i x I ishes. Investigation m Pish Coiiin>l K e stu iu e Piiblicalim; I .S. lV p a t t- m e ntol the Intcnoi v\ashmgtt>n. I C . p ) i*. o -1 CP CP CG043r4*57239 I ?>*+ 7240 \ ;v V ;. ' ^ *V| * CHENEfa costa iiOW 1511225 frimai. B io ch tm . P h a rm a co L 17: <89 i,E, Guidoni, A.. Mao, C. C., .A. 1977. New concepii on ihc Simun of acumi of benzodiaze.L ift S c i 17167 86 i1..I) R .Ilosli.I..Jnhnsion,G. A. Ihnslon,I II |9(i8 Theh>perp*> imoispinaininloneuim.shyglysud iclalcil amino aculs. E xp . iR n 5U S 62 127. Obaia. K., Takcda, K., Shinozaki, H. 1970. Furtherstudyon pharmacologi calpropertiesofthecerebellar-induced inhibition of Deiler's neurons. E xp . B r a in R e i 11:327-42 128. Nicoll, R. A.. Padjcn, A. 1976. Pen tylenetetrazol:An antagonistofGAIIA alprimaryalTcrenlsoftheisolatedfrog spinal cord. fiieu r o p h a rm a c o lo g y IS: 69-72 Ann. R e * p h a rm o c o l Toxicol. / 9 7 7 / 7 ;JS 7 --4 0 9 Copyright Q 1 9 7 7 by A n n u al Reviews Snc. AU rights rtserv td THE PHARMACOLOGY OF EXPERIMENTAL MYOPATHIES 'V M ic h a e l B. L a s k o w sk i 1 a n d U 'olf-D . D e tib arn Departincut ofPharmacology, Vanderbilt University,School o f Medicine, Nashville, Tennessee 37232 ggs? A large group of disorders affect striated muscle in man. The direct approach to the study of these diseases presents major difficulties. Itiopsics can be useful in diagnos ing a muscle disease and in confirming its progress. However, at a time when muscle weakness and other neurologic abnormalities become obvious, the myopathy is usually well advanced. It is also impossible to provide adequate controls to follow the development of the myopathy in man and its reversibility with experimental techniques. Consequently, a wide variety of animal models of human muscle dis eases have been developed. These models include myopathies occurring spontane ously in highly inbred strains of mice, hamsters, goats, and other mammals. However, a new and rapidly expanding area of inquiry involves the pharmacologi cally induced myopathies developed with a wide variety of drugs. This review is concerned with this second area of research, that of pharmacologically induced experimental myopathies. A survey of the literature of the past ten years reveals many attempts to reproduce pharmacologically human myopathies in animals. Three areas have b^en particu larly active. These include experimental myotonia, experimental Duchcnne's mus cular dystrophy, and general neuromuscular disorders. MYOTONIAP H A R M A C O L O G I C A L L Y I N D U C E D Of all the pharmacologic myopathies that have been attempted, experimental myo tonia bears the closest resemblance to its human counterpart. When rats are injected with 2,4-dicblorophcnoxyacclic acid or fed a diet containing 20,25-diazocholeslcro!, muscle fibers develop abnormal membrane parameters closely resembling those in patients with myotonia. A similar abnormality is observed in the myotonic goat. 'Dr.Laskowski'presentaddressisDepartmentofPhysiology,St.LouisUniversitySchool ofMedicine,St.Louis, Missouri 63104. 'The surveyofliteratureforthisreview wascompletedJune I,1976. ... <D 0 0 ? J . 7 5 T - i a ;v ^38$ LA*. ^tVSKI * DETTBARN OOV 1511226 S im ilarities Between H u m an a n d D ru g-In d u ced F o rm s o f M yotonia In man, myoionia is characterized by sustained contraction of muscle produced cither by a voluntary contraction, percussion with an instrument, or electrical stimulation o f the muscle (I, 2). The irritability of the muscle is markedly increased after insertion of a needle electrode. Electromyography reveals the typical "diveiHimber" bursting of muscle action potentials, but the myotonic effect is reduced by repeated contractions (" warm up period"). Myoionia is particularly evident in myotonia congenita which is an autosomal inherited disease (2). Myotonic effects arc produced experimentally by a wide variety of compounds including the monocarboxylic acids (J), cholesterol derivatives (4), veratrum al kaloids (5). acridine (6), several amines (7), and indoleacelic acid (8). Closer exami nation at the membrane level reveals that only a few of these agents, monocarboxylic acids and cholesterol derivatives, produce effects approximating those seen in myo tonia congenita (3). The effects of two drugs in paiticular have been examined in detail. Thcse"arc 2.4-dichlorophenoxyacetic acid (2.4-0) and 20,23-diazocholcsterol (20(25-D). 7/ie Myotonic Effects o f 2.4-Diclilorophcnoxyacetic Acid 2.4-0 is a tnonocarboxyhc aromatic acid which was also a widely used weed killer (4) Accidental ingestion of tins ding by animals and mail has led to symptoms of insomnia (10). When given to rats (200 rng/kg i p.), 2.4-D produces a myotonic cloctiomyorram (EMG) pattern within 30 mill (II): Similar effects arc observed when normal diaphragm muscles are pet fused in vitro with 2.3 ttiM 2,4-D (4, 12). I licse studies have revealed several major changes in muscle contraction produced by 2.4-0: (u) Within the first few minutes of exposure to the drug, the peak amplitude of a single contraction is increased scvcralfold. Shortly thereafter there is a ten to twenty fold increase in the duration of contraction. ( b ) These effects are augmented when a tetanic burst of stimuli, instead of a single pulse, is given to the muscle (r) A conditioning or "warm up" stimulus reduces the myotonic effect of a subsequent stimulus. (s7) I he prolonged contraction is associated with a prolonged scries of spontaneous repetitive muscle action potentials (12). These major observa tions produced in vitro by 2,4-L) closely parallel the abnormal muscle contractions observed in human myoionia (2) and in the goal (13). The Myotonic Effects o f 20.25-Diuzocholcstcrol 1 lie second drug that induces a myotonic-like condition in rats is 20,23-diazocholesteiol (20,25 1)) 1 his drug inhibits the enzyme desmosicrol reductase (14), resulting m decreased plasma cholesterol and increased plasma desmostcrol, a precursor of cholesterol (15) 1 he myotonia-inducing side effects of this drug were first observed in patients being treated for hypercholesterolemia (16, 17). Muscle cramping and spasm, together with EMG patterns similar to myoionia, were reported. In the experimental animal model, the drug is given in the diet or through an esophageal lube for 3 to 8 weeks (4, Ift) Several other cholesterol analogues including 23a/ocholcsicrol can lie injected s c. for a similar period to produce the same effect (19-21) Like 2,4-dichlorophenoxyacelic acid, the 20,23-D animal model closely 7242 EXPERIMENTAL MYC HIES parallels human myotonia with respect to abnormal contractile properties, as well as changes in electromyography: M e m b ran e A b n o rm alities in E x p e rim e n ta l M yo to n ia The underlying membrane abnormalities in both inherited and experimental myo tonia are similar. Myotonic fibers of both man and the goat have an increased membrane resistance (3). In addition, the mean resting chloride conductance is significantly less than in normal fibers, while the potassium conductance is in creased. However, muscle resting membrane potential was unaffected (3, 22). The myotonia produced experimentally in the rat with 2,4-D or 20,25-D displays similar changes in membrane cable properties (4, 23). There is an increase in specific membrane resistance and a fall in chloride conductance. Like inherited myotonia, muscle fiber resting membrane potential is normal or somewhat higher than normal (3, 4, 23-26). However, unlike human and goat myotonia, the myotonia produced by no 20,25-D does report on the enfofetcdt iospf l2a,y4-iDncorenaspeodtapssoituamssicuomndcuocntdauncctea.nTchee(2e3ff)e.cWt oenhpaovteasfsoiuunmd conductance may depend on the type of myotonia-inducing drug employed since several monocarboxylic aromatic acids other than 2,4-D increase potassium conduc tance (3). An abnormally low resting chloride conductance by itself could account for both increased membrane resistance and the spontaneous firing of action potentials (27, 28). When untreated rat diaphragm muscles were exposed to low chloride solutions, the muscles fibers became myotonic within S min (23). Membrane resistance in creased with no change in potassium conductance. Spontaneous firing arid the warm-up phenomenon (reduced myotonia with exercise) occurred during the first half hour. A short burst of stimulating pulses produced an augmentation and a prolongation of tension. Unlike the resting membrane potential in nerves, a major component of membrane conductance in skeletal muscle is chloride conductance. In frog muscles that do not display myotonia (3), chloride provides approximately 70% of the resting membrane conductance (29). Chloride is an even greater factor in mammalian muscle where it accounts for up to 83% of resting membrane conduc tance (30, 31). S ite s o f A ction o f M y o to n ia-In d u cin g D ru g s v The foregoing evidence indicates that the primary defect in inherited myotonia is an abnormally low membrane conductance to chloride ions, and that many of the properties of myotonic fibers can be reproduced in vitro with low chloride solutions or with various drugs. It appears that a wide variety o f chemical agents with little structural similarity are all capable of producing a very specific effect, namely the reduction of chloride conductance. What is needed is a careful examination o f the altered cable properties produced by each drug and a comparison of these effects with abnormal membrane characteristics in inherited myotonia. As previously men tioned, the monocarboxylic acids in general and 2,4-D in particular arc capable of reproducing the myotonia-like condition in animals (3). On the basis of potency in blocking chloride conductance, Bryant A Morales-Aguilera (3) have determined the chemical requirements for agents that block the chloride channel and hasc sug- 0007176 j 390 LA VSK| A DETTBARN UOVI 1511227 gested a steric block of the channel. Much less is known about the binding potency of cholesterol derivatives other than that 20,25-diazocholcstcrol also produces a rcJuced chloride conductance and increased membrane resistance (23). Most stud ies of membrane biophysics during experimental myotonia have been devoted to only 2,4 D and other monncarboxylic aromatic acids. On the other hand, biochemi cal studies base considered almost exclusively the inhibition of cholesterol bio synthesis, piiuiuiily 20.2S-D. Consequently, it is difficult to make meaningful comparisons of their respective sites of action on muscle membranes. While the simplicity of a common mechanism of action for both groups of drugs is appealing, it appears that 2,4-D and 20,25-1) may act at different membrane sites. In sarcoleinmal membranes isolated from 20,25-diazocholesterol-treated skeletal and cardiac muscle, the amount of cholesterol is reduced and desmostcrol is in creased (32). The myotonic cll'ect is highly dependent on membrane cholesterol, since including cholesterol in the diets of 20,25-D-treatcd rats reverses the myotonia (19). Membrane cholesterol levels have not been reported after 2,4-D or other monocarhoxylic acids. ^-- Most re|Hiris show that N a *\ (` -stimulated ATPase activity increased in 20.25-1)-treated rats (15, 18. 32. 33). with one exception which demonstrates a decrease in activity (33). Sarcoleinmal Ca-slimulaled ATPase activity was also increased after 20,25-1) treatment (IS, 32). In contrast to the action of 20,25-1), 2.4'diehloroplieiio\yace(ic acid had no clfeel on N'a*, K +-slimulaied ATPase but increased the activity of basic /i-nitroplieiiylphosphalasc (/r-NPPase) activity (34). There is cviJenee that this sarcoleiiunal enzyme may regulate the gate for passive (lux of K ions (34) Increased activity of /-NITasc increases K ` elllux from leuko cytes (35). This may explain the increased K conductance observed after some inonocai boxy lie acids (3) llie activity of p-NITasc in human or myotonic goat muscle lias not been described, nor has the efTcct of 20,25-diazocliolcslerol been evaluated with respect to p-NPPase activity. Until this area is further explored, no unifying hypothesis can be pul forward. Clarification of this point will be essential in determining the importance of altered potassium conductance. A logical step in analyzing the mechanism of myotonia is to look for morphologi cal abnormalities. Unlike human myotonic dystrophy, myotonia congenita is not assiH-iated with any generalized structural abnormalities. In the goat one minor change was an increased density of T-tubules (37). Similarly, rats fed 20,25-diazo cholcstcrol did not show abnormal morphological correlates of altered function (38). However, a nonspecific toxic side elTcct of 20,2S-D was observed, possibly due to the drug's effect on muscle sterols. The Relationship o f th e A t n r to D r u g - I n d u c e d M y o to n ia The myotonic response docs not involve the neuromuscular junction (39). Myotonia can still be induced pharmacologically in the presence of curare (II, 40). Rats treated with 2-5 azotholcsterol demonstrate a tendency toward neuromuscular fail ure which may contribute to muscle weakness, but this is separate from the typical myotonic cllccl seen clinically and in vitro (21). However, an intact nerve supply may be required for the development of experimental myotonia (41-43). 7243 EXPERIMENTAL M\ Once established, the myotonia produced by 2,4-D tvas not affected by subsequent nerve section. However, section of the sciatic nerve 7 days prior to administration of 2,4-D prevented the typical repetitive discharges and the waxing and waning of activity (41). Cacciu et al (43) have observed a similar dependence on innervation for the myotonia produced by 20,25-D. However, when treatment with 20.25-D was extended for several weeks after denervation, EMG and muscle contractile behavior closely approximated myotonia (44). A difference is that typical myotonic potentials were rare. The alteration in membrane cable properties following denervation is highly complex. Muscle membrane depolarization begins within a few hours after nerve section, followed after 3 to 4 days by increases in membrane resistance, capacitance, and time constant (43,46). The increase in membrane resistance has been attributed to a reduction in potassium conductance (47-49). It is during this period of in creased membrane resistance that 2,4-D no longer produces myotonia in diaphragm muscles (42). An explanation for this may lie in the observation that untreated muscles in chinridc-frec media display typical myotonia activity (27. 23). This spontaneous activity is abolished in high K. * solutions where K conductance would be lower (28). Thus the relative insensitivity of denervaled muscles to 2.4-D may be due to decreased K f conductance. However, much more work is required with both 2,4-D and 20,25-D treated animals before the subtleties of neural regulation of myotonia can be defined. The pharmacologic model of myotonia congenita closely parallels its human inheritable form. While a wide variety of unrelated drugs produce myotonia symp toms in rats, those few drugs that have been closely studied show a common action on muscle membrane involving altcicd chloride conductance and possibly potas sium ns well. A more thorough examination of membrane transport proteins may also reveal a common biochemical change in the sareolemma. Myotonic changes in man precede the histologic changes in dystrophia myotoniea (2). The slight histopathologic changes which have been reported in myotonia congenita are very similar to early changes observed in dystrophia myotoniea (3b) Recent studies show that chronic treatment with a myotonic drug, 2,4-D, began to reproduce some of the histopathologic abnormalities of dystrophia myotoniea (50). The significance of the myotonia model may lie in the long-term effects of these drugs and the possibility of providing an unimal modcMor the more severe disease, dystrophia myotoniea. P H A R M A C O L O G IC M O D E L S O F D U C H E N N E S M U SC U L A R D Y STRO PH Y Vhc development of experimental models for progressive muscular dystrophies, of which the most debilitating is Duchcnue's, has been much more difficult to achieve than the model for myotonia congenita. Myotonia is primarily a membrane-related dysfunction with minimal histopathologic abnormalities. Ducliennc's muscular dys trophy is far more complex in both its etiology and its pathology. At the present lime no animal model, cither genetic or pharmacologic, accurately reproduces all 0007177 . 392 L a SKa KI^DETTBARN DOW 1 51122L aspects of Duchenne's muscular dystrophy. However, a first approximation to the disease has been achieved with several drug-induced myopathies. For an animal model to reproduce accurately th e pathology of D u c h e n n e 's dys trophy. several minimal criteria must be met: (a) Initial histopathologic changes in biopsies of Duchenne's patients include grouped fiber necrosis surrounded by nor mal muscle fibers (51) (>) As the disease progresses there is a wide variation in fiber diameter and an infiltration of endomysial connective tissue and fat (51, S3). (c ) 1here is a fluctuating increase in serum levels of many enzymes including crea tine phosphokinase (CI'K). glutamic-oxaloacetic transaminase (GOT), glutamicpyruvic transaminase (GI'T). and lactic dehydrogenase (LDII)(S2). ( d ) The proxi mal muscles of the pelvis and lower extremities are the first affected (53). Some of these criteria arc met by the genetically developed dystrophies in a wide variety of animals, of which the mouse and chicken have been studied most exten sively (54) An alternative approach to simulating Duchenne's dystrophy in a ge netic model has been the recent development of the pharmacologically induced experimental myopathies. Two general approaches have been followed. The first was based upon the initial studies of Hathaway el al (55), which involv$d''irculatory obstruction with or without treatment with various monoamines. A second ap proach has been strictly pharmacologic. Either pharmacologic model meets several of the above listed criteria for Duchenne's dystrophy. In addition, they offer greater control in analyzing the basic mechanisms that produce the myopathy. The Vascular Model of Duchenne's Dystrophy A unique and diagnostic characteristic of a muscle biopsy from a patient in an early stage of Duchenne's dystrophy is the characteristic grouped fiber nccrosi. (51). This has led to the suggestion that the circumscribed area of necrotic fibers surrounded by normal muscle may represent the field of perfusion by a dysfunctioning terminal blood vessel (55). This vascular hypothesis is supported by histopathologic abnor malities in blood vessels of some patients in the early stage of the disease (55, 56), but others have not been able to confirm this (57). Complete ligation of the femoral artery and vein yields no histologic abnormali ties in the muscles of the lower extremity of rabbits (55). However, the relative ischemia produced by injection of 20p.-80)i dextran panicles has led to a nearly complete reproduction of the histopathology of Duchenne's muscular dystrophy (55). Two weeks after an injection of the panicles, early changes such as grouped fiber necrosis and phagocytosis occurrcJ, and areas of regeneration were surrounded by fibers of normal appearance. Hie muscles of animals sacrificed 3 months after the initial treatment showed characteristic middle- and end-stage lesions such as proliferation of endomysial connective tissue, fatty infiltration, and a wide range of muscle filler diameters. Occasional thickened walls of arterioles and some occluded vessels were also noted. These observations suggested that the histopathologic pic ture of Duchenne's muscular dystrophy could be reproduced experimentally by ^producing a relative ischemic condition in muscle. ^ A similar development of necrosis and phagocytosis at the light microscopic level _ was reproduced by aortic ligation by itself in rats (58). However, a thorough exami- 'T . EXPERIMENTAL MY" ' T H i E S ^ ^ j nation of the myopathy at electron microscopic level revealed significant differences from the ultrastructural changes seen in human Duchcnne's dystrophy. The authors also noted that aortic ligation was selectively destructive to solcus muscle when compared to gastrocnemius muscle. This selectivity was presumably due to the higher oxidative metabolic demand of soleus muscles, since increasing the work load by contralateral denervation or tenotomy exacerbated the myopathy. The experi mental methods of producing the partial ischemia dilfur greatly between the two studies. Consequently, until the ulirastruclurc of muscles treated with the microsphere approach is examined, the "relative ischemia" hypothesis cannot be fully accepted. Ligation P/ns Serotonin Model Hceausc very few abnormalities are seen in arteriolar walls of Duchcnnc's muscle biopsies, a new approach was developed combining aortic ligation plus the vasoac tive agent serotonin (S-1IT) ($9). The rationale to using this combined approach has been given added significance in view of the observation that in Duchcnnc's patients, the platelet uptake of serotonin is gieatly diminished (60). Serotonin itself has been suggested as a causative agent in carcinoid myopathy (61). Contrary to earlier observations (59), rats given 5-1IT alone display some histopathologic abnormalities (62;. In the rat, normal circulating levels of serotonin arc loo low to contract vascular smooth muscle (63). However, when the isolated rat hind limb was per fused with 2 to SO mg/liler serotonin (5-IIT creatinine sulfate) a decrease in blood llow in the femoral artery occurred (59). The combination of aortic ligation plus 20-75 mg/kg 5-111', was given to rats for 5 days to 3 months either acutely or cluonically. hither method alone produced no muscle necrosis, but in combination, a histopailiolic picture reminiscent of Duchcnnc's dystrophy was produced (59). No sliuclural abnormality was observed in blood vessel walls, leading the authors to suggest that a functional inadequacy rather than a structural lesion is responsible for the relative ischemia. Aortic ligation in the rat previously has been reported to render skeletal muscle more susceptible to vasoactive agents (64). The same charac teristic pattern of muscle lesions has been reproduced with aortic ligation and another vasoactive agent, 3 mg/kg norepinephrine (59, 65). Additional evidence for the "functional ischemic" model of Duchcnnc's dystro phy was obtained by examining plasma enzyme level* in rats given serotonin after aortic ligation (65). Aortic ligation itself produced some increase in plasma .-nrymes which returned to control levels within 72 hr. Administration of serotonin produced a marked increase in creatine phosphokinase (CI' K). glutamic-oxaloacetic transami nase (GOT), glutamic-pyruvic transaminase (Gl'T), and lactic dehydrogenase (I.DII). Enzyme levels returned to normal within 43-72 hr, but repeated injections produced a return to elevated enzyme levels (65). These data suggested that a second requirement for the animal model for Duchcnnc's dystrophy was achieved, that of increased levels of muscle enzymes. Damaged liver as a source for these enzymes was ruled out because there was no increase in alkaline phosphatase. Ligation plus norepinephrine was also dTective in increasing enzyme levels (66). rretreatment with phenoxybenzaminf and chlorpromazinc, both a-adrencrgic blockers, pre- 0007178 )94 U S K C .IADETTDARN OOW1511221 venicd the elevated enzyme levels. Prelrealmenl with imipramine which blocks the prelcrminal amine uptake pump (67) had a paradoxical efTect. Low doses (1-2 n<gAg) maintained low plasma enzyme levels whether noradrenaline or serotonin was studied. However, high doses of imipramine (20 mg/kg) actually increased the plasma enzyme levels elevated by norepinephrine (66). This interaction between imipramine and norepinephrine must be explored adequately before firm conclu sions can be nude with respect to overstimulation of a-receptors. In apparent conflict with these results, Mclmcd & Karpati (68)observed that phenoxybenzamine plus aortic ligation significantly increased the ischemic myopathy. It is quite clear that funlicr studies arc required to explain the fundamental ditfcrcnce between the myopuilnc effects of aortic ligation between the two groups. It is likely a difference in ligating techniques, but the question must be resolved before detailed phar macology is explored, lit addition, it will also be necessary to provide a more complete pharmacologic analysis in the prevention or exacerbation of histopa thologic changes. Depletion of amines with rcscrpinc, blockage of synthesis with p cltlorophenylalanine, and destruction of aminergic nerve terminals with fi ll) droxydopuimne all must be thoroughly tested. S It lias been emphasized recently that increased scrum cnzymclevcls actually precede significant muscle necrosis in Duchcniic's dystrophy (69). Significant abnor malities have been observed in dysttophic muscle and erythrocyte membrane com position and tranS|Hirt. It is conceivable that an expressed genetic error in membranes may be the earliest stage in the pathogenesis of Duchcniic's dystrophy (69, 82). It should be noted that peak increases in enzyme levels occurred 12 hr after 5-1IT. that is, well before the first lesions were apparent (66). Much work needs to l>e done to clarify early abnormalities in membrane transport and composition during the initial hours after drug treatment and before the development of lesions. V asoactive . t n iin a a n d the D evelopm ent o f M yopathies Two more specific pharmacologic approaches have been attempted. The first com bined iiiiipramiiic and serotonin. The second studied the myopathic effects of the monoamine oxidase (MAO) inhibitor, pargyline. Imipramine is a tricyclic an tidepressant which blocks the uptake of primarily serotonin but also other amines into nerve terminals (67). I'arkcr & Mcudcll (70) have succeeded in reproducing the characteristic histuputliology of Duchcnnc's dystrophy by pretreating rats for three days with imipramine (10 mg/kg). On the fourth day serotonin was given (100 mg/kg) This 4-day procedure was repealed each week for eleven weeks. The combi nation of drugs closely reproduced the early and midstage lesions of Duchcnne's dystrophy with the addition that proximal muscles were selectively affected. This procedure also raises plasma enzyme levels of CJ'K (71). When untreated muscle was pcifuscd in vitro with imipramine and 5-1IT. uC-labeled serotonin was taken up ) tunes faster than control. However, oxygen uptake and the amino acid analogue y-aminoisobutyric acid were both significantly reduced (71). When rats were injected with the MAO inhibitor pargyline, soleus muscles con tained a lluroresccnt material generally characterized by the Falck-Hillarp tech nique as catecholamines (72). Duehcnne's dystrophy is the only human myopathy 7245 EXPERIMENTAL MY HUES I known to contain catecholamine-fluorescent fibers (73). It is not known, however, * whether the ligalion-plus-S-HT or imipraminc-plus-3-HT myopathies contain fluorescence. The pargyline myopathy displayed grouped fiber necrosis and. with seven-day treatment, significant connective tissue formation in the endomysium (72). It is very much unlike Duchennc's dystrophy in that only the solcus is uflecicd with no lesions produced in the gastrocnemius (72), or diaphragm, or quadriceps muscles (M. I). Laskowski and W. D. Del (barn, unpublished observations). Dener vation reduced the myopathy, which svas assumed to be "neurogenic." This ap proach is questionable since both motor, sensory, and sympathetic innervation are iionsclcctivcly removed and, of course, muscle activity and metabolic requirements are significantly reduced. In none of the above-mentioned experimental myopathies has any significant ullrastructural examinationX>f the muscle, blood vessels, or nerve been reported. It is clear that before the 5-1IT model can be accepted as a replica of Duchennc's dystrophy a thorough pninl-by-poinl comparison must be made with biopsied dystrophic muscle at the fine structural level. Recent reports have suggested that an abnormal axoplasmic flow in motor nerves may be responsible for several of the myopathies produced by vasoactive amines (74, 75). Two MAO inhibitors, pargyline and phenelzine, were reported to increase the rate of fast axoplasmic transport (75). The increased flow rate was partially pievenlcd by prelreatmenl with a-methylparalyrosinc. Aortic ligation with or without 5-1IT produced a similar increase in axoplasmic transport (74). However. Komtya & Austin (76) found that ligation of the iliac artery plus 5-1IT did not alter flow rate although the myopathy was fully developed in the gastrocnemius muscle. It should be noted that in the reports of increased fast transport mentioned above, confirmatory histologic examination of muscle lesions was not described. Reso lution of the controversy will add valuable information as to whether or not a trophic material from the motor nerve might be augmenting or reducing the my opathy. Recently a " neurovascular hypothesis" has been proposed to explain (he mecha nism of the myopathies produced by vasoactive amines (77). A single genetic deficit was proposed, that of reduced effectiveness of sympathetic vasoconstriction. Ac cording to this hypothesis, the normal reflex vasoconstriction during exercise is impaired, resulting in an overperfusion of unused musejes (78). Reduced blood flow to exercising muscle in Duchenne's patients has been observed (79, SO). Such pa tients also display reduced oxygen tension in exercising muscles (81). While the hypothesis is simple and attractive, much more work is required at the phar macologic and ullrastructural levels before it can be accepted. Sympathetic innerva tion of biopsies of Duchenne's patients must be examined at the electron piicroscopic level. In addition, more thorough pharmacologic analyses must be made of the " functional ischemia*' experimental myopathies. What is required at this point is very basic and thorough work to help clarify the origin of ischemic lesions and (lie way these are enhanced with vasoactive amines. Such studies will provide pharmacologic models that will be an even closer approximation to Du chenne's dystrophy. This approach will certainly provide a potentially productive area for drug development and testing. 0G07179 396 LASKOWSKI & hETTBARN DOW 1 511230 G E N E R A L N E U R O M U SC U L A R D ISE A SE S Investigation of the involvement of the neuromuscular junction in muscle disease had been active before the development of electron microscopic techniques. Dased u|X)n preliminary work at the light microscopic level, ultrastructural investigations have revealed a frequent involvement of the motor end-plate in a wide variety of muscle diseases (82). Zacks (83) has written a thorough review of this topic. In a majority of muscle diseases, motor end-plate degeneration is secondary either to a primary myopathy or to motor nerve degeneration. Myasthenia gravis is a promi nent exception (83). Less frequently occurring diseases that specifically affect the neuromuscular junction arc carcinomatous neuromyopathy (84-86), amyotonia congenita syndrome (87), Isaacs' syndrome (88), Coxsackie 0 virus (89), and a variety of ucuroioxins (83). , Experimental myasthenia gravis is being pursued in several laboratories. Such studies involve the myasthenic dog (90, 91) and rats made chronically autoimmune to ACIi receptors (92). In recent years vve have been actively pursuing studies of end-plate inediated myopathies using a variety of cholinesterase inhibitors and cholinomimetic dings (93-97). Rather than developing a model for a specific disease entity, this approach permits an examination of the delicate nerve-muscle relation ship and explains how altering this relationship can initiate muscle lesions. Neural Control of Muscle Properties 7 lie now classic woik of Duller, Ecclcs & Ecclcs (98, 99) revealed the significant influence that motor nerves have over the structural, functional, and biochemical properties of muscles they innervate. The trophic function of nerves was originally defined as a function that is not mediated by nerve impulses (100). However, only a few of these trophic actions arc independent of nerve impulse transmission and muscle contraction (101, 102). The level of muscular activity influences contractile properties, content of specific enzymes, its sensitivity to ACh, and its ability to accept further innervation. The structural and functional integrity of skeletal muscle depends on the presence and normal function of neuromuscular transmission. Prob lems may arise from pharmacologic manipulation of neuromuscular transmission and tiny lead to changes in the development, maintenance, and integrity of the end-plate and muscle fiber. The physiology of neuromuscular transmission can be affected in several ways. The amount of the transmitter acetylcholine which is released may be increased or reduced by a wide variety of drugs (103-108). Postsynaptic sensitivity may. be changed by altering the number of ACh receptors. Pharmacologic manipulation of the input resistance of the muscle fiber membrane will alter the threshold for generation of muscle action potentials. Variation in one of these factors or in combination with one another can be contributing causes to neuromuscular degeneration and can be altered experimentally (109-113). Some of the changes that arc seen after denervation may be due to disuse rather than loss of the trophic function of nerve. Other experimental procedures will interrupt various aspects of nerve-muscle interaction, while leaving the neuromuscu lar junction and its neivc struciuiully intact. Some of these approaches such as the EXPERIMENTAL MYOPATHIES ^ 3 9 7 use of vinblastine and colchicine block axoplasmic transport without interfering with the synthesis and release of ACh or the propagation and transmission of impulses (114). The clTccts of these compounds are almost similar to those of denervation. Total disuse of the neuromuscular junction can be induced by chemical application of local anesthetics (102) inducing block of conduction and transmission 'without changing structure and function of the neuromuscular junction. Such treat ment also induces phenomena seen after denervation. Cholinesterase Inhibition and Transmitter Release Altered acetylcholine release or a reduction of its hydrolysis by loss in cholinesterase activity is observed ns early palhophysiologic^changcs in murine dystrophy (11$. 116), chicken dystrophy (117), atrophy (I IS), some experimental myopathies (03), and some forms of human muscle disease (119, 120). There has been considerable debate as to whether the nerve terminal or the postsynaptic region of muscle is the primary site of action of anti-ChE drugs (121), and whether cither or both of these sites are allecied by neuromuscular disease processes, such as rjutscular dystrophy and myasthenia gravis (112) Few reports have been published on the effects of anticholinesterases, especially orgauophosphorus agents, on miniature end-plate potential (MEIT) frequency and end-plate potential (HIT) quantum content. Neostigmine increased MEPP fre quency over a narrow concentration range but at higher doses decreased frequency (122, 123). Edrophonium was found to have similar efTccls on ME1T frequency (123, 124). These drugs, however, contain a quaternary ammonium ion which by itself is capable of increasing MEPP frequency (125). Indirect estimates of quantum content during neostigmine, edrophonium, and ambenonium, drugs that facilitate neuromuscular transmission, indicate 'hat quantum content is elevated (123). Re cent work has shown, however, that the method cf paralyzing the nuscle for intracellular recording afTccts the estimate of quantum content in edrophonium (124, 126). Paraoxon, an irreversible organophosphorus inhibitor of ChE activity when in jected into rats, causes (a) an augmentation of spontaneous transmitter release, (A) a reduction in quantum content, and (c) spontaneous and evoked antidretmc nerve action potentials (94). Similar efTccls were observed when paraoxon was applied in vitro (94, 127). A critical question is whether paraoxon produces these effects directly or acts indirectly through its inhibition of cholinesterase. The strongest evidence indicating that paraoxon acts through ChE inhibition comes from the experiments with pyridine-2-aldoxime melhiodide (2-PAM). Reactivation of inhibited ChE with 2-PAM reduces the paraoxon-augmented MEIT frequency and eliminates antidromic nerve activity. Another organophosphorus compound diisopropylfluorophosphate (DPI*) was without effect on MEPP frequency (128). However, these experiments were conducted while basal MEPP frequency was artifically accelerated with 10 m.M K ion. Paraoxon depolarized muscle membranes by approximately 15 mV (127). This depolarizing effect was partly reversed with 10 * M tetrodotoxin (1TX) and totally abolished in combination with u-bungaroloxin. The depolarizing effects of pa- 0G0V180  398 L A S K O . -iCI & DETTUARN fiOWI 1511231 . raoxon are due lo the combined depolarization of ACh receptors and extrajunclional receptors controlling Na-conductance channels. Paraoxon has no cITcct on the K-conductance channels (127). Teirodoioxin reduced the paraoxon-accelerated M O P P frequency b u t did n ot alter spontaneous release in control preparations. It has been reported previously ilui IIX blocks nerse and muscle action potentials while leaving the depolariza tion release mechanism intact (129). Consequently, an intact action potential gener ating system must be present for paraoxon to yield its full cITcci. Whether the faster MOPP frequency is produced by reinvasion of nerve terminals with spontaneous nerve impulses or some other mechanism remains to be determined. Acute Cholinesterase Inhibition and Muscle Fiber Degeneration Irrescrsitde inhibitors of the Chi: activity such as paraoxon, DFP, tabun, sarin, soman, and p.tralhion, as well us teversible inhibitors such as physosligmine and neostigmine, inhibit AChO activity at the neuromuscular junction. This loss of en/ytne activity blocks the hydrolysis of ACh and leads ip increased activity and stimulation of the skeletal muscle fiber. Reports from Several laboratories have shown that all of the above-mentioned Chi: inhibitors arc myopathic { 130--135). Ihe cjihcsi evidence ielating Chi: inhibitors to necrotic lesions of skeletal muscle was in a ic|x>rl by Carey (1TU). Since then this observation has been repealed by several mvesligalois; however, not all of them were aware of previous investigations (9), 131-134). Injection of paraoxon produces a progressive myopathy in the rat diaphragm, soleus, gastrocnemius, and quadriceps muscles. The diaphragm is the most severely aHeelcd of these muscles (133, 135), followed by the soleus and the gastrocnemius muscles. The earliest lesions noted were focal areas of abnormality close to the surface of the muscle filier (93, 133). On ll-L slain this area appeared to l>e more basophilic. The trichromc stain demonstrated an area of red-staining, and the normal basic pattern of mitochrondria, usually identified with LDII and NADII reactions, was disrupted by clumping of highly reactive material. These focal changes progressed to a generalized breakdown of fiber architecture, charac terized by a loss of staining quality followed finally by phagocytosis. Longitudinal sections indicated that the early changes in a focal necrosis affected only a small segment of fiber lengths. The later stages affected progressively greater lengths of muscle fibers (95). Following i p. application of soman (mcthylpinacolyloxyphosphoryl fluoride), local necrotic lesions arc seen scattered throughout the diaphragm muscle. Some fibers display cd severe lesions characterized by loss of cyloarchiteclure and phag.icytosis (131) Within 2 hr of an injection of DFP or tabun the earliest light miscroscopic changes are seen characterized by localized eosinophilia, swelling of the sarcoplasm, and loss of siruiions in several muscle fibers. Approximately 12 hr after the i.v. injection a complete but localized necrosis has developed in the affected fibers (133). A delayed neuropathy beginning at the nerve terminals develops within three weeks after exposure to DI P (136, 137). At this time muscle contractile strength is returning toward normal. 7247  ; EXPERIMENTAL MYO. ..HIES Ultrastructural Changes Produced by Cholinesterase Inhibitors Motor nerve terminals showed varying degrees of changes within JO min to 2 hr after injection of soman and paraoxon (131. IJ8). Some nerve terminals appear relatively normal with the exception of slightly swollen mitochondria. These tiers e terminal abnormalities are more severe after soman than after paraoxon. Mote obvious changes are seen in the subsynaplic area and the surrounding muscle fiber. More severely alfected nerve terminals display myelin figures, membrane enclosures, and an increase in the number of large coaled vesicles. Soman. parao.ua, and neostigmine initiate the formation of vesicular structure in the primary and second ary subsynaplic cleft (95. 131, 139). Occasionally some of these arc seen in the sarcoplasm. Many of the cleft vesicles are similar in density and size to synaptic vesicles but with considerable variations in diameter. The severity of lesions in the subsynaplic folds varies even within the same muscle. Normal subsynaplic clefts with few cleft vesicles are seen side by side with subsynaplic clefts with many cleft vesicles and a widening of the cleft itself (95. 131). The Clili inhibitors, in addition to the changes seen in the region of the end-plate, cause changes in the muscle liber itself. Muscle surrounding the motor end-plate shows a disi option of cyloarchilcciural organization. Initially the first changes are in the iiiilochroiidiia which show swelling leading to lysis of the central crislac. Myelin figures beneath the cud-plate are frequently observed while the region more distal to the end-plate is less affected, flic nucleoli of the muscle cell nucleus are enlarged and move to the periphery of the nucleus. 1 here is an increase in the sarcoplasmic ribosomes, the sarcoplasmic reticulum becomes dilated followed by a loss ofstriaiinn of the myofibrils, and later total destruction of the myofilaments and fragmentation of 7. bauds occurs (95. 131). Muscle Lesions Developing During Chronic Cholinesterase Inhibition Treatment of rats with paraoxon over a period of seven consecutive days produces a progressive myopathy (93,93, 133). In the diaphragm a maximal effect is seen after 3 days of treatment. All stages of the myopathic process were maximal at this point and 6.591 of all fibers were found to be affected, fly day 7 the muscle appeared to be relatively normal. In soleus muscle the maximum nuipbcr of lesions was seen at day 3, but only 0.7% of the total fiber population was afTccted. The gastrocnemius muscle, unlike the diaphragm and the soleus, exhibited maximal lesions on days 3 to 7. The total number of fibers involved at this time was 0.8%. In all muscles studied, maximal loss of ChE activity occurred during the first 30 min after the initial paraoxon treatment. Within 24 hr of each injection of paraoxon, the enzyme activity in diaphragm and soleus muscle had recovered to about 30% of control activity, while in gastrocnemius muscle the ChE activity had returned to 67% of control. There is a marked difference in the susceptibility of muscle type to the paraoxon action. The muscles tested are distinct from each other as to predominant f:ber type, rate of firing, speed of contraction, and inherent metabolism. The myopathy :s much more severe in the diaphragm, which is predominantly a slow contracting, tonic or 0007181 402 L A S i w WSKI A d e t t u a r n bOW 1 51123 antidromic firing and the MEPP frequency. Il also completely prevents the myopa- iliy, unless given after a critical lime, usually between 60-90 min (133-133). Ilcini- cliolinium has been shown to reduce the amount of ACh in the quantum released and reduces significantly the severity of the myopathy (93). Ily inhibiting ChE in vitro, cholinergic as well as adrenergic stimulation is achieved. All preganglionic nerve fibers, as well as motor nerves, arc cholinergic and the preganglionic transmitter is ACh. Under conditions when ChE is inhibited, simultaneous stimulation of postganglionic cholinergic and adrenergic and motor nerves will occur. This results in an increased release of ACh or norepinephrine from postganglionic fibers. Under these conditions not enough blood may be supplied to the most active muscle fibers ami thus relative ischemia might result. This could lead to ischemic changes, leakage of muscle enzymes, and eventual necrosis. The cITccis of prolonged cholinergic vasoJilation in combination with reduced adrenergic vaso constrictor tone during the periods of increased ChE inhibitor-induced muscle activity could lead to functional ischemia in certain hyperactive muscle fibers and not in oiheis. Less active muscle may have normal oxygen requirements, but may receive ihe same blood supply. The functional ischemia could appear during the period of the ACh-induccd muscle fiber activity. Thu could also explain Ihe pre vention of necrosis by denervation due to immo of the dencrvalcd muscle. It is generally acccpieJ that two groups on the enzyme are essential for hydrolysis, the csieratic and anionic site. The anionic site interacts by ionic binding with the cationic head of the ACh molecule. There is good evidence that part of the chemical forces binding ACh to the enzyme are more or less similar to those between ACh mid its receptor. It is therefore not surprising that drugs that inhibit ChE may, to some extent, also react with the ACh receptor. DFI\ for instance, in higher concen trations than needed to inhibit enzyme activity has a curarc-like action which may block neuromuscular transmission (128). In contrast to ChE inhibition, this latter cITcci is reversible on washing. Furthermore, it has not been shown as yet whether this higher concentration is due to direct action on the receptor or on the ionophorc which controls the permeability once the receptor lias been activated, an effect not unlike dial of local anesiliciie. The myopathic elects of the ChE inhibitors can be explained entirely on the basis of their inhibition of the enzyme und the increased action of ACh on pre- and postganglionic receptor sites. Other ciTects that have been described occur at a much higher concentration of the inhibitor. Desides their inhibition of ChE, or interaction with the ACh receptor and or Ihe ionic conductance modulator, organophosphates may have direct effects on Ihe muscle. We have observed that paraoxon directly depolarizes muscle membranes (127), and subsarcolemmal abnormalities of muscle fine structure arc occasionally seen not associated with an end-plate (93). This unspecific action as a cause of the myopathy could not explain the selectivity of the myopathy, since only 6-8% of the fibers are afTccled. The importance of studying the myopathies produced by cholinesterase inhibitors derives not front the accurate reproduction of a specific human disease but rather from the insight they provide into die myopathic clfccts of altering the delicate EXPERIMENTAL MYOPATHIAS ^ 40) nerve-muscle relationship. With this approach it is possible to follow a highly controllable disease process from its origin at the end-plate within 30 min after the drug, to its completion with necrosis and phagocytosis after 24 hr. The crucial questions may lie with the mechanisms which spare most fibers, rather than the disease process itself. C O N C L U SIO N S Within the last ten years there have been major developments in the field of experi mental models of human muscle disease. Recently models of Duchcnnc's muscular dystrophy have been pursued most actively because of the particularly sinister nature of the disease. It is essential now to define carefully the mechanism' u:x`crly ing the generation of these myopathies and to clarify major areas of controversy. This will require a multidisciplinary approach combining physiology, ultrastruelurc, and biochemistry, using the same drug under the same experimental condi tions. Duchcnnc's dystrophy is a multisystem disease. The significance of pursuing the pharmacologic models rests with the development of drugs important to com bating muscle degeneration. The models for myotonia present a different problem. The pharmacology of these experimental myopathies has been examined in Considerable detail. A teview of the literature demonstrates that membrane physiology has been examined thoroughly in one model and biochemistry in another. Again, what is required is a multidisci plinary investigation of each model toward the end of defining whether a single common defective mechanism is responsible for the myotonic state. I'itmlly the myopathies induced by cx|H;rinicntal alteration of nerve-muscle rela tionships have been developed to analyze the basic pathophysiology of muscle degeneration. The most actively studied of these have been the experimental myopa thies induced by cholinesterase inhibitors. Muscles respond to insult with a limited arsenal of physiological and histological alterations. In-depth multidisciplinary analyses of the very earliest of these myopathic processes can provide insight into some of the basic mechanisms underlying muscle fiber degeneration. L iterature C ited 1.Thomsen.J. I87J.TonischeKrampfein willkrlichen beweglichen Muskeln in Folge von crchlerpsychischcr Disposi tion (Ataxia muscular?). A rch . P iy ch ia tr. X e r v e n k r . 6:706-18 2. Merritt. II. H. 197J. A T e x tb o o k o f v X e u r o b g y , 311-36. Philadelphia: Lea & Febiger 3. Hryant, S. II., Morales-Aguilera, A. 1971.Chlorideconductance innormal and myotonic musclefibersand theac tionofntonocarboxylicaromaticacids. J. P h y tio l. 219:367-8) , 4. Rudel, K.,Senges,J. 1972.Experimen tal myotonia in mammalian skeletal <* muscle:Changes inmembrane proper ties.P J iu e g e n A r ch . 131:324-34 J. Ulbricht,W. 1909.Theelicciofveratridine on excitable membranes ofnerve and muscle. E r g e b . P h y tic l. P e l. C k e m . E x p . 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P h a r m a co l. 30: 69-72 138. 1askoscski.M. II..Olson,W. II.,Dett- barn. W-D. 1976. Motor end-platede generation coincident with cholineste raseinhibitionandincreasedftequeney ofminiature end-plate potentials. Peil. P w c. 35-KU) 139. Engel, A. G ,Lambert, E. IL, Santa, T. 1973. Study of long-term anti cholinesterase therapy. Ellecls on neuromusculartransmissionandmotor cnd-plaic fine-structure. N eu ro lo g y 23: 1273-81 140. Lytle. R. II. 1970. Increased synaptic areaofneuioniuscularjunctioninneosiigminc-lrculcd rats. A n a l. /fee. 166: 339 141. Masland, R. L., Wigton, R. S. 1940. Nerve activityaccompanying fascicula tionproducedbyprosligmine.J. N -e u ro p h y u u t. 3:249-75 142. Ilarslad, i. A. II. 1962. i'rcsynaplic effectsoftheneuioniuscular transmit ter E ip e r ie n lio 18:579-81 1 VM >$r EXPERIMENTAL MYOPATHIES HJ. Randic, M., Straughn, D. W. 1964. Antidromic activity in the rat phren ic nerve-diaphragm preparation. J. Phyuot. 173:110-48 144. Veng.T. P..Li.T.II. 1941.Studieson the neuromuscular junction XXIII. A newaspectofthephenomenaofcseriuc potentiation and post-lelanic facilita tion in mammalian muscles. Chin. J. I'hysiol. 16:37-56 145. Van Deer Mccr, C., Mceter, E. 1956. The mechanism of action of ami* chnliiicstciases.II. Theelfeclofdi-idopropylduorophosphate (Dll') in the isolated rat phrenic nerve diaphragm preparation. Acta Phyiicl. Pharmacol. A terl 4.4)4-71 146. Hiker,W. F.Jr..Roberts.J..Standaert. V. G.. Fujimori. II. 1957. The motor nerveterminalastheprimary'focusfor drug-induced facilitationofiieuri-mus- culartransmission. J. Phumiac-j.'. AT.? Ther. 121:286-312 147. Ulabcr, L. C.. Uowman, W. C. 1963. Studies on the repetitive discharges evoked in motor nerve ar.-J skeletal muscleafterinjectionofai-.uchr'.u-.cvcrasedrugs.Dr. J. Pharmacol. 2'.1 M V 4 Q 0Q 7L8G \*5- V3T 7252 ISTITUTO NAZIONALE PER LO STUDIO LA CURA DEI TUMORI 20IJ milans via yanaxlaa. I lalafoso 2390 SERVIZIO DI EPIDEMIOLOGIA by Berrino Franco Introduction, 03 CO o The Brianza di S eveso is an area of about 9 >300 hectare, 20 Em North from Milan, subdivided in 11 small towns of 6 to 37,000 inhabitants, with a total population of 220,000 on Dec 31 , 1976. The ICMESA plant is located in the territory of Meda, near the border of this town with Seveso (fig. 1). The.age and sex structures of the Oct 1971 (census year) popula tions of some towns of the area are shown in fig 2 ; these figures have never been properly updated; moreover an adequate description of the population distribution inside each town is still lacking, so that the'best available denominators for the present study were the Dec 1975 populations of each town with the 1971 age structure. * This report is part of a feasibility study for the extension of the Cancer Registry of Lombardy to the Brianza di Seveso. At our knowledge so far no official study on general mortality has been conducted. 7253 OOO.l/fcc 2 D w 68009G According to the first epidemiological data on acute dermatitis in the emergency period and to the territorial distribution of chloracne cases in children, the towns of Seveso, Meda, Cesano M a d e m o and, to a lesser degree, Desio, seem to be the most heavily contaminated by TCID; both acute skin lesions and chloracne,however,have been observed in all the towns of the zone. On the other hand, apart from these very rough crude incidence figures on dermatological diseases and a few data on obstetrical diseases , no meaningful population based morbi dity rates have been produced. Quality of information. In most towns the available information on causes of death is of rather poor quality, because the demographic offices do not regularly keep copy of the death certificates before sending them to the Central Institute of Statistics (ISTAI) in Rome, and the forms on which some information is recorded are not standardized and in some instances not regularly filled in. In some cases the quality of registration became somewhat better after Oct 1975, when new rules for burials were issued. So far no official recommandation has been given to improve and standardize the death registration. As the ISIA statistics can be disaggregated only to the province level, it would be very important to get back from the Institute the original informations. As for the more interesting towns, fairly good information is available from Meda (after Jan 1976) where the three causes of death are routinely registered; only the "prin cipal" cause is recorded in Seveso and only the terminal one in Cesano M a d e m o . Moreover, with few exceptions, the causes of the deaths occurred outside the town of residence are not registered and have to be searched for at the municipalities 7254 DOW 680097 excellent death registration system exists in Seregno and a useful one in Desio and Saronno, where the m ain hospitals serving the zone are located and where most hospital deaths occur. Hesults. Table 1a,b,c,d show the age distribution of deaths by sixmonths periods. The only significant increase in mortality after July 1976 occurred in Meda in the age group 55 t c `74. Such an increase is not apparent in the other towns. In Seveso and Cesano M e m o however a possible mouvement in mortality may have been masked by a marked excess of deaths occurred in the older age groups just before the ICUESA episode for unknown reasons, possibly following springtime complications of some winter epidemics ( in Seveso these excess deaths have been coded mainly as ill defined cardiovascular diseases). Table 2 a and b show the causes of death in Meda and Seveso by groups of causes (intermediate A classification) and by six-months periods before and after the ICMESA episode. Five leukaemia deaths in 1977 in Meda are far in excess from expected ( F . 02); 2 cases were specified as acute, 2 as chronic and 1 was not specified; the age at death was ... respectively 54 , 72 , 75 81 and 35. Morbidity studies should be soon undertaken to find out the meaning of this observation. Moth in Meda and in Seveso a significant increase in liver tv > < . cirrhosis deaths has occurred, as if intoxication w s m ^A hasUw killed people with impaired liver function. This effect does not seem.to be due to a changed attitude of phisicians towards death certification because liver cirrhosis diagnosis were Doiy 680098 4* equally distributed among phisicians and no excess has been observed in Desio. Table 3 shows a comparison with the mortality for liver cirrhosis that would have been expected from the mortality statistics of the nearby Province of Varese, where the causes of deaths are routinely collected by the cancer registry. f 00034752958 reOn 0003460 GG0039 MOO cono PB.OVMC* D o w 680100 F i g. d 9 F i Y y ramici5 o| 4 "t"ooans A* i IA * m a w i >n ini -r n O* 7258 0003461 ; rTatole i s,t - Aje. apc**^c \yra'te.i C /iOOO^ parioeii^.. _ lalolle i c t ol - -Ag e s p e c ific 'Tal.ty_r.il'es. ( - / l Q O . J _per\o4 i 1___ "~ g F 5 E y B 5 o ~ ~ h n M X , A 9 ? -y ? he. 3 i , i 3 ? 5 4 ? 5 __ftje___ ppjL3 'n_ --JaH-.Jiwe Jwly-Jbec 0- 4 3- 4 4 1 4 6 0 ... 2 fl . .. , 0.7 0 .3 1 5 -4 4 * J3% . o."? - 0 .5 . 19 0 .5 0.4 f IST ?" .-]uly_=Jbv _.Ja*sJ*.C O -------- o.$. --CT5---------'"Cr-Oi-------- - m 1--` O - 4 o .a .. tw 4 5 - 5 4 ..... 1 7 2 8 _. i ' -4 1 5 3 5 -7 4 1145 . ' 5 .7 11-6 35 7 .6 1 6 .5 4 .1 _ 6 .3 3 0 .5 2 _ 6 .3 _ 4 5 .1 2 /i J5 ' 6 .3 _4 5 . 7 75* 404 - 6 .4 . <3 * - 4 ^ 0 * Zj L S** ?4 - 3 53.4 TfttaL 1 6 3 3 ?. 5 . 8 3 . 7 5 . 3 I f t : V . *. ; __N*c_^J*ei^JLf<_kjam_I..3 4 - 5 _r_R^_c*4 :LM.<.e_LeLs-: . 3 1 ..5 .=l?3 ^ fJh-- 9 5 2 _ t e * . i l l i -- it 79-A - 4 38 b ) - lE S AJ!a_rU EB-N Aqe la lati#* T 5T S : --------- *3 T ----------- " S f t Jan J*lr - W Ja - Ime _Jly-_be. 0 -4 3325 0 .6 1 . 2 ___ 0 ,_^_ ft _0 .6 5.4 5 - 6 5 7 4 .... 1 5 8 SO 0-2 0 .6 0.5 0. 2 .L . __ d - 2 __ 0 . 3 0 . 3 . 0 . 6 - 45- _ 55- S 2 Lfl ... 22*6 2 .5 5 .2 4-7 _ 3 .8 3 . 4 ----- 4 . 7 -- -- 8 . 1 i . 2 ___ 8 . . 5 _ . a . 5 _ . 65- 1437 ?5 ______ 4 6 5 ____ -S fa i. * .3 3 5 3 6 . ________ _____ 21 A 7 0.2 3 -3 2 0 .0 .8i A 3 .7 ,2 4 .7 8 .1 2 0 ..0 .__ ,, . 4 1 1 . 8 ... _5 4 . . 8 .7 0 . 2 .r 3 .7 3 .4 _ 3 .5 . 7260 -W3 CO I Ci r- i K . kK 1 . 4 L I * N 5 ut w* S * 0 J; * il O J S* ja "* * 'S -' f i 1 i r Tl * n * J *< ? $ o o w 680104 :I CM I O CO CO CM t y 1> o o o o . bpe1lri*osdes* -iwEn oVME tSso, ^ ^ * 3! 6S) i^x a*404,.IbSy? up spUodecaujsoelsy 1 *~aw M .^ t4> . .'i 1 rlM 1 M 1 M 1 | t > ) | l.-H.i 1 ......................................... ............................... . NW OiCQM . 1H .! 1 I t* IV I M M it iN<*n. . ^ ....................................... ........... i" ; ; .......: z i ' . ................................ : | r| .. N v ol <S3 S t-%;* . i iS t .. T . V 1 , ., . . ^ i i H/i * "**. V .. ! i . : ti . . 1 .1' . ! v> moo 1. * n n n 1 i; n i I : : i 1 - 1. * .! 1 . 1 ; ;1 i, i, 1 i 1 1 i' 1 - i o T oi O\o ^-, K ! ` *a 1 h" 1 tI H -H 1 Tl T 1 H 1 1 (I) |I 1 M , 1 I \o Kl M CM : 'g, ....... ;...................... i i 1 ' . , ti ., ; M t i | I I , ^ t cm 1' : ! ' ! ' : 1! ' 4 ION N tf o\oo 9X i? VO . . . . . . . . . . . . . . . . . ; : j -, - , I l 1 1 1 ! 1 1 1 ' i 1 j j : - : 1 1 i ' ' ' h> ) Tt t ; -rt ..!....i..^iT *e -i. ri .1).. .4......4.......M....!...1. .i 4 J, ....i... i* U f 1 I ^ :. :. ., .! ...,..... !...:. : i'i! -:j- ;; : t: - I. :! |i- ........ 'I ' j i' O r n i t i *1 m M r i n H i. i t n 1. 1 ! 1 I ' l " !...i. i !... ! 1 !.. ! : i 1' i M l1 i l1 i - ; ; i! in H in h i' i " u *> ^ m in Ct%M : . . .: i mH :. 1.t.** 'j . .. . '... . i. ,i. .'i.!4! t! rt. rl11 '11 1 "* ! 2 r* ** . i. ! .....,........... 1. .: ; 3* i. . . i; . i ,. . . m ti n *o H . 1 m ..... ...... ------- i -!.. . ---f-. f........ .-4. j. -1 ..J.....j... .... -i-. . ..* i i 1. i 1 1. . 1 by g r o Iew**esa ibe bebifotrre k3otwe*(*ao^je r _ .2 ^ l V < . . +*^ . ; .................... : . . . . ! ! i co t i n ri i i . * i ........................... : !! '.i t h ....................................: . . . ; : ..................... h v i.u h n i i ! i io t i i ;! i . n. u> w Usoi , ^o 2i Ir *~=nasi s_ o ol I 3 , o . - a1>- 1w5. fXi* |A < <r .......-*: ............1................ " f1 ' . : ...I. , .........................` 1 *J 4 3f e------. ^ .to.l...^.o.1...j^........2*..n...St..|I...31.. S1 ^^ ^^ ^w <<<<<<< J " se. `* ' 1*14j S ti *^ri u! 1 !.. t1 I i 1 A 4* b> l Table 3 " Deaths for liver olrrhoale (ICD 571) In the Seveso Zone by 6 months periods before and after the ICME3A episode (July 10, 1976); males only. The expected numbers have been obtained from the age specific mortality rates observed in the nearby Province of Varese in the same period. Commune Meda Year Semester obs. exp. LIVER CIRRHOSIS DEATHS _____ 125________ 1 1976 1 St 2nd 1 at 2nd 1977 1 st 2nd* n.k. n.k. 2 1.81 4 3/61 3 1 .20 Total after P July 10. 76 value 10 4.82 <.05 Seveso obs. exp. 3 1 _3 % 5.18 .3 __L L 3.45 5 1.15 9 4.61 <.05 Seregno Desio obs. exp. obs. efcp. n.k. n.k. n.k. n.k. 2 3.61 3 3.06 3 7, ^ 751 2 2.41 2- 6 -- 6 i12 2.04 --- 1---- 1 12 9.64 8 8.17 NS NS * 4months only (July to October) -4 CO n,k, = not known NS a not significant C0T039MCXJ 0003466 13V 13 io/ 7264 Strictly Confid SOiiSOWH WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE CENTRE INTERNATIONAL DE RECHERCHE SUR LE CANCER INTERNATIONAL AGENCY FOR RESEARCH ON CANCER CO 0 JOINT NIEHS/IARC WORKING GROUP on the COORDINATION OF EPIDEMIOLOGICAL STUDIES on the LONG-TERM HAZARDS OF CHLORINATED DIBENZCDIOXHIS AND CHLORINATED DISENZOFURANS Lyon, 10-11 January 1978 AGENDA 1. Opening remarks - Dr J-. Higginson 2. Appointment of Chairman and Vice-Chairman 3. Adoption of agenda and agreement to working procedures 4. Presentation and brief discussion of working papers (a) Chemistry and toxicology (b) Epidemiology 5. General discussion about epidemiology 6. Future research: recommendations and international coordination 7. General outline of meeting report 7265 0002273 DOW372504 ORGANIZATION V S n. ; . I. ,'J$? urt^AMSATION V ONCIALE OE I A SAHIE CENTRE INTERNATIONAL DE RECHERCHE SUR LE CANCER INTERNATIONAL AGENCY FOR RESEARCH ON CANCER Joint NIZUS/IARC Working Group on the Coordination of Epidemiological Studies on the Long-Term Hazards of Chlorinated Dibenzodioxins and Chlorinated Dibenzofurans Lyon, 10-11 January 1978 LIST OF MEMBERS pec6'v * auat* iSG L/ t w Dr A.O. Arstila Professor and Chairman Department of Cell Biology University of JyvSskylA Vapaudenkatu 4 40100 JyvaskylA Finland Dr O. Axelson Department of Occupational Medicine Regional Hospital S-S81 85 Linkaping Sweden Dr P.J. Baxter Employment Medical Advisory Service Health fi Safety Executive Baynards House 1 Chepstow Place London W2 4TF UK Dr F. Berrino Servizio di Epidemiologia Istituto Nazionale per lo Studio e la Cura dei Tumori Via Venetian 1 20133 Milan Italy Dr L. Bisanti Uffici Regione Ospedale Desio Milan Italy 1 Dr R. Frentzel-Beyae Institut fir Dckumeiitaticn, Information und statistik Deutsches Xrebs/orschungszentrum la Neuenheimr 'Feld 280 6900 Heidelberg 1 FRG Hotel Residence 4 Hotel Residence Hotel Residence Hotel Residence y Hotel Rsidence r 7266 0002274 Cr A. Hay Department of Ar.ir.al Physiology and Nutrition University of Leeds XirV.stall Laboratories Vicarage Terrace L~c-ds L55 3HL Jx Professor L. Jirasek Universit Karlova FVL 2. Dermatologicka Klinika U. Nemocnice 2 123 08 Prague 2 Czechoslovakia Dr G. May LA Welbeck Road Bolsover Derbyshire S44 6DH UK Dr J.A. Moore Acting Associate Director Research Resources Program National Institute of Environmental Health Sciences PO Box 12233 . Research Triancle Park North Carolina 27709 USA Dr G.F. Parozzo Commissario Straordinario Uffici della Regione Via S. Carlo 4 Seveso (Milani Italy Professor F. Pocchiari Director General Istituto Superiore di Sanit Viale Regina Elena 299 00161 Rone italy Dr A. Poland ... . Assistant Professor of Oncology McArdle Laboratory for Cancer Research University of Wisconsin Medical School Madison Wisconsin 53706 . J. USA Hotel rsidence Hotel Residence Hotel Residence & Hotel Residence Hotel Residence Hotel Residence 7267 0002275 DOW372505 Professor C. Rupee Depar tr.ent of Organic Chemistry University of Unea S-901 37 U.-ea Sw eden Dr V. RiihinSki Department of Industrial Hygiene and Toxicology Institute of Occupational Health Haartmaninkatu 1 00290 Helsinki 29 Finland Professor I.J. Selikoff ^ Mount Sinai School of Medicine \ Environmental Sciences Laboratory \ Cummings Basic Sciences Building 10 East 102 Street New York N.Y. 10029 / USA Dr R.R. Suskind Director Institute of Environmental Health Kettering Laboratory University of Cincinnati Medical Center 3223 Eden Avenue .Cincinnati Ohio 45267 USA Dr J.G. Vos Head, Department of Oncology Laboratory of Pathology RijHs Instituut voor de Volksgezondheid Postbus 1 Bilthoven The Netherlands Dr N. Wald University of Oxford Department of the Regius Professor of Medicine Radcliffe Infirmary Oxford 0X2 6HE UK 4 Hotel Residence 0 Hctel Residence /H Hotel Residence Hotel Residence Hotel Residence Hotel Residence 7268 0002276 90Q2/,PMOn Secretariat i Dr H. 2artsch, Cnit or Chemical Carcinogenesis Dr J. Kiggir.son, Director, IARC Dr J.E. Kuff, Unit or Chemical Carcinogenesis Dr T. Kuroki, Unit of Chemical Carcinogenesis Dr R. Montesano, Unit of Chemical Carcinogenesis Dr J. Parizek, Control of Environmental Pollution and Hazards, Division of Environmental Health, WHO, Geneva Dr R. Saracci, Unit of Epidemiology and Biostatistics Hotel des 3eaux-Arts Dr L. Tcmatis, Chief, Unit of Chemical Carcinogenesis Mr J.D. Wilboum, Unit of Chemical Carcinogenesis DOW372507 J 0002277 m 151 7270 // fJ 4\/ ***.'* * **!/.*&y ; / : / - ' y2 / ?r 7 i t i ( " j t - ' - 1 *-- v IARC MEETING ON DIBEMZODIOXINS AND DIEENZOFURANS INDEX 1 M. L. Long_____ j M. J. Traynor - 2030 B. B. Holder - 2030 R. L. Johnson - 2040 JANUARY 10-11, 1973 LYON, FRANCE FR0M: John H. Davidson 3/13/78 COMPARATIVE TOXICITY OF DIBENZODIOXINS AND DIBENZOFURANS John A. Moore TCDD --------- Chlorinated dibenzodioxin toxicity has been extensively characterized o O K^ SE CO sjn CO \h i H -1 07 oO v jl using the 2,3,7,3-isomer (TCDD) as the prototype. Toxicity in laboratory ; animals is characterized by a progressive wasting (mobilization of _ peripheral fat stores, increased triglycerides, fatty liver); and by hypoplasia of lymphoid tissues. Liver is also a target organ. The severity of effect varies between species with rats, mice, and rabbits N markedly affected; hepatic effects in guinea pigs and Rhesus monkeys are minimal by comparison. Classic chloracne is not observed in laboratory species, but analagcus integumentary effects are seen in monkeys, rabbits, and hairless mice. (Table 1) TCDD is classified as a teratogen; however, the dominant prenatal effect is fetal toxicity or death. Existing reports on t.he mutagenicity oi' TCDD are not definitive. Recent studies have reported that TCDD is a carcinogen in rat/. Additional lifetime studios in rats and mice and experiments in mice designed to determine cocarcinogenicity are nearing completion. 0002358 7271 .Immune suppression has been demonstrated in rats, mice, and guinea pigs 4 exposed to TCDD. The cell-mediated component is primarily affected, # with humoral immunity also depressed at higher doses; low doses may be stimulatory. The developing fetus and pre-pubertal animal appear to be more susceptible. An increased susceptibility to bacterial (Salmonell challenge is observed in mice that received 4 doses of TCDD that caused no gross or microscopic pathology. T8TG TCDD is reported to have an extended half-life (24-31 days), liver and fat being the main body compartments. Significant species variability in the amount of TCDD stored in liver occurs with greatest liver storage found in rats and mice and lesser amounts in guinea pigs and Rhesus - -monkeys. OTHER DIOXINS / The acute toxicity of other dibenzodioxin isomers has been systematically studied in guinea pigs and mice. The degree of toxicity is dependent on the location and number of halogens (Table 2). Extreme toxicity (LD 50/30 + 1 mg/Kg) is found in tetra, pentn, and hexachloro congeners that have halogens in the four lateral (2,3,7,8) positions. x The toxicopathologic pattern of disease caused hy the chlcrodibcnzo* */ dioxins was found to be similar in a given species. Chronic toxicity (2 year) studies of the 1,2,3,5,7,8 and 1 ,2,3,7,8,9-hexachlorodioxin 7272 isomers in rats and mice are nearing completion. 0002359 D O W 37318 V 3 n The acute or chronic toxic dose for hepta or octachlorodibcnzodioxin q has not been established. Significant body burdens of these dioxins do occur as a consequence of prolonged exposure. The interactive effects of subtoxic doses of various dibenzodioxin isomers have not been studied. DIBENZOFURANS The acute toxicity of 2,3,7,S-tetrachlorodibenzofuran has been evaluated in rats, mice, guinea pigs, and Rhesus monkeys. The LD 50/30 is similar r\D to TCDD in guinea pigs and is about 40 fold higher (1 mg/Kg) in Rhesus ' monkeys. The toxic dose in rats and mice was not established using doses about 40 fold greater than fur TCDD (Table 3). .4 Experiments in guinea pigs showed the acute LD 50/30 for 2,3,4,7,8pentachloro and 2,3,7,8-tetrachlorodibenzofuran to be similar to the 2,3,7,8-tetrachlorodibenzodioxin and dibenzofuran. The pattern of toxicity of all the dibenzofuran isomers studied to date was found to be indistinguishable from that produced by the dibenzodioxins. Chronic toxicitystudies have not been conducted with the dibenzofurans. 7273 0002360 TABLE 1 PATKOLUbY summary t h y u s INVOLUTION SPLEEN REDUCTION (WHITE PULP) BONE MARROW HYPOPLASIA LIVER MEGALOCYTOSIS/DEGENERATI ON BILE DUCT HYPERPLASIA TESTICLE DEGENERATION RENAL PELVIS HYPERPLASIA . URINARY BLADDER HYPERPLASIA ADRENAL . CORTICAL ATROPHY (Z.G.) HEMORRHAGE INTESTINAL ADRENAL ASCITES CUTANEOUS LESIONS GUINEA PIG +++ + ++ + +++ ++ ++ ++ + ++ - s MOUSE +++ + + +++ + ++ __ + -- ++ - MONKEY +++ + + -- +++ ND (FtHALE) + -- + +++ 7275 0002362 n U L ^ oQ / CO CM Tob'i e 2 E stim ate d S in g le O ral o f CDDs * C h lo rin a tio n 2,3,7 2,3,7,8 1,2,3,7,8 1,2,4,7,8 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 1,2,3,4,6,7,8 1-N02-3,7,8 l-MH2-3,7,8 l-t:02-2,3,7,8 l-N!I2-2,3,7,8 lic | / l : q G u in e a P iq s M n iolc/h c] >300,000 29,444' 2 3.1 1,125 '72.5 70-100B . '60-100B >500 >30,000 >30,000 47.5 194.2 >1,180 120.41 0.005 0.009 3.15 0.185 0.178-0.255 0.153-0.255 >1.400 >90 >99 0.129 0.575 Mi c e y n n le /b q - >3,000 233.7 337.5 >5,000 25 1,250 >1,440 - - - >2,000 >4,800 >10 0.88 g 0.94 ^ CO 2: H*-' 2.11 00 3.19 * >3.67 - - - >5.4 >14.2 JJSTGiEMOa ^ S p o a r n i 3 n - K a i ' b c r M e .h o d P Estimated Range D u e to V a r i a b i l i t y in R c p l i c a t e s . *M c C o n n e ll, E ., M oore, J . , H ascm an , J . , and H a r r is, :M . 1977 i 7276 - 00?363 TABLE 3 I GENERAL TOXIC EFFECTS OF HALOGENATED DIBENZOFURAN5 IN GUINEA PIGS3 * OOV/373186 Dose Chemical (ug/Kg) Mean Time Mortality to Death (days) Comments TCDF o-' 0 1 5 10 15 0/9b 0/6 0/6 6/6 6/6 - - 11.8 (9-16)c 14.5 (12-19) Norm,al growth Decreased body weight gain Body weight loss in 2; other decreased weight gain Immediate fi progressive weight loss Immediate & progressive weight loss PCDF ^ /; \` 0 -* i 3 10 30 TBDF 1j\r 1 l \ 0 .47 1.58 4.74 0/9 0/6 0/6 . 6/6 6/6 0/9 0/6 0/6 1/6 15.84 ' * 6/6 - 15.7 (13-19) 9.2 (8-11) - - 26 11.7 (10-13) Normal growth Decreased body weight gain Decreased body weight gain Immediate fi progressive weight loss Immediate fi progressive weight loss Normal growth No effect No effect Docreasnl body weight gain progressive weight loss in animal that died Immediate & progressive weight loss 3A11 animals received single oral dose in corn oil, controls received only corn oil. 7277 Numerator indicates number dead, denominator indicates number tested. 0002364 Mean time to death; number in parenthesis is range. Selected References Kociba, R., Keyes, D., Beyer, 0., Carreon, R., Wade, C., Dittenber, D., Kalnins, R., Frauson, L., Park, C., Barnard, S., Hummel, R., and Humiston, C.: Results of a two year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. Tox. & Appl. Pharmacol., submitted 1977. McConnell, E., Moore, J., and Dalgard, D.: Toxicity of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) in Rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. & Appl. Pharmacol. +3 (1): -3- -- /7S-/77, / 97/. McConnell, E., Moore, J., Haseman, 0., and Harris, M.: The comparative toxicity of chlorinated dibenzo-p-dioxin isomers in mice and guinea pigs. Toxicol. & Appl. Pharmacol., >n p ress, mQ77. y y ; 3 3 S - I S Moore, J.: Toxicity of 2,3,7,8-tetrachlorodibenzo-para-dioxin: /1 7 * . Chlorinated phenoxy acids and their dioxins. Mode of Action, Health Risks and Environmental Effects. Ecol. Bull. 27_ (Stockhol in press, 1977. Moore, J., McConnell, E., Dalgard, D., and Harris, M.: Comparative toxicity of three halagenated dibenzofurans in guinea pigs, mice, and Rhesus monkeys. In preparation, 1977. Rose, J., Ramsey, J., Wentzler, T., Hummel, R., and Gehring, P.: The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Tox. & Appl. Pharmacol. 36: 209-226, 1976. Vos, J.: TCDD - Effects and mechanisms. Mode of Action, Health Risks and Environmental Effects. Ecol. Bull. 27 (Stockholm), in press, 1977. -DOW 37318 ,/ 7278 0002365 \3>S> 7279 . D O W 129547 Stri.<. w i i y OH f 5H i H\- i l l I O O ' * f IARC MEETING ON DIBENZODIOXINS AND DIBENZOFURANS JANUARY 10-11, 1978 LYON, FRANCE H COMPARATIVE TOXICITY OF DIBENZODIOXINS AND DIBENZOFURANS . John A. Moore ^ TCDD' Chlorinated dibenzodioxin toxicity has been extensively characterized using the 2,3,7,8-isomer (TCDD) as the prototype. Toxicity in laboratory animals is characterized by a progressive wasting (mobilization of peripheral fat stores, increased triglycerides, fatty liver); and by hypoplasia of lymphoid ^tissues. Liver is also a target organ. The severity of effect varies between species with rats, mice, and rabbits ^ .markedly affected; hepatic effects in guinea pigs and Rhesus monkeys are minimal by comparison. Classic chloracne is not observed in laboratory species, but analagous integumentary effects are seen in monkeys, rabbits, and hairless mice. (Table 1) TCDD is classified as a teratogen; however, the dominant prenatal effect is fetal toxicity or death. Existing reports on the mutagenicity of TCDD are not definitive. Recent studies have reported that TCDD is a carcinogen in rats. Additional lifetime studies in rats and mice and experiments in mice designed to determine cocarcinogenicity .are nearing completion. 7280 0001572 2 D O W 129548 .Immune suppression has been demonstrated in rats, mice, and guinea pigs exposed to TCDD. The cell-mediated component is primarily affected, 0 with humoral immunity also depressed at higher doses; low doses may'be stimulatory. The developing fetus and pre-pubertal animal appear to be more susceptible. An increased susceptibility to bacterial (Salmonella) challenge is observed in mice that received 4 doses of TCDD that caused no gross or microscopic pathology. & TCDD is reported to have an extended half-life (24-31 days), liver and fat being the main body compartments. Significant species variability in the amount of TCDD stored in liver occurs with greatest liver storage found in rats and mice and lesser amounts in guinea pigs and Rhesus - -monkeys. OTHER DIOXINS / ` The acute toxicity of other dibenzodioxin isomers has been systematically studied in guinea pigs and mice. The degree of toxicity is dependent on the location and number of halogens (Table 2). Extreme toxicity (LD 50/30 + 1 mg/Kg) is found in tetra, penta, and hexachloro congeners that have halogens in the four lateral (2,3,7,8) positions. The toxicopathologic pattern of disease caused by the chlorodibenzo- dioxins was found to be similar in a given species. Chronic toxicity (2 year) studies of the 1,2,3,6,7,8 and 1 ,2,3,7,8,9-hexachlorodioxin isomers in rats and mice are nearing completion. 7281 0001573 fiOW 129549 3 The acute or chronic toxic dose for hepta or octachlorodibenzodioxin has not been established. Significant body burdens of these dioxins do <? occur as a consequence of prolonged exposure. The interactive effects of subtoxic doses of various dibenzodioxin isomers have not been studied. DIBENZOFURANS The acute toxicity of 2,3,7,8-tetrachlorodibenzofuran has been evaluated in rats, mice, guinea pigs, and Rhesus monkeys. The LD 50/30 is similar to TCDD in guinea pigs and is about 40 fold higher (1 mg/Kg) in Rhesus ' monkeys. The toxic dose in rats and mice was not established using doses about 40 fold greater than for TCDD (Table 3). Experiments in guinea pigs shov/ed the acute LD 50/30 for 2,3,4,7,8pentachloro and 2,3,7,8-tetrachlorodibenzofuran to be similar to the 2,3,7,8-tetrachlorodibenzodioxin and dibenzofuran. The pattern of toxicity of aTT the dibenzofuran isomers studied to date was found to be indistinguishable from that produced by the dibenzodioxins. Chronic toxicity studies have not been conducted with the dibenzofurans. 0001574 7282 DOW 129550 SUMMARY 1. The pattern of toxicity produced by chlorinated dibenzodioxins and dibenzofurans are indistinguishable. Other chemicals, notably certain halogenated naptha!enes, biphenyls, and likely the azoxybenzenes, also cause a similar pattern of toxic response. 2. Toxicity is dependent on degree and location of halogens with halogenation at the 2,3,7,8 position required to cause marked toxicity. The interaction of various dibenzodioxins or dibenzofurans is unknown. 3. Toxicity caused by-these chemicals is characterized by chronic wasting, fat mobilization, and altered lipid physiology; lymphoid hypoplasia and immune suppression; sebacious gland metaplasia and hyperkeratosis, and a hepatic toxicosis that is species dependent. ' 0001575 7283 TABLE 1 ,PATHQLUY SUMMARY GUINEA PIG_ _ _ HOUSE THYMUS INVOLUTION ++ + + ++ SPLEEN REDUCTION (WHITE PULP) + . .+ BONE MARROW HYPOPLASIA ++ LIVER MEGALOCYTOSIS/DEGENERATION - .+ + + BILE DUCT HYPERPLASIA ' + + TESTICLE DEGENERATION '+ + + . + + RENAL PELVIS HYPERPLASIA ++ URINARY BLADDER HYPERPLASIA ++ ADRENAL CORTICAL ATROPHY (Z.G.) ++ HEMORRHAGE INTESTINAL ' ADRENAL + '+ ++ ASCITES - ++ CUTANEOUS LESIONS MONKEY +++ + + +++ ND (FEMALE) + +++ D O W 129551 Chlorination 2,8 2,3,7 2,3,7,8 1,2,3,7,8 1,2,4,7,8' 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 1,2,3,4,6,7,8 1-N02-3,7,8 1-NH2-3,7,8 l-K02-2,3,7,8 1-NH2-2,3,7,8 Estimated Single Oral of CDDs * Guinea Piqs vq/l:q v mole/kq vq/kq -M-i-c-e to CO cn to v mole/kq >300,000 29,444' 2 3.1 1,125 '72.5 70-100B *EQ-10QB >600 >30,000 >30,000 47.5 194.2 >1,180 ' 120.41 0.006 0.009 3.15 0.185 0.178-0.255 0.153-0.255 >1.400 >90 >99 0.129 0.576 - >3,000 28317 337.5 >5,000 825 1,250 >1,440 - >2,000 >4,800 - >10 0.88 0.94 >14 ' .' 2.11 3-19 >3.67 - - >5.4 >14.2 a Spearman-Karber Method ^Estimated Range Due to Variability in Replicates. , McConnell, E., Moore, J., Haseman, J., and Harris, M.: 1977 r TABLE 3 ( GENERAL TOXIC EFFECTS OF HALOGENATED DIBENZOFURANS IN GUINEA PIGS3 * ' O, Dose Chemical (pg/Kg) TCDF 0 1 5 10 15 Mean Time Mortality to Death (days) 0/9b 0/6 0/6 6/6 6/6 - - 11.8 (9-16)C 14.5 (12-19) _ Comments M to to rn ^ CJ Normal growth `Decreased body weight gain Body weight loss in 2; other decreased weight gain 'Immediate & progressive weight loss Immediate & progressive . weight loss PCDF c ... 0 1 3. 10 Q/9 0/6 0/6 6/6 15.7 (13-19) 30 6/6 9.2 (8-11) Normal growth Decreased body weight gain Decreased body weight gain Immediate & progressive weight loss Immediate & progressive weight loss TBDF ' 0 .47 1.58 4.74 . 0/9 0/6 ` 0/5 1/6 - - 26 15.84 6/6 11.7 (10-13) Normal growth . Ho effect. No effect Decreased body weight gain; progressive weight loss in animal that died Immediate & progressive zi'rr c - v , / # ^ S l ^ 0SS aAll animals received single oral dose in corn oil, controls received only -corn oil. . ^Numerator indicates number dead, denominator indicates number tested, c Mean time to death; number in parenthesis is range. Moore, J., McConnell, E., Dalgard, D., and Harris, M.: 1977 129554 Selected References _O K o c ib a , R , Keyes, D., Beyer, J., Carreon, R., Wade, C., Dittenber, D., Q. Kalnins, R., Frauson, L., Park, C., Barnard, S., Hummel, R., and Humiston, C.: Results of a two year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. Tox. S Appl. Pharmacol., submitted 1977. McConnell , E., Moore, J., and Dalgard, D.: Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. & Appl. Pharmacol. 43 (1): 1-13,1977. McConnell , E., Moore, J., Haseman, J., and Harris, M.: The comparative toxicity of chlorinated dibenzo-p-dioxin isomers in mice and guinea pigs. Toxicol. & Appl. Pharmacol., in press, 1977. Moore, J. : Toxicity of 2,3,7,8-tetrachlorodibenzo-para-dioxin: Chlorinated phenoxy acids and their dioxins. Mode of Action, Health Risks and Environmental Effects. Ecol. Bull. 27 (Stockholm), in press, 1977. Moore, J. , McConnell, E., Dalgard, D., and Harris, M.: Comparative toxicity of three halogenated dibenzofurans in guinea pigs, mice, and Rhesus monkeys. In preparation, 1977. Rose, K, Ramsey, J., Wentzler, T., Hummel, R., and Gehring, P.: The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Tox. & Appl. Pharmacol. 35: 209-226,1976. Vos, J.: TCDD - Effects and mechanisms. Mode of Action, Health Risks and Environmental Effects. Ecol. Bull. 27 (Stockholm), in press, 1977. 0001579 7288 DO W 372610 MM 053308 NOTICE: POOH COPY DUE TO DEFICIENT ORIGINAL i Symptomatology, morbidity ar.d mortality Kjerier.ce of chlorinated phencxyaci herbicide (2,4-D; 2,4,5-T) sprayers in Finland. A clinical and epidemiological studv. INTHCSUCTIGM In 1372 there were widespread rumours in Finland that chlorinated phensxvacids used for fighting brush vegetation by rcacsice ar.d'railroad tracks, along pcwerlines and cn forest plantation areas, had caused chronic ailments including cancer among the applicators. The Finnish National 2 oard of Health established a working group to investigate the allegations ar.d the Institute of Occupational Health was assigned to conduct a nationwide survey on the health effects of herbicides 2,4-D and 2,4,5-T. Herbicides 2,4-0 and 2,4,5-T have been extensively used in Finland since the latter part of 1350's. In 1971 consumption of both compounds was about 50 metric tons active ingredient but there has been a considerable decline in recent years. The regular preparation to fight brushwood contained 2,4-D and-2,4,5-T in a 2:1 ratio mostly as water soluble emulsified esters. Spraying took place mainly from a portable, manually operated or motor driven "knapsack" equipment, to a lesser extent fren a moving vehicle. It can be postulated that exposure to the chemical must have frequently boor, quite large because the sprayers did not take special precautions to avoid inhaling aerosols nor to protect / against skin contamination which often was heavy due tc leakages in the equipment. Annually a population cf 0002309 7289 DOW3726U ?h*re are few sources of information describing toxic manifestations in the context of occupational application of chlorinated pher.cxyacics. Fetisov (1365) relates that exposed personnel suffered from unusual fatigue, headache dizziness, gastrointestinal disturbances and irritation on the skin ar.d mucous membranes. In the end of 1950's is veil as early 1360's a series of publications report a* on fairly acutely developed and sometimes incapacitating peripheral polyneuropathies from systemic and local cutaneous exposure to 2, 4-D (Goldstein et al. 1959, Todc 1362, Foissac-Gecoun et al. 1362, Berkley and Sages 1363). There have been no further observations of the like since then which raises the possibility of the occurrence of a r.eurotoxic impurity. A large oral dose of chlorinated pher.oxyacics administered to mammals causes gastrointestinal disturbances, apathy, incoordination of limbs, myotonia and ataxia (Drill and Kirazka 1953) and suicidal intake of 2,4-0 has caused similar symptoms in man (Brandt 1971) . In his case of a surviving patient toxic changes occurred in the liver and kidney function; when the patient recovered a reversible alteration in colour vision and impairment of memory were also observed as well as a persistent neuropathy in one limb. A whole complex of pathological symptoms and' signs including chloracr.e, liver disorders, neurological and 'behavioral changes, hypercholesterolemia and porphyria cutanea tarda are known to have occurred among 2,4,5-T and trichlcrcphenol manufacturing personnel (XA3G 1977). These effects are new mainly attributed to a toxic byproduct and zentum.in.inc 2,3,7 ,S-tetrachlorouibi.-nzod ioxin (TlTBB) . In / the manufacturing process of 2,4,5-T seme TCBD will be invariably produced. In 1971 a maximum allowable 72 DOW372612 J r.c-.r.Ui - i n of 7'.ZD in 2,4,5-' vos :.:t et 0.5 ppm in " ir.ltnd. Prior to 1971 ne monitor t.-.g of TCDD hoc been onrri-jd out in commercial 2,4,5-T containing herbicide pr>;g-.raticn.s. 7o investigate TCDD contamination in rc-trcspect in 1973, a search for eld 2,4,5-T containing preparations vas cone and "CDD vas analysed. 3 commercial prt-pnraticr.s -ire found, representing large lots and several manufacturing firms from the years 1957-71. TCDD content vas found to be at most 0.47 tic/'tg and generally ituch less' (Table 1). Analysis was done gas chrcmatographically according to El.idee (1971) and although the sensitivity and specificity of the .tothod do not .test modern requirements, the results are thought to deviate only to exaggerate true concentrations-. SYM?7CMAT-O.CGY EXPERIENCED 3T 2,4-D AND 2,4,5-T APPLICATORS In a nationwide hunt for reports on adverse health effects frem brushwood killing herbicide exposure the public was informed to send their data to the Institute of Occupational Health. A further inquiry was then performed by the investigators with a through questionnaire. The question naire was also widely distributed by several employers to their personnel engaged in herbicide application. The investigators received nearly 2 0 0 reports of adverse effects. The leading symptom was headache which was reported as having occurred during the spraying operations by every second respondent; in half of them headache had persisted for longer periods of time post-exposure. Approximately one in ten respondents mentioned transient symptoms like dirtiness, fatigue, abdominal complaints and irritation cn mucous membranes and the skin. Skin aCcurtesy of ?. Paesivuo, M.Sc., The State Institute of Agr'.cultural Chemistry 0002311 7291 DOW372613 iff.-ction 3 al.->o of fairly common occur:ace (7 per u-.nt of responder,ts) and oncer closer scrutiny r-aforod to obvious toxic reactions. There, were no reports of ohloracna but a few notified cases of persistent popular skin disorder are presently being reinvestigated. There were no personal histories reminiscent of the syr-.ptcr.atic peripheral neuropathies described by Goldstein and ccwcrkers (1953) and others. A survey of subjective symptoms associated with brushwood spraying produced a fairly uniform symptom complex. There were no clear indicators of a TCSD effect which could be anticipated to manifest itself as chloracr.e. There may be several plausible explanations to the common occurrence of headache chemical and non-chemical, ar.d the factors may interact: physical strain, heat load, compression in the shoulder-neck area by straps, exhaust cases ar.d* unpleasant ccour to mention the most obvious ncn-herbicidai reasons. l"et, the investigators were left with an impression that in many, if not in most cases the most suspect ace.nt was the herbieical preparation itself, generally emulsified esters of 2,-4-0 and 2,4,5-?. Clinical investigations were directed primarily to the nervous system, accordingly. CLINICAL INVESTIGATIONS Two groups of herbicide applicators were clinically examined. Cr.e, encompassing 29 males and one female, constituted the most heavily exposed croup in a certain railroad maintenance district and the persons were selected cn the basis of the duration cf exposure ("local group"). The ether comprised 13 males of 50 years of age or younger who wore selected from the nearly 2 0 0 who reported subjective symptoms from .exposure ("symptom group"). "Symptom gr:;:p" persons wore individually chosen according to symptomatology 0002312 r\ D O W 3 7 2 6 I4 i I itt :ntion to 1 . severe acute C*-*t."i-SCUS symptoms and 2 . unexplained, r-iToijtc-at c :xr.d behavioral syrigterns. Due to the urcsr.cv of the examinations nr.d voluntary stoppage of brushwood striving in 1972, nearly a year and in r::,y eases a rxch longer cericd of tine had elapsed since the last exposure. Clinical investigations induced a routine physical examination supplement with a neurological examination by a specialist neurologist. TZG was recorded and electrcnourcmyography (Z:"'.G) was performed (Sespaidinen 19 75). Clinical chemistry included S.S.3., Kgb, Kcr, V.3C with a differential count, GOT, G?T, alkaline phosphatase, serum creatinine, urinary albumin and glucose, 2 hour sugar loading-test, serum ?3I and T3 binding capacity, serum cholesterol. Spireme try was performed and SCG !12 laces) recorded. Thorax and cervical spine were X-rayed. Several examinations in the battery were accpted to enhance differential diagnostics. As expected, some persons were found clinically ill (elevated blood pressure, CHD, cardiac ir.sufficie.ncy, emphysema) but these illnesses are hardly attributable to toxic agents. There were no notable skin disorders and the signs of pathology in the function of the liver, kidneys, blood forming organs and endocrir.es (super metabolism and thyroid function) were singular and mild. A special emphasis was placed on a thorough neurological examination, Z Z Z anc TXMG. There were several observations of subclinical pathology in both groups which cid not, however, constitute any uniform clinical picture nor conform to established clinical observations of a marked neurotoxic effect. F.F.C and Hk\".G findings were compared with those representing an industrial peculation cccupat tonally ncr.expcsed to neurotoxic compounds (Setp.illinen end Tnlonen 197-J). In the "local group" E:::;C y -abnormalities occurred with the same frequency as with 0002313 7293 DOW 372615 the :o15 , ir. t!-- "symptom group" slijh-.ly -ire ft-.yu-intiy. ^~G abnormalities, which for -.he .-is*: port classified cs "iicht", occurred ir. both groups approximately two times .tere frequently than -.m.or.g the controls. The difference vis not statistic?.!!/ significant (0.G5*p<0.10; binomial distribution). It is obvious that there car.r.ot be any conceivable control population for a copulation deliberately chosen for symptoms. Therefore, the findir.es of the "symptom group" must bo evaluated individually in the light of pcssible underlying factors arc between the individuals of the croup. EZC abnormality found in the "local group" bears more weight and was thought to warrant a medical follev-up which is being performed through inquiries to the persons ar.d local health authorities. Sot a single person in either group was found to exhibit unequivocal .signs cf neurotoxicity. Since the examinations took place long after the last exposure to herbicides, only late ar.d persistent sequelae can be thought of becoming detectable, ."edical histories did r.ot suggest, hewever, that clinically manifest neurological disability would have actually occurred in the early post-exposure period, either. :-:C?.TALITY STUDIES Coinciding with our studies an epidemiological investigation in Sweden on the mortality of the Swedish State Railways herbicide applicators vas being carried out. This personnel was exposed to mixtures of herbicides, for the most part combinations of chlorinated phenoxyacids with atrazlr.e and aminotriaaole with diuron or mor.urcn. The results of this investigation pointed to en increased risk cf respiratory cancer among the aminotriaaole exposed workers while those exposed to phenoxyacids and combinations did r.ot exhibit increased cancer mortality (Axelscn ar.d Surdeli 1S74). A recent re-analysis of data has indicated 0002314 4f r-'-W/.-r, th-tc t a *;rt incv.-Ui-.d r i s k -f o-r.o.rr 2: --o*: v :-* t-h.* - z `. r.o Lim t o - h _-r.o : : y a o i c s , a n d a f o l l o w - i p h a s 1;c o n -- n t i/ion Iscp. and Sun-fell 1977) . It vcs n e c e s s a r y 2l;o in F i n l a n d to i n c l u d e aa rpicemioiugic study into the investigation on the health effects of the herbicides. 9T9c?G/v\oa MY7I3IAL ;0:D "ETKGDS The cohort study carried out by our institute consists of two parts, a retrospective follcw-up study which covers the years 1955-1971, and a prospective follcw-up study covering the years 1972-1976. The data were collected in 1972 fre.-n the personnel records of four enslavers which are the twin brushwood Hilling herbicide users in the country, the Finnish State Railways, the State Highway Authority, the State Forestry Authority, and a state cvr.ed electric power company. The material includes workers who have sprayed brush vegetation for at least two weeks' time during the years 1955-1971, irrespective of their employment status at the time of the investigation. Included in the study are also those who have retired or chanced work later. The data were collected by the employers. They were obtained from old payrolls which unfortunately were not always complete enough, for instance, they did not always reveal the precise length of time in exposure. Therefore, in some cases we had to rely on the memory of the foremen and the clerks who keep the records. However, after checking the data it was reasonably certain that no -systematic bias would affect the results. The material consists of I960'men. The list of names and d.itcs of birth was collated with the population data register in the "aticnal Pensions Fund where every living Finnish citiaen is registered. The death certificates of persons found to be dead were obtained from the files of " the Central Statistical Office of Finl -.r.d. N'ine p-trsor.s could not be traced. The mortelitv of the herbicide 729 0002315 wrayi-rs s c-j.-y-r'.i w: ah verbal >.`.y ci the i-i-i1 Finnish male itinn in the ccI'm.-iton'inr time p-;ri:cs. 7ho material was divided into two cohc.rts accorcir.g to the years when the ixtesura had cecucred for the first ti-.e. The categories used were years 1955-1365 arc lit 6 - 1971. `.-.'e have no information of their exposure to herbicides after 1971. Separate analyses were race fer both cohorts and the whole material. Statistical calculations era based on person-years. DOW372617 ?.rs u i t s The number of deaths during the retrospective'follow-up of 1355-1971 was 45, and during the prospective follow-up of 1972-19*6 it was 49. The deaths and the corresponding person-years are presented in detail in tables 4 and 5. The observed numbers of deaths frem cancer in both cohorts follow quite closely the expected mortality during the retrospective study period, in the prospective study period they remain significantly lower. The mortality from all natural causes is in both cohorts and in both study periods only abcut'a half of that of the male population in Finland. Deaths from unnatural causes are at the same level as those of the general pcpulaticn. The mortality of the cohorts do not differ remarkably fren each other. In the prospective follcw-up the mortality differs between the workers of different employers (Table S). Amcnc the employees of the State Railways the frequency of deaths from cancer was about the same as the expected number, I whereas there occurred no deaths from cancer among the personnel of the Forestry Authority although six had been expected. This group had also a much lower mortality from all natural causes than the ether populations, only one fourth of the >;:-:r:ectod nunter. The number of violent deaths was about the same as expected among the employees of all four employers. In the retrospective follow-up the 72 9 fi 0002316 ii T-. j '.`.'..'iiin mplcy-rrs -vro less pro.-.ovnc>id -lith: h 2 sm.iilur i J.;.icy could to observed- The workers of the State "ail-ways wore on the average ever 10 years older than those of the Forestry Authority (Table 2}. DISCUSSION D o This mortality stocy did not reveal any increased .mortality from cancer or from natural causes on the -hole. In fact, c o the considerable undarmortality in comparison with the general male population seems somewhat puzzling. The spraying of brushwood by foot carrying a heavy gear as most of the subjects had done, particularly the employees ro az 00 of the Forestry Authority, must be considered physically heavy work. It is not uncommon that working populations, particularly when they are highly selected by work requirements, present during the occupationally active age considerably lower tcrtality than the general peculation (the so-called "healthy worker-effect" C-icMichael 1376) !. In an other investigation by the institute on the mortality of clerks and engine drivers at the State Railways, these croups presented figures closer to the death rates of herbicide sprayers than those of the general population (JSrvinen 1976). Of all natural causes of death, cancer mortality could be expected to be little affected by the healthy worker-effect. This appears to be consistent with the findings from the observation tenca 1 9 5 5 -1 9 7 1 . The lack of cancer dcjths amcno L.Ui Authority spraying personncj-- d t-i. g^l 972-1976 is unexplained. This group represents the youngest and largest population (Table 3) in the study. Scciintly it was reported as a clinical observation in Sweden that in a material of 87 patients with Resor.chyr.al tumours treated by a regional clinic for malignant disrz.c-os / over the years 1S7C-75, 7 cases were thought to be expos s-a 7297 0002317 DOW372619 ;t to rr.l 'r.oti i phon;::yac i'.5 m o time previo .isly i'-rir-i1 i'J/7). -Zr.ti localisation me 1ignant neoplasms cbtermed in cur study do not reveal a single mesenchymal tumour and present 1 distribution rather typical of cancers in those aye croups in Finland. The findings of the epidemiological mortality study .oust be critically examined in the light of >:r.c-n factors in chemical occupational carcinogenesis. Although the exposure to chlorinated pher.oxyacids during spraying has probably been frequently heavy, total length of exposure has in most cases been a few .eeVis only. Furthermore, the follow-up period since the last exposure is at most 20 years and in most cases much shorter. In the conditions concerned, only strong carcinogenic effects of exposing compounds could be anticipated of beceninc manifest. Washer effects `could present themselves after a much longer period of latency. v:e cannot, therefore, exclude the possibility of anything lias than strong carcinogenic effects and a further follow-up of the population is warranted. 7298 0002318 -on, 0 :-.-i t.r. ':.>)1, L.: nuxityror :-:h canc-.-r. laV.artidr.ir.z--.a 7; r.ili) 3127-1313. 0 .5licr., O . a.-.d Cundell, L..- 'v.-rbicica exposure, mcrtality and tumor ; d i:.eid:.-.cn. An epidemiological invostig-tion on Swedish railruad ^ workers. ..'orV.-iir/.iron.-health 11 (197-4) 21-23. Brandt, Herbatox poisoning. A brief review and report of a nuw case. Cce-ixr. laeg. 123 (1971) 503-303. Drill, V.A. nr.d Kir.ttka, T.: Toxicity 01 2, 4-dichlorcghoncxy,-icctic ^ ^ DOW372620 acid and 2,4 ,S-trichlorophonceeyacottc acid: a report on their acute end chronic toxicity in docs. Arch, incustr. hyg. cccup. -c-d. 7 (1953) SI-67. Elvidye, D.A.: Analyst 96 (1971) 721-727. Fetisov, M.I.: Problems of occupational hygiene in work with herbicides of 2,4-D group (in Russian! . Gi=. sanit. 2 (1955) 23-31 . Foissac-Gegoun, ?., lolievre, A., Basin, 3. and- K.'.rot, ?.: Folyr.evrite cores usage d'un cesherba.-t: l'acide 2,4-D. Lille medical 7 (1962)- 1049-1051. Goldstein, it.?., Jcr.es, ?.H. and Srcwn, J.R.: Peripheral neuropathy after exposure to an c-st-ar of 2,4-D. J. Arr.er. ncd. ass. 171 (1959) 13C6-1309 . Hardell, L.: Malignant mesenchymal tumours and exposure to pher.cxy ^ acids - a clinical observation. Lskartidnir.cen 74 (1977) <=_ o 2753-2754. -ARC: IA?.C Monographs on the evaluation of the carcinogenic risk of chemicals to man, Vol. 15, 1977. JSrviner., E.: Vetur imiostutkimus osa 4. Xuolevuus ja tyCkyvytte.-.yys. Tyoterveyslaitos 1976. (In Finnish) McMichaei, A.J.: Standardized mortality ratios and the "healthy vorkas effect*. Scratching beneath the surface. J. occup. mod. 13 (1976) 163. Soppaiaincn, A.M. and Tolcr.cn, M.: Neurotoxicity of long-term exposure to carbon disulfide in the viscose rayon industry: a .-.curo- physio'.cgicsl study. Kork-er.viron.-health 11 (1974) 145-153. S.jprilainu-n, A.M.: Applications of neurophysiological, methods in occupational mc-cicine. A review. Soar.d. j. work environ, a h.Mlth 1 (1375) 1-14 . 7'-':d, RyL. : A case of 2,4-D intoxication. Iowa st. med. see. 32 (1963) C6j "ibi 7299 0002319 DOW372621 Tabla 1. TOTT contamination in seme 2,4,5-T containing herbicide preparations applied in Finland in the years 1357-1371. rp.paraticn Y^ar of ^plication Active ingredients Vesvkcnfaho SO Tributen TT 5r*jsh.pcn Spray 201 /3 VesakonhSvite F 500 1367 1971 1363 1960 1956 1357 iior.r.esiyr Seguan 1965 1362 2 , 4-D + 2,4,5-T B " _ WM _ N-- -- 2,4,5-T ** B TCTD contamination nr/kg (calculated fr 2,4,3-T) 0.31 0.20 0.10 0.31 0.14 0.23 0.21 0.47 y 0002320 DOW372622. o .rs by i-r.plc-yer in the toyir.r.ir.g Zr.player ."c-dian, years State P.ailv.-sys State Highway Authority State Forestry Authority Power ccnsar.y 47.3 33.0 33.S 38..3 Interquartile rar.ro, years 33.0 - 56.6 29.0 - 47.0 24.1 - 44.8 24.6 - 49.2 Table 3. Person-years by employer curing the follow-upperiod of 1372-1976. S.T.plcyer State Railways State Highway Authority State Forestry Authority Pcvor cc.r.pany Person-years 2050 2410 . 4561 433 0002321 Table 4 Mortality of 2,4-D a n d 2,4,5-T exposed workers during the time period 1955-'.0'/1. Expected numbers of deaths arc based on the death rates of the total Finnish .i.ulc population. Cause of death (iiiju, y e a r s ) Exposure sta rte d in the years 1955-1965 Observed number o f d e a t h s (O) Expected number o f d e a t h s (K) Pcrtsonycciiru 1966 -1971 (O) (K) Persionyea vu Maliiir.ant neoplasms J5-24 25-34 35-; 1 15-54 55-64 65-74 75 and over - 0.1 1030 1 0.3 i/an 3 1 . 1 204(1 1 3.2 1560 4 6.7 1060 1 1.5 112 "- Total 10 12.9 7590 guaBasGB8>Baaias&stauiizsae8iiB3sBna All natural causes of death 0.1 - 0.1 1 0.4 - *1.2 2 1.0 - 0.0 1110 750 000 653 292 1 3 3. u 3674 assissi 15-24 25-34 35-44 45-54 55-64 65-74 7 5 and over - 1 7 5 '7 6 " 0.4 1.7 6.2 14.4 25.0 6.3 - - 0.3 - 0.5 3 2.5 4 6.1 3 7.2 * 0.0 " Total 26 54.0 3UULUL s c1u0a r . a1i6j s. 6 To t a 1 (O) CKI i*criiuny d ii' j (M 0 . 2 1 0.4 4 1.5 1 A u n a .5 i 1.5 -" 214.1 1;'j i it 2213 1352 113 ~ 13 6 . 4 11272 BSSSSafcBSBSiSilSBBS - 0 .;; l 2 . 1 1 6 . 0 3 2 0 . 5 10 3 3 . 0 u 6.4 -- 36 7 1 . 4 u a s c s&a; s u u i4 a -.-ti.ti 0002322 ^3 C-O. 9 o 9 Z I m O G