Document G5280g91mKJJnEpJKpgBQmrkY

M r\n ..< *Lc. S c i t .>tyf6My c'tITS-Si ) v / " -</ _f - / /' April 22, 1953 Drigadicr General Fred J. Felder (t3 . ) u . o . A m y Munitions Ccr.mar.d Chemical-Biological-Radiological Agency vlTIco ol* the Commanding General U. U. Department of Defence Army Chemical Center, Maryland cn Cj vU CJ1 >o, o Dear General Dclcor: At the roqueat of Dr, l/arrcn C. Sliaw, Agricultural Rea oarah Rervico, United Ctate3 Department of Agriculture, v.'e are supplying herewith three coplea of the information available to ua concerning the toxicity of 2,4-D (2,4-dichlorcphcnc,-:y acetic acid) and 2,4,5-T (2,4,5-trichlorophcnoxy acetic acid). This Information was prepared by Dow for a petition to the Food and Drug Administration requesting a tolerance for 2 ,*>-D in aeparguo, (Incidentally, the tolerance of 5 ppm aa requested was established by tho FDA). Included In tills Information aro references 0 and 9 which give data on tho toxicity of 2,4,p-T. In addition to tills information, we would like to also mention tint vjo havo boon manufacturing 2,4-D and 2,4,5-T for over ton years. To the boot of our knowledge, none of the uorisnen in these factories have shown any ill effects no a result of working with thcco chemicals. Further, many millions of pounds of those chenicalo have been used h e m and abroad without causing a single proven caso of illness to livootoclc. There havo been very fow complaints and alleged illnosseo in humans duo to 2,4-D, however, vjo havo novor been convinced that those instances were in fact caused by tho chonlcai. In view of tho low toxicity obsorvod in othor marxialian species, our toxicologists boliovo those caooa havo been related to 2,4-D by coincidence. 17831- T^Sf-es -2- iCnowlns that thcao producta aro used by all corta of people undor varied circunatancca, wo ccnaidor tho few number ei1 Gllccod injurlea to bo phenomenal when related to tlio larco quantitlca or product manufactured and sold '.ve aro lad to havo this opportunity to servo you, and trust tho Information wo havo provided will bo of cssictaneo Sincerely, O O G. E. Lynn Director of Hoclotratlon Bloproducta Department Jo g c e : Dr* Warren Q Shaw, A.n.C., U,SD,A* be: W. W. Sunderland V. K. Howe (No Enclosure) 17932 Biochemical Research Laboratory The Dow Chemical Company 1332 V ? ^ / - OFFICE COP'/ 199 BUILDING WIPE SAMPLES FOR CHLORACNE CONTAMINATION, 1966 TO D A T E / y / ) . y - J ^ r File: TI7 .4-12-3 Date: 5/5/66 6/ f / t eBy:^ y L. G. Silverstein Date ______7 -- b(p___________ Date ___ T _ T ________ PROBLEM The chloracne hazard in 199 Building requires continued monitoring of the cleanliness of the reactor room and surrounding areas by means of wipe tests. PART I CJ1 <in3 <1 ininnO ,<E CD CO o CONCLUSIONS. 1. The positive samples in all three sets of wipes taken in 1966 indicate the definite need for continuing the protective measures which are in force in the building. Proper use of gloves, clothing and shoe covers is still necessary. 2. Wipe testing indicates that the need for continued, frequent, thorough cleanup still exists. There is an improvement from the January to the February and also to the March samples but the latest series indicates that cleanup is still needed and the "ups" and "downs" in cleanliness testify to the need for continued good housekeeping. 3* Because the floors seem to be the most likely spot for significant contamination, the removal of shoe coverings at RESTRICTED: for use within The Dow Chemical Company only. 7U3 DOW 1547794 Biochemical Research Laboratory T17-4-12-3 Page 2 the door between the reactor room and the still room must be observed strictly. Contamination was found on the stairway to the lunch and locker room, although none was found in the lunch and locker room Itself and contamination was also found in the foreman's office in the first 1966 wipe samples. This indicates laxity in the observance of shoe cover protection. 4. Wipe samples covering the areas indicated in Table 1 should be continued at least on a monthly basis during trichlorophenate production at 199 Building. 5 . Wipe tests for the period 4/19/65 to 7/23/65 are presented in Part II of this report to complete the documentation of wipe testing since the chloracne problem began. DISCUSSION The method of taking wipe samples and interpreting them has been described in the previous report on 199 Building contamination, File T174-12-3, dated 5/6/65. A positive response on the animal indicates contamination which should be removed. A negative response indicates a clean area. The severity of response may be taken as an index of the dirtiness but any positive response should be considered significant. Past experience has shown that working areas can be maintained free of detectable contamination by wipe test. Table I gives a detailed description of the locations in which wipe tests were taken and the animal response on wipes taken on 1/17/66, 2/14/66 and 3/29/66. q34 Biochemical Research Laboratory T17-4-12-3 page 3 DOW I 547795 These three series of tests indicate that cleanup procedures do improve the situation generally, although in a couple of instances there was an increase in response in the second series over the first which means that continued frequent cleaning is still in order in thi3 building. The findings of activity in the foreman's office, on the stairway to the lunch and locker room indicates that there has been some laxity in the past in confining the contamination to the reactor room. This probably resulted from not using shoe covers or not removing them as soon as the men left the reactor room. The existence of activity in the reactor room on controls and even on the operator's desk and chair is sufficient evidence that the protective measures now in force must be continued. DOW 1 547796 Biochemical Research Laboratory T17-4-12-3 Page 4 Table 1 Location 199 BUILDING WIPE TESTS 1966 ----- Animal Responses 1/17/66 2/14/66 3/29/66 Turbidity meter Floor E. panel board Floor W. panel board Floor inside Door A Floor inside Door 20 Floor inside Door 2 Floor, caustic-methanol pumps Floor inside Door D Floor between still and reactor room Floor and door, S. side of still enclosure Floor and door, N. side of still enclosure' Floor and benches, locker room Floor and benches, lunchroom Floor In laboratory Floor, foreman's office Floor by sink inside Door D Operator's desk and chair Operator's tool boxes Valves and floor, oil draw-off station Valve handles, S. side of still enclosure Valve handles, W. side of still enclosure Stairway to bottles Platform above Phenate tank Severe Severe Moderate Moderate Slight- moderate None Severe None Slight- moderate Moderate None Moderate Moderate Slight Moderate None Moderate Slight Slight- moderate Slight Severe Moderate None None None Slight- moderate Moderate Slight None Slight None Slight None Moderate Sllghtmoderate None Moderate. Moderate Moderatesevere None Moderate None None None None None Slight None Slight Slight None None None None Slight Slight continued on next page 179^6- Biochemical Research Laboratory T17.4-12-3 Page 5 Table 1 - continued Location Plant mechanic's bench Pipefitter's bench by still enclosure Stairway to lunch and locker rooms Laboratory hood Sovereen's shoes ------ Animal Responses ------ 1/17/66 2/14/66 3/29/66 Slight- None moderate Moderate (Floor) Slight Moderatesevere None Slight None _* ^ -li J.t / l-->*1 Biochemical Research Laboratory T17.4-12-3 Page 6 PART II WIPE TESTS FOR CHLORACNE CONTAMINATION AT 199 BUILDING APRIL 19, 1965 - JULY 23, 1965 This Part documents the wipe testing done in a period not covered by previous reports on this subject. CONCLUSIONS 1. The still enclosure in 199 Building was demonstrated to be substantially contaminated by wipe tests on 4/ 19/65. 2. The operating areas were free of contamination on 6/ 1/65 indicating that good housekeeping can be attained. 3 . A positive wipe test in the oil draw-off area indicates that this area is susceptible to contamination. 4. A rental sparkler filter unit was judged to be free of acne hazard on the basis of VPC analysis only. CCOD DISCUSSION The wipe samples discussed in this Part were taken during a period of time not covered by other Biochemical Research Laboratory reports on the subject of chloracne wipe testing on 199 Building. The samples reported herein are primarily of historical value because of the changing nature of the situation at 199 Building. This completes the written record of wipe testing in the trichlorophenol area from 1964 to the present time. >0.11n v t l * U M U JI Biochemical Research Laboratory T17.4-12-3 Page 7 DOW 1547799 Table 2 describes samples taken in and around the still enclosure. All of these were positive indicating that contamina tion was present at that time. It has been assumed that work within the enclosure involved the risk of contamination and appropriate precautions are taken at any time a person must enter the enclosure. These samples only substantiate our assumption. Table 3 describes 20 wipe samples taken on 6/I/65 which pretty well cover the operating area and possible exposure points. All wipe tests In this table showed no response on the animal, Indicating that the areas were clean on that date, and also indicating that clean-up procedures can successfully keep the operating area free of contamination. Table 4 describes wipe tests in the oil draw-off station and oil dumpster. One floor sample showed substantial activity on the animal showing that contamination can get to that spot. The dumpster was free of contamination on 7/ 1/65. Table 5 describes both rabbit, tests and VPC analysis on a series of wipes taken on a rented sparkler filter which was to be returned to the vendor. Wipe samples on 7/1/65 after the first cleaning of the filter demonstrated that cleaning had been inadequate In terms of removing acne contamination from the interior of the filter. VPC analysis showed agreement with' the animal test. Wipes taken on 7/14/65 after another cleaning showed that the inside was still severely ^contaminated. Wipes taken 7/19/65 showed a substantial decrease In the amount of contamina tion by VPC analysis only, and those on 7/23/65 showed no detectable dioxin by VPC analysis. The filter unit was returned to the vendor on the basis of the last VPC analysis. 17939 Biochemical Research Laboratory T 1 7 4-12-3 Page 8 Table 2 WIPE SAMPLES TAKEN AT 199 BUILDING APRIL 19, 1965 1 Floor inside north door of still enclosure 2 Inside surface, north door of still enclosure 3 Wall panel - first one east of north door 4 West side of lower decanter 5 West wall, north half 6 Floor sample, filter area RABBIT RESPONSE Slight Slight-Moderate Slight Moderate Slight Moderate oofilfrS l MOO 17840 Biochemical Research Laboratory T17.4-12-3 Page 9 DOW 1 547801 Table 3 WIPE TESTS - 199 BUILDING JUNE 1, 190^, 1 . Malecki's shoe 2. Floor by W. panel board 3- Floor by E. panel board 4. Floor by N. door still enclosure (outside) 5- Floor by S. door still enclosure (outside) 6. Floor between still and reactor rooms 7- Door (N.) to still enclosure 8. S. door to still enclosure 9- Floor area inside Door D 10. Floor area inside Door 2 11. Floor area inside Door 3 12. Floor area inside Door 20 13- Floor area - locker room 14. Floor area - lunch room 15- Floor area and first three steps to lunch and locker room 16. Valve handles and chain at oil draw-off station - Door 2 17. Laboratory floor - 199 Building 18. Laboratory hood (inside) - 199Building 19- Door and door handles to laboratory - 199 Bldg. 20. Door and bottom of turbidity meter enclosure Rabbit Response None None None None None None None None None None None None None None None None None None None None Biochemical Research Laboratory T17*4-12-3 Page 10 Table 4 WIPE TESTS AT 199 BUILDING JULY 1, 1965 RABBIT RESPONSE 1. Floor area - oil draw-off station - north of gutter Moderate-severe 2. Floor area - oil draw-off station - south of gutter None 3 . Oil dumpster - top and sides None cn u 00 0 rc 17842- Ms: 3 CO *-3 CO VK O 23 *s td 2 a -3 HM H 03 oS >^ ta0Ma->)*3 en Sample Description -- 7/1/ 65 _.flabblt--- VPC* 7/14/65 7/19/ 65 Rabbit VPC VPC 7/23/ 65 VPC Outside surfaces None <0.3 i Inside surface of door Filter plates None Moderate < 0.3 18 <0.3 <0.3 Inside walls (exit end) Severe 75 Severe 58 8 <0-3 Inside walls (door end) Severe 16 Severe 42 5 <0.3 Auger and puger trough Moderate- 30 Severe ModerateSevere 6 9 <0.3 Movable parts - plate rotator Moderate- <0 .3** Moderate- 56 4 <0.3 Severe Severe *Micrograms 2,3,7,8-tetrachlorodibenzodioxin per sample. **7 Ifreported at 1.05 retention time. Probably accounts for severity of response which is not normally produced by this low level of dioxin. M3 OOtHd- -Ip-mtr urI-->o*otH3u- K- i pa <uv DI tfDu CD o CD oF tTu) O eosi^s W fu fl><Ort Biochemical Research Laboratory T17-4-12-3 Page 12 UNIT INDEX Wipe samples for chloracne contamination in 199 Building revealed the existence of contamination in many areas which indicates that continued Intensive clean-up and the continuation of the strict personal hygiene and housekeeping efforts is still required to avoid further chloracne problems. DISTRIBUTION G. G. Goergen, Aniline Section, 172 Building A. F. Lueck, Aniline Section, 199 Building D. C. Dunkelbarger, Aniline Section, 199 Building R. E. Walker, Aniline Section, 199 Building H. L. Gordon, M.D., Medical Department, 607 Building A. W. Wilson, Safety Department, 401 Building LGS:sJl -- iJre'"~inT."'i^'nirti:u'i um s'cuv j r r t'"~on`ly . Pago Industrial Hygiene Chemical Exposure Data Entry Loy Field Survey TWA Exposure And Excursion Exposure Data Report no. / 7/ -/ ~i,0 ~Z> / Wr?S/ /\/)\ c: .Date U J [pO Building no. f<N I n d e x no. Units of Data Ll L Respirator Worn? M a s t e r no. Job Classification o Job Ko. n Chemical Kamo mn DR// (oP)0Cf 2 b DR// TWA/Exc. o TWA/Exc. / -cjr -- jon Cr~J>1 i IW i I 3Lf 09LZ l~MOfl TELEPH O NE > -P l4 E C n M M O N W ^ g ^ R G ,Xll I4u0 G EO FFR EY T. M ANN, M. D ., LL. C H IE F M ED ICA L EXAMINE! GEORGE W . THOMA. M. D. M 'T . C H IE F M EDICAL EXAMINER H A RO LD L. BEDOOC. M. D. A S S 'T . C H I E E M E D IC A L E X A M IN E R C H A R L ES O. BARCLAY, M. O A S*T . C H IE F M EDICAL EXAMINER N O R FO LK D IV ISIO N 4* DEPARTMENT OF HEALTH O F F IC E O F THE C H IEF MEDICAL EXAMINER 4 0 4 - 4 0 0 n o r t h I 2 tm S t r e it Ri c h m o n d Ih SIDNEY KAYE. M. S c . TO X IC O LO SIST J . DOUGLAS REID . SC. D. M ICRORIOLOO ICT M ARION V. W A LLER. M. S c . R o L o airr THOM AS D. JO R D A N , B. A ., LL. G A DM INISTRATIV! A SSISTA NT October 22, 1956 & The Dow Chemical Company Midland Division Midland, Michigan Dear Sirs: He have had several cases of sudden death in children with no apparent cause indicated after autopsy. Each of these children had a possible exposure to Esteron 245. Would you kindly advise us of the relative toxicity of Esteron 245; signs and symptoms developed following exposure, pathology seen at autopsy and also analytical procedure for identity and assay in tissue. Any assistance you may give us in this natter would be appreciated. Very truly yours, SK/st S idney Kaye, P h .D . AUg June Apr lY.ar M a rV Nov May May Sent July June Jan July July July 1 t?o 0 1950 1950 1950 1949 1948 1948 1948 1947 1947 1947 1947 X 5T w 194c 1945 Auc Mar Dec 1951 1949 1948 -eb Feb 7*."--q rjr Dec Oct June Dec Nov June June May May Oct Apr kar Oct Sep w Aug 1952 1952 1952 1952 1951 1950 1849 1949 1949 1949 1949 1949 1948 1948 1948 1946 1946 1946 Willard Hagle Arne a M a n c h e s t e r Willard Hagle Dudley Herman Jack Maraen Dexter Ames Ivan Gillis Willard Mosier .Michael Cox Richard Baldwin Alfred Emeott Leonard Huaock W m . A . Sni th Ea. O'Toole Edward Smith 14oii WEED Y^IJS^^,7 ^ t ^ * ^ ^ c ^ C ) y 1526-137 1204-123 1526-137 1204-100 1527-143 321-202 1204-149 1204-170 315-345 324-141 1204-209 1204-224 1204-198 1204-201 1204-111 Irritated eyes Burn left eye Irritated eyes Burn left eye Burn left eye Irritation booh eyes Burn left eye Bilateral Conjunctivitis Irritation left eye Irritation of eyes Left eye irrit.ted Left eye burn Eyes irrioaoed Left eye irritated Left eye burn cs c=> -- * \t CO C-. He in 1z e l m a n Joe Ortiz Joe Ortiz 2-4-5 T 1204-201 1204-217 1204-217 Irritation left eye Slight irritation Irritation left eye c: 2-4 D Louis Eocisky H. L i v i n g s t o n Vincent Mako Harold Mahon E. Dra v e s Leonard Parsons W m .R . M e A n n a l l e n W m .R . Me A n n a l l e n Lawrence Dice Floyd Schreiber Lawrence Dice Lawrence Dice Irvin Bristol Pat Walters James Toles Phillip Sanders Andrew Wilson Clinton Skym 1526-111 1526-113 315-241 1526-125 167-117 1526-138 167-103 167-103 1527-111 1204-151 1527-111 1527-111 1204-198 1204-220 1204-182 1204-192 56-145 1204-210 Irritation of eyes Burn of eye Irritation eyes Burn rt eye (with HC1 Fh Burn left eye and nose Burns both eyes Irritation both eyes Irritation both eyes Irritated left eye Irritated left eye Irritated left eye Irritated right eye Both eyes irritated Eyes irritated,facehneck Irritated rt eye Burn left eye Burn eyes Rt eye irritated 17847 THE DOW CHEMICAL COMPANY M ID L A N D M IC H IG A N V-/ March 3, I953 U U It Dr. E. W. KInnaird, Administrator Bourbon County Hospital Paris, Kentucky Dear Dr. Kinnaird: . This is in reply to your letter of February 17th asking for information regarding the identification and treatment of inJuries which might be caused to persons handling 2,4-D and 2,4,5-T. 2,4-D and 2,^,5*T in their various forms are used as growth control regulators and herbicides. They are among the least hazardous of the agricultural chemicals used in the field today. The LDc q of these materials varies between 500 and 1000 mg./kg. or body weight for several species of animals and hence, the likelihood of systemic intoxication through oral ingestion is quite small. If they should be swallowed in substantial amounts we would expect them to cause irri tation of the gastric mucosa, headache, ataxia, and possibly, an Increased tonus of the extremities. These symptoms would be expected more or less on the basis of animal studies, but insofar as I know - no experiences involving human subjects have occurred. As with any other chemical the first thing to do following ingestion is to induce vomiting. This can be accomplished by any of the well-known means. Symtcmatic treatments should then follow. The compositions of these materials as encountered in agri cultural practice do not present appreciable hazards to either the eyes or skin and they lire not absorbed through the skin in hazardous amounts. In one or two instances over the past 7 or 8 years, we have heard of a person who has demonstrated a typical hypersensitivity to skin contact, but when one con siders the multitude of persons who have had contact with them, this could probably be expected. Patch tests conducted by accepted procedures have not indicated that these materials are likely to induce sensitizetion of the 6kln. If some mild irritation should develop through accidental prolonged contact, symtoraatic treatment would again be the rule. During the past seven or eight years we have manufactured and sold millions of pounds of these materials without en countering any difficulty. More millions of pounds have been ? c c c C909AZ | MW1 Dr. B. W. Kinnaird 2- March 3, I953 used in the field and the lack of any serious difficulty what soever is, we believe, very excellent evidence that the materlals themselves present no hazards of real concern. If I can be of any further assistance to you in this matter, please let me know. Sincerely yours. V. K. Rowe Biochemical Research Department 2-280 Building VKR/bb cc: H. H. Gay, M.D.^ 17949 MOfl ry TAILS AOORIia DOWCHIMCO BRANCH SA LK S O FFIC E S JEW YORK CI TY SAN FRANCISCO PHILADELPHIA LOS ANQKLCS WASHINGTON SAINT LOUIS CLEVELANO HOUSTON CHICAGO ATLANTA ,, SEATTLE DETROIT BOSTON The Do w Chem ical Com pany MIDLAND MICHIGAN April 17, 1951 n e u . u t o ir ilr. F. 7. Reagel Engineer of Materials Missouri State Highway Department Jefferson City, Missouri Dear Mr. Reagel* Eiis is in reply to your letter of April 2, 1951 to our Hr. L. L. Coulter requesting our opinion with regard to the possi bility of skin irritation occurring from contact -with 2,V-D, 2,L,5-T, and TCA (I presume you mean Sodium TCA) Vie hare studied these materials rather thoroughly in our laboratory on animals. We have had an appreciable amount of work dona at an outside consulting laboratory on both 2,U-- D and 2,L,5-t using volunteer human subjects. The results of these tests nil show that it is very :uilikely that 2,U-- D and 2.L.5-T would cause appreciable skin irritation in the normal individual. It is possible, of course, that a rare Individual say occasionally exhibit a sensitivity to these as well as to any other chemical. Such occurrences arise everyday with many common household chemicals - soaps, for example Ton have mentioned infection in this case. Certainly, none of these materials would cause a bacterial or fungus infec tion by themselves. However, irritation of any sort car certainly open the door to infectious organisms. Sodium TCA is perhaps sore likely to c suse skin irritation than either 2,U-D or 2.L,5-T for 2 reasons5 first, it is used in much higher concentrations! and second, it is inherently mere ir ritating. Appreciable contact with Sodium TCA usually results in a burning sensation which encourages the operator who has carelessly or accidentally gotten the material on him to wash. However, irritation can certainly occur if the material is carelessly handled. This material would not cau.se infection by itself. We do not believe that if m y of these herbicides are hurdled in a reasonable manner tr/ t they arc likely to cause difficulties. This, we believe, has been borne cut by their very *0 r. F. 7. Heagel brille. widespread use with hut an exceedingly low incidence of complaints. If we can he of further service to you, please do not hesitate to call on us* Sincerely yours, THE DOW CHEMICAL COKPABT V* C* Rowe Biochemical Research Department CCt Tech. Serr. 4 Dev* Attni L. L. Coulter, C. 5. Branch Legal Department Attni G. J* Williams Hedical Department Attni H. H* G a y ^ Agri. Sales Attni B. L* Smith 17 14o0 April 20, 1961 P.O.Box 469 Kelly S. Segars, M.D. Iuka Clinic 114 S. Pearl Street Iuka, Mississippi Dear Doctor Segars: I have your letter of April 18th, a copy of which I have enclosed so that I can point out to you ay difficulties in understanding. You speak of "trichlorophenoxyl acetic add** and I know of no such acid. I assume that you probably mean 112,4,5-trichlorophenoxy acetic a d d " which Is widely used as a brush killer. We have had a great deal of experience with this compound which we have manufactured in huge quantities and we have done a considerable amount of experimental work on animal s and on humans. This a d d of itself is mildly Irritating and In strong concentrations can produce Irrita tion of the skin, but it takes a considerable length of time of direct, Intimate contact even to do this. As a matter of fact, I was not aware that the a d d form of this herbicide was on the market, since It Is coomonly marketed as either the salt or the amine form. If this Is indeed the material to which you have reference, then I can only say I am very puzzled as to how the man could have possibly received a burn, unless the contact was with a solution of this mater ial at high temperature. I would be Interested In any further comments you may have, especially to know the circumstances under which the alleged burn was produced. Very sincerely, 00 co ^ Harold H. Gay, M.D., Medical Director Medical Department HHO:ccf enc. J.STUART -McQUISTON, M.O.. F.A.C.P. R. D. HUNTING. Jr..M.D. R. M. QUETSCH, M.r. W. M. KOONTZ, sr.':. 1328 2nd Av..S.E. Cedar Rapid, Iowa 52403 June 20 , 1966 Tow Chemical Company Midland, Michigan Gent lemen: A patient we currently have in Mercy Hospital with symptoms of anorexia, weight loss, joint stiffness and wasting of muscles in the upper extremities attributes these symptoms to a presumed exposure to 2,4,5-T in the form of "Esteron". In the spring of 165 he used heavy applications of Esteron to the bark of g^JP'trees that he wanted to kill and in the spring of 1966 he cut these trees down and was cutting them up' for fire wood when he developed acute onset of the above symptoms. Approximately three months later, he still com plains of some of these symptoms for which no other cause is apparent. The weight loss and some muscle atrophy and weak ness in the upper extremities is apparent, without anything else very remarkable on physical examination or laboratory testing. Is there any remote possibility that 2,4,5-T could haveleft a residual over this long a period of time and that it might have something to do with the symptomatology? Richard M. Quetsch, M. D RMQ :ajl C C : Iowa Poison Information Center Ees Moines, Iowa i I o N S Coow 1445 c 0 M M U N 1 C A T I O N \ 6 ? LD NY S )R. GAY / @ > 607 BLDG G 0 M M U N 1 C A T I O N S OOW^ IN TRAVELING WITH SOME Ofy.'DUR CUSTOM SPRAYERS I HV BEEN T O LD 'T H A I SOME OF T IC PERSONNEL WHO HV BEEN HANDLING THE SPRAYING OF 2UD AND 2U5T FOR THE LAST TWO OR THREE YEARS HV STARTED TO ERUPT IN BLISTERS ABOUT THE S IZ E OF A HALF DOLLAR ON EITHER THEIR ARMS, LEGS OR FA C E S . THESE BLISTERS EVENTUALLY GET LEATHERY ON TOP AND THEN D ISAPPEAR. WLD U PLS ADV WHETHER THERE IS ANY RECOMMENDED TREATMENT FOR T H IS . DICK VAN HO'JTEN "1! V n 3 S l l JCM K, = - - . : v'mv.'n tO X> to C Vo c c x K L I I C. A T i C N S C 0 M M U N 1 C A T I 0NS c 0 M M U N C1. A I July 17, 1957 \f&LL\Zi MOQ U. K. Bcher, M.D. Director, Poison Information Center Childrens Hospital Society of Los Angeles ^6l4 Sunaet Boulevard Los Angeles 27, California Bear Dr. Bucher: Your letter of June +th addressed to The Dow Chemical Company regarding the information on 2,A-P herbicides and their effect upon humans has been referred "to me for reply. As you undoubtedly know, they are quite a number of herbicides based upon 2,4-D (2,4-dichiorophenoxyacetic acid) and its derivatives. We have investigated a number of them quite care fully on animals because of the expected exposure of livestock to them. A reprint of our publication on this is enclosed. It will give you the basic information on ingredients and formulations on the market, whether ours or someone elses. Unfortunately, in one respect and fortunately in another, I am unable to give you information regarding the effect of human ingestion since I do not know of any such cases. It is conceivable, however, that such could occur and I realize the reason for your inquiry. The information'gained by animal studies causes us to comment as follows regarding 2,4-D and related type herbicides: 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,^,5trichlorophenoxyacetic acid), MCP (4-chloro-o-toloxy- acetlc aoid or 2-roethyl-^-chlorophenoxyacetic acid), and Silvex (2,4,5-trichiorophenoxypropionic acid), whether in the form of the acia.. salts, or esters, are all low in acute oral toxicity, but ir ^wallowed in cuantity, they can cause more or less serious e j e c t s .''"*'Pgsgd on animal studies all of these materials in s u m tent doses are capable of causing gastrointestinal irritation, injury to the lungs, liver, and kidney, and greater or lesser degrees of myotonia, particularly of the extremi ties. It should be emphasized that many formulations of the basic ingredients listed above contain oils, such as kerosene or fuel oil, which may well cause more serious effects than the so-called active ingredients. This, cf course, depends upon concentration. DOW 1 27794C W. K. Bucher, M.D. 2 July IT, 1957 Certainly the first thing that should be done in case an herbicide of the 2,4-D type is swallowed, is to induce vomiting as quickly as possible and call a physician. The physician should consider gastric lavage to remove any unabsorbed material. Further treatment will have to be symptomatic since there are no specific antidotes for these materials known. * If the preparation swallowed contains oil (such as an emulsifiabie preparation or one designed for dilution with kerosene or fuel oil before application), considera tion must be given to the effect of the oil--. The danger from the diluent oils, primarily, is respiratory embarrass ment caused by pulmonary edema and pneumonia. In such cases the administration of oxygen most certainly will be beneficial. Chemotherapy would have no effect on the so-called "chemical pneumonia",but it may have some bene ficial effect in preventing or controlling possible sub sequent bacterial invasion. Oil laxatives should be avoided. Liver damage may be minimized by promptly plac ing the patient on a diet low in fat and high in protein. The usefulness of lipotropic drugs has not been investi gated. Kidney involvement is rarely sufficient to merit special treatment and for those rare cases where the kid neys are seriously involved, the treatment should be the same as for toxic nephritis of other etiology. I trust that this information will serve your purposes. Sincerely yours, V. K. Howe Biochemical Research Department 12-63^ Building VKR/ba : K. H. Gay, M.D.u' 17956 Jun- IS'37 Mr. R. L. Keith General Claims and Land Agent Rutland Railway Corporation Rutland, Vermont 1448 DOW 1 27794 Dear Mr. Keith: It has been requested by our Agricultural Chemicals Sales Department that I write you regarding the toxicology of various herbicides which are alleged to have caused a hemolytic anemia in a man spraying one cf your right-of-ways. As indicated before, we have no knowledge or suspicion that any herbicides which we manufacture would have any hem atological effects upon persona. In order to reassure you of this, I am enclosing reprints of technical articles having to do with this subject. One of the articles is entitled, "Sunaaary of Toxicological Information on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of the Hazards to Livestock Associated with Their Use." This article published in the American Journal of Veterinary Research gives results of a rather exhaustive study. We observed no hematological changes in the studies we made, I am also Enclosing a reprint entitled, "Toxicity of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trlchiorophencxyacetic Acid, A Report of Their Acute and Chronic Toxicity in Dogs," b y Drs. Victor A. Drill and -Tomiharu Kiratzka. You will note that in their studies cn dogs with these two base materials that they also observed no hematological changes, even at dosage levels which produced appreciable mortality. We conclud from these results that 2,4-D and 2,4,5-T are net hematclogically active. Insofar as Radapon is concerned, I am supplying you a cat;a sheet which we have prepared as an interim report cf the toxicological woric we have dene on this material. A de tailed report has been filed with the Food and Drug Adminis tration and as a result they have seen fit to set tolerances for its use on several agricultural commodities where it might occur in human food. An expensive report cf this work is being prepared for publication. In the brief summary, however, no point is made of the hematological studies although such were cade and no effects were observed. I trust that the summary information will be adequate for your purposes. It certainly shows that Radapon (dalapor., sodium salt) is exceed ingly low in toxicity, and hence, presents little or no hazards under normal conditions cf operation. Certainly no hematological effects would be expected from exposure tc this material. 17S57 DOW 1 2 7 7 9 4 3 Mr. R. L. Keith -2 June 24, 1957 I have discussed this problem with H. H. Gey, M.D., our Medical Director, and he concurs with my opinions in this respect, I would suggest that IT you have expert medical people who would like to discuss our clinical experience with these materials that you have than contact Dr. Gay at the Medical Department here in Midland. His phone number is TEmple 2-2311, Extension 243. If we can be of any further assistance to you in tills matter, let us Know. We would be glad to hear from you at any time. Sincerely yours. V. 5. Rowe Biochemical Research Department 12-634 Building VKR/vmv cc: A. A. Lawrence D. L. Conner H. H. Gay, M.D.^ E. T. Coon V. K. Rowe (2) .x~*O1*7 . DOW 1 277848 D O W CH EM ICA L OF CA N A D A . LIM IT E D SARNIA. ONTARIO March 17, 1966 Mr. L. S. Valters, K.D. Assistant Professor of Medicine Queen's tJnlrsrsltgr Kingston, Ontario tear Dr. Valtergt Tour inquirr of tbs toxicological properties of 2,^,5-trichlorphen ol has teen forwarded to Dr. H. L. Gordon of our Medical Departasnt, Midland, Michigan for his acknowledgeeent. Z an certain this arrangenant will be to pour satisfaction. njh cc: Mr. E. L. Gordon, M.9. The Dow Ctesloal Conpany Building 607 Midland, Michigan Bremner, N.W. Baker, G. _ M. K. Miller Technical Service 1 Developaent 17959 CD *,H rj- tH PRCCNANCY UU1C0ME 0NCEPTUS DEATHS MISCARRIAGE (RATIO) STILLBIRTH (RATIO) TOTAL (RATIO) IVE BIRTHS HEALTH DEFECT (RATE) COIIG. HALF. (RATE) IHD. HALF. (RATE) INF. DEATH (RATE) rtlTAl. UNFAVORABLE TOTAL FAVORABLE TOfAL M. CUHCLPt I0IL7 | M M aI. UHFAVURABLE (PATE) \I HUAI. MODHIGH 4f. u u * -c,/ j t a CRUD*E.F.R.E.QU.ENC.I.E.S AN.D.RATE_S OR R..AT.I.O.N.P.E.R..0*00? \ * X k J pr^^i----- C^j (S*a -cc^/Llij *Xo urf.t'-- f yylM V rC <r#-^> EXPOSED TCDD ALONE ANY LEVEL OTHER DIOXINS ANY DIOXIN C V vnT(tOu TOTAL 4E0KD y-' tYR ^ 1YR TOTAL 1yr : y> iyr TOTAL 0 <8 (00.0) (100.1) 03 (00.0) (18. t> 0 21 (00.0) (126.5) 25 13 0 (123.0) (116.8) (00.0) 170 (I9.8) (19.0) (00.0) 29 50 0 (113.6) (135.9) (00.0) 36 (112.5) 0 (23.0) 11 (137.5) 19 05 ' (151.6) (133.1) 7 15 (22.1) 123.5) 56 too (176.7) (157.0) 213 (119.3) 33 <10.5) 216 (137.0) 298 (123.0) 18 119.8) 316 (112.9) 2 18 (0.8) (100.1) 0 11 (00.0) (01.3) 01 (00.0) (06.0) 01 (00.0) (21.1) 0 16 1 150 11 166 11 (69.3) 7 (31.7) 1 (05.0) 2 (09.9) 0 191 202 32 2 (87.0) (100.0) 21 1 (57.1) (50.0) 20 (05.1) (00.0) 61 (16.3) (50.0) 21 2 311 18 360 20 26 (01 .3), 19 (59.1)' 1 (03.1 ) 7 (21.9) 21 296 320 26 (82.0) 11 (31.7) 2 (06.3) 2 (06.3) 12 305 317 52 (01.6) 155 (06.8) 30 (17.1) 87 (10.7) 3 , I0 (01.7) (03.6) 9 (11.1) 39 (21.8) 36 120 601 1665 637 1785 207 (05.5) 117 (10.3) 13 (05.4) 18 (19.8) 156 2266 2122 0 (0.0) 11 37 37 (177.9) ' (160.2) 107 231 71 (177.0) 110 2 (100.0) 20 60 (I86.8) 361 60 136 (102.3) (101.3) 373 737 366 (180.2) 2031 502 (ini.1) 2760 |UUSA/i d . *> (3. A 1*-! 5 M 0~tsi-c^ 5' . Cj0 - Ja^ V? iin l o 03 03 3> r-A Most Dangerous ' Pemeton (Systnx) (QJ*) P I Syston (OP) Mqvinpbos (Phosdfiq) (QP) Parathion (QP) ) J # 5cbn^an(OMPiU(QP) TBPP (OP) 7 fhorate(Thimet)(OP) ?lpjp!9f. C9P} TAIlf 1 UTUUTIO ULATIVI ACUT* TOXICITY HAZAtO Of rUTIClOH TO IFftAYMEN* Dsugerom LM Dangerous Aldrin (OCC) Rldrtn (OP) Carbophenothiop (Tritfsion) (OP) DPVP(OP) Pelnav(OP) Ploldrin (OCC) PNOC(N) PNOSBP (N) gndrin (OCC) mUm Bthlon (OP) f Methyl Dembton (Meta-Systox) (QP) Methvl Pmathlor. (OP) f - V Z . Nicotine (M) BHC (OCC) Binapacry) (Morocide) (N) Chlordane (OCC) Z S H S f f f Co-Ral (OP) Diasinon (OP) 4 1 * # * Dlcapthon (OP) Pichloroethyl Ether (M) Plmethoate (OP) Pipterex (Dyiox) (OP) Endosulfan (Thlodan) (OCC) Fentfaion (fiaytex) (OP) Cuthion (OP) Heptachlor (OCC) Lead Arsenate (M) Le st Dangerous ArmroUe (M) Captan (M) Carbaryl (Sevln) (C) Chlorobenzllate (OCC) 2.4-D (OCC) Diquat (M) P D D (TDE) (OCC) . PD T (OCC) Pilan (OCC, N) 2,4,5-T (OCC) IPC (M) Larathane (N) Kelthane (OCC) Malathlon (OP) Pentachlorophenol (M) Phosphcmidou (OP) Sodium arsenile (M) Zectnn (C) Lindane (OCC) Naled (Dibrom) (OP) Ruelane (OP! jBMBhena tf>cc) *f^A85 V-C13 (OP) Vapam(M) pHtftk9m Maneb (M) Methoxychlor (OCC) Mlrex (OCC) Morestan (M) NAA(M) Perthane (OCC) Phostex (OP) Piperony! Butoxide (M) Ronnel (Korlan) (OP) ' t o l - M S ' Rotenone (M) Simazino (M) Sulphenone (M) Tetradlfon (Tedion) (OCC) Thlram (M) Zlneb (M) Ziram (M)______________ 1 . *Fjom H . R. Wolfe and W- F. Durham, Prociedtngt o f th* Second Eattem Washington p tr f (liitr and fatticid* Cot\f*rence, Washington State Pplvprxlty, pp. l^ -i|| (1888). f r y : PCC r prgmpchlorine compoupdj QP - prganic phosphafej C - P|it>eqtatflj N " nitrp compound; M - fnjicpl- qj CO 1 MOO* MN073280 DOW 1551800 a -Q tH I \ 1S i 3 H i .* X 'S ^/ ~f V j3 !A`-i rj :5 M *> r--f:T 71 r-\ O}.A:s3 '.i r\: ..:: a: " n : . -- J r*! 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X:.*l>3GC`-TiOC4JrT.-i. f-f<L.O <-*1-}\o.-i --34-4 '.3/J r. rG-lO:] r.>44 L^ O u o 3 3 p. \ n o ao C HC 4 wl c . t> Cl 'o \o e o C O 'S & nro r1rClCJI OS OO HX3\J. O -rl!-1 -nad o u g j3ro G 3^ ,1-Jp?G CO oGi oQui fcdmo0 Qo K OJ o X) --3 3 M O >&o Moni; O .?u > Q H ^4 o o co CD <30 r-  DOW 1 551762 l -i 4W .W ,'*i ***i*4UtJ^K> '**V.A .t*TA<.'/. I V ; L. . * ' > il J-V i--J ;-'V Y -''7 (rjLiLVJUvZ.ilL ^ L i P A K l Y MIDLAND MICHIOAN **j ,>Tn.'%% V !'*V * v d , . . U/-*- . J . J j i. ./ rrod bari evotz : ; f ; ii- iv;L Cr.ei:.l.:a.l G.v.:ipa.;y w .b * j i j v*i*v' bear .'rt*a ; I ara u r i tino: -/ou a t ti.o roqueso o f o u r d.cloa Doparcela, n.. re La t vo lo cae o c a p L a in l ..if X i\ a,'id Hru. G r i f f i tr. w.-.c &1 i a yod in.ul a nz'Jtn w;1 tres y uro f a r ;.,**-La.-: nlu.:-wn ar.fi r c.vor H vj bal:, uns .ontaralr.nlo<i b-, -..ino o f y o u r tru-o.'cs t . td.-e ox- uar. Loar, i l i:i l i e d a i l f t-U clr fia;- aru oausou dama-yo. L; *0 i r a o t e a i l y i... i:vs t i tora-... ..re o a l La.. w. l. i .;c t a z i a i t y Lo fi.ro. u f '.ii- boboa r a r e whior. -/rra Lovoivoc. i.r. L.Lv. cario, au i andarsi aro rano i y Sutcu-afr l a ~10 and dn t o r o n b'iv. In i-A-. J . d. b a r r i., .r and d, W. rio^c-e rab L ob od a r tic le ir th Awariaor. J.surrel :*V vantary, VeVario 1.1(5, pri./uo -h J n til.i- .id , V-1-D ..-xleit. y l\**;cirtfn GorbiVi : poo l e s o f i 'iu;. 1 b-allove Ir; U : l r ira: tur 00 to.oy vouai ib c uoci Vira a r .lt and f '-und trttt c o n o o a tra L i-in o o f >00 ppcr; .,-r c u r a n e r o Uan.per .us t c a naraber v f v .o a L o n . XI ?v> l..y j .e a i 1 , a t V.o d i u r i i b l r vi* em l a i f i a l i lo p r a p a r a v i w cuoi, a ?1 y.... uoro .usi:..;. uva ld bo n e r o loxL; r,Ir.jo a l l Li. yv i f lo very lo .x ia i f i s o a n p o a l a i i y w o n lapor.coU in ..u nacer. Iva, \; I- .-li I \*fr' . wJ. . MsJ . .,. 'ujI ..O v -x -iT-Vj -jf -*^ `1> ; *- 'j1 -**j); y ci 0jc<il'*-1m-Ji - i j > bffoc- .>i* Viro nf tiro l.vw ^eaeriLee prob 1on derbi rifili dieran and 1; . a L i . a L 1 > JIG IVa c w 3 i 1 h i r r;./ r e o la b i e c - e r c a . 1 L > V :.tL C-*i.*k>0 0 L i! i ; ,1w \ . x .* 0 -0<a a t - d y n f L . e n t . l \ - 1 . , f r ia 1w a ; '1 %* ^ ao ,i -**-- * a;'. tJ : ^ V 1 , ! < / .. |> v ca u n i 1 0 1. c a> ; >L i. -;'i V. '.j e o r . e v c*c* - ; bb-.;V.. tt ::..aav. ..! a-., u . f ' . . M e a ' / i r. ve. .eIat i ra iv:'d .ree; ca-i n (' 1 ;: o.'-n*.->; v.- e w a te r r. ; 1 ..ai : . '.A.ia U ' j w r r . i.aX ..*i li.:. Z. o -SJ VN ro 00 o !.. w:,ric wnivj i; ..ac b e e r e r** -n`\. '^ .'", ' ...a'.-,e >, '+- ..'Stri' roj.;b a;. ;red ;rroerr. !'at 1..nr -rf al' v a b o v e o.ri-a.;. l--nOo:;:: >' n ..i ^ tvai ic U:..;, ar. li-. ac-.e b'iep :-.,a i';..- .ave i .: .cui e ' 0 t ;:o ; ! .: . , b , ,-A l.'i'.'Lt.`i. a - l..'v a r '>1 ac (l ri-:;; co (; 119S4 e 9 * T s s I MOO 1- hv. I-Tod iiari.yv^tj -t- .dinar;. dwrujtor.u) wora to/.ia 1:; our: jet trail or.a creator than I j>pa an chat the uhi4ic.lXiera uoed In proparatt j ;o nunn tin ysi are er.tpluyir.,..; are tv;x!c in cunuont ra tiei.o 'eater than 10 ppn. it would noon:, bnorofurej tr.ni the appreciable .u.,inre5oaf. inn -f a ar.ni.1 utr':-ciu cr.ula well m n ;it in nerl.: u I " 1*7 to nqunti.n 'Lit'. buibjir..; frorw l;.e o -.p*- etf the* Latter frou tno Griff it..- it w-.-j Id nnp'/ai* -.nt t:.oro oust nave boo:; quite a aubotanl lai n^junc of t..u -.erbijlclal pro pa rat!or. dumped Into toe otrocw or there would uvt ..ave been cao foaoinv and the vhi ter. In;; of two water. It would bo uy opinion, .Prod, that ountaainavi./) L*> auuh a do t o o an V.r": G.'oeribo nould w.:i ' ..are fcuer. re ap-. n s 1bio for the offv.r.-s dneor i b a d . X re :;rn tfc&t L ;unr:\ -..y: ir V:.: wrr* i r ,r K .. a nrit* `av rble rap t >1 b ..b Li?:'*-.' avs1 n t V K, H\ 2*1 * l ;'*</.* V" ii/'ba 0. ::(!<y w. /. ^<'K':V:l, V. K. Rowe (2) T 2 .30-3-30 - T2.30-4-30 Corroaponderioe 17965 14oG UN<> 7 3 2 8 o DOW 1 551662 s rw i\ I L , / , ) ... _t i -- - -- * Y:\\ : u ik P rln i ti ... . gaprinceVWHamSUeet. ^!3 rccurliiia iK? . 634-8840 Pf'^lKordeiafls We can"c^sskW0orsalesrep-fr ilo C e t i etti:ar CZ3 llCOfciin ta ) O L'Ocra? tC3 \ ctorry. o tcAocnatra t# t e e errerei .. . v.._euel e r r c e i e , I ucoll credei L -` - ... . . e e ; : - l e r c e c i tLo w to clery Ciccarci C eto* ;' c !. . ...... -. \. _.;.3a L'j # Ccl--e 22 errici a tja jer le e ? i. --' '.. -\ ; .` __... ; i:.)Zo e l i t e cerer ceca erry ceaiLczi V` -1.. '.'. ly.i ^ . .., ::.i..j U_,,:.... ;.:. ;, e ce e - tiri cirriy Ceco Leci ce te eel crei ae?eCroC7eproetieeme li0. -.rc-e, e ;'_j '. . . / l e i r e 3. c e ero Crii cicca rtt rrcC* 1^222.3 *Cj L j ;v;:".;:V:' X rry to e e,::.'rei Co c r t e i e E'zzrzzjt LVj i l ...ero cCrlcj ero ta eleo becvcaflcmco7AU,cycurlitirdts era <e 2#(r-J e~ io C o ecitla ero e s ili ila t e castra'. L\v Cado co evo eri treo r.'rio to firi eoa caca cf all tfco cvrecol Q3*i9 - 23h0lj>JJ x j z o prcloot n t e ce irajray t e a t e eroe teca cerere lve1. CriIlova era, w bva teied -- fcr vitti te era et teeo croccialo c ruller eri culler cf coircrj cf faro Irci, r e lcr.i3 croccicij ca utility er seolaiCa rl^ria-cf-'rry, lt la o pertica t e e seiaiccd-.o frea t e racy taffi.' I e v i pescemily era ca taircy-eea cceylaicto icraltrtlca-cattla ex i i v m f c m l Certa tiro to tki usa cf a #^ D ex Q,k,52. 17SS6 MN073280 I DOW 1 551663 V* 'V f M .ty 1^, 1`1' 2i. 14-1 l-2(i 1 4l?O 'SJ C ^l LAP.4 iC A L c : O LA P A A V MIOLANU. HlCHIQAh 440 Mr. S. \ . ( .< ;:/t-r!.iin O. \1. ..'ini! and ii;< 12 i:.i o t Is .Str*-*t M..r j.-.v ;Jt:. 44,,-ii' CJ1 cn H* cn s* o Dear Mr. Chambe rI>m : I have i.cL-M t' |rl*viiIyu 'with information concerning the health ha.:ari.s, if anv. iur the handling >>f our products 2,4-1) acid, Formula b! heron idc ami Kuron herbi ide, Ihope tlie followirg will he .ivictj1ati- y>ur mrpo.ic.s Ku r<.n' Iferhieide 00 O The em losed n.:ihi,nentitled, "Sum marv of Tn.xicoloi;:cnl Informa tion and l-'r.'.ilii ai l.nnMder itious in the Handling and u.se ot Knron , A brush Kilh-r 1ormwl ithuT', and '`Karon Toxicological Information ami Suggestions u ry.iniiiu Treatment tor Persons Swallowing Kuron , shnuh: provide '.on with the desired information. 2,i-l> Avid loth til.* single iiise and the chronic oral toxicity, on the 2,4-D acid >s reported m live two additional reprints entitled, "Summary ot Toxiidentic.1 Intor::ation or. 2,4-i) and 2,4,5-T Type Herbicide and an lavaIllation ot tin- fl..cards to Livestock Associated with Their Use.-." by \ . K. lu-v.r, and T. A. Ilyinas, and "Toxicity ot 2, 4-'Hchlorophen xyaceiw ;d and 2.4. 5-Tr irhlorophenoxya etic Acid", by Victor A. Prill, and i-miharu 1iiratv.ka. Kye and skill irritation studies on rabbits iudi at'that the undiluted acid may cans: marked irritation von cornea! injury while strong solutions mav cause serious eye damai.e. \ single short skin contact should cause at most only minor irritation. However. pr donyed or frequently repeated skin contact mav lead :> moderate irritation even a superficial burn. The acid aium r.*::'Iv is not likely tc'be absorbed through the skin in acutely loxu iimoii:'.: Hust- ihi: aedd are likely to be irritating to the no.~>e '.! h.real}..-f. on% ;ii. , !'};- 1'w i !.i:;\ii.i! i o11:paiy POW 1 551641 * ) ,' * .* ,X A iX`. < i.mde i1i *'y ; !``f ['O K .\1ULA 40 he_rh'.cidc The reprint entitled, "Summary of Toxicblogica! Informa>iun on 2,4-D ami 2,4,:'-T Type 1ierhieidos and an Lvnluat ion of the Hazard*, to Livestock Associated with Their Use", also gives the single dost.* oral toxicity on this piuxiuct. ''kin and eye studies on rabbits indi cate that the undiluted product is capable of causing serious eye damage. The eye injury should be greatly reduced if the product is diluted with water. The product may cause mild skin irritation upon prolonged or frequently repeated skin contact. F O k M U L A 4b is r.ut likely to be absorbed throng!; the-skin in acutely toxic amounts. Suggested precautions both ,4-11 acid and F O R M U L A -Id herbicide A study of the above information and long human experience both in the manufacture and use indicate that neither 2,4 -L) acid nor F O R M U L A 40 present any unusual health hazards from handling. The precautions, therefore, are those normally recommended for materials with signif icant eye ami sl-ir. hazard. Where exposure is likely, suitable eye protection, such as eye glasses with si.de shields >r their equivalent should he worn to protect .the eyes and suitable protective cl.thing , such as clear, overalls with long sleeves should he worn *.o protect the skin. * Should exposure occur; the eves should be flushed immediately with plenty <d water lor at Rm s ! J^ minutes and medical attention should L.* obtained prfinptly. Fur skin exposure, ihe <.i:>!uaminuteci clothing should be r e mmed and the contaminated slur, washed with soap end plenty ot water. The clothing should be1washed before re-use. Any skill irritation or injury should rec:ivi medical attention. In addition to the above, y-uf.inav find the eitclosed reprint entitled, "Health hururus Associated with Handling and Use ol Herbicides", by \ . K. Uowv, id general interest. rh i.u k you tor your inter-.-'! in mir products. It 'a e can b< of further assistance, phrase icvl ir-e to 'ok. Yours sincerely.. V." ., .//;! ' f 1 ' * Marl A. WoI ; R eS'arl; T< i.oi ui ; loin n e m o ni r ;;< !. I!ib . :. ;; .i:,;' F l u 11 > u - ( -i y-7969 .ut MIDLAND June 15, I960 SZ0 Hn M. 0. Norris, Jr, . Oklahoma City, Oklahoma. ce: L. L. Coulter ' F. Fletcher ' ; Dear Mark: Your ROI Doc, No. 43531, May 12, I960, regarding preliminary results or toxicology tests with herbicide formulations had ; several interesting features In It,. You mentioned that Ni Sstev ^ 2^5 jxt 100 m g /kg fed for a 60-day period to sheep -- -- produced no harmful effects. However, Kurqn was fatal .after V A* <*a i *Y dises at 100 m g / k g . ;; I am wondering if you have any'' ^5'; reasons why iiurvn should bq so :toxic. Do you think this ; .should be rechecked? Of course, I realize that only one test animal was used for dach of these experiments. However, we have several new. salt: formulations of. silvex-- pne in particular, M-1.845; which- v:e would also like to have checked by Dr. R. D. Radeleff at Kerrville, Texas, if you think this w.-rk can be done. We would.he interested in having.it done, if you can make arrangements for it, and we will be glad to supply additional samples of chemicals needed. Please let us know. cn cn O' 00 Sincerely, c Paul M. R.tty Agricultura;. Chemicals Development vm 17970 \ / 1470 MN07328Q John H. Hickon, ..on. tiiskon and Herron Attorneys at. Lav; Power Block liolena, tontana iHvif: V\r{ cn or Ul ax OJ Bear'Mr. illEk-fij This la In reply to your letter of October 17th Inciuirlr^ ao to what atuciloc wo have nacio on varloutt 2#4-D and 2,4,5-T type herbicides on human subjects;. First* I wish to apologize for the tardiness In ^ottlnii an answer.back tp you. Your letter arrived about the time 1 loft for. tontuna on a month's hunting ''.trip and I.huv* haddifficulty gettingcaught up ainccmyroturn. . I was In Helena twice duringth#trip having mopp 'd to leave some trophies with Bob Miller ami to visit with a friend, liarry Joelin. ii3d I known of this I;would have been glad to diflcuso it with you there. ` ;o In direct answer to your question 'we have not eonuuawd com parable studies on human -subjectsv^kp^tayd,' however, conducted tests on human skin with a number of such products and ti*.buve had a lot of human experience in tnanufacturin^, bunding, sale and use of these t^tcrlalo ovur many years. -They uri acion,/, the cafeot agricultural chemicals known ^The hazards associated with their uso as rocomaondod are nbtiayprdoiuble, This, of ; course, does not mean, that a parson could not, if he vjor-j a mind to, swallow enough of .fchd material to produce, serious in jury or even death* To have Inhaled- onpugh of the. material to have caused death aeeuio lmpooalbid,.>in^>:,:.: You note that the olulm was made that death was a result of usin.;; spray for a very short time of a product culled 'Lew Volatile Brush Killer to. 2 *. I do not have any specific information on Low Volatile Brush Killer Mo. .2, but 1 am told it Id a product typical of the commonly available (inters of 2,A-D and 2,A,'j-T. If this is the case, then the above comment apply} if not, then of course, they do not. I would certainly appreciate it if you could give mu more noto11s of the operations and the alleged ultimate oaui'ij of death a a U U a might shed some further light on the bade. A notailed descrip tion of the claim, any available medical reports or the like would bo helpful, 17971 '* vT * J . 11 Jliclren, 3 ^ . 2 - . ltober li, 1]>6.I If WS can b-j of any further help, please let me know* 'Incerai? Voura, :;' ;.; V. K ."Rtte . Biochemical iiaaearoh laboratory.' .; 1701 Buildins *} '.V; ''* *.'/ .*>. * ' .->; * .\ . . , ' .V -:;. j r.-.v . ^ ynn/jd ;:. '. V;: . : 7 .> ..col D* Koehlineer :- - - - - V. K. Rowe (2) . .. T2.30-0-30 .V:^7^-J2.30-13-30' ^ Correspondence DOW 1 551554 i i I i 1 i 1 O fllV 1 551491 ,..'.'J IQ g l y 'J l j 1477 u u`j u. t_DOL/ C. E'l LI ivi 1it*z-i L C. Q !>] L-*-i J Y MIDLAND MICHIGAN Hr. G. B Missouri !1:jl-- -y Bepart;...ont 2 Kansas C jcuri Nj r~a. -->U>*.-',,J--1 Va ro 00 o Hr. U u : ; D . Graia of onr St. Loula Office hac informod r.i-o that one or your et:ployooc ha a capar lene ed olcln irritai; ion cf tho feet after Locoming i;ot wLrllo uolng a 2,4-D - 2,4,5-T formulation Hr. Crain al co aoked co to paco on to you tho Information we have regarding the clcn irritatine properties of such materiale. Bosulta of rather extensive laboratory work tocother with experi ence in tho manufacture and use of these type materials ha a led uo to cLo following conclusions: (1) 2,d-D and 2,4,5-T have essentially the 3acie effect upon tho akin. (2) Concentrates ouch ao the commercial formulations are somewhat irritating to the skin If exposures are pr o longed or frequently repeated. Occasional contacts of short duration are not likely to result in any irritation. (3) material diluted with water for spraying io not a p preciably irritating to any except perhaps the hyperoenaitive individual. (*i) Material diluted viith diesel oil or kerosene will be distinctly more irritating than viator api-aya. Diesel ell or kerosene alone will cauoo serious irritation If confined to the skin beneath clothing or shoeD. (9) Patch tests on 200 human subjects uith several com mercial formulations, including Dow Esteron Brush Killer, have indicated that these materials az*e not likely to induce hypersensitivity nor to cause irri tation under ordinary conditions of use. Tho difficulty you have encountered is of particular interest to r.o since it 13 most unusual for skin irritation to result from the use of th^se materials. I would certainly appreciate very much receiving all the details you can give me as to the caterlala used, r.ianner of application, to what was the worlaan exposed, duration of exposure, when with respect to hi3 exposure did he first notice or 7Q < .iii'fVU . 13. i fool Z: 1 i' OS ci, a C- J to a,.:i___ L j - - J i o , 19323 i1...i ci' :d injury, Inforcai;ion with a J :o:j coriouo cao tl:o injury. DOW I 551492 AG.'iln, 1 i.: rooioto coy infcreation ycu can Givo no mid if I con o cc -- J *-- 11y at aoy tico, plaooa do not Loufcafco to call en co. oincoroly yearn. V. IC. nono Eicctiocicai goo corah Eopartcont 2-220 Euildioj VlLli/JC Enel. ce: II. D. Crain, at. Louie Office II. L. anifch A. L. Diotaoi a i. . ``` W ' i1* . ''"ft ! Dova C H L iU IiiL CADLCi DOW PANAM i JTC lW iC aU U IC B LIM ITED 1714 EASTMAN HOAD M ID L A N D . MICMIOAN U.O.A. May 15> 1533 I ^ j. .t/. u 1478 -, Mr. Murray P. Humes General Purchasing Agent International Packers Limited 135 S. LaSalle Street :' Chicago 3i Illinois Cl ci 00 CO Dear Mr. Humea: " Hr. Dali haa turned over to me your letter of May 5 regarding your request for more Information on our Eateron Uruoh Killor. You have seen my letter to your Brasilian representative and I can add very little to it a 3 to any possible irritating canee or effect on the o p e r a t o r ^ stomach from using cither our Eoteron Brush Killer or our Esteron 245. Those matarlalo have been very widely used in both this country and abroad and the letter from your Mr, Irwin io the first caca we have ever had reported to our attention where it was thought there was a nauseating effect caused by tha use of Eoteron Brush Killor. In view of this, I feel certain there is some other factor which brought about the described 8ffoet other than the use of our brush killer. N1 VJ JO 03 o It has boon the general experience of commercial opera tors in this country that the use of Estoron Brush Killer is more economical than the use of Estoron 245 alone. This is particularly trua when controlling brush in an area in which you have cany different kinds of opecioo growing. However it is also true that some species of ~ 17975 brush are more susceptible to Estoron 245 and are almost completely unaffected by Estoron 1010 which is the 2,4-D ester in our brush killer mixture. It is also true that there are some specloa of brush that are quits ausoeptlblo to Estoron 1010. In general however Eateron 245 is more, effective on a wider number,cf .species in controlling brush than Eateron 1010.r Due ;to the economics in^tho '* ^ cost of these two m a t e r i a l s H o w 3oteron.Brush;Killer. waitliPI developed and -his ?ip^oved: vwyj^sa tisfao ~ J**"3 mfm . .y.v'.*'j,f-**;a'. iiv,rvi' mm DOW J 551490 `* fy I itA'iA}k*X j 11' t ^1/i /-I (UUUUUU UUU^/^ibUt^j^M'frtAUUkillAU'iUUAUrf.. `.'/1.5 /33 no for t::o particular oltuaticn tnt prcvailo In Brasil, 1 can not year cyicaticn. In fcho firot place, I do not t;.:-:; ilia coooico with which you ci'o troubled end % ciid-Lapu then 2 would have la no onporionoo In trying to eradicate) then with any of ou* natarilo. I could cua'Dcotjtint if you oro interested in detcrainlns thio Gnaotly, you have your local nan carry out ocno c::pcri nento u a i m all throo of our catorialo-- -Eatoron 1010, Eotoren *)i> end Eotoren Bruch Killer-- -to determine which io moot catlafcotory for the cpocioa with which ho io bothered under bio conditions). Very truly yours, W. B. Ilullison Aoaiotant Technical Director gb cc D. B. McCackoy Hontovidco Office 1. R. Ball lark A. Uolf- id(3v j c m e m i c a l c o u p a m v 3 C I5 O LIVO 3TFE E T ST. L O U IS O M IS S O U R I Jay 1, 1953 1479 MN07 328 DOW 1 551488 Dr. V. K. Rowe BIOCHEMICAL LABORATORY A. L. Dietzel MISSOURI STATS HIGHWAY DEPARTMENT Kansas City, Missouri Dear Dr. Rowe: Mr. G. B. Major, Maintenance Engineer of the subject organization, Inquired the other day concernin'? the effects of 2,4-d or 2,4,5-T in formulations on the skin. One of their employes using our material satu rated his clothing to the point where the solution got into his shoes and caused burning effects. This employe has been away from.work for several weeks. We would appreciate very much your writing Mr. Major giving data as to whether-the materials mentioned is apt to cause dermatitis which I believe is the injury claimed by their employe. Information as to treatment, et cetera, will also probably be appreciated. We would like to receive copy of your letter. Kindest personal regards. Yours very truly, IIDC :HG Hubert D. Crain Agricultural Chemical Div 17877 J,. % > *> i I ?. <s. tM d ce v*t**r*ii: 1r. :*if ,.i ~ **\ V*> _ _ :'? ,.:1w::::d r.ocziicmJ IAN07 32 80 / 551234 r-.vl:.\- Crli'i'.i.-h i.:,-;. a iLJ aaci <ttor vi rdrjod t.`-v-a1*i*r*Oc1a1r.n"* o . v.a U - i j. . J^ J t - ; * , o i.-. . . . . - to a h l:. . i L :. ar,- vlvatior; vi.5.ih tuo ear; levo *.v:. I.r; loir .. i a ~ l h tir.a : i : `.v 'i'r n l a i t . .V-: r-h rota irhotl a:. ri. !.<.:i<; .--t %ividH*i *-'V>.JVS*V.'*...-!>*I J..i. ...SrVUf%..^Z-'J*-*1. -- *'* i` 1 *1*iI .-.I-.;y- u n d tru i:a.i(; :vd i:v-v; rtO a;rj ;r eora Uuro dar;.-jr-r.a * :li' i n v cir;/ Lar.JLu t o t Lao u :;:,'j j l a i. Ly t ... . * U *i S.* - * * . V - r l ^ r . a : 3 %i . .i ,S .. . i, , <i . i * W V * i ` t . . a J, U . U `._yJ l . : -- * - I J ' 1 . . U i VvJ l . u - i* V v - .tl ; J l V. V.Clit _ t * ! * ! . i. * i .J JL ti flirta rl i ; ........ */'% _ , 4 "*I.* ** ,c -/4.-i )* i. f * ; . - a t i r a t a v 1. vifU l h V* V ..;*1* Jr . y r a u s U '- o c i '*- 1 w V- 1 u .) 1 \ 4'h!, ?**>' rv<, i ^ V l> V> ^ V *, + > . < * * V* Vi-s f" ' ,* la >t< k > v.; . * /* i'o r --a 1.*n i i t t i i t a - d th i . \ SS `j x. . i * * y vili e r t i V oi*VXi..).*v.ii **.a H .; -b ` v.i nrirS 1`i t ' *i ' ' T** r-:. j c ia r >'*n J o r h . ** *1 1. w < t / . ,> a. J.1i i. \j W * j 1*:2 tie d . to 4V-*t ,, t r t !v'^ 1 ^ il: .Wvtrl: ;ihi;j j.ar. L'oa; ac.-abuatod ir; ...ur (uai i;l..;Jr:n; i-atv..ra- .f . * r r y <- w L*j' i -- . V r ! *1 s ^ *i U X V* \j> t. i .Li v_`i ^ -..'..f. , r* i:.:va : : a a r . i f `a i . l U Lr. d a t a r : a J -iO .: t; ;::J.a il ;' Yjj/js. and liat,t. 2 ei,:i-r oat Si* if i i - a l i i . i i oroat /* V r*m r* ' -- b ..i 1 ij , b* ft % * o d - t j ( , r ;r a a DOW I 551235 J) -J. n in "%* -. '1 4 W--- W . . * . 4 i " . i..* ,:... ;i ^ L.: - ;.:n or ti i* * * J w <i J w! .- `j o.:;ild u^li * J v . W t . , ..J^ J ^ **' *f-i*\ *j .n .j on i*j - N ,f .-i ii-. h 7 -. r \ ^ ~i\ -J *7 -; l.J11: Gjr ro ::nor;^ j o I.1 ' 1 \f' . I t * ' >* A > *w ^ *.* ".hire : j L-l . to Le . a fine '.ika.Lr Harbor I4 8 6 / - .7 J? 0 - /u V v ^J /.;:: m *: : i-.*'.u *.>v. a ;t*;/a w..V J oij t ;ie - ' >**v ; . r rr\ I J i ./ l i . . . jiv;!I.---j,tt ;#*j;.U:..n*' y <<'J-iJ.'.T.j 'ii J1.C'.'U.s fif'd U 7!,j Cupt . . J U i. ^ v* U 1 . v i i called :.:e and that they I.-ci rth-auckor; Harbor Dench like to till!: with ecreone about an eron 10-10, Es-fceror.1 2*}5 (Bruah Killer). The % the: ; u:ron County Ho^/V'ooy^iFFj on hac* "T>rivei\ a m ro the road arid ro:.:e o f the. material drifted GO or .01. M- fO nho the field. The cattle were pastured ei a:;d 30 others-'aro nick. he stated, ho : -joiitc-.ot-.*d by idnonn ~ Harbor Beach r r.., 1 called Harbor Ecaoh 521 c.:zd w U J iI V C U nor v'.'.: is the.ace of Albert ilehiauclcer. He is ./.it, " : ;v.' p e r s o n and ho related to re:: sasen- ir:fo.or.v.tlo:: an he had to Plant Protection. r t f V.ir he stated that they were searching their tr~abie and that they were unable to 1 dore:' if there could be any association between .j 1...2 eras.: control opeso u .l . He further n r ttatv Vehe;J.n?.rian wan -piny to be there Friday he the Icoal roterlnai ian 5 i'i'. ilonry Jurgens of . :::.r : ci the near;in so of this c:u.of I fo3v -let 1; to ;:avcetl.-r.t.: A'. first rmi:c to find cut 17980 DOW 1 5 5 1 ^ 1 3 Therefore, after 8:00 a .m , I contacted Dr*. Ilymats, Dr. .Horton, Dr. Johnson, Larry Eouthwlck, anci Ed Coon, As a re sult cl* those discussions, Ed Coon, Dr. Ilymae, Dr. Loasure and myself IsTt for Harbor Hea d : . Ho arrived there shortly after lunch a;id tallied with the people concerned. It seems that the right -:..f-v;s.y had been sprayed by the Huron County Head Commission ..it,, a proper mixture using 5 gallons of Hsteron 10-10 and 2 1 / 2 /gallons of E3toroii 245 in 3.000 gnllens of w a t e r . The material h.ad been appli ed to the road s i d e * the north side of the pasture on July 8th. The eatt.'e became sick and started to appear ill on or* about July 15th. T7/o were' stated to have died durinG the week of July 24th, am1 -ne was killed for autopsy just before we had arrived at tire del .acker farm. This animal was down and since death, vsis imminent, she was killed for the State Veterinarian and camples taken, presumably for chemical and microscopic analysis. V/e also visited the carcass and took portions of the liver, kidney, spleen, rutr.ni contents, and lower intestines. The only evidence of abnormalitios tv at we could detect upon gross examination were areas of marked hemorrhage in the small Intestine and in the bladder, and an apparent lac.1: of fermentatIon in the rumen. The urine v?e.s not bloody. Ko` snple of tho rumen contcats, 1 tier oV' tilo .lev;or intosilines wW>4',/if* brouglK the e.rC,'ji.Vny Oi* vA.ly oh th, when 7. parsi>o.:i3.1y free;:,V,> or late v".*x!*T`O\iu*m of the 1.17 81 BNN \ 551214 - 3- wore placed at this time in formalin for subsequent microscopic examination. On August 1, I personally delivered to Mr. Luce at the Analytical Laboratory the frozen camples for analysis. Vie examined the hard which Mr. Schmucker had in the barn and in the feed lot. He had taken them out of the pasture several days before and was feeding.them hay. It appeared that three of the sick anlmal3 were down in the barn and one of them we were able to get up but the other two never did get up. One was reported to have been up and eating earlier in.. the morning. Her mouth was clean* her nose was wet* her.eyes were dark ar.d somewhat protruding and mucous membranes were.a light chocolate color. The rumen was not active. There were no skin lesions vlsible on any of the animals. Some of the animals obviously were very weak, particularly in the hind quarters and walked stiff legged . The hocks of .two of the group had a peculiar stiffness in which the ankles of the:hind legs protruded forward and the animals app-eared to be'walking on their tees. There was considerable unsteadiness in a quite a few the anima?.3 in the herd. The calves appearou to be in good health except for one of the larger ones that apparently was effected the same as .the effected cows. Tno other calves .seemed, very frisky, About half the cows ana tho herd bull appeared to be :ln excellent condition o rwith no r,i gre r;yi.vplwms of 'illness. All of the animals had boon ex am in ed by a v b r ; r inurlun In Harbor liuieii* hr. Henry Juivrss, 1798? a n d a e c u - : : ' ;.. . ; . ,..o. a r a n v.n w e l l a s a S t a t e D r. I'.u li-j D r . ba:.-. u c . j ... . ... . . . v ' j i# Jii.} we a;r r i v o d . V/o kN. U.O w*)./U-.**e* i.*w J uil'J 1 loro :. ............ :vo?j> ci::d-suggested that -lr. Schnmcker drench all ..,j h . M I i.iM;leais that evening.. I'..: s .ru.;.;.j the premises inri noted that there v;as a waste pru from t h 2 .liaron. liilling Company in Harbor Beach, which 1551215 w.a-: whits aqueous. suspension of flour and middlings which Mr. Sobro:-.:.or rad said that lie had been using for years.- This m a t e rial is h ruled away fro) the mill' by a number of trucks' ..working continuously making 'delivery-to'farmers all over that area. Upon questioning the veterinarian, he stated that there were no other cases of sic!-: cattle 'around the area and that a number of farmers had been using this liaste product *aaa source'.of food for their stock. It would seem that if this material ware re-, sponsible that others would have-had similar trouble. Dr. Leasure toured'the pasture along the roadside that had been sprayed and generally, over=the pasture area and found no evidence of poisonous weeds to which the trouble could be associated.- Over grazing or other evidence which would suggest anything unusual was also absent. He stated that if there was any drLft from the roadside spray, it was only very slight and in his opinion was of no consequence, le examined the bean field referred to by hr. fchmucker and.said that there was little if any damage to it although a trace of Injury could be found in certain . ; :.uo ono.circumstance which tic.Q chronologi cally d to -I:- poisoning,.and that uaa the occurrence of heavy ....Li i n flash flood that went through thn pastures ol2 July It waa iremodiatGly after this flood that the ill.;-:;;; of oho cattle became' apparent. Whether or not this had anything; GO 0.0 !with it is totally At fcho present time, we ness of a rninbo:r of Hr. Gclirnuclcer which cceurrad. In our opinion, way related to ithe cpraylng along County r;oad Co:;.;.ilSSlon. DOW 1 551 216 Dictated 8-JI-55 BY V.K.R Trar/ecriibad 8-5-*55 BY J.C.W. cc: G. J. William T. A. Iiynas K. Leaaure K. C. Esrrona J. E. Johnson " . ^"*V'* 0 -- ;' - T d a ,~> * 0 2 * 7 o a /K ( // ,, '/ Y. / J e r r y . /; / . J If t- ? 1 . $})///& C-J'f-s/f/Z HOW J 5S U 9 7 j/si s'i JfU /H * '."/. f i/s S/~.' X /, - / >7. y- 'V t- ^ ` , Z<--/*/r/ S.-`*r. S.'r/ j'/~ . // - - - 7 -J ^ S ' ' _ / /'"/Ji r~/~ /{cz-i-i'i* >- i y `" / / f/c & itU a i '"/ J . : ^ s / ff ' . ! )i/ M ^ ^ ^ ^ / / /s yz 7' ' /Z ltc ^ C ' Z u< ^ . ^ s f Jf'Zf //' 'lo _ > /r -. ^ * sfLi/st *i*:r " 7 7- --. /; ^i-L ^-d^- y J '' /y/J, j '1 &sS* : -.K , Jl* 4*7 /'s * * X ? s 'fe.k. / T&vCe&i' c iy J ' , / / / . /.i/'', !^//~yJ~ y.-y, -'// . i/ yJ/J\ f t -<//* s(U , e r c / v J J / / ' y * A\ (' 'Js/7 4/- J \y'l -l iy jL-'.fiZ-4/J . / //< / yy "* /- y / -S/S./ ,* ///// V .' . y//-(- y ' < ///c J J i ^f--a -r* yy* ~~s r J -/'-^ y/ J /J / ,/s . s ^ s. ' < /` / . y Z % <yVjiTJy'z-/J / /j y;./ - < y/ 'y/ / / * -:- y'-y1'7/ ", 1',,. % u J > W . * * ? . j r * / ,v- ../ ./ y '/ ,/ .y - . xwuo t.^\ i ff i TMu ' ^*\ ' - * * > * * , / v- ^ ,, /p sviy // //< 7 /7 "z-citM,?i-.i-' <sjT'4t}d / ei<r ~7- J-1*' J y / s^ /y y y ! y j'- A 't 'J'y ..'<//J .S i''- .'v <f ^//.cS _o/ ^y ; s%cd..fju,- <z<'i,y .s/Jy^(/.-yi . // // y - 1< Y / // Y t'/ i .-V'i' yey ^^\..". /'' y i f V. z *f Jr^<S.JS. / y y^ri-..' IT S 85 ' DOW 1 551198 V s. r- A A OQZOM RRTJ Q I llflfl# April 16, 1950 1438 Mr. P. E. Adley, Manager inductrial 'y^iono Operation Relaciona and Utilities Operation lanford Atomic Producto Operation Genera] Electric Company iiichlund, Washington Dear Mr. Adley: Dr. Irish has asked me to reply to your letter of April 7th inquiring as to toxioological data we might have with respeot to humans exposed to 2,4-D in the form of our product, Esteron *14. Frankly, I am very happy to report that I have no toxicity information on humans. So far as I am aware there has never been a case of poisoning in humans from the use of any 2,4-D or 2,4,5-T product. We have had a few cases of skin irrita tion and with each of these, when it has been possible to study the particular problem, it has been found that the per son was hypersensitive to the fuel oil used in diluting the product prior to application. These cases, however, have been rather rare when one considers the extensive use of products of this nature. Thi3 experience with humans over a number of years has verified the results of skin irritation and skin sensitisation tests we had conducted several years ago by a consulting laboratory. They found that Esteron 44 itself was neither a primary irritant nor a skin sensitizer. In regard to precautions to be taken when one is applying the material for prolonged periods day in and day out, it viould orly be reasonable to provide sufficient protection to pre vent the continuous wetting of the skin and continuous inhala tion of spray mists. We are all aware that oils themselves can cause considerable pulmonary edema and injury to the skin. The type of protection used will be determined by the nature of the particular operation involved. If one were to apply the material where he could not avoid being in the spray drift, then I would recommend the use of protective clothing and a respirator designed to remove mists; further, the operators should clean up afterwards. On the other hand, if the appli cation is of such a nature that there is very little exposure, G l. DOW 1 5 5 1 1 8 9 e Hr P .aley -2 A p r i l 16 , 1958 clinical prceciitlono I on cure would be adequate. I feel quite confident that if you c::era'iGO good judgeccnt in these situa tions that you will encounter no difficulty. I hope those ccacento will be adequate for your purposes. Sincerely your3* V. K. new CicchGDical research Laboratory 12-634 Euilding VKn/ba cc: V. K. Rowe (2Y ' T2.30-34-20 Correspondence 17988 T 2 . 3 0 - y - So' DOW 1 551183 MIDLAND August 2 3 1 19137 Richard J. Darker Legal Department 47 building 36: 2: 0 Nv V/^ rvj oQ o TOXICITY INFORMATION ON S3THEON 44 AND ES THRON 45 SUITA-.-; FOR TRANSMITTAL TO ATTORNEYS IN THE CASE, OF RADEL 73. DOU The data you asked for In your letter to Mr. Rowe for the case of Radel vs. Dow may bo summarized aa follows: TOXICITY OF ESTERON 4-, Active Ingredient The toxicity information on isopropyl eater of 2,4-dichlorophenoxy acetic acid, the active ingredient of Eatoron 44 may be summarized S3 follows: Ingestion Single oral doses of this material were administered by intubation to rats, mice, guinea pi&3, and chicks a3 an olive oil solution. The doses were calculated aB milligrams of the substance per kilogram of body weight of the animal. Table I shows the L D TM values calculated by sta tistical analysis of-'the data obtained using the method of Litchfield and Wilcoxon. TADLE I CALCULATED SINGLE ORAL LD,-, TALLIES FOR ISOPROPYL Spec] oa LD,. Value3 In MyaV/kj. of body wt. Rats Mice Guinea pigs Chicks 700 540 550 1,420 19/20 Confidence Lim In mgm of body 570 to OoO 4u0 to 740 450 to o70 i ,1 '-.0 to i,790 DOW 1 551184 f 7 4 M j . . > k aargcn .`ui .Uw u J i v )'lj i Skin Contact hopea ted application of this material to the o h m of rabbits aa the undiluted material rasv.itod In but oil;hit Irritation and all ;!;t euc.r_a on repeated prolonged contact (2 4 hours a day for 12 bays). There wan no evidence lr. these tcsto that absorption thi'ou,;h the I - -t ... had occurred. Enteren 44 formulation The toxicity information on this formulation nay be summarized a follows: J.r.yoctlcn v/hen Eatcron 44 wan administered to rata as an olive oil solution, an LDrg value of opO mgm./icy. of body weight was found.J (19/20 confidence U n i t s were 300 to 1,000 mr^u./icg. ) It Bhould be noted that the fort;uluLIon .as essentially the same acute oral toxicity as tne active ingredient. Shin Contact Esteron 44 vihen applied to the shin of rabbits in undiluted form was found to cause moderate hyperemia, edema, and eventually r.ccrcsiB. This response is typical of kerosene when tested under similar conditions. Tne formulation, diluted to use concentration (1$). had no significant effect on the Bkin of rabbits. Furthermore, there vierc no Indications that tills formulation v;aa absorbed through the olein in acutoly toxic amounts. Human okin testa done by the Industrial Toxicology Laboratories of Philadelphia on a similar formula tion containing another ester of 2,4-- Jlchlorophenc. if acetic acid indicates that Estercr. 44 should bo neither a primary irritant nor a sensitizing agent Very wide experience m the manufacture and use of Ibis product confirms these conclusions. 17 f >c o ichard J ;L-'w' *i -frc> , . * h !\a v- l j, i'Jl_j BOW 1 5 5 1 1 8 5 fU/T1wLJmOr.m.rU ri-,-r.?' rT^TA..A"--T>i\p.'/1i ;hr, A c t i v o In rod lent co tcaiclty information on the polypropylene lycoi butyl ether ester of 5-triehleropt cotte acid, the active ingredient of ilar.or on 1o o '.l-.t .larined as follows : In.Tontion MU-six fciiia material wan fed to labora tor?; an Hiala by intubation aa a ooU;tion In corn oil, the roilowing LDc.-s values were obtained by calcula tion. ^ necica LDr~ valueo in 19/20 Confidence Linios mgro^/kg. of body v/t. in r.i;7n. . of body t . Rot Rabbit ilice Chick 40 710 9f0 1,190 jilo to 740 500 tc 1,000 6 7 O to l,o!0 C'oO to 1 /-jfij Work on guinea pigs rouulted in a LD_~ value in the range of 500 to 2,000 mgm./kg. ^ Eye Contact . The instillation of this material onto the oyei of rabbits resulted in moderate pain, alight con junctival irritation, and slight transient corneal injury. Skin Contact Vfnen the undiluted material was applied 1.0 the kin of rabbits for repeated prolonged periods of time (24 hours a day for 12 days), a mild but definite irritation developed. In these tests no evidence of absorption through tno skir; was observed. t Inhalation Rats auojooted to essentially saturated vapor of tilo ma ieri, 1 ;enero.ted at 10JC 'aere not observed to be aff e01ed adversely. ri: /;* u **. 4 r >:j\ ~ lr - J 00W 1 551186 jtoron ju forr.v.;JLiMcri The tcurlcity inform t Ion cri thli formulation ia uu.-roarisod ao folio va: I:~. a . f .li Whan rota v:aro fod oln ;lo dcaoa of unis rcrmuls- felon byv> a o j j u ^ y Ii . in. tv uu ;b; uation cai uu anc i . 'i clive oilu i j l usov l uLitu.ii ov -n. *,j ut i caall;culated LOro 'value of boo in' body woeight was fotihd The 1<J>/G0 confidence 1;^: ;;i were calculated to bo uOQ to 1,000 fSye Contact The undiluted formulation liaa found 'tc ba slightly irritating and caused alight tranoler.t corneal injury when Instilled onto the eysa of rabbity. Dilution of thia fonnulaticn to a 10/5 concentra tion resulted in only voi'y alight rain and transient irritation of the eyelids when cectob In a similar manner. Skin Contact Thu undiluted formulation caused cl.ight tc rode rase irritation and slight edema when in repos Lea ;u\> longed contact with the akin of rabbits, especially VJhcn confined. Strong solutions (10*) caused crly or ighfc irritation and a "chapping ` unde:- similar conditions. In testa conducted by the Industrial Tohicoic..^ Laboratories cn too human subjects, th ferme la- tion wan found to he neither a primary akin irritant upon a single prolonged contact, ncr a sensitising agent. (The material was applied cnce to the backs o r forearms of lOO* unr.eiecteu male and female subjects each for an osur-u period cf five days. The sensitising dose was cr pliou about three v/eoka late r . } I;TEPRE7ATTC P based upc-; the accve data, the following inter prta tiens car. be naue. DOW 1 551797 N073280 14 j O MIDLAND August 3, 19p9 Nels II. Mun3on Insurance Manager Lcjal Department h7 Building T M 3 is in reply to your letter of Aucust 3 requesting information to send to the Fireman's Fund Insurance Company in regard to alleged injury to cattle from the use of Dow Esteron 2,^,5 Brush Killer. I believe/ at this point, the best thine; we can 3 is to supply the insurance company with reprints of the attached articles. One entitled, "Some Effects of Herbicides on Pasture and on Grazing Livestock".by Grigsby and Farwell, and the second entitled. "Summary of Toxicological Information on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of the Hazards to Livestock Associated with Their Use" by Rowe and Ilymas. You mi;jht add in your letter that we concur with the opinions of the claim adjustor, Mr. Tubbs, that it Is very unlikely that a brush control treatment had anything to do with the illness of these cattle. If we can be of any further help, let me know. V* K. Rowe . Biochemical Research Laboratory 12-o 34 Buildlnj Phone - ME 6-237o 141 THE D O W CHEMICAL C O M P A N Y MN073280 MIDLAND, MICHIGAN August 3, 1959 <1 CO 05 V. K. Rowe ; Biochemical Research Lab 63^ Building I am enclosing a letter received from Fireman's Fund Insurance Company regarding an alledged in jury to cattle resulting from, the use of Dow's 2-4-5 Brush Killer. Inasmuch as we are not Involved at this state but possibly could be involved, please tell me what I can pass on to these underwriters who are attempt ing to protect the defer.dent who used our brush killer. Nils H. Munison Insurance Manager *' / KILL ALL' J i l l / 1 7 , lfiv'": 1492 .1 joansou . ...* and L . J - ./ . .w J. .. I .ul AM3:a*irUil: Vi.iluryje Y. L. done !i. ii. H e l m yvo A c t i o n i-'i l i . '- i i u r o o r . l SL 7, 0 *H.V/v. o ' *.:ioci;eraic ni UCCCiVe: La uc:ra ;o.-; u.in rcl .v -t. i.O o IAC X.L) X<iv.iiCi3 rvi'l- *.o i.*1'/-v.:.dj.4-;> 4.'re 4.v.i...i.fJALiiJ to aOp rove in the L;ei.i ;:.oriai i'y. u.e Yep..ru.!ta: to ii:;o ".11r..5S 1 as an nqu:: tic i.rrbi cie'e _ih cert ain i,,..nrcii c.n 1aJ;s . You indiea let 1.0 .*xi-L.'. 70v;e hat you lit.ec'c'd v'l :31.atvfWCiit or. the label intii c a i1hj v:iit-a t~r,.*.. Vi'a tor 3UCii V.coaled 3:ibc-s would bo fit to c.!r in Or n r.XtCO'il:itivf' 30:.'0 4.uscructi::ns ior treating iI.t. ..A or, ijvii;i, as ii o i lifU; , to i;i3:finale t;:e eue.'.'iea. residues. ', cn * >*4. z o> O 00 Ni <J3 V*J ro CD o We have nncie eevorol taeir io tiecfv;uiiio ti- persistence >, KUl'OSnl CL ill 1:7:0 Wiior i a H o m i n g i o n s an ri ni.: 1 in certain instance: s i l v e r * .vi 11 errs: st throu0.i tnc- x'. ac L a v e no r e noon t o t e time oilini,; e-si c u e o i Cii 1 v o x i h w a t e r . Although we c.o n o t re- c o n e nd. K u r o s a l CL V.' U .. O V' . ': we do r.m be lie-vo that there :v, any hunan or animal He-.-*1.> hazard from ihe occasional use of wnuer treated witu ihe recom mended concentrations of Xuro&ul CL. In support oi ,,ho con clusion we have available a 70-day dietary ceding ~uuiv in rats. Z aci enclosing duplicate copies of this information which can be supplied to the Canadian authorities ii you loci that this would be helpful. V.'o have recently set up a 2- year dietary feeding study r.l s i l v e : (active ingredient of kurosal SL) with rats and cogs and, usual, we will be glad to furnish the Canadian aovernxeni the results of those tests when they become available. lie trust this informal, ton will be of assistance to you mid i: you have any further questions, please advise. S in c e re ly , Original higr.r.-d By: G. L. LYNN G. 1. Lynn Eioproducie i:ep*>riinc-nf nhc: y. : 82*0NH ' V '1 Y FOR DC:i EEnSOIJIJEL ONLY NOT FOl OUTSIDE DISTRIBUTION ACD Project Status-Report - Document No. 21529 SUTUADY OF AOtaTIC HERBICIDE INFORMATION . ON KUROSAL SL (11-1045) AND KUROSAL Q (H-1847) w.:,-;- :;'byA:V;V. -: Paul M. Ri tty 3 fc ; :.Agricultural Chemical Development The Dow Chemical Company Midland, Michigan ; February 8, 1961 Copies to: J. E. Johnson K. C. Barrons L. L. Coulter W. P. Falsey j. W. VanValkenburg R. C. Dosser G. B. Truchelut R. Gantz 0. H. Hammer G . E . Lynn j. S. Wilson P. M. Ritty E. M. Lutz A. W. Swezey D. L. Conner ACD Circulation H. A. Nation R. L. Warden J. W. Gibson R. N. Raynor J* R. Fisher M. G Wiltse E. S. Saunders E. E. Wiffen R. A. Schwartzbeck V . K . Rowe W. L. Corbin A. J. Watson W. R. Mullison J. L. Spalding H. H. McIntyre T. S. Foster J. E. Greenfield W. J. McCoy F. S. Furman D. K. Surgeon R. 0. Fletcher L. L. Martuch . J. K. Leasure W. W. Allen B. Seeker H. L. Smith R. W. Colby 17SSS OUTLHE FOR AQUATIC IEilDICIDE STATUS REPORT i ;Summary Introduction ; Compound Identity Corrosion Studies Analytical IZethcds Toxicolosy Mammalian Toxicity Fich Toxicity Weed Toxicity Residue Problems Water: Odor & Taste : Fish Fleoh Effects Patent Status Compound Herbicide Activity Aquatic Weeds Controlled Speed of Activity Aquatic Environment Effects 1961 Promotions Plans Test Marketing Areas Aq . Chem. Development Plans Specimen Label f' O- 8 iM 8 & " l SUMMARY OP AQUATIC K . r<> Z INFORMATION ON KUROSAL SL (M-1845) :: OJAL Q (M-1847) Paul M. Rltty SUMMARY The potassium salt formulations have shown control of a wide variety of submerged aquatic weeds. When applied according to directions, herbicide activity has been equal to the ester form of silvex, Kuron. These salt formulations control a wider range of aquatic species than those which are controlled by 2,4-D formulations. The toxicity picture of Kurosal SL (M-1845) and Kurosal 0 (M-1047) are better in regards mammalian toxicity and are considerably better in regards fish toxicity than are those exhibited by Kuron. Residue problems such as water odor and taste threshholds are higher and there is less effect on fish off-flavor. Dow has no patent status in regards to Kurosal SL and Kurosal 0, since these are covered by previous Jones and ICI patents. INTRODUCTION The potassium salt formulations have been evaluated in various areas of the United States for the control of submergent and floating aquatic weeds by a number of experiment station, fish and game, and federal and state representatives. Because of many of the inherent safety factors in the potassium salt formulations, interest has grown in various areas of the country. Much investi gational work has been completed to date on the potassium salt and,, prior to the release of the pot as slum salt for development, on the sodium salt and the ester formulation of silvex (Kuron). Much of the information is interchangeable, however, the major portion of this report will deal primarily with the potassium salt activity unless otherwise stated. COMPOUND IDENTITY 1. KUROSAL SL (M-1845) The composition of Kurosal SL is as follows: Silvex 53-3 % KOH 11.1 % Versene Water 0.05% 35.6 % -2- The formulation contains six pounds of acid equivalent per gallon, and has a specific gravity at 68p. of 1 .3 5 1 * Kurosai SL, with the pH adjusted between pH 7*3 and pH 13, showed freezing points somewhere between 0 and 3F. Crystals do not form upon freezing, and the formulation reconstitutes to homogeneous solution at 15F* to 17F* Even when the solution has been seeded with potassium salt crystals, no difference in freezing or in reconstitution temperatures has been noted. Stability of Kurosai SL is constant, p r o viding the compound is not allowed to evaporate. If evaporation of the water content occurs, the compound becomes somewhat thickened. Since Kurosai SL is a metal salt, flocculation does occur in hard waters of 200 ppm or more. Flocculation may also occur under some conditions in waters of less hardness than 200 ppm. Therefore, the formulation does not lend itself to pre-mixing before spraying. Studies with the sodium salt of sllvex show this material to be very persistent in water systems in which there is little water exchange. In earlier studies on the sodium salt of silvex, a bioassay experiment indicated the continued presence of the sodium salt of silvex in various waters for the duration of the test.whether the systems had been kept under refrigeration or in the warm greenhouse. CORROSION STUDIES No problem to date has occurred in tests on tin, galvanized iron, or black iron. The possibility of corrosion in the vapor space above the stored liquid should constitute no problem. The material will be shipped in unlined drums. 2. KUROSAL (M-1847) ' The composition of formulation Kurosai 0 is as follows: Silvex potassium salt 2 2 .85g 815AA-RVM Attaclay 77*2# This formulation contains 20# acid equivalent. In physical and chemical property studies on this formulation, the stability of these granules appears to be excellent. There . has been no formation of crystals from sublimation. Kurosai G is adsorbed to the attaclay particle surface. Release of chemical upon contact with water is believed to be accomplished almost immediately. There are no problems in regards to storing this formulation. The material will be shipped in fiber drums. CHr <0 173S9 ANALYTICAL METHODS A method for determining potassium silvex In water by the use of Infrared determination has been perfected by the Dow Spectroscopy Laboratory. The procedure makes it possible to detect 0.02 ppm in one liter of water with an accuracy of plus or minus 0.01 ppm. There is no simple water analysis that can be made at the treat ment site. However, this should not pose a problem since toxic amounts of potassium salt will not occur in a treatment area when directions on the label are followed. TOXICOLOGY Mammalian Toxicity Acute oral toxicity studies on rats, using.the potassium salt, showed no marked differences between this salt and the previous work on sodium salt on which extensive tests had been run at an earlier date. According to V. K. Rowe, there is no possibility of skin irritation expected by swimmers in the area where the potassium salt is being applied as an aquatic herbicide. Reports from the toxicology lab have suggested precautionary labeling for Kurosal SL (M-l8^5) as follows: "This material has low acute oral toxicity. Eye contact may result in severe irritation and severe damage. It may cause moderate irritation and moderate damage upon prolonged skin contact. It apparently may be absorbed through the skin in acutely . toxic amounts. It presents no problem from inhalation of vapor or fumes." The warning statement, which will be on the label of Kurosal SL, the liquid formulation, will be as follows: "Warning - Burns the eyes. May be absorbed through the skin. Do not get in eyes. Avoid contact with skin and clothing. In case of contact, flush eyes immediately with plenty of water for at least 15 minutes and get medical attention; for skin, flush with plenty of water. Wash contaminated clothing before reuse." In regards to precautionary labels for Kurosal G (M-l8^7) the following statement ha3 been made: "This material has low acute oral toxicity. Eye contact may result in mild irritation and no damage. It may cause minor irritation and no damage upon prolonged or repeated skin contact. It apparently is not readily absorbed through the skin in acutely toxic amounts. It presents no problem from inhalation of dusts." No additional warning statement will be needed for Kurosal G. DOW 1:S5'l681 -4- Flsh Toxicity Twenty-four-hour exposure of Lake Emerald Shiner to 0, 5, 10* 25, 50 and 100 ppm of Kurosal SL gave no mortality at 2 5 'ppm* and 100^ mortality at 50 ppm. In additional tests on fish toxicity, an acute 72-hour toxicity tests showed that the toxic level for Kurosal SL appeared to be greater than 150 ppm. However, in another 3 1 -day toxicity test performed in 55-gallon drums, primary dilution of Kurosal SL was fed to the fresh water flow at a rate to give a final concentration of 10 ppm acid equivalent with a continuous flow-through rate of approximately two liters per minute. Results showed a slight indication that Kurosal SL may exhibit some long-term toxicity to brown trout at 10 ppm, however, additional comprehensive studies would be required to verify this. In this 3 1 -day toxicity test, no deleterious effects were exhibited by fathead minnows, brook trout or yellow perch. Toxicity to Aquatic Weeds As will be seen later in this report, the,weed toxicity of Kurosal SL and Kurosal G is equal to that obtained through standard use of our present Kuron formulation. A wide variety of species of aquatic weeds are controlled or effectively reduced through the use of the potassium salt of silvex. RESIDUE PROBLEMS Water: Odor and Taste It is well known that the Kuron formulation in relatively low parts per million can cause an off-flavor in water. Studies conducted by Winston prior to July 1959 found that the Penla Heavy Aromatic Naphtha used in the Kuron has an odor threshhold of 0.2 ppb (billion). Studies completed on February 15, 1961 showed the odor threshhold of Kurosal SL to be 1 .9 ppm (million), much higher, and safer, than the odor threshhold of Kuron, or more specifically, of Penla Heavy Aromatic Naphtha. Fish Flesh Effects Test fish (perch) from a 31-day toxicity test were subjected to routine taste procedure tests for the detection of off-flavor in fish flesh. Fish exposed to Kurosal SL had a very slight off-flavcr that was detected only by the most sensitive tasters. On the whole, it was the consensus of the taste panel that what off-flavor there was was not objectionable and that it would not be noticeable if there were no control fish with which to be compared. 18001 -5- PATENT STATUS A literature search has found that the potassium salt is covered in some early Jones and ICI patents covering the aryloxy, aliphatic . and phenoxy propionic acid derivatives. Therefore, Dow does 'not have a patentable position on the potassium salt of silvex. We are licensed to sell herbicides under both the Jones and ICI patents, however, and no royalties would have to be paid This means, however, that any company that can obtain silvex acid could manufacture and sell the potassium salt. At the present time, The Dow Chemical Company enjoys the position of being the only manufacturer of silvex acid. Should the demand for silvex acid become increasingly great, however, there is a possibility that other chemical companies could manufacture silvex acid in this country or that greater pressure could be applied from foreign manufacturers. Until such occurrences happen, even though other companies would purchase silvex acid for the purpose of manufacturing potassium salt, Dow would enjoy increased sale of silvex acid. en H** or* o to COMPOUND HERBICIDE ACTIVITY Aquatic V/eeds Controlled Numerous reports of test3 by co-operators have reported that the following aquatic weeds have been controlled at various rates. Listed below are the aquatic weeds, scientific and common names of only those weeds reported from test3 which gave accurate reporting. Many other reports have contained comments concerning the "ccntrol" of other aquatic weeds, not mentioned, but the remainder of the dat, in the reports were subject to question. Hence, only those listed below are results of accurate testing and reporting. Scientific Name Anacharis spp. Cabomba spp. Ceratophyllum.s p p . Eichhornia crassipes Eleocharis sp. Jussiaea grandiflora Jussiaea sp. Leersia op. Myrlophyllum spp. Najas spp. Nasturtium officinale Nelumbo pentapetala Nltella sp. Nuphar advena Nymphaea odorata Potamogetn capillaceus Potamogetn crispuo Common Name Elodea Fanwort Coontail V/ater hyacinth Splkerush Water primrose Primrose willow Cutgrass Water milfoil Naiad Watercress Water lotus Nltella Spatterdock White water lily Variable pondweed Pondweed ioUU/i -6- Scientific Name Common Name DOW 1 55I6B3 Potamogetn epihydrus Potamogetn pectinatus Potamogetn sp. Ruppia sp. Salix sp. Sparganum eurycarpum Sparganum spp. Utricularia spp. Sago pondweed Southern pondweed. Widgeon grass Waterwlllow Burreed Bladderwort Speed of Activity Reports received from 1959 and i960 test results showed great variability in the time needed to control any of the above species. Time varied from one week to three months before complete control was obtained. On an average, it appears that control can be expected in approximately six weeks after treatment. Aquatic Environment Effects No accurate results have appeared from the tests conducted prior to this report in regards to extremes of herbicide activity due to pH. We have pH ranges in the reports ranging from 5*5 to 9 .0 with apparently little noticeable effect on herbicide activity. We do have numerous reports, however, indicating that any time that Kurosal SL was pre-mixed and applied then as a diluted spray, the activity was not as good as occurred where the material was introduced directly into the lake as the concentrated chemical. Hose proportionaters or Venturi metering systems apparently can be used without seriously affecting the activity of the chemical. Water temperatures were reported on tests applied at 65P. water temperature ami abo\e. We have no Reports of Outside Information indicating chemicals applied to colder waters than this. However, undoubtedly, some of the northern experiments were apwl i-.-ci to waters cooler than 65 P. without deleterious Tec t;o . Generally speaking, laboratory information shows wate:.- temperatures of 5 5 0F - and above should be safe minimal limits for application of the potassium salt of silvex. 1961 PROMOTIONAL PLANS Test Marketing Areas At the present time, test marketing of Kurasol SL and Kurasol G will be conducted in the following areas of the country: 1. Michigan - Test marketing in three regional fish commission areas of southern Michigan in recreational -7- waters. Main emphasis will be establishing dealers and users who-will be do-it-yourself applicators, that is, lakefront lot owners. Kurosal G will be promoted for this purpose. Also in Michigan, we will be working with a custom applicator who will sell Kurosal SL on a custom applicator basis for the treatment of large portions of, or total, lake areas. 2. In the Northeast -- We will work directly with two large custom applicators, one from Boston, Massachusetts and the other from Philadelphia, Pennsylvania, whose areas cover the North Atlantic seaboard states. c Kurosal SL will be the major product moved through S them; however, they will have access to Kurosal G if * their custom application work calls for it. - 3- State of Georgia - SPADE will work directly with the 'fl State Game and Pish Commission who are the major \)rr* custom applicators for the State of Georgia. Probably both granular and liquid formulations will be promoted through them in the State of Georgia. 4. Possible distribution may be set up through present Dow distributors in San Antonio, Texas, and Norman, Oklahoma, working with custom applicators and with both formulations. Agricultural Chemical Development Plans for 1961 1. Nation-wide investigations of Kurosal SL (M-1845) and Kurosal 0 (M-1847) will continue as was conducted during i960 in order to obtain more meaningful data from the test area3. Reporting forms will be sent to each of the Ag. Chem. Development field specialists for distribution to co-operators. Continued effort, soliciting state and federal game and fish commissions, public health agencies, and fish and wildlife personnel, a3 well as experiment station investigators, should be continued for this year so that a more complete group of data can be obtained for possible nation-wide sales in 1962. 2. CRI Intra-Company Sampling - A plan is now underway for sampling to Midland employees of The Dow Chemical Company who have lakefront lots in an effort to obtain additional information on the use of the granular formulation by lakefront cottage owners. Results from such a test program through the Midland employees should give a wide range of aquatic conditions in cold and warm recreational waters within the State of Michigan. 1 O1 CONCLUSION As will be seen from the foregoing information on Kurosal SL and IfnriWAiQSCafc1X /VIJf we have two formulations v/hich nave shown considerable ability as aquatic herbicides, increased safety factors over presently available formulations of Kuron or competitive compounds, and a reduction in these formulations of some of the inherent hazards found in presently commercially available aquatic herbicides. V/e need additional good records of field successes with these two formulations, tte should aggressively pursue the development of these products during 19 6 1, so that we can obtain additional information as to the market desires of the aquatic herbicide field. MIDLAND Muy 22, 1959 Milton Irvlno Agricultural Chemical Sales Abbott Road Building CH cn h* IO MN07 32 8o TOXICOLOGICAL INFORMATION ON KURON FOU OUTSlDc, DISTRIBUTION This is the Information I promised you over the phone. It is my understanding that it is to be sent to a company who will be using Kuron to formulate new products. I hope the following information proves adequate for your purposes. KURON Toxicity Kuron has a low acute oral toxicity. It doo3 not present, a hazard from ingestion under the usual conditions of handling and use. However, Injury could occur If large amounts were swallowed. The LD,-- values for a number of species are as follows: Species LDrn In g/kR of body weight Rat Guinea pig Rabbit Mouse Chicks 1.07 0 . 8 `j o.Qp 2.lk 2.0 Kuron presents'no unusual hazards from skin and eye contact. It may be expected to oauuo, at moot, only mild Irritation when In contact with tho eye. Skin contact., for repeated prolongod periods of time, may cause, at most, only a mild irritation. It apparently is not absorbed through tho skin in toxic amounts. In addition, results of human skin touts with Kuron diluted to 1.0 percent with water indicate a high degree of uoauranco that Kurcn should not cause skin irritation nor skin sensitization when used as directed, Buggontod Precautions for Handllnp; Kuron H a r d y ;v Suitable eye protection such as safety glasses may';Uo\woni t protoot the eyoo simply to avoid,,fch disoojnfortii oqcur upon oontaqt ; i^aflonablCdf^ non should I x i p r u a t l o e q ^ a y o i a iflkii}^diffiqa; `:v J... / 551275 Milton Irvine -2 May 22, 1-555 Recommended Plrnt Aid Measurea Should the eyes become contaminated veltri Kuron, they should bo H u s h e d with plenty of water. Any irritation or injury that devolopa should receive medical attention. Grossly contaminated sldn should be flushed with plenty of water. Grossly contaminated clothing should not be re-used until cleaned. Any skin irritation that occurs should receive medical attention. We plan to write up a suitable bulletin on Kuron for outside distribution. It should be available In about 3 ^ 5 weeks. Mark A. Wolf Biochemical Research Laboratory 634 Bundin': Phone Mil 6-2776 MAW/Jd c c : V. K. Rowe M. A. Wolf T2.30-40-20 rU *** i 1501 MN07328o DOW 1 551264 T Y |1 *t O^ i 1..j <*s Dr. J. V. Clauder lyj'; spruce street Philadelphia l't, Pennsylvania Dear Dr. [\lauder: An pox* cur telephone conversation, I air. writ.in; you I'ejard.lnj your d o i m for us a skin Irritation ;md ci:ln sensitization atudy on humans with a product of euro Lcnotm ao Huron. Thio product ia an o m u l s fiable composition of the propylene glycol butyl ether eotcra of 2 ( 2 , 'r,3"trichlorophcnoxy)propionlc acid. This material lias s h a m considerable promise aa an aquatic herbicide which may be uaed in lakes, etc. where owimuern riilijht come in contact with- it in diiutod form. It will be used for ouch purpooca in concentrt Iona ci ppm. Ao you icncw, our general policy for marketinj a product which L-iay come into contact with the general p u b l i c , even in dilute solutions, is to have data In our fllea reyardlni; its skin irritation and sensitisation proportion on humana. Our laboratory teoto on rabbito with thin material have ohown that a concentration cf I# may cause a trace of irritation wiien contact io prolonjod and i*opented. At 10#, it ia mildly irritant upon prolonged and repeated contact and undiluted, it ia moderately "T'lfcatinj. It io not absorbed fchroir; h the shin in toxic amounts. This ia typical of most herbicides cl* thio type which aro commonly uoed and handled today. This material io not markedly difforenfc than some of those which you tooted oomo yearo ayo foi* un in conjunction with the late Dr. Shelanoki. Ao wo agreed, I ou^ceot a pilot atudy at concentrations of 1# (10,000 ppm), 0.1# (1,000 ppm), and 0.01# (100 pom), all in vjater, pluo whatever additional concentrtiono need bo checked on humans to dotorwino the maximum con centrt.ten not llkoly to cause primary Irritation. f v;ant the sensitisin'; applications to bo made with no hi ;h a concentration aa practicable no as to establish aa lorje a margin of safety an possible. An wo agreed further, the study will bo cf the repotod insult typo on bO human subjects and tho coat to un will bo Vf'y ,0, # . i80.ee DOW 1 551265 Dr*. J . V. lO.au6.or -2 - January 2, V-y/j Aa you know, I usually do not aend sln&lo compounds cut but /alt until wo have a sroup to study, however, our people ai In a hurry for studios on thio one and I cm wcndorlns If you could do it promptly. We want to have a report by March 15, 1959 da aona decloiona resardln^ it must be made then. If you have any questions, please do not hooltate to coll me. With best personal res&rda- Very truly youra, V. K. Rowe Biochemical Research Laboratory 12-634 Building VKR/vnv cc: V. K. Rowe (2) T2.30-40-20 T2.30-40-3 Correspondence -r< * *\ December 5, 195& 1502 Albert M. Kligman, M.D., Ph.D. Associate Professor of Dermatology Hospital of the University of Pennsylvania 36th and Spruce Street Philadelphia 4, Pennsylvania Dear Dr. Kligman: y*i PJ SQ o Some months ago you visited our laboratory and discussed stein work with Dr. Irish and others. I am sorry I was not here at that time for I would have enjoyed such a discussion. Since then we have been planning to uce your services when the opportunity arose. With this background, I am writing to discuss with you the possibility of your doing for us a skin irritation and akin sensitization study on humans with one compound. This material is a product of ours known as Kuron. It is an emulsifiable composition of the propylene glycol butyl ether esters of 2(2,^,3-trichlorophenoxy) propionic acid. This material has shown considerable r..lcmioe as an aquatic herbicide which may possibly bs'a used in lakes etc. where swimmers might come In.contact with it in diluted form. As a general policy before marketing a product which may come in contact with the general public, even In dilute solutions, we want to have data In our files regarding its skin irritation and sensitization properties. Our laboratory tests on rabbits with this material have shown that a concentration of 1# may cause a trace of irritation when contact la prolonged and repeated. At 10#, it is mildly irritant upon prolonged and repeated contact and undiluted, it is moderately irritating. Thio is typical of most herbicides of this typo which are commonly used and handled today. In thinking about what might bo an appropriate study, It seemed to me that we should check decreasing concentrations of Kurcn in water to find the maximum concentration which will not cause primary irritation in humans and call thin concentration, (X). I would propose then a repeated insult study by the flheitanaky and She/lanalcy method on 30 humans applying 20# of (X) 9 times in 3 weeks and then challenging two weeks later with 10# of (X). Thio would 1 8 0 1 0 give us basic information on tho primary Irritant properties and also on the possible capacity to produce sensitization, DOW 1 551263 / r .r r* i d i j.Maxi, M.D rm .D. _0 When used as an aquatic herbicide, thin material is applied so as to give a concentration in the water of about .3 1/2 4 ppm, fence, concentrt lone, which I suspect can bo tolerated repeatedly by human; W. . JJ .T_L.11 be far above this figura tut I believe this i3 desirable. In other words, I would like to ;t it in as high a concentration an we can without show iix-^ irritation3 . I v/ould lilce your suggestions as to the appropriateness of such a teasing program. If you have a program which you thinJc might be bettor, do not hesitate to suggest it. In any event, I would like your suggestions and m estimate of the charges for such a study together with an estimate of when it could be completed and reported to us. I might inform you that it is not our usual procedure to send one compound for testing. Usually I save these up until I have several so that they can all be done at once but thi3 time our folks are in a hurry and I do net have any backlog for such testing. Your early reply v/ill be appreciated. Sincerely yours, V. K. Rowe Biochemical Research Laboratory 12-634 Building VKR/vrav cc: V. K. Rowe (2)' T2.30-40-20 Correspondence ' zr 18011  DOW 1 551258 bvj,..; j j , i c u -j llju MlDLAiiD October 10, l i . \ lj 7 150G s O Nj Va / /V) Od O V . Ko Rowe Blochen Lab 634 Bldg cc : Harry McIntyre C. Eo Otis subject: rnic-ic:! cm sv/irirsns t o iojroit Thank you fcr yo r cements on our proposed labe 1 copy for use of Kr.rcn In the control of aquatic weeds. I7e have forwarded a revised proposed label to Harry McIntyre and 1:3 ancici e that he will a.pply for registration in Washington ve shortly. Once we know how we stand with tlie folks in mklngtcn, we can better determine what our approach shea .J be in regard to skin reaction to to. If we get accept .nee reasonably soon so that we can forsee a good program , we will then contact you and it would seem to be a good thing then to take whatever steps ara necessary to put us in a bettor position to defend ourselves. Meantime, until v/e have a little more assur- ance of how thio thing might g o , it might be just as well to postpone these tests, Yours very truly, L. L. Coulter Agricultural Chemical Development - wb ?" p-irr. T J.3 0- i O - J c 1507 DOW 1 551251 MIDLAND January 31* 19^8 L* L. Coulter Agricultural Chemical Dovolopxont Acariejan lecl'cn aulldlnc iiu n a 'j ? o ;i a q -j a t i c ' u s e d c o w t h o l This io in reply to your letter of January C^Jth in which you aciad for cur eozzonto on the subject problem. Frankly; wo are ccncorr.od about the paooible liability which cay well roouit fren tko ucu of Huron or any other herbicide for aquatic weed control* I will try to put the various problems aa wo coo then in the follculnc paragrapho* ns CO 1* P l D h * Tiie data that hao boon obtained by A, w. win3ton, Jr. in the waoto Dlcpouai laboratory indlcatoo that a ocncontra- tlon of 5 p*p.o, io probably tolerable for at loaat a few days to tha lata Eaorald Shiner Me have no data that I an Qtiaro cf rolotivo to the affect upon fieh# and other valunblo equable U f o fop that Ratter* frem prolonged ex posure to concentrations off 8 p.p.ra. Whereas a concen tration of 5 p*p*o night ba suitable for corfcaln opocieu for a feu dayd 9r.ua liauo no data to ascure uo that 2 p.p.o. would fca cafa for doolrablo opcoloo over a prolonged e*pooura nuah aa would occur In tho use of Kuron. If a lot of floh wora to did oubooquont to treatment with Kuron* I fool qulta confidant that It would be bloaed ra'cardlenc of uhothor It wap* In fact* responsible* or not* 2. rathorn * You have rained a question about bathers. I do not ho llovo that tlioro lo cuoh likelihood of batliora being ad versely offootad by 2 p.p.n. of Kuron, However, wo do not have data which could bo used advantageously to prove ouoh a point If oonoono wore to claim akin irritation, rash, or whatnot from water which had boon fcreutod. In my opinion tho probability of difficulty occurring io rather email but if an allocation ucro Dado I am afraid wo vtould nutbo in a strong defensive poult ion. *. h* Coulter rfC*I.. f/ '.>j i1 c_yv`` 551252 5.. Formulation I d> not know wow tho material will bo upplied but, 1 would ualto ovary offcrt to avoid oij., ;>urtl;rularly oil klou oat a lc:.; odor thronhold. .-no of tho easiest ways of oroabliv.; tyuorvIona in to treat water with a notarial which can readily bo debo-ctod by, odor, l*unhcrtaorc, xsoi;t oil:, arc- fa x* core toxic to rich than, aro the herbicide of tho 2,**-r: type. In fact, concentrations in the notgnborhood of 1 p.p.n, of oils ai'C in ooibo Instance qjito lethal to rich. . *l. Municipal Water .yntorar., 1 do not know whether it i piannod to uuo Huron on Irapouirucci watora tioslgncd for Ivesian concuraption a 1 n o t . X would bo ceneuhut apprehensive about each `.aterr., not bocause I believe they will'constitute a real unuard, but because of the furor throughout the country today in ru ga* to contaoinaticn of various fooda and drlr.hr ..ith onvinicala. In certain arcaa Inn/<;undod waters arc uned directly for drinking purnoBoo; in others; they ara filtered and chlorinatod prior to uno. The effect of nubh trcatcer.t' upon a water containing Huron io unknown, whether or not it would produce offensive odora should bo investigated, fosc.r.dly, it could be alle/^d that tho treatment wf suck waters amounts to undesirable contarairiaui<,n of tho drink ing wator of the public. Again, I da not believe we would bo in a good position to defend ouruoivo:.' should ouch a t i c otlonn bu undo. k * Livestock and m i d l i f e . I would also v/ager that the question will corse up an to whether water from a treated lake would be suitable or safe for domestic 11 vortoxic an-.i wild aninuis tr; drink. At>ainr there 1;: no- quovtion in ray mind but that ruth water would be cafe* However, wo have r.o data to prove It an. our opinions would bo our only defense. I doubt, however,If this will be aa nori.:us u problem ns: tliat of water* be in w e d for human consumption. I co n o t wish to bo an a la iw ln t in ro toi t h o r.o wf / . u r V r a q u a t i c w e e d c o n t r o l b u t I d o t h i s . '.: i i r t v.o wi i.- ul d a - . I , ; i v e r i o u n t h o u g h t tv, t h o p r o b l e m v '-.1 .a, may a r d e o a.: a ; v , w i t o f *h u s e . My w'-ri f o b l i n g l a t h a t we n ' - u k i b-..* vyr\* caat.'i.rwn a b o u t ; ;r .M ' - t l a y t h i n p i v d u c t f u r " : u r.u u v . U . i ....... l !' Li' Gouitor -j - armar? _ , IO1-3 nuifolcnt cctpcrlcr.ao hnn feecn c^slnncl \yj covo:T:::ontal a fenolo a and tho Ulio ubo oioi fco toriato ito uno to ann;-:as acca 'ci ttxo questiona ehch I fcavo raiaod in tho puroCT.pha efcova. In ony ovant, 2 toiiovo v:o nhould reocesena arcioni; tho trc-ataor.fc uf petabla uator cyateca I u i n ha Elad to dloauoo tioso pr-bieca uith yoa et any titao. DOW 1 551253 V. II. Howo lioahoDical Foncareh Laboratori I?-ij34 Eaildinc Phona - 8701 Vltt/ba eoj P, u. Plotchor 12. L. eccitar K. C. Earrono J. W. Eritton D, D. Iri^b V. K. Rowe (2) T2.30-40-20-- kifv : .) S-*V/V/ V . . -- JO -V Pj-;L Lab o Uv; _ cc: \ia t.710 `0 v/ild; .o Uo Kie lcl EVALUA'JlJCJ Cf U L__D CC-jTiiO.Tj lJ.'ITil ICUHOII :-: ^ v/AJQ '-.i -ji During th o la , two years wo have had some very excellent resul Cij i_n 1I.fi:.,,.j hautcra states ucins Kuren (Dowanol eater of 9-2,9, 3 - t r . iohlcrcphonowypropionic acid) at a concen- treeion c. i /o. ...i warto par million for control of aubmergent aciusclc w ;d0 The normal procedure ia to apply Kuron to the pend ni:rf: :o just ha'.Lew the surface in sufficient 'quantitie, iriltl a coneontration of two parto per mil.- lion in h volume of pond .i.'.oor< l:e do not know how leap . -i however, v;a do have come tocta froi,i acta 1Id applications- made this year and know that it r .v ; as lonq as a month and probably last lorn:oi'a 1 .lory work is continuing cn this aspect. In any event , it is aonarent that wo will want to inten sify our efforts in ijovelopirv" this use, at 'least, for certain phases of aquatic wood control during 195*3. Ac tually there are a lot of questions that develop in con junction with the addition of any towicant to water. These deal with the effect of Kuren cn the taste of water In tiu event that it is used in a reservoir,' the effect on chlorination, the taste of fish, the towicity to other organisms, etc. Since the performance of this product seems to be sufficiently established to promLso at least some commercial use ultimately, >v;e wonder if the Robert A. Taft Sanitary Erysineerini'; Center ml^ht be a ,'jood organ ization for us to contact in conjunction with some of these problems. Before doinf; so we, of course, should decide what work can be done hero and what specific projects wo would be interested in having them work on. Since you MN07328Q CJ1 or M ro 00U t j 7 / 2 `V ? * I 7 / 3 ,/ 7 ? I ^-- --------------- * n 10J n RESPONSE OF DOW CHEMICAL U.S.A. Q TO NOTICE OF REBUTTABLE PRESUMPTION O o AGAINST REGISTRATION AND CONTINUED REGISTRATION OF PESTICIDE PRODUCTS CONTAINING 2,4,5-T o CO a\ CO cn Exposure Analysis - Humans Realistic Evaluation of Pesticide Application and Use z --, U J o LU UJ O' T h e f o l l o w i n g is q u o t e d f r o m P o s i t i o n D o c u m e n t 1, , F e d e r a l R e g i s t e r , A p r i l 21, 1978, p. 17 13 7, S e c t i o n I I I (3) "in order to determine whet h e r a r e b u t t a b l e p r e s u m p t i o n should be issued based on reproductive and fetotoxic effects p u r s u a n t to S e c t i o n 1 62 . 11 ( a ) ( 3 ) (ii) (B) , the W o r k i n g G r o u p CO f> TI T z < UQJ Q_ C UJ O4 U Cl must determine whether or not an ample margin of safety exists between the levels of 2,4,5-T and/or TCDD which produce reproductive and fetotoxic effects and the level(s) to w h i c h humans can r e a s o n a b l y be a n t i c i p a t e d to be e x p o s e d ." ______________ * alro i n t & r c y tTiTg^ta luTueNLLat ~Jhe f o r k i n g G r o u p --- --- ----- ------ --- ^ ^ "ZkjuC * j3i mi fr-s-- out tjiart-- t-h cy 'h a v e b e i m -- 1 e q ui 1 ed1T U r U L u n s i U e v 'th 'tkv p o t e n t i a l e x p o s u r e to that p o r t i o n of the p o p u l a t i o n at risk (women of child-bearing age) because of social changes which have given these women the o p p o r t u n i t y to n o w be employed in o c c u p a t i o n s such as "pesticide applicators, o p e r a t o r s - o f ii ^ LU ? s highway construction and maintenance equipment, foresters a nd c h e m i c a l f o r m u l a t o r s ." With r e spect to r e p r o d u c t i o n and 18017 a n i m a l s t u d i e s , a n d (4) ju d g m -e-nt bftirsd'iq-xyn b o th l o g i c and scientific perspective^whether an ample margin of safety i n d e e d d oe s o r d o e s n ot e xist^ C Tpn t a t i o a-gr birltiwii 1j e o n s l u d o d :# /f//zifLifdi**. ^ S '- ^ # 1 . 7 ^ O ccv .4. 4 , 5 -T-- fo r TCDD)-- de^es -po t o o w i r 1 i.n t)ve -humaft eed supply,^-- ThirTr^gTuT^mTirgi n nf- m il~rfyi_L r n rir g.roat-CT-- lliail -t'liaL esTiuidlid U)'1LJV . E PA ' s e s t i m a t e for e x p o s u r e of a p p l i c a t o r s is grossly exaggerated. An ample margin-of-safety does indeed exist b e tween the levels of 2,4,5-T and/or TCDD which Z~~U. if p r o d u c e r opi'o-du e t -ivo andjt-o-y f e t o t o x i c e f f e c t s and the levels to which humans can r e a s o n a b l y be exposed. O ro CJT CD CD to B. O r a l E x p o s u r e r ^r/ ffi. fi Ct E<</Tl lit if .. * \ -- <11-- / C E ^ y considered range and pasture grass uses with their "worst case" assumptions meat and milk derived from laboratory type studies (EPA #37) in which cattle were fed f tEtCu. " % 300 p p m 2 , 4 , 5 - T in t h e \ d i e t f o r \ 2 - 3^wa^Ks7)v A l t h o u g h ther-k _ 3)' calculations resulting t'herefrom were deemed as ample margins jJ r y j* of s a f e t y by the W o r k i n g G^oup, it is i m p o r t a n t to n o t e that the m a r g i n of s a f e t y is e v e r \ g r e a t e r w h e n one c o n s i d e r s the levels reasonably to be anticipated. Hin H` liHI-f.Mf.AI. t.OMPA/JY SJ O to cji CD 1 NO CD CD A. D a U rf /* -<n7*-- -r**7-ftht 0 , c f (j*,L ^ />^) &Jr {un aya. <a/ ^i^ yS; _j)=i\tJ." >yad)N.r'j - tC ^ ^ * ? f -^Ct. o faa 5 Z.y.S-Tj j h _ Z&-: u**~<*r " ^ -/?ifdi^t-t ^Vdl^Zu . /C a ~ Y^C& tA-L / \ a.6->id*r<jj ^ - ( g u r t^ ix i^ U x i. o *^ -71 SVTTA-lst L4*L C P/3 '* C&n&*46*'K.Jy /tta/^cA J 4 A*Ck^4, fM ^ +*~ 3O o j < UjT-DTMJLgU|*JJc ^ 'Z' s^ s4L4J-^/AJC%y M&~'h xa**-*+D<4. K 7" mAX. . axu. / X louCLoL ^as-zJl 3 r`/.`5 '- 7* ~' T** /L&. 0^ /aZr?u~*y' *4*- . &- A, < u Z<*^ ^a-* 7. ^ CCtr->>~~ DU))*-C. ,. 0OX.. c U ~ 3. *-//? ^/z? 0^ ^ t*/. *7 ~ / X. ?* ^>1--OJ ^ Q S&+==7&C ^4ya*r- ^n+cuMtr < y y W jjst 0-*Cs\i. Xsr-- '/IAT&~C**1. 2S fri^KoC ?7 y 3?***- ^ 96220^ i!i>. ili 11 ( (D j s/Uh <U JZ. */,S - 7" y^yrvC A r atsT U ^ m aJ** sV ?U A <W /<- -^ n , 2 ^U rC uJi^ JE & t l /Q2.4*a^A+* cl^AZ*- ^sh. L^Jau^j ^ ~(tJ~ L*u~meU<v /yyLu^r & ^r^&CT /t. ^-4<v*^ <^w ^ > yUj-jjJ^y> O-^&L jtZL*&i+U*n~ y~%J*<LJt. s/Lh Js\2 ^<-A. Ca7 /4*u*+nL'^L C r 7*rf^C+*. yai. u^ZZa^, sfi^cyZ&2i ~n. J' - -" - u<J X.Y.&-7 AA*. -^.aJ^CLfaJ ^rruJCAnC^ ^n. WKHmT y^mju~ {/*/ Afe.//^s/OX. / i4f " - y/v^L co /-A. o -c^a^i i + 9 1 1 Z /ytuuW tj sr>lu, sC&u/L 2. % - 7 Kfc Z ^ 4 >LlZ12-. s ***xi. f f / ? O/ /dl<.j|'<1</i ^ U , u/ 10020 aV.' Table IV SU:'J-WRY: NORMALIZED (SPECIFIC) RESIDUES OF 2,4,5-T ON TREATED GRASS Location Formula tion Applied lb 2,4,5-T/A Applied a.e. California Michigan North Carolina Texas ESTERON* 245 ESTERON BK VERTON* T VERTON . T ESTERON 245 ESTERON BK ESTERON 245 ESTERON BK VERTON T VERTON T ESTERON 245 ESTERON BK VERTON T VERTON T Trademark of The Dow Chemical Company **A few intervals are approximate. 12 6 6 1 12 6 12 6 6 1 12 6 12 2 Average |atA. CO CO to Averag e Specif ic Rsidu e pp.Ti/lb 2,4, 5-T/A Applica tion-Harves t Intor/ai V.!<soks * * 0 1 .!2 4 3 16-iS I*. 27 42 !27 39 11 7 52-54 __ 65 -- 15 12 4 _-- 63 6 23 16 9 2 ND 49 32 26 21 10 2 HD 112 63 :34 13 178 91 *41 18 6 8 4 5 _ __ -- 123 96 46 109 100 107 96 112 92 34 30 4 25 il7 5 21 i: 5 2 24 35 JL - 48 I s 27 53 122 9 i ij. 24 8 142 r~ 54 :27 20 1 2 1 <1 1 13 12 3 _7 7 <1 <1 <1 <1 <1 <1 <1 <_1 2 ___ -- ND :d ND ---- ND 0 : c\ S96SS(y^o a / pjLcO 5Z! _L- . 9^ " ^ A ^ t ^ ^ ) < v ^ZAa U~ /If 0 .jfatu'A***+ &L* C.*^l.L. /C^CZtC ^c-*c4. V. 7~~ 7 rfiT l -rgy^u ir ^n ir j ^aZU Z1 ^ H. / < >y<i./rt.^ctfvo t>t / y 7" ^_ aMW* fJJufAJrf-' t ^ d A Z m ^ ^i&C '. 2 a ^ T 3 2 Z aLcZTe/h' J " <^4 ASo ~ f~ C A a t Q ^* y t~ * . /U < * ^ ^ L l^ A , j ^-I_ ^0. 7" ^4Am . 3 St*y'/< ^ //t<3 "7~CC/> j ^ k ^fc/. -7. < s - r j .r /jmAj ~tZ** /X*.***-< 7 ~ * ju S . E**JU * 71,H.**-T sr^<n- 7~c>2> X*A hyA- &f 'Zi,-*rt*CUl `^T&UZh ) pZsust-*<c ytxwxX- 3.J. Yf S~" 7~. yX&Lf yy\MA-*u X-d / u W < l<4< 7. XsC Oj fj/ o U. 18022 DOW025967 States as a result of the present uses of 2,4,5-T^ mari(iS$f-safe;feY im pl ie d b y ^ P A . e n e n th o u g h cxThsider^sj by C. Perniai and I n h a l a t i o n E x p o s u r e In considering exposures by dermal or inhalation routes, twfc*- KJCX-- if JVi,I ^and the W o r k i n g Group assumed c o n d i t i o n s in w h i c h a pregnant woman, weighing 60 kg during the critical days of organogenesis, is carrying a b a c k p a c k s p r ayer treating a right-of-way or spot treating a pasture or rangeland, or is c l e a r i n g b r u s h wi t h t r a c t o r - m o u n t e d low b o o m s p r a y e q u i p ment in rangeland or right-of-way, or during aerial a p p l i c a t i o n of 2 , 4 , 5-T is e x p os ed d i r e c t l y b e n e a t h the s p r a y plane. EPA's estimates for exposure of applicators to 2 , 4 , 5-T are g reatly exaggerated due to a series of erroneous a s s u m p tions. The m ajor source of error is the linear e x t r a p o l a tion of data for contamination by each of two thiophosphate insecticides applied as fine spray for m o s q u i t o control, or by a contact-type herbicide applied for broadcast "burn down" of ground cover, as representative of what might occur with 2 , 4 , 5-T appl i e d as a systemic a u x i n - t y p e h e r b i c i d e to weeds, brush or trees. The calculations also assume that all major contamina tion is due to contact with diluted spray during application^ 1 DOW025968 O 4+enrt-e-r the g r e a t e s t p o t e n t i a l for d e r m a l e x p o s u r e o c c u r s during handling of the concentrate, such as in loading or m i x i n g . E P A ' s e r r o r is c o m p o u n d e d by a s s u m i n g that v a l u e s for total exposure to the c o n c e n t r a t e in the dilute spray are due only to the dilute spray, and by extrapolating from the dilute spray (e.g., 0.06% fenthion) to a 4% 2,4,5-T -solution'. - Consideration of such mediating factors would appear to be essential to make reliable estimates of the potential for exposure of applicators of 2 , 4 , 5-T. The information s u m marized in Appendix V gives the following reasons for the gross exaggeration of human exposure analysis presented in the P o s i t i o n D o c u m e n t 1. n a) the linear extrapolation from data for contamination with totally different chemicals applied under d is similar conditions; assumption that all applications of 2,4,5-T were at the high rate of 4 lb/acre although most are a t to 2 0 __CktJc /CjiLLe-n. -& /oytli**-*' ****+** lb/acre; -and "ifi t h -- a d i l u t i o n -o f 'jrrlO hi ft nd uux *cn Vo CT J 3 ?3J w h e r e a jso-o yni,, mns-f-- 2- -3rS~-di'l'QTetl 1.100 and-- iirss---oofrten appli iedu nm r- . n Oil Ox oCL!***'&*.AA^<v . assumption that applicators would be wearing only very light clothing although most states require pesticide a p p l i c a t o r s to wear m i n i m u m p r o t e c t i v e clothing, and the fact that even wearing simple protective clothing 025969 o % 10 as w o uld be required by following the label p r e c a u tionary statements would decrease potential exposure AcCtL d o w n to as t ew as 2% of the " u n p r o t e c t e d " v a l u e s ; (4) d i s r e g a r d i n g c o n s i d e r a t i o n that the g r e a t e s t p o t e n t i a l e x p o s u r e is on the h a nd s p a r t i c u l a r l y wh e n h a n d l i n g the / concentrate which can be avoided by simply wearing gloves;. 0^ y&u. 0^2J<A^Ctrvc La, -^4. ^ O^-iL tbc" yf Cl2 ^ j j o j u CZAc.*-iA. L\<dtU.\j ^ZsU~eAt*tJL. g ) tX^Ay4-b~~c.aAZ-a^<. j~o srru^-<A. ol JzAx. oU^s^iAzZ^, syruXJL* (r7) Vd^w^-rvy*^*. sC&U" riy^2-*o--a~i<s^* '^**2 leess ^yCiVS^ dL. Js 3 O a uf JZU. 7r- 'f0i <U. ^ s7tU+**^++*<s ^ZJuX~+^ O . *>0 tiSU<&*L /tf-t*^ <>ux- ' /UfW ^fackH*~} fa ^ (. 'f("f 1 a O rvaluation oC the procedures and c a l c u l a t i o n s u n d e r t a k e n by the EPA W o r k i n g Gr oup leads to the following modifications: to en co o -- /^ r r o r s ^ i s } a s s u m i n g r a te of a p p l i c a t i o n and d i l u tion of s p ra y --^afto 8 times j -- ^liisregard for protection supplied by even simple . c l o t h i n g -- u p to 50 times^,-.v ; -- ^ 3 v e r - e s t i m a t i o n of n u m b e r of h o u r s p e r d a y e x p o s e d -- 2 tu""4 "Lijucii Eis^or in qijiivf'i^y assuprd~~aT>^rbed^TirottRlL s k i a --- -s /0^ - UA ' -- * E r r o n e o u s a s s u m p t i o n of e x p o s u r e b y i n h a l a t i o n of"--- D , / ^ s p r a y droplets, particularly for ground applir Amo Z3 c a t i o n -- -to 10 t im e s , ^ "jy-c a M m . j r f*if<i >^c Xn- Z.V.* +2X i* u r Xa-a* &-v~C*- ^ 3l . swwit /o o P a r t II o f A p p e n d i x V p lerit's a r e v i e w o f - i n g e s t i o n studies with 2 , 4 , 5-T in human data from the exposure of workers manufacturing 2,4,5- data from workers obtained ' during actual application :T. F r o m the m e t a b o l i s m and pharmacokinetic studi demonstrated that urinary levels (amount excreted 24 hours) can be used as a W a s u r e of th absorbed by ex- HQ C O Q o / v T - (j^ J S , SU a i^ iS I _^lfrrri- s&tu-eiiXe ^<AJ-ic- 3, Y.^~~7 < W 4t+-rUL+*-<* ^1+-**- 4^swa-Ufc^ ^ ^ ^^ / J Z -~ * A . dj4yz*^r^ 7, V. 5 - 7 < 'liyO^t^j &&- /w^V<-w*. /Q*Lvl&Ux + j -o ^z^4^-7i<aZ!xLis Ja ~ T ~'/>t*'ifiiAr\t-it C.&-+1. j< yU^XJ^L "i JL-<CtyruAX- -TU. LrC -cra*~j, y ^ v ^ Cib^tr*-t-<L A^yc^z-*L i/siyrrxajL (^syyx*irl&^ syturtu. a 2.Y.S-7. ^ Y/ -- / ^ou^a^, A-^cJn^a--cAt* .s^Lsis-CoUy /7CA~ J & c ^t-ZZL<Z. y^^x^<-cty\t. O . O (o I sC<-~*-YLtsi, yAjre-iscut e *-*u. " Ol V u AT O .o 3 yl/-t<7r ^UlM^CJOvc^^T" LaaJ. /f"< ^^^ui/uCo-uo Zv --vt>+-A- * " *1 ^ 8 0 2 8 C^o-v^ay 6308! ~? y ? ? ~ n y j _ y7 V<??J. A <7/ X / ?- t ^ ? ,rv ,;>'^r^ 7,' '7W 77 ^ y ?y ^ y $ jv y ' ' ^ -_s V# 'r * / :TZb^iy' ~'^nZrZr J_ -& '/ `f '**'' -i i > V t / 0 "P - Z 1? jy> * r7 ^ rvT ^ ( 7 Q 7) / ^7 t v ?~k c - 0 ^c/ tP j r ^ -y^-ryv^ j*->-(U\sq7 o^ ~ r> ^ y' 7pyT?i*-0 ^ T r 7 1 / yt-vt^p -?**-r*i`t> >?'A/' yyy*?' J L - & ' y x Jo ^?~>J^P>~rp^-dy9 (A^rX'yyqO v*i*yi~c~ty~d)\l' ~ * Q ^ iy'/ L**' t o 'o ~>~y2y '*rf ^ r y T Y TT~r**jy*' -rc-9yp ny%' ( '/i '^nyr^roh/ ***' yr-r^y^^>^-t>^y0 ty ^ -- 7 7 g > >22ZrnA '^rvy -O A^ O r y ' ,6^i(>7jy^'*~rrvy~tn? 'ryZ' *v 'U^V^' -J <1d=*=t~ '~-f<>~rrjrT*uA-truy' Jo G/?'' -Z*1'*/' Jf -tf^x 7^ i/-=> </vrtny ^ / hi Qq O \ .i. * oeo'81 ` 7*TJ 1 "X/Jy ^ { (__ O V ^~" z r ^ r r r r 1^ ^ v/S *:*f7 ~ r > v y L>r' 'rryr-^rr' l&-yy(! v w / 9 0 -p ^Tr" *~yjrxr>.;yx/t,O -ypT-V vty '7'tZyjO *y ~Lpt-g i r mrd/t * -r-pz* -T^pZ' J?z>~0' 'tyj' <-*-ny_ * yT<>tf t7ryf' ~v^> * i "V Y nffy r 'ry^' ~r>^-e yzrt**T/ jy*rxt/ rpZ' 2 w-tf' j^yjtP 'Tpv^' T-P *ry/` 'v*& ' ^T** -t*rr^?-> -j*f-vy&jvy" <va ; rjtJtv YT*srp J*3aaa*cp y^f'/PrU'*^Q) 2*.-- ' TM-- c/tpT^zj/' -yy > T W ^ -cA+dp' 'Wn-vy' '*', y 7 e~>*pnrrrpt>AyA? Tr-qr>rfyr ./ / , <-acrwe? `yyp?~^Vy nqnrp xp^vpyaJiyy >,, 0 C & 2 m \* i 7*AA /*<*' A- (fjf 'rfi'iA Z & ry+ i ,X<f O A. * ^ ^ IFflQ Z J* e P F /o o*tj j $ 7 3 ^ , C&aa-%x-*L. I A . ^~ ***-1 22vt * t/.is^Ti. cLo-a.+-^t M-*s au~L n/t-n. 0a<j /r- ctusSS^ . /}aLsLot&an+A't' /a><x/<M W J-t-n-ck -a^-U Asid* jr , c. / */ kt&~ -Are<1U(Lf*, 7^-,, TVt -r/- ^ + X C s U S c Z a. cjU*- -/*/? '. S i sttt /*Jt La ZZL d*td --c^L^f , lV*feflL / Y 0"`be*-***- ( /C 0 M * J L A- /W W ! sa^*-V j p*S -- jLr**nrtiii^iii, n -- r G> /WA| ^*A*>V *-* C ibt~T^*TJ-=l'',*1^ --- ^ax^^o^L 0 ^fidcCn. s4/vb*A. &A*0 u JruXLt - s*. /ju.As^*. P A t ^AAU. /c yit7T^ , CO TABLE V TERATOLOGY - 2 ,9 , 5 - T 0 . Pf 1Fno z Xt~L / f 7 -?- \ AUTHORS Collins Wil l ia m s . 1971 y^FORM OF SPECIES'1'2 , 9 , 5-T Hamster Acid ROUTF Oral DOSE LEVELS MG/'<G/nAY 2 0 j@XS0.1QP 'dRTifcY il /LL., 1970 - Rat Mouse j 30 ppm 2,3,7,8-TCDD :....... - - Courtney Moore, 1971 Mouse Rat Ac i d Ac id SC (5$, 1 0 0 ,1 2 5 ,1 5 0 SC 1 0 , 2 2 @ S 0 , 2 5 0 Khep.a McKin ley , 1972 Heubert Dillman, 1972 Rat Mouse Mouse Ac i d , ^ Salt, \ Esters ( j Ac id Butyl Ester Oral Oral Oral 25 @ , 1 0 0 , ] 50 8,15 ,4 5 ,6 0 ,5 12,74,(@ ) ?CLL, 1971 Mouse Acid Oral @ ( 5 ) 6 0 , 9 0 , 13i Emerson i L , ; 1971 Sparscmu EX &l . , 1971 Binns Ba l l s , 1971 Rabbit Ac ID Rat Ac id Oral 1 0 ,2 0 .@ ) Oral 1 , 3 , 6 , 1 2 , Rat Sheep-- Ac i d Ac i d Ester Ora l 5 0 ,1 0 0 S O ) Oral 1 0 0 ,(> 1 0 0 ) O = Mo- EFFECT DOSE LEVEL FOR EMBRYO AMD FETAL TOXI CI TY. 3033 i . 1 Q A Qj 5 y ^ , fi.* * ./O q \ ./? % *r^ ^ 1 y ^ ^ t . D l f ~ e . i t * T L * < A ~ lAJ*dl***^ r/\s+ ja f s&yi*j-+X~ 7*4***i t-.'u^. y*ArA/**cAu*. _^w^j- j. in_ C U ~f*k nSRT?:-' o f s O i> ^ j A**^*C*. J~ C OC> sur*+A. yi^trfLt^&L. &U cl-t*~*<- g f Z .Y .S -T t*V*-iL X&4- ti y^C- ti '&< J jzt<_ /twt~ /stiMiL sUriti-cf '$*fj S*s&c*AA- 7V *y*"*4. tt n> <cC ^ a_ d^ou, c>jt 0.03.% fL^ `'^j ^L*\J^6Ja JZ*~u. .sU*. Q* ******-*1 ^ ssCo-c****. u^a*3 d*4~^A- <*-*<- s 4 O-^jLtU^ 9-'*- ^t^***^o ^Cjh-h do-^u. th & Lc sL ^mAt shahid. 2.0 s>*y-4j /A<^ ^ n . 3,*/,- 7~^ CK. yyyliL^c^u C&ir*4A-iLiSi4JL N Cti^ i n i ^ i r l i ^ ^ d ^ L ^ l A - O y t t ^ . " 2 0 J 0 . 0 3 * - S>~7O -jjhh- *0 -^huT * e n f t 7+<*%> a, d i* * * 3 ~ ^ o J s u La J . O jO ja 18C34 4 in c c MT,Mit AJ fV o. V V9. c<9 IaJ LJj/b^at t* &<&k , y^2A.L^^ / A u * * ^ L-'Oxjx-o^t w . yCtJr a~ jd^ot^aU&u JU* s*v**^~*^ OL~4>(.<^<A-&j1 ^ls\svpt*-J-^s~t. L*+X^- ^QsC*^c-<. t /\L A < 4 -n .A t. ^jtA U . t i L ^rzy^L y ^ B ^ n . ^ C ^ ^ tS Z J r vL ^.S-T O . O S ^aytr-*<_ ~7~C>), ^tt--irix*mji> :j. ^cof1 sC^u^o*~ Ao >-*./}f-fyi^L+ yi ' . 4 . ^ x r^Jmt=*7&3EEL tt hh-, L rn*-a. tft V ?, `/ . ' S - *7>~0 a -~* 7~c & _ L.>-**/ yAjl*t>+.~4.-- 7->TX9---TOg" .> / /^ ^ggg * x r -4.C /< f t r r 'T T r ^ a . O O O S X./ 7 X - s^yyx,Ls<-~4y***r*~~aj r~ ol^ ^ L --6 O . O / X O. O S X-/o rz^ 5" X /o - 7 "^3 / ^ j itj s& ?*+ f9Asu-e*- &> y t^ L yjjL^r^'i*' CrOAsLsa^-^- /yt* -- ~ J^L 2 / O. O i - ZOOO-^ t r U . ^n. 2.Y.S- T >, / 5 X = >fO O Q y^td. T C 2>l> ,, /6 t-^ /Ca-rotS-i, . jcL. -1: O C i- " >o "i-1-*<!V i.V.Mr \*jV <a V % P '2 i ^ /O^-t+U-d. s?4. , o u ji- f it * * - 5 } /V*0l ^n. --xv-j.. C i y t* v t 4 * <A^f^-r - t * ^ * r j " / t ^ . ZV M -4 M^t, /+>&** *m u *i ,4-, s~ Z.^'S-T M / 9 7 / - 7 ^ - 7 3 j fi-ru- M e sZ~*. cL> h*rU -M ** Z u. / } ^-rr\ t +*u.* .d jc& i. ^O irC ^y ~u. / T U ^ n e ~~ ^v/^ys^T ^a-i. iM , ^ n J j ' f j S ' - r P & e*s- # / S o ) . ^L u^cJy. J-t<*- - c ^ 1-<X (U>4*CtjXjL^ ^91# ^ i ' j y / ^ r* i1""* "^7^1* i-- -7^ V i f7L= =^Ss>~ 35 . /i-- ii^ r. > ^ / - U * . ^JuT "ft,V/? "/ I ^ i~ i i- ^ ^ 1 / ^ i - . S^ti. ^C-^'h. 0L<^ a^c*^a<'*'t-^ ^CtUr p\ syyv0-u 2 t*/M " T" S&l+M. ^L, 7 S&t. /CcrU< /A^O^ZU^ // - tZZ~uzj' \."Ti-FI o/ 3 y^sf\^^LC ^T\. /-- 34-AjL-h ^CZ 2m L*<-+*^aJL / sC^TLa ZZ-^ ^a sfrj s M J sfryi4-x~ V j ^ s7r3 **'+i /? f r ~V Z-(c*-) * - r n ll ( b j r t ' /O ijL*Z y j y\.Zsas /.ct--v-*- i JjLA^y. Cleric. Z i ? . * - T Osf yKS3 sis^6-J*A*^ir^-'uZx*t<. ti ^s^^sL^t TZAscs-- ^ s+ fti.-3 x^ ts 6Cr~s>^*-' ?sw-*- tJu- f ' 2T X9 / ^*-a/ t A stiswk/' cL*s^~**-+~+ l4-AUyn& *<-<->, a^ztt*** .A'i Zi* ^7 "7"C D > <trMgi!^jTT , s/j&&y-t-L **nL ^***-^+***-1. J^ `- /X O s t& J * * t & I * *-U y tls ^ C L 0 T * ~ > - . `- iC f ' 17~<r c^- s n + r ^r^L 7~A^- /i />iv0-- "^/O^! /Cky/T ^yyr^vt ' v yOr+^tctU 0/ r A / W - t , rf /--AspaA~/zr Z?f~ /UJ4F+T.S ^ Jirtj / UU#>u^t- ysi*~r*fx*eJ? , Z* ?. y- 7" o~'^*-/si- 7~<?> `j S*<*< ^ *>?<.fi 5r-- 7" <SHT~ J00O~ Z TABLE Via Safety Factors For Selected Drugs Drug Dilantin (anticonvulsant) V a l i u m - '( t r a n q u il i ze r ) Phenobarbital (anticonvulsant) Meclizine (antihistamine) Norethindrone (progestin) Norethynodrel (progestin) Vitamin A Acetazolamide (diuretic) Morphine (analgesic) Aspirin: teratology reproduction - Acetaminophen (analgesic): reproduction Safety Factor* 20 ' 17 0 * 10 12 50 4 40 7 666 3 <2 <6 *The n o -effcct dose level in teratology studies d ivided by the therapeutic dose for humans. See Table for further data on the drugs listed. J Cjj * 4 vrij S a f e t y F a c t o r s F o r S o l **' r f t ! Jirn ^ Prin: ..N.-o-- K-f -f c-e t--D o-s-e ____ ______ T l i c r a p c n t i r r)M* S p e c i c. s Uo n t o | ) i I . m e 1 11 ( . m e i c o n v u l N . i u t ) 20 UUe O ral V.i 1 i u m ( c r . n i i j u i l i : : o r ) 170 M ice O ral Pi e. n-t> i r l < i t j ] ( a n t i r . O n v u l * 1111) 10 K .jb b U O ral i.ii,hrv'iic;-:ic _Teratogenic N o-rf feet Doae ___ II Fj _ ^ c L Ob s e r v a t i o n s K e f o r c m :i? 10U laji/k-; 1 2 5 iii; : / I : j : 1 5 0 m j j / k i; C l e f t p a l a t e M u r r a y o c a h Tx. A p p l . P i i . i r m i c o L . I ' J 7 8 , I n 1*r 100 mc/k - 1 4 0 m " / k ;; C l e f : p a l a t e Ml H e r e t a l . TAI,b 1 2 , 5 2 - 0 1 , T'j 7 3 - - 50 im;/kr. S k e l e t a l d e f e c t s M e C o l l e t a l . TAP 1 0 , 2 4 4 - 2 5 2 , "l'.'47 M e c l i z i n c ( .H i t i l l i S t a m i in.') 12 P.;i l O r a l 3 in|*/kj; - 2 5 n i '. / k | ; C left p alate mlcriisLonilr. Kint;, C .T .G ., S c ie n c e J A I , 3 5 3 - J 3 3 , 1 `V O N urctliiu.lri no ( p r o ^ t s t in) D e r e t l i i n a d r c l ( p r o t e s t i -.0 I'Uam in A At 1. La a l .h i t i e (<J 11 r :L i . ) M>r pi t 1 t i e ( . m i l It ) 50 40 7 006 Mi c e Or.i 1 0 . 3 n j '. / k i * Ml Ce O ral 0 . 1 r) nif; / U| ; : i i c l- O ral - H it Mi.-r D idary AO mj' / l . j ' SO .`Oil mi*./ kj ; - 1.0 - Andrew cc a l . T e ra to lo g y 5, : i y ('T y > i - 0 . 0 ni: ' / kj ; Crania 1 O iilley et al . T e rato lo g y re ta rd a tio n 3. T r)-} '? + /i')7 Q | 400,000 m /kr. K x enccplialy i (licit p alate M lcro filith a 1.nia C i remJ e t n l . C . K. Soe . B io l . ]Vftt i)6 i 15 0 m;',/kr. 1.1 ml* d e f n r r . i t I c r . i l . a y t o i l i*l a 1 . S c i t n r t K-y, 306-30.1, iyr.3 " jnu n i ' . / k r. Fxciir.i'plin 1y .fnbaniu son et a l . A rt a P b n rt. Toy.ieol. 31. |0>- 313, I'ill A`i|i m m : U T i i f n l ' ; ; y 3 P it O ral 200 mr./kj; 225 repro.lnc l Ion <2 M ice D i e t a r y l i f I ' er . L: ; a L 1ii wi ' i i L t]t*S>: , 1 1 0 n i ; / I n : GO CD Ac tarn in>|>hcii. ( n u i l ;h -m ' ; : e - p r o d u c t i o n <6 M ice ' M. t i . ci l o n l l i e i m - c f l v l sc- l i v e ! i n l e r a i o i u p . y s l i h J i c s . 'r.M1- Tnxi1:0 I*/.V.'11J A('j`Iit.-l Pliar.-r.acnlop.y. D i e t a r y IM f ei: t s a L 1 n w i - . i t l o s e , 1 1 0 i u i : / k j; *1l i <>np: . o: i c L a l . K** j *i t o f 0 . .i. C.ii.|(.iny, ( n o p a l ; 1 j 2.It. .1 J a l . i ) W r l p l i t . II. M. :'h o -:")_*. i mo? TAP 11, Wi i . - . i n . i i . 2 M O - : 9 J . 1*0.7 tap i i , A>(y k. Corrected working copy 1.0J 3. CONFIDENTIAL - SUBJECT TO INJUNCIIO N D .C , E.D. M l. 4 -4 -7 8 ; D O W /E P A AGREEMENT 9 -7 9 O O V V 0O 1 2 1 6 2 WHAT THE 2,4,5-T R PA R MEANS TO YOU D O V V 0 0 1 2 16.1 1. On April 21, 1978 the United States Environmental Protection Agency scheduled a Rebuttable Presumption Against Registration (RPAR) review of the continued use of products containing the herbicide 2,4,5-T. In view of the importance of 2,4,5-T for the control of unwanted brush and weeds in forests, rangelands, and rights-of-way areas, as well as for weed control in rice, we know you are interested in information on this RPAR action, and the threat its possible loss represents to liou as a farmer, rancher, rights-of-way manager, or forester. The RPAR Process An RPAR is EPA's formal review of the benefits versus risks of con tinued use of a pesticide. This action begins when EPA concludes it has found some evidence of a potential health or environmental hazard (usually from laboratory studies) on the basis of certain risk criteria as defined in the Code of Federal Regualtions. Notice of an RPAR does not represent a ban or cancellation of the product's use. Once an RPAR is issued, producers and users alike have a period of time-- usually 105 days-- to both refute the risk evidence cited by EPA and provide information (usually in the form of letters) illus trating the importance of continued availability of the product for agricultural or other uses. Producers of a product have the primary responsibility for assembling evidence to challenge EPA's allegations of possible health or environmental risk. 2*. Evidence attesting to economic or other benefits derived from use of a pesticide must be provided primarily by those experienced in its use including scientists, grower organizations, agribusiness, and individual users. DOW0012 164 After assembling all available risk/benefit information, EPA will determine whether the compound is reasonably, safe as used, whether additional limitations are needed, or whether it should be taken off the market. In order for the agency to make an intelligent decision, EPA must have information from users. 2,4, 5-T Background '* Since its introduction in 1948, 2,4,5-T has been a mainstay in the battle to control trees, brush and weeds which 1) threaten the growth of rice, conifer seedlings, or rangeland grass, and 2) create hazardous situations on road shoulders and utility rights-of-way. During this period, the toxicity of 2,4,5-T has been exhaustively studied by scientists worldwide. The consensus of these scientists is that it is not hazardous to human or animal life under normal conditions of use and even under conditions of substantial misuse. Despite this enviable safety record, special interest groups which oppose the use of virtually any of modern agriculture's chemical tools have been exerting strong pressure to have 2,4,5-T banned What these groups fail to understand is that so far there is no alternative-- chemical or non-chemical-- that achieves the public benefits of 2,4,5-T. DOW 0012 165 .3 . The 2,4,5-T RPAR - - Dow believes that the 2,4,5-T RPAR proceeding provides an effective means to resolve-- once and for all-- the issues raised by those who oppose the use of this important herbicide. Dow is dedicated to working towards a scientific resolution of these issues. Consistent with our belief that 2,4,5-T does not pose a hazard to either human or animal life, Dow will present a great deal of scien tific evidence to SPA to rebut alleged health risks. and You Economic and other benefits information must come primarily from you, the user. To keep these products on the market, it is essential that letters be sent with information showing the importance of 2,4,5-T to your farm, ranch, forest operation, or the rights-ofway you manage. Detailed information on the following points will be most helpful: 1. Usage of 2,4,5-T containing herbicides by crop, acreage, or weed control objective. 2. 2,4,5-T Effectiveness in your programmed use. 3. Availability, comparative effectiveness, and cost of alterna tive herbicides or cultural practices. 4. Effects that loss of 2,4,. 5-T herbicides would have on your ability to grow rice, raise livestock, or manage forests or rights-of-wav. D O W 0012 166 4. 5. Your estimate of the dollar impact the loss of 2,4,5-T might: have on your local economy. Where and when to send Benefits Information Benefits information should be sent to EPA. Although the deadline is not clear, information should be sent as soon as possible in order to have the greatest impact. D o n 't assume "the other guy will save it." If possible it is best to send three (3) copies with the reference notation: 0PP-30000/26 . Mail to: Federal Register Section Technical Services Division (WH-569) Office of Pesticides Programs, EPA Room 401 East Tower 401 M Street, S.W. Washington, D. C. 20460 Your input is vitally important to a correct resolution of this issue. ACT NOW 18 C CONFIDENTIAL - SUBJECT TO INJUNCTION D.C., E.D. M l. 4 -4 -7 8 ; D O Y //EPA AGREEMENT 9 -7 9 10 J4 G D O W C H E M I C A L U . S . A . V May 30, 1978 M IDLAND. M ICHIGAN 48640 # D O W 0o 12 l54 i \\\ J . N. D . W. C . E. C . T. J. C. R. E. D . M. D. B. J . R. R. W. P . 0. Bean - Seattle Cummins - Geneseo Jentes - St. Louis Lichy - Philadelphia Ryder - Minneapolis Ullom - Minneapolis Baldwin - Phil, Hale - Atlanta Huffman - Atlanta Steele - Lubbock Ludwig - 9001 Bldg. B . C . Byrd - Wayside L . E . Hammond - Chicago J. B . Grumbles - Dallas M. L. Smith - Seattle P . M. Ritty - Kansas City L. E . Warren - Davis R. D . Fears - Ci ncinnati R. P . Harrison - Memphis S . M. Hunter - Charlotte P . VanDerSchaaf - Geneseo M . L. Traynor - 2030 B l d g . W . H. Mullison - 9001 Bldg. R. C . Hunter - 9008 Bldg. 0 . K. Jantz - 9008 B l d g . J . A . Gray - 2030 Bldg. R. E . Hefner - 9008 Bldg. C . S . Williams - 9008 Bldg. R. D . Moss - 9001 Bldg. R. W. Colby - 9008 Bldg. J . H . Davidson - 9001 Bldg. M . L . Leng - 9008 Bldg. D . D . McCollister - 9008 Bldg W. J . Stearns - 9008 Bldg. W. G . Wright - 9008 Bldg. 2,4,5-T RPAR - BACK-UP LITERATURE As mentioned in my communication of May 15, 1978, literature pieces are being developed to support your efforts towards (1) informing cooperators, users, etc. of the issues involved in E P A 's RPAR action and (2) soliciting benefits letters. While some of the ma te ri al is still being printed, I thought you might be interested in seeing "proofs" as well as c o m ple ted materials so you can begin developing strategies for use of these materials. Copies are enclosed with this letter and include: 1) A set of transparencies which briefly covers the RPA R for use in talks before user groups, etc. The same f o r mat is being used for preparation of 2x2 slides which will be sent in the near future. 2) A copy of a 2,4,5-T "backgrounder" entitled "RPAR Review of 2,4,5-T Herbicides". Prepared by Wendell Mullison, this piece was designed primarily for univ er sit y/ ext ens ion personnel, but can be distributed to anyone who might have interest in the material contained therein. This l i t e r ature piece is being copied in quantity and additional copies can be obtained by contacting Mazie Sowles (517636-1020. AN OPERATING UNIT OF THE DOW CHEMICAL COM PANY D6W0012 155 2 3) A proof copy of '"What the 2,4,5-T RPAR Means to You", This release, wh ich is being formatted into brochure form is intended primarily for farmers and other users as a solicitatio n for benefits letters. As such, it can be distribut ed at meetings, through grower organizations, etc. I t is anticipated this brochure will be available in qua ntity within one week. Again, place your requests th rough Mazie Sowles. 4) Also enclosed is a proof copy of "Fact Sheet 2,4,5-T R P A R " . This is a press release and is being distributed to various media from Midland. However, you may wish to use it as a supp lement to or in lieu of the above mater ial s This item is als o being print ed in quantity. Best of luck in you r efforts towards stimulating those benefits letters. A good idea of the types of comments needed in benefits letters is contained in the enclosed DuPont r e lease "Update in the Benomyl RPAR". As to possible strategies for g en era ti ng letters - in ad dition to direct solicitations by mail and personal conduct, one idea which has substantial merit is to give a brief talk before users or user groups followed by passing out pens and paper so that individuals can prepare a response during the meeting. Letters can then be collected and posted to E P A . Needless to say, the paper used for such letters should not contain Dow letterhead and the mailing to EPA should be through a third party sponsor. According to reports, this technique has worked quite well on occasion with other RPARed compounds. One final comment - on no occasion should benefits letters show Dow as a "ccn on the letter. Some letters of this type are inevitable, but it is important that we do not encourage W a s h ington supporters to copy us (or show that they are copying u s ) . We will be able to track the volume of the response by direct inquiries of EPA in Washington. L . L . Sm i t h , J r . Product Technical Specialist Agricult ural Products Department nbe enc . 10 J o GCS 2000 MOCI# 2,U,5-T RPAR - REVIEW OF EPA'S RATIONALE AND THEIR CALCULATIONS FOR EXPOSURE TO 2,U,5-T AND TCDD by Marguerite L. Leng, May 1, 1978 / / EPA's rationale for issuing a Rebuttable Presumption Against Registration for 2,U,5-T is presented in their Position Document No- 1 dated April 21, 1978^. We have compiled a file of the 179 cited references including EPA memos from their Cancer Assessment Group and of their exposure estimates as calculated by their Criteria and Evaluation Division. In their exposure analysis, EPA states that " th e c a n c e l l a t i o n o f u s e e o f 2 , 4 , S -T on f o o d c r o p s in te n d e d f o r human c o n su m p tio n a n d f o r u s e a ro u n d th e home, r e c r e a tio n s i t e s , a q u a tic a r e a s , an d d i t c h b a n k s in 1 9 7 0 w as th o u g h t to have e lim in a te d th e p o te n tia l exposure to th a t p o rtio n o f th e p o p u la tio n a t r i s k (women o f c h i l d b e a r in g a g e ) . S o c i a l c h a n g e s o v e r th e l a s t fe w y e a r s , h o w e ver, h a ve g iv e n women th e o p p o r tu n ity f o r em p lo y m e n t i n a r e a s t h a t on ce w ere c o n s id e r e d o p en o n ly to men. S in c e women o f c h i l d b e a r in g a g e a re now em p lo yed in o c c u p a tio n s su ch a s p e s t i c i d e a p p l i c a t o r s , o p e r a to r s o f highw ay c o n s tr u c tio n an d m ain ten an ce eq u ip m en t, f o r e s t e r s , and ch em ical fo rm u la b o rs, th e y have become p a r t o f th e p o p u la tio n a t r is k w i t h p o t e n t i a l e x p o s u r e t o 2 , 4 , 5 - T a n d o r TCDD." (p. 101a - 102 of typescript copy received by certified mail on April 2 h , 1978) Teratogenic Effects EPA's press release-issued on April 12, 1978 stated that " th e p e s t i c i d e a n d i t s d io x in co n ta m in a n t h a ve k i l l e d f e tu s e s o r c a u s e d b i r t h d e f e c t s , su ch a s c l e f t p a la te , among la b o r a to r y m ice, r a t s , h a m sters an d b ir d s in t e s t s co n d u c t e d b y t h e N a t i o n a l I n s t i t u t e s o f H e a l t h a n d o t h e r r e s e a r c h e r s . " On the basis of many cited studies, they concluded that no-effect levels had been established for teratogenic/fetotoxic effects at 20 mg/kg for 2,U,5-T and 0.03 yg/kg for TCDD. Although it was mentioned occasionally in the text that the doses were administered daily, few of the tables specified that dosage rates were in milligrams or micrograms of chemical per kilogram of body -Federal Register Vol. 1*3, No. 7 8 , p. 17116 to 17157, April 21, 1978. - 2- DOW 0C02 5>10 weight per day, nor that the treatment was continued during up to half of the animal's pregnancy. Exposure Levels for Pesticide Applicators To simplify calculation of exposure by skin absorption and inhalation, EPA assumed that all applications were made using a product containing U pounds of 2,1+,5-T per gallon diluted 10-fold with water. However 2,1,5-T low volatile esters such as in ESTERON* 2 U5 Herbicide are relatively insoluble in water and require addition of surfactants and/or oil to form emulsions with water. Some applications are made in oil alone, particularly for low volume sprays. Dermal exposure was estimated for different types of application equipment based on available data for other products, chiefly insecticides. Workers were assumed to be wearing very light clothing with little protection from the spray. However, 2,1,5-T is generally applied to foliage in early spring when target brush and trees are growing vigorously, or in the fall with thorough coverage of dormant stems. Use in forests is limited to a brief time in the spring between leafing-out of deciduous trees and bud-break in conifers, or during the dormant period in late fall to early winter. Female applicators would not likely be wearing shorts and halter tops at these times, as apparently assumed by EPA. The letter of transmittal accompanying EPA's memo on exposure-- stated: "We h a v e m o d i f i e d t h e W o r k in g G roup'8 d e s c r ip tio n o f b ik in i c la d p e rso n s e n te r in g in to sp ra y e d a r e a s . In g en era l, sp ra y a p p lic a to r s o f 2 ,4 ,5-T are v e ry s e n s itiv e to r e le a s in g sp ra y d e p o s its in to p o p u la ted se c tio n s o f e ith e r rig h ts-o r-w a y o r f o r e s ts . T h is i s in p a r t due to in te n s e p r e s s u r e s w hich have been b ro u g h t to b e a r by th e p r iv a te s e c to r on p u b lic u t i l i t i e s and fe d e r a l and s ta te f o r e s te r s . The m o d ifie d ex p o su re e s tim a te we have p r o v id e d nam es no s p e c i f i c e x p o se d p o p u la tio n because in our op in io n , id e n tif ic a tio n o f an exposed su b -p o p u la tio n to 2 ,4 ,5-T sp ra y, even under ran gelan d s itu a tio n s where r e s p o n s ib ility i s Trademark of The Dow Chemical Company -- Reference No. 161. Exposure Analysis, 2,1,5-T, dated February 1978 from Gunther Zweig, Chief of Chemistry Brance, Criteria and Evaluation Division to Harvey Warnick, Project Manager for 2,1,5-T in the Office of Special Pesticide Reviews. The calculations were made by Carroll Collier. 18050 DOW0C02 541 -3 - m ore d i f f i c u l t t o a f f i x 3 i s a t t h e h e s t 3 v e r y d i f f i c u l t ." tt! i The following calculations for different spray situations are also presented in a summary table attached to this review. 1. Backpack Sprayer EPA's worst case calculation is for use of a hand-pressured backpack sprayer to treat brush in right-of-ways or for spot application in pasture or rangeland. Based on data for fenthion insecticide applied as a dilute spray in water (0 .06%) by workers wearing short-sleeved, opennecked shirts with no gloves or hat--3/ , they estimated that a woman weighing 132 lb (6 0 kg) would be contaminated by 0.l8 pints (1/3 cup or 86 ml) of 10# 2,H,5-T spray solution in an 8-hour day. If 10# of this was absorbed even after washing, she would receive a dose of 6.8 milligrams of 2,^,5-T and 0.0007 micrograms of TCDD per kilogram of body weight. It does not seem likely that a woman would continue to get this much on her skin each day, particularly if the formulated product is applied in oil or as an oil/water emulsion. Furthermore, she would not likely be doing this work, day after day, during several months of her pregnancy. (in the Dow animal studies, dosing was done daily on days 6 through 15 of the 20-21 day gestation period in rats). 2. Tractor Mounted Low Boom Sprayer EPA next estimated a somewhat lower exposure in or on a woman operating a tractor-mounted low-boom sprayer for clearing brush on rangeland or rights-of-way. Based on exposure studies for paraquat applied with this w type of equipment as a 0.12# solution at a rate of 100 gallons per acre,-- EPA concluded that a female applicator would be contaminated by 0.0U8 pint (23 ml) per 8-hour day. Using the above 10# absorption rate for 2 , k , 5 - T applied as a 10# solution, her dosage level would be 1.8 mg 2,U,5-T and 0.00018 yg TCDD per kg per day on this job. Again, no -- ^Reference No. 166. Wolfe et al. Mosquito News 3^(3): 263-267 197^ . 11 / A u 0 -- Reference No. IU7 . Steiff et al. Bull. Environ. Contain. Toxicol. lU(3): 33U-3U0, 1975 ^ X Dowoeoa 542 It consideration was given to how frequently nor for how long she would be doing this during her pregnancy. 3. Aerial Application For exposure during aerial application, EPA assumed the woman was standing directly beneath the spray plane. Estimation of dermal exposure was for contamination on all areas of bare skin, listed as head, neck, shoulders, forearms, hands, and thighs (such as when wearing shorts and a halter top which is not likely during 2,U,5-T spraying in early spring and late fall). Based on data for "malathion in oil sprays, applied at 0.U6 pounds per 0 . 7 6 gallons of water(?) per acre;"--^ EPA calculated that application of 2,U,5-T at U pounds in 10 gallons of water per acre would deposit 31 mg 2,U,5-T on her skin. If 10# was absorbed, she would receive 0.051 mg 2,U,5-T and 5 x 10~^ yg TCDD per kg of body weight. Compared to no-effect levels of 20 mg/kg for 2,H,5-T and 0.03 yg/kg for TCDD in teratogenic studies, daily exposure at this rate represents a margin of safety of almost UOO-fold for 2,U,5-T and 6000-fold for TCDD. If the 2,k,5-T was applied at usual rates of treatment (0.5 to 2 lb/A) the margin of safety would be proportionally greater. Nevertheless, EPA concluded that this exposure is not safe enough for 2,U,5-T although it is safe enough for TCDD (in spite of the fact that TCDD is always present in 2,U,5-T). EPA also calculated the inhalation exposure for aerial spraying, again based on data for malathion.--^ Since 2,^,5-T is applied as a medium to coarse spray, they assumed that only 2# of the spray droplets would be small enough for inhalation, but that all the inhaled material would be 100# absorbed. They also assumed that the spray would be inhaled for 8 hours per day rather than for only 2 hours after application as in the malatkion model. Even so, the daily dosage level by inhalation was only 0.023 mg 2,U,5-T and 2 x 10-D yg TCDD per kg of body weight, with safety factors of 870 and 15,000 respectively. Again, this margin of safety was inadequate for 2,U,5-T but adequate for TCDD. --^Reference No. 167* Caplan et al. AMA Arch. Indust. Health lU: 326--332, 1956 -- Reference No. 1.68. Akeson and Yates, undated and unpublished. DOW0C02 5$3 -5- fumulative Exposure EPA also estimated the cumulative doses for dermal and inhalation exposure in each situation. Exposure by inhalation was judged to contribute only negligibly to the dose received by dermal exposure from application with a backpack sprayer or a tractor mounted sprayer. However, the contribution by inhalation from aerial sprays was deemed to be additive. The cumulative dose was estimated to be 0.07^ mg/kg for 2,lt,5-T and T x 10-6 for TCDD, with margins of safety of 270-fold for 2,U,5-T and ItOOO-fold for TCDD. This was deemed adequate for TCDD but not for 2,U,5-T. Once more, no con sideration was given to the fact that the woman would have to be standing directly beneath the spray plane and inhaling the spray for 8 hours, each day, during the critical stage of pregnancy. Oncogenicity EPA's U/12/78 press release also stated that " 2 , 4 , S -T a n d d i o x i n h a v e ca u sed leu k em ia o r lu n g , l i v e r , o r o th e r tu m ors among m ice and r a t s in s t u d i e s c o n d u c te d b y t h e N a t i o n a l I n s t i t u t e s o f H e a lth y Dow C h e m ic a l, a n d o t h e r s . " They did not differentiate between effects alleged to have been caused by 2 ,U,5-T and those caused by TCDD " s i n c e m a n u f a c tu r e r s a r e u n a b le t o p ro d u c e 2 , 4 , 5 - T w i th o u t TCDD." It should be noted that the RPAR was issued before EPA's Cancer Assessment Group had even reviewed the two complete reports for Dow's 2-year feeding study with TCDD in rats, as delivered to them by me more than 6 months ago. On the other hand, they apparently accepted at face value all the "data" from poorly conducted and reported studies by others. Fox' example, they concluded that Allen's study-- demonstrated a h6% incidence of tumors in a total of only 50 rats in five groups fed 5 to 5000 parts per trillion TCDD for up to 65 weeks. In that study none of the 10 control rats developed tumors but 6 of them died before the study was terminated at only 22 months. ]_/ Reference Ho. 109 VanMiller et al. Cheraosphere 9 : 5 3 7 -511!*., 1977 .'o DOWO002 44 - 6- The French study with 2,U,5-T in two strains of mice was also discussed extensively in spite of the fact that the authors stated their results were Q/ inconclusive.-- We need to submit a progress report on the Dow 2-year feeding study with 2,U,5-T in rats to supplement the negative study in mice reported by Innes et al. of NCI.--9/ EPA also cited two preliminary epidemiological studies from North Vietnam^/ and Sweden,-- ^ " n e i t h e r o f w h ic h i s s u f f i c i e n t t o b e t h e b a s i s o f any firm c o n c lu sio n s co n cern in g a ca u sa l c o n n e c tio n b etw een 2 ,4 , S-T an d c a n c e r. B ut in v ie w o f th e r e s u l ts o b ta in e d in e x p e rim e n ta l a n im a ls, th e y w a r r a n t n o t i n g . " Unfortunately, they did not have time to include comments on the Dow Study submitted in March 1978 which demonstrated no adverse effect in 20U employees exposed in the 2,1,5-T plant for intervals of 1 to 10 years. All exposures were during the years 1950 - 1971 when the TCDD level was higher than the current 0.1 ppm limit in 2,1,5-trichlorophenol and al1 products derived from it. Conclusion EPA's appraisal of the hazard of 2,l+,5-T is exaggerated. Your comments are solicited on the above review, and on any portions of the position document not covered herein. Please send any comments or additional information no later than June 15 1978 to Marguerite L. Leng, Building 9008, The Dow Chemical Company, or Box 1706, Midland, Michigan k 8 6 k 0. 8/ Reference No. 105 Muranyi-Kovacs et al. Br. J. Cancer 33: 626-633, 1976. 9/ Reference No. 107 Innes et al. J. Natl. Cancer Inst. U2: 1101-Hll, 1969- 10/ Reference No. 120 Tung et al. Chirurgie 99: 1+27-^36, 1973. 11/ Reference No. 108 Hardell, L. Lakartidningen 7^: 2753-2751*, 1977. Situation Backpack sprayer; rights-of-way; spots in pasture or rangeland SUMMARY OF EPA ESTIMATES OF EXPOSURE TO 2,lf,5-T.AND TCDD IN A 60 KG WOMAN Treatment Daily Exposure If lb/gal diluted 10-fold with water applied to wet. 0.18 pint; 1/3 cup; 86 ml. 2,1+,5-T (Margin of Safety) mg/kg/day vs 20 mg/kg 6.8 (2.9)* TCDD (Margin of Safety) hg/kg/day vs 0,03 Ug/kg 0.0007 A3) Tractor mounted low boom sprayer; rights-of-way; rangeland. If lb/gal diluted 10-fold with water applied to wet 0.01+8 pint; 3A fl. 07,; 23 ml. 1.8 (11 )* 0.00018 (167)* Aerial application U lb a.e. in on person directly 10 gal water beneath spray per acre as plane, wearing medium to very light clothing, coarse spray. and who remains there all day. 3.1 mg (dermal) 0.31+ mg (inhalation for 2 hr.) Cumulative 0.051 0.023 per 8 hr 0.071 (392 )* (870 )* (270 )* 5 x 10~6 c. 2 x 10 6 7 x 10"6 ,. #Inadequate margin of safety (for daily exposure at these rates, during critical stage of pregnancy). (6 0 0 0 ) (15000) (1+286) , ii i t< i b"4 M.L.L. (5/1/78) i V-1 C"1 sfs soooMoa m] #DOWOC02 533 I l o n ;-d ')i>v- -o , ^ ..-- ------ D.C., E.D. MI. 4 - 4 - 7 3 ; D O W / E P A AGREEMENT 9- 79 DOW C H E M IC A L U.S.A. 109t 9005 Building May 1, 1978 MIDLAND. MICHIGAN 48640 E. H. Blair, 2020 G. E. Blau, 1707 R. B. Bumb, k j W. B. Crummett, 57** J. H. Davidson, 9001 J . E . Donalds, 9008 P. J. Gehring, 1803 C.A.I. Goring, 9008 J. A. Gray, 2030 R. D. Hart, 2030 R. E. Hefner, 9008 F. D. Hoerger, 2030 B. B. Holder, 2030 0. K. Jantz, 9008 J. E. Johnson, 2030 G. G. Jones, 9001 R. J. Kociba, l803 R. R. Langner, 1803 L. A. MacDonald, 9008 \ cc: R. H. Schroeter, Los Angeles M. R. Wessel, 2030 D. D. McCollister, 9008 R. J. Moolenaar, 1702 R. D. Moss, 9001 W. R. Mullison, 9001 J. M. Norris, 9001 J. N. O'Connor, 2030 P. F. Oreffice, 2020 C. N. Park, 1707 M. E. Pruitt, 2020 V. K. Rowe, 1803 B. A. Schwetz, 1803 L. A. Shadoff, 51k D. P. Sheets, 2020 L. L. Smith, 9008 P. G. Stamison, 9008 W. J. Stearns, 9008 M. J. Traynor, 2030 J. R. Vaccaro, 1803 C. S. Williams, 9008 From: Marguerite L. Leng, Senior Registration Specialist Health and Environmental Research, 9008 Building 2.U.5-T RPAR - A GAME BY EPA'S' RULES The Rebuttable Presumption Against Registration (RPAR) process is a game played under EPA rules with a stacked deck. Contrary to David Menotti, EPA's new Associate General Counsel, it is not an open two-way debate of all available information in which the pros and cons are reviewed in public before a regulatory decision is reached. Instead, EPA publishes all the "risk" information in the Federal Register whereas rebuttal comments and information eventually become available for public inspection only in an obscure Washington office during normal working days. Meanwhile, a chemical such as 2,^,5-T is indicted by the press and public outcry continues. What happened to DDT can happen again. The EPA Administrator still has the last say, regardless of the outcome of "public" review of data or of legal decisions following public hearings. A N O PE R A T IN G U N IT OF TH E D O W C H EM IC A L C O M PA N Y t*A 3 Viaw<^ A L'O' 0OV/0C02 534 .I E. H. Blair, et al. 2- - May 1, 1978 As noted in the attached EPA press release, issuance of the 2,U,S-T RPAR vas prompted by "som e 4 5 0 l e t t e r s f r o m c i t i z e n s a n d e n v i r o n m e n t a l g ro u p s such a s th e C itiz e n s A g a in s t T o x ic S p ra y s (CATS), p r o t e s t i n g u s e o f th e c h e m i c a l . " The U.S. Department of Agriculture is also succumbing to the same pressure due to complete absence of similar input from the other side. Positive action is needed now to avoid a moratorium on continued use of this valuable herbicide pending a final decision by EPA to possibly restrict it or ban it. Jack Anderson's column on Monday, April 2h (attached) exemplifies how the RPAR action can be distorted. EPA's press release also provided fuel for our opponents. Their " c a l c u l a t i o n s sh o w t h a t a n a m p le m a r g in o f s a f e t y may n o t e x i s t f o r p e rso n s a p p ly in g th e p e s t i c i d e o r p e o p le e x p o se d to a i r c r a f t s p r a y i n g o f i t . " Costle's assistant Barbara Blum stated " F in d in g o f b ir th d e f e c ts and ca n cero u s tum ors among la b o r a to r y a n im a ls e x p o se d to 2 j4 t 5 -T i n d i c a te t h a t i t may c a u se th e sam e p r o b le m s among p e o p l e . " EPA's conclusions about carcinogenicity are based on poorly conducted and reported animal studies and on "preliminary" epidemiological studies in North Vietnam and Sweden. However, they still have not reviewed two extensive reports on Dow's two-year study with TCDD delivered to them six months ago, nor a recent study demonstrating no adverse effect in Dow production workers. Appended hereto is a review of EPA's rationale for issuing this RPAR and of their calculations on exposure of applicators to 2,U,5-T and its TCDD contaminant. Although their Position Document admits that these chemicals are not accumulating in the environment, they reserve judgment on the hazard of TCDD in food until more data are available from their monitoring studies. With regard to teratogenic/fetotoxic effects, they concede that no-effect levels have been established in animal studies with 2,U,5-T and TCDD, but ignore that these dosage levels were administered daily for up to half the duration of pregnancy. They also appear to require a 1000-fold margin of safety even for their exaggerated estimates of exposure in women of "child-bearing age" during actual application of the herbicide. I urgently solicit comments from all recipients of this letter, either for incorporation in our formal rebuttal or for independent submission to EPA before the final deadline of August U, 197^. Other Dow products are also at stake in this "game." RPAR's are scheduled to be issued shortly for 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). Next on their list are silvex (2,U,5-TP), erbon and ronnel. 2,U-D and dinoseb (DNOSBP) are currently under pre-RPAR review and others are likely to follow in domino fashion. Copies of EPA memos or other cited documents are available by calling Agnes at 636-1350. abc 86 0T DOW 408789 OD LO O CD DW 4Q879 REBUTTAL TO EPA'H INTE V.P RET AT IO OF DATA REGARDING THE HEALTH HAZARDS OF 2,4,5-T Dr. D. G, Crosby University of California, Davis Davis, California and Dr. J. B. ICnaak California Department of Food and Agriculture Sacramento, California July 1970 ********x*tr***************** ************** Table of Contents I I n t r o d u c t i o n .................................... 1 II The " T r i g g e r s " .................................. 1 A. General C o m m e n t s .......................... 1 B. O n c o g e n i c i t y .......... 2 C. Teratogenicity/1'etotonicity............. 2 III Human E x p o s u r e .................................. 3 A. General C o m m e n t s .......................... 3 B. Occupational E x p o s u r e .................... 5 1. Oral E x p o s u r e ......................... 5 2. Dermal E x p o s u r e ....................... 5 3. Dermal I n t o x i c a t i o n .................. 7 4. Inhalation E x p o s u r e .................. 8 5. Cumulative E x p o s u r e .................. 3 C. California E x p e r i e n c e .................... 8 D. Current R e s e a r c h ............. 9 IV C o n c l u s i o n s ...................... .'............ 9 V R e f e r e n c e s ...................................... 10 18059 DOW 4 0 S 7 9 1 I. Introduction 2,4,5-T (2 ,4,5-trichlorophenoxvacetic raid) and its derivatives long have been important to California agriculture and forestry* According to the 1972 California Pesticide Use Report (1), almost 108,000 lbs. of the chemicals were applied cnually in users of uses throughout the state at that time. In recent years, permit use has declined to less than 20,000 lbs. (2), but "2,4,5-T" still retains the. herbicide of choice for forest and range- applications. For both practical and toxicological considerations, it is important to specify the exact chemical species referred to as "2,4,5-T." The free acid 2,4,5-T seldom is used, but rather its amine salts or lowvolatility esters. The physical anJ chemical properties of these com pounds differ widely, alth.ou.iUi the derivatives eventually are converted to true 2,4,5-T in tie environment. The molecular weight change can be especially significant: 2,4,5-T (aciu) containing 0.1 ppm of 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) would produce a commonly used propylene glycol butyl ether ester containing o;ily 0.07 ppm. of TCDD. Unfortunately, the RPAR generally leaves doeut regarding which form of 2,4,5-T is being referred to. During several decades of widespread use, 2,4;5-T herbicides wore considered undistinguished in their toxicity to animals-- so much so, tliat they were sold extensively for farm and home use and have remained without any reported episodes of human harm in the state. However, in the late li'uO's, observations of to'v.toyenicity and fetctoxicity .in laboratory animal:: fed TCDD-conr.nmanncca 2,4 ,3-T introduced a decade of public confusion end furor. The deep resentment over the Vietnam War (in which 2,4.5-T butyl ester was used as a aefollant) , a widely publicised industrial accident in Seveso, Italy, and die susceptibility of TCUD'c unusual toxicity to dramatic and imaginative reporting com bined to set the stage for the present rr-gidatory controversy. Indeed, there is r.o question that TCDD is an extremely toxic and dangerous substance, as demonstrated by the extensive (although var iable) investigations of the past few years and outlined in the RPAJl document. However, present-day n-anufacturing methods produce 2,4,5-T generally containing less than 0.05 ppm of TODD, and the present com mercial herbicide and its derivatives can be-- and presently are-- used safely in CalifoTiiia. This document is nor. a recommendation for the use of phnnoxy herbicides or ior any propi.etary product but is intended to offer alternative interpretations of data used by EPA for a F.ebuttable Presumption Against Registration and continued registration of pesticide f products containing 2,4,5-T. II. The "Triggers" A. General Commrntn. An RPAR (Rebuttable Presumption Against Registration and Continued Registration) has been issued against products containing 2,4,5-T because there is evidence tnat 2,4,5-T and/or its dioxin contaminant -1- 18060 (TCDD) nay cause a significant increase in benign or malignant tumors and teratogenic and/or fetotoxin erfacts in certain sensitive species of laboratory animals. This section of the response, dealing with the toxicology of 2,4,5-T and TCDD, will atter.'c to bring about a clearer understanding of the relationship between published toxicological studies concuctcd m the laboratory end the use of 2,4,5-T in the environment. B. Cr.cogc.nlcity, At present, most 2,4,5-T oncogenicity stadias rust be considered to be primarily academic. Of the four investigations cited in the R?AR, one represented daily oral dosing of 2,4,5-T (containing 30 ppm of TCDD) at 60 mg/kg via gelatin capsule or subcutaneous injec tion of the 2,4,5-T to very aged mice, and two others were concerned with feeding mts pure TCDD. The fourth and rest pertinent study (3) involved ad libitum feeding of 2,4,5-T containing lees than 0.05 ppm TCDD to two separate strains of mice at 30 ppm in the diet over most of their lifetimes. The results showed little or no significant difference between treated and control animals, although there is indication that 2,4,5-T actually may protect against oncog enicity. Tee most notable feature among X.VU./G mice was the very significant increase in longevity among the treated animals. This longer life-span clouds the results, as any increase in tumor inci dence could be attributed to the greater susceptibility of old individuals to cancer (in mice as well as people). The TCDD oncogenicity data are widely considered to be preliminary and very controversial (4), although they snow that both TCDD and 2,4,5-T exhibit no--effect levels. IV.fortir-.ataiy, the key question of oncogenicity by dermal exposure-- essential to the SPAiT argument-- remains unanswered. C. Teratogen!citrv/Fetatoxicity . There. cesms to be little controversy over the fetotoxic effects of TCDD. In fact, it waa this property of contaminated 2,4,5-T which was brought to light by the early studies (5). As with oncogenicity, however, nest investigations have bean more academic than practical-- high -level or pure TCDD, DDS0 injection, eta. However, a more real istic test with 2,4,5-T containing 0.5 ppn TCDD (about 10 times present levels) at 1-24 r.g/k://day to rats or 10-40 mg/ieg/cay to rabbits during key periods of pregnancy snowed no effects; beta fetotoxir.ity and mat ernal toxicity were observea at the environmentally ur.rc-.atiStic dosage of 100 mg/kg/aay, however. Unfortunately , the maternal toxicity observed :ln a number of the tests where toxicant levels produced fe.totcxiuiry obscures the-, results, as poisoned mothers generally are not expected to produce healthy'off spring. Again, no-effect levels for both TCDD and 2,4,5-T were observed, but tests utilising dermal exposure have not been reported. -2 - DW 408792 18C DOW -408793 D. He-cffect levels. Data in the IlL'Ail document rcveaL no-effect l'.vtIs for TDID and/or TCDD-containing 2,4,5-T i.n relation to mortality, oncogenicity, and teraT.ogenicity/fetoeo:d.eity . Significarli: mortality increases (compared to controls) aid not occur with pure TCDD below oral doses of 0.5 ppb (0.1 ug/kg/week); the oncogenic no-oifect levels in lifetime feeding studies were between 0,001 and 0.01 nilcrop,rams/leg/day for pure TCDD (corresponding to 20-200 mp./kg/day of ,4,3-T containing 0.05 ppm TCDD); and no teratogenicity or fetotar.icicy were observed with pure TCDD at 0.03 microgran/kg/day (rats) and 0.1 microgram/kg/day (mice) or with 2,4,5-T (0.05 + 0.02 ppm TCDD) at 25-150 v.g/day (rats) and 20 mg/kg/day (mice). Although c.loe outran >]ation of toxicity data f `eats to humans generally is unsatisfactory, tlic lowest of the .alues-- correspon dir.g to daily ingestion of 20 mg/kg of present-c d s - ! (1.2 grams/day in a 00 kg person)-- seem .likely ts be acceptable. Dermal aiui inhalation exposure data are inadequate to sec no-effect lev 1X3 III. Hunan Exposure A. General. Principal application.': of 2,4,5-T in California in 1977 (2) were on forest cimbericini and ou rangw land. Ti.e m.-nimma application rate uns cn th order of 2 Ibs/acre or actual 2,4,5-T. Ir. a tynlcal appli cation-- for lini.;: noncroi on farase land (''c'.nifev releasef)-- iicrbicide is applied as a tingile aerini spray of emula ifiad low volatile estera of 2,4-D and 2,4,5-T in water (cortespandi.:!;' co 2,4,5-T at 1 Ih/acre) (Paole I) . There are no "flagmeiv1, r.pp'iiar.;:.ion is by enc.j.osed heli co?ter evito largaiy uniahabiteci araaa , spray is mir.ed and loaded cami-automatically (r.ithough "liraiced contact s r.ossiblc) , and th '.erbicida io applird only under county perw.it and under in.~pection. tail: composition of r/riCi-JL iiiidicidi: s pray, Cor.: tj.tuent lbc/acre Viater 2,4-D propylene glycol butyl ether ester 2,4,5-T propylene glycol butyl ether ester solvent aud emulsifier 100 1.52 1.45" 1.26 a*quiv. to 1 la/aera 2 ,4 ,5-T il ug TCDD) Despite a permitted TCDD level of 0.1 ppm (based on 2,4,5-T content), the concentrate actually contains less than 0.05 ppm TCDD (typically 0,035 ppn in out analyses). Spray contains let's than 0 .5 parts nor billloti for each lb/acre of 2,4,5-T, corresponding to less than 25 nicr'^rams of TCDD per acre as released from the sprayer. ^ O p IQi OuU In herbicide. s.prny, TCDD is very 'nr.tnfcl.fi i;<j rrunl.F.i'ht on lp.nv.ns, noil, an:: other surfaces (<'). [The persistence referred to in the RPAR doc ument was due to a 1nooratory study with pure TCDD and without the energetic ultraviolet portion of sunlight (7)]. The ElV.-cIteu 196'1 work of Getzcndaner and Hummel (unpublished) also demonstrated rapid TCDD loss from 2,4,5 T-troatcd grazing land (rrass). TCDD becomes tightly bound to 3oil due-^o its unusual electropositive nature (<>). Consequently, dispersrd, environmental degradation, and adsorption reduces the concentration of TCDD to well beic.w the 25 microgran per acre figure and allows the rough estimate of potential exposure shown in Table II (9). TAT-T.E IT. ESTI.LATED MAXIMUM PROEARLE TCDD LEVELS RESULTING FROM A NORMAL HERDICUE APPLICATION (9) TCDD Maximum Cone, (ppt) 1'robabie Cone, (ppt) Leaf 10 Soil/litter Vater E-iota (fish) Milk 1.1 io '4 1 0.1 ? (1 pc/ern") (100 pg/kg) (0.1 pg/i) (1 mg/kg) 000 pg.l) 0.2 0.05 io"8 !0~4 0.01 ai-'rom a 1 Ug/ha application of herbiciue (0.2'ppm 1CDD) The chemical analysis for environmental and food residues of 2,4,5-T is routine and allows reliable measurements at part-per-h-illion levels (10). Such tiathorls have been used to demonstrate the environment dissipaticu and breakdown of 2,4,5-T (10,11). Millie TCDD analysis at these levels also is relatively straichtfurward (6), the methods required to detect and to overcome interfering: substances at part-per-trillion (ppt) levels of TCDD become extremely complex (12,13,14,15). Despite isolated and poorly verified claims to the contrary, tue best analytical methods presently available anywhere generally are conceded to be reliable only down to about 10 pot. Tills would preclude measurement of TCDD in most environmental samples (Tabie II), and indeed, surveys have failed to find any TCDD which might be present os a result of normal applications of 2,4,5-T (14). Considering the points presented above, the restriction of 2,4,5-T applications in California to professional (licensed) applicators, prin cipal application to uninhabited locations, and cancellation of uses on food crops, around the home, in recreation areas, and near water, it is most unlikely that the public will receive measurable exposure to TCDD, let alone exposure above the no-effect level. The two cited instances of epidemiological investigations of 2,4,5-T effects in humans involved massive exposure and questionable circumstances and data base, and jus tify the RPAR document's conclusion that "neither of these studies is sufficient to be the basis of any firm conclusions concerning connection between 2,4,5-T and cancer." However, a nor?, detailed epidemiological study Involving railroad workers who had used only phenoxy herbicides in comparison to these who had used other herbicides revealed no relation of tumorii'enesis to exposure to the phenoxys (16) . DOW 408794 .. * . p fi-*; ^` B.___ Occupational Exnccurc. ! Oral Exposure. The continuing FDA nationwide. "Marker Basket Survey" Kau railed La show 2,A,5-)' present in any food composite at or above -0.02 ppm, and a specific search for TCJJl) in riilk with the he.sc existing method also was negative (i.;). The estimate provided in Che RI'AR is based on a single highly exaggerated and artificial oaimalfeeding ejtperir.onc (17), so the FJVva document's "theoretical exoosure of an average veran'1 at 0.7 micrograns/k-r/day is meaningless, 'iha Working, Group c>7rectly considers that tne difference between the noadverse effect level of 2,4,3-T for teratogenicity (20 mg/kg/duy) and this exaggerated oral exposure, constitutes an "ample margin or safety" (a safety faeror of auouc 3G,GG0). DOW 408795 2. Dsrr.nl Exposure. In the absence of published data, the estimated der mal exposure levels cited in the ILHAP. necessarily are based on application of chemicals other than 2,4,a-T. However, the IUV.K expo sure estimates are born simplistic and unrealistic when applied to 2,4,5-T. First, 2,4,5-T is a restrieteu-usa chemical in California and oust be upoiied under a county permit and airect supervision of a licensed applicator: this means appropriate spraying conditions and personal protection-- the example of "exposed shoulders, forearms, hands, and thighs" is not applicable. There will be no "exposed popu lation directly beneath the snray plane**' except in an unforeseen accident wnen virtually r.o pesticide ray bo considered safe. The description of the other applications is ecivaliy unrealistic: no more than 151 of the body surface will normally la exposed to spray (18). While data on skin-absorption of 2,4,5-T by man have ret bean found, Kaibach (IS) e l:-.;mined the penetration of the clot: aly-rsiatcd 2,4-D to bvi. 5.81 of the dose (:.<'.a_i.'.u.nii-3Vca to cue forearm). A liquid e3tt.r of 2,4,5-T might be expected to penetrate more readily, but aalathien was absorbsto an extent of 6.2% and eshion to only 2.3%. It is probable that the Working Croup's estimate of 2,4,5-T skin absorption is high by a factor cf at least cwn. Considerable effort has been expended toward the development of reliable data for estimating the amount of pesticides to which a worker is exposed during mixing-loading and spraying operations. Exposure is measured by using clotn patches placed on the skin or clothing of the worker and by measuring the amount cf metabolites excreted in 'urine or feces. Cholinesterase activity before and after application of U--? insecticides also provides an estimate of both exposure and absorption, In all cases, the: measurements must be cade under actual use canaltica . Act:,ding to the estimates of Houghton (26) an orchard sprsysan collects pesticic: residues on his clothing equivalent to roughly 6 times the level deposited on foliage during spraying. For example, axinphosmechyl applied at a rate of 3 lbs. pur 100 gallons of water to peaches gives a deposit on Che foliage of 3.0 micrograres per enr* o f leaf surfr.ee (27), so the applicator would collect about Id ug o f cainphosnetkyl per cm- cf skin or clothing. If vs nasrme that onJ." 15% of the cornel surface of a 60 kg woman is exposed ar.d a cf 10~ of the dcrosic is absorbed from the exposed area, the calculated uose amounts to 0.09 ng/kg. Tai.a is only 1/7. of the dose estimated in tin: Position Document Lor spray applicators using a lew-boom sprayer. -5 - 1808 However, in a field study by Knaak et al. (28), workers mixing and loading phosdrin with closed systcns and' applying the insecticide with a tractor-mounted low-boom spray rig, actually collected only 0.5 ug/em^ on cloth patches attached Co their clothes during a 7 hr. work-day. On a mg/kg baais, this amounts to 0.16 mg/kg total deposit for the 60 kg woman, or an actual dose of 0.C02 mg/kg through exposed skin rather than the 0.09 mg/kg calculated value or the RPAR value of 1.8 mg/kg. The estimates made by the Working Group for tractor-mounted, lowbooa spray equipment also are inconsistent with the study of Staiff, et al. (25), cited in the Position Document. The Working Group assumed chat 0.048 pint3 of diluted material would be spilled on the skin of the worker. According to tne RPAR, this amount of 2,4,5-T spray (Table 28) would contain 10C9 mg of 2,4,5-7 and less than 0.1 ug of TCDD, although the 3tudy cited in the Position Docu ment states that a maximum of only 27.2 eg of pesticide was actually found (3.4 ag/hr. x 8 hrs. + 27.2 mg). In 1976, Knack and Schlocker. (23) extracted the coveralls of mixerloaders at the end of the working day and analyzed the extracts for organophoephate pesticides. The total amount of pesticide extracted from each coverall varied between 1.5 and 10.5 mp,. The residues found on the clothing of these workers are typical of these found when workers use open systems for mixing ar.d loading. The 4,086 mg of 2,4,5-T uuggested in the Position Document (Table 27) would result in 2G0 ug of 2,4,5-T per cm^ of clothing if the amount were equally distributed over the coveralls, whereas the analysis of clothing from men mixing and loading gave actual values of only 2 to 3 ug/cet^. These gross discrepancies between the values calculated by the E?A and these actually found emphasize the inaccuracies which can result when "worst-case" estimates become multiplied. According to the cited study by Wolfe et_ al. (24), maximum fenthion exposure amounted to 6.3 mg/hr. using a backpack sprayer. Because n worker's exposed skin amounts to only 152 of his total skin area, the total pesticide collected over an 8 hour work-day must be multi plied by 0.15 (6.3 cg/hr x 8 hr x .15 7.5 mg/day) to give the total amount of pesticide available for absorption. The 7.5 mg/day must be multiplied by 0.1 to give the actual amount absorbed (0.75 mg/day) or 0-.0125 mg/kg/day of 2,4,5-T accoruing to the Fosition . Document's method of calculating dose. This value is substantially lower (544x) than the 6.8 mg/kg proposed in the Position Document. On the basis cf this new estimate, the safety factor would be 1600 for 2,4,5-T and 25,000 for TCDD, certainly adequate to protect the health of the worker. The exposure estimates for aerial application presented in the Position Document were based on a study involving malathion (30). In a recent study conducted by the California Department of Food and Agriculture, 2,4,5-T wno applied bv helicopter to forest areas at a rate of 3 ibs. per 10 gallons of water per acre (29). Under these, conditions, foliage residues amounted to 10 ppm (0.11 up/cm") irorediately after spraying. A woman wo iking in this'area during spraying would collect about 2 r.g of 2,4,5-T on exposed skin and clothing. Since the exposed skin represents only 15Z of the total -6- DOW 4087.9G 18Gu5 D w 408797 body surface, the amount available for nbcorpcion represents 0.3 mg, arm even if 10JS of cl.ir: residue ia absorbed, die 2,4,5-T dose received amounts to 0.000> m;;/kg. The safety factor for 2.4,5-T under these conditions amounts to 40,000, and the factor for TCDD is approximately 1 billion. 3. Dermal Tonicity. The Working Group diptinguishec between terato genic effects of "2,4,5-T" and those of TCDD in estimating margins of safety. As the 2,4,5-T values already represent herbicides con taining usual levels of TCDD, the additional data on pure TCDD are extraneous and misleading. Unfortunately, no data appear to be available on the teratogenic effects of cither TCPD or 2,4,5-T applied dentally, and so the oral no-effecc levels are used in the RP.'.R to calculate margins of safety. However, a large store of experience as well as any handbook of toxicity values indicate that chronic oral toxxcity figures cannot be extrapolated successfully to dermal exposure, except that the dermal values always show distinctly less effect. The acute LD:g of TCDD in the rabbit has been measured for both the oral (.0.115 DS/kg) .and dermal (0.225 mq/xg) routes (19). The oral toxicity of technical 2,4,5-T butyl ester in the rabbit is 712 mg/kg (range 500-1,000) (21), hut corresponding der..:al values apparently nave not been reported. The oral LD^n cf 2,4,5-T acid in most mammals is on the order of 500 ag/kg, while the aermal LD-q of the closelyrelated 2,4-D Is 1400 rg/kg (20). Toxicity values from injection in DMS0 would bear no relation to surface application of an aqueous solution to ir.Lact skin. At low 1.-velis, TCDD becomes bound tichtly to protein and would be expected to find difficulty in penetration. LviJerae cf persis tence in shin may be found in the toxic skin reaction-- called chloracne-- whicli appears even at very lew dermal exposures to TCDD [detectable on a rabbit ear with a few nanograms (19)]. Although chloracne has been observed in factory workers exposed to massive levels of 2.4,5-T (22), the lack of a record indicates it is not prominent even among high-risk workers such as aerial applicators. Following r.n industrial acaidenC in Scvcso-, Italy, chloracne was observed among Che population exposed to very high levels of TCDD, as might be expected. However, abnormal births (or aborted fetuses) among some 2DO (allegedly) directly-exposed pregnant women were "not disproportionate to tne usual incidence of abnormal births" accord ing to the F.t'All document. Thus, even vastly greater exposure Chan ever could be envisioned for the RPAft's "60 kg woman" did not produce the effects reared In Position Document 1. Considering the safety factors already incorporated into 2,4,5-T use in California, the undoubted large overestimate by the V.'crking Croup of possible exposure and dermal toxicity, and the lack of terato genic or fetotoxic responses among even highly exposed humans, Che highly conjectural calculations of the 5lFA.Il document are obviously erroneous. -7- 4 . Inhalation F.:*^ocutu . Many of the problems which plague calculation;, of uur.iui exposure ale: apply to inhalation, although oral roxicitics perhaps arc more rcnson-ibiy extraI'tl.uod to the; respiratory route; again, use or "unprotected persons directly beneath Che spray pl.-me" as the basis of ail safety calculations in simply unrealistic. Other assumptions a'..so are probably quite in.-.scurnco tor a 60 1; ssrron; 2,4,5-T is nor. applied at 4 lba/ccre but at one to three lbs/rcre; the effective ei.meter of respired pestle '.dr particles is generally no more than about 10 mLcrons (rather then 60 microns), so the availability trom a coarse spray wcuid bo greatly dir.inisned; respira tion race would generally not excuse 1 nr'/br for a 60 kg woman (31); and Che lung absorpeien race certainly would hoc be 10CZ of the available particles (31). Thus, the rinse level calculated by Che Working Croup could be as much as 100 times too high. Toxicity values, for pure TCDD again have little meaning compared to those cf TCL'D-contaiaing 2,4,5-T. If we assume a dose level of even 2 ug/kg/uay compared to a no-effect level of 20 mg/day, the safety factor baccess 10,000. Even the P.PAR document's value of 0.C23 mg/kg/day for 2,4,5-T provides a daily dose of only 10-"" g/kg/ of TCDD-- why is this considered unacceptable when 5 times this amount of pure TOD would provide "an ample margin of safety"? Inhalation exposure to 2,4,5-T certainly is not a new phenomenon, either during application or onnufactu:u, and there is no history of complaint. based on tonicity values and practical experience, OfHA bus recently set the time-weighted average air Stanciard for human inhalation of 2,4,5-T at 10 mg/m~ (35). 5. C::ulotiva l.'xrn *ura. It in apparent that oral and inhalation exposure tc botn 2 , 4 and TCDD will be negligible compered to possible dental exposure. The greatest dannai exposure nay be expected from use of c back-pack sprayer (0.0125 isg/Ug/dav of 2,4,5-T and 1 x 10" w*/kg/dny of TCDD as "verst cases"). Ccnslderinr the Working Grcus's n.o-adverse cumulative: effect values of 20 rag/kg/cay and 0.03 mg/kg/day for 2,4,5-T and TCDD respectively, the argument or acceptability reems to revolve about what (unspecified) nargir. of safety CPA considers acceptable. We prenone that the minimum saiety factors of 1,DQ for 2,4,5-T and 30,000 for TCDD represented here are acceptable. C California F ience. 2,4,5-T and it3 derivatives have seen large-scale vise in California for about 40 years. While c-aalytlcal figures are not available, it must be assumed that for most of this period, TCDD levels were much higher than they are at prescur. The California Department of Public Health has maintained records for herbi cides in general curing much of this period (Table III) and for specific herbicides during the most recent reporting periods (1975-77) (33). California physicians are required by law to report in detail any pesticide iliac.'3. The reports for 1975 , 1676, and 1S77 show no observable illness attriL'uted to use of 2,4,5-T, despite the fact chat chloracne is a readily recognized symptom. Likewise, neither the ofnor state agencies applying 2,4,5-T nor the professional applicators themselves have, observed toxic effects in workers. DOW 408798 Kj V DOW 408799 TABLE III SUMMARY OF PHYSICIAN'S Oii REPORTS'1 Year 1977 1976 1975 1974 1973 IS72 1971 1970 1969 1960 1967 Iota! Pesticide Rnwortc 1B7.S 1752 1762 -- 1451 1505 1477 1493 727 G34 1400 Total Herbicide Rc.rcrr*^ 225 216 213 -- 200 243 201 214 76 65 189 total 2,4,5Reports 0 0 0 -- -- -- -- -- -- -- -- a State of California, Department of Public Health, Center of Health Statistics, Berkeley, California, 1978. Estimate. The Wildlife ar.d KotoraL Resources Committee of the California Senate heard extensive testimony in March end April, 1978, on a bill (SB 1357) to prohibit aerial .application of 2,4,5-T and "other TCDD-containir.p, materials" and than voted the bill down. In view or the California use restrictions, the con ditions of actual registered and peav.Ltted uses, and the complete lack of verifiable evidence of deleterious effects even among high-risk human popula tions, California experience with 2,4,5-T does not presently justify suspension or use. D. Current Research. The University of California, California Department of Food and Agriculture, and the. U.S. ioresc Service presently are engaged in research on the environmental distribution ana fate of 2,4,5-T and TCDD in both laboratory and practical field exaeriments. ;'nc principal purpose is to assess tiie possible exposure levels of humans and wildlife, and to determine possible bipaccumulaticn and food chain transfer, especially of TCDD. Preliminary results suggest that TCDD may not survive normal spraying operations. IV. Conclusions During the past few months, a number of careful, thorough, detailed reviews of 2,4,5-T and TCDD h.iuard3 have become available (4,34,35,36). Their consensus is that, under present restrictions, use levels and patterns, and TCDD levels, 2,4,5-T ar.d its derivatives can be applied safely. The concern of the regulatory agencies and the emphasis of the RVAR. document on occupational exposure is appropriate-- there is, after all. no evidence of public danger under normal use conditions-- out calculated "worst-case" extrapolations based on ocher "worse-case" eotimatas soon become unrealistic. -9- There i:; no question that TCL'D is itself a very dangero.s GubeC'nce. VJith the lack of explic.lt huaai-ixpcsme data for 2,4,5-T, prudence aemar.ds close control over its release, occupational awnrenr-.a of ch-. hazards, end free choice by individuals-- especially women oichild-bearing age-- of their occupational exposure to it. However, in vicv of the facts end interpreta tions presented here, this should not preclude continued important use of such a valuable chemical tool as 2,4,5-T. V, References 1 California Dzoartraent of Agriculture, "Pesticide liac Report 1972", Sacramento, CA. (1373). 2. California Pensrtncnt of Food and Agriculture, "Pesticide Une Report 1977," Sacramento, CA. (1378). 3. Kurahyi-Kovccs, I., et__al., Drit. J. Cancer 33^, 626 (1976). 4. Dost, F. N. "Toxicology of Pheroxy Herbicides end Hazard Ascar.ament of Their Use in Reforestation, " Forent Service, USDA, CaliforniaPacific Region, San Francisco, CA. (1373). 5. Courtney, K. D. , c,;: al.., Science ICS. 864 (1970). 6. Crosby, D. G. and A. S. Wong, Science 195, 1337 (1977). 7. Ilelling, C. S. at al., J. Environ. Quai. 1_, 171 (1973). 8. Hiller, G. C. ef s i . . Bull. Environ. Contt. Toxicol. JS, i l l (1977). 10. Radosevich, S. R. and W. L. Wintcrlin, Hoed Sei, _ 2 5 423 (1977). 11. Crosby, D. G. and A.. S. Wor.g, J. A&r. Food Chem. 21, 1052 (1973). 12. Baughman, R. and K. Meselson, Adv. in Cham. Ser. 3.20, 92 (1973). 13. Bcrtoni, G., et cl., Anal. Chea. 50. 732 (1378). 14. Buser, H. R., Anal. Chen. 49_, 913 (1577), 15. Hahle, N. H., et Bull. Environ. Coatam. Toxicol. 1S_, 123 (1977), 16. Axeison, 0. and L. Sundeii, Work Environ. Health Lj^, 21 (1974). 17. Leng, X. L., Down to Earth 2_3, 12 (1373). IS. Federal Worktng Group on Pest Management. "Occupational Exposure to Pesticides," Washington, D. C., 1974, p. 122. 19. Schweiz, B. A., et al., Adv. in Chen. Ser. 120,, 55 (2973). 00880P yv\oa pr pQ DOW 408801 20. NIOSH, "Registry of Toxic Effects of Clutafc.il Substances," 1977 ed. Vol. II, DuEW, Cincinnati, Ohio (1977). 21. NaS, "The Effects of Herbicide in South Vietnam," Washington, D. C. (1974). 22. Poland, A. P., et al., Arch. Environ. Health 22_, 315 (1971). 23. Knaak, J. B. and P. L. Schlecker, Pesticide Residues in the coveralls of mixer-loaders experiencing cnolinec te.-ase depression. Unnublished report, Califernia Department of Focu and Agriculture, (1976). 24. Wolfe, II. R. , J. F. Armstrong, and W. F. Durham, Mosquito Mew.: 34, 263 (1974). 25. Staiff, b. C., S. W. Comer, J. F. Armstrong, and H. R. Wolfe, Bull. Environ. Conran, Toxicol. 3_4, 334 (1973). 26. Houghton, E. P.., Unpublished Report, California Department of Food and Agriculture, (1578). 27. Kilgore, W. W., Food Protection and Toxicology Center, U. C. Davis, Davis, C.\. (1977). 28. Knaak, J. B., T. Jackson, A. S. Fredorickson, L. Rivera, K. T. Middy and N. Akcsson, Monitoring of Pesticide application workers, using closed mixing loading systems in C'.?iforaia. Unpublished report, California Department or rood and Agriculture, (1577). 25. V. Chancy, Unpublished Research, California Department cf Food and Agriculture. (1973). 30. Capita, P. E., D. Culver, and U. C. Ihralcn, AMA Arch. Indust. Hewilch 3.4, 326 (1956) . 31. Casarette, L. and J. Doull, "Toxicology", Macmillan, N. Y. (1975). 32. OSHA, Federal Register 39, 23540 (1574). 33. California Deportment of Public Health, Occupational Health Section and Center for Health Statistics, Berkeley, CA.' (1973) 34. Rnss, R. T., Statement before the California Senate Wildlife and Natural Resources Committee, April, 1578. 35. California Department of Food and Agriculture, Report on the A.crial Use of Pnenoxy Homicides, Sacramento, CA. , April 6, 1978. 36. "Vegetation Management with Herbicides," Vols. I and II, Forest Services, U3DA, Pacific Northwest Region, 1978. 18070 - li- 1103 U ...est 2, 1977 SUE JC i T: TCLD - d i o x i n : u t a g e i i c i t y V- < <-ra/ Fr.:.'.'. Rucli P e r t e ! , Ph.O OSPR, 7- Mary Reece and Harvey Warm'd; v.:-`i~. Hi A t tach ed i s a r e v i ew o f t he m u t a g e n i c i t y o f TCDD and r e l a t e d h e r b i c i d e s . The evi dence i s weak f o r h e r i t a b l e g e n e t i c e f f e c t s s i n c e the le v e l o f t e s t i n g is meager and the r e p o r t e d t e s t s have major d e f i c i enci es . However, TCDD appears t o have t h e p o t e n t i a l t o a c t as p o i n t (gene) mutagen ( Hu ssei n, e t . a l _ - > ( 1 9 7 2 ) and a chromosomal mutagen from the in vivo cytogenetics studies. Of concer n, i n t he l i g h t o f t h e D8CP pr obl ems, a r e s i m i l a r e f f e c t s on t e s t i c u l a r a t r o p h y i n r a t s due to c h r o n i c exposure to t he butyl, ether 2, 4, 5-T (Yefimenko, 1974). The rev iew of the Hussain paper by Dr. Malcolm is o f the q u a l i t y of v a lid a t io n s t h a t I would l i k e to see f o r a l l v a l i d a t i o n s . <7 C C <r' CO ocn CO t\D. .. .,o-;.... Jr -h i PA F . . n ? 0 i, I H . . . 3 74 Jam es M arlin (R, N.C.), a former professor of chemistry at Davidson College, proposed that this zero-risk concept has its root in the Delaney A m endm ent ( 1958) to the Pure Food, Drug, and Cosm etics Act.. T hat am endm ent essentially adds, to re quirem ents that a food, drug, or cos m etic be safe and effective, the m an date that the substance also be gener ally free of risk. Now suppose that to achieve zero risk, regulations state that an effluent contain a no detectable (N .D .) level of Chemical A. Suppose, further, that the latest monitoring instruments, capable of showing concentrations in parts per billion, indicate " N .D ." levels o f this Chemical A in the effluent; thus, the discharger is in com pliance. T hen, XYZ Instruments Corp. announces a new generation o f instruments that can delect Chemical A to one part per trillion, and this instrument, which now must be used as the monitor, de tects two parts per trillion, m axim um . The discharger is now in violation, and perhaps subject to adverse actions, regardless of what, if any environ mental, health, or welfare risk such a minute concentration of Chemical A may pose. Rep. Martin the D elaney syndrom e This is one of the m any problem s that the chemical industry faces-- problems brought about, and aggra vated by w hat C ongressm an M artin called " the Delaney Amendm ent mentality that pervades regulators." N aturally, one would like to minimize risk as much as possible. However, w hether zero risk is a desirable goal, even if it were technically and eco nomically achievable, is a question that could have philosophers in deep dis cussion for a long time. But no m atter how one m ay fe d about the m atter of risk, one thing is c e rta in --the present regulatory trend certainly has the chemical industry going through in teresting times! JJ 640 Environmental Science & Technology Z099960MOQ EPA suspends the major uses of two herbicides AliiennmkweboregmtewenenceylnisvfouinsrgpeesinntrsAyiolssnperoaa,fy2Oin,r4ge,s.5,-apTnrdoamnmdpistSeciadlvrtrehixaeges In M arch, the EPA took a step un render the herbicide less toxic, more precedented in its nine-year history: it safe. placed the herbicides 2,4,5-T and Sii- However, ongoing anim al studies vex under an em ergency suspension continued to docum ent the deadly po order, tem porarily banning the m ajor tency of even very low levels of dioxin. uses of these chemicals. EPA again held hearings in 1973-74 Both herbicides are unintentionally to consider cancelling 2,4,5-T 's regis contam inated with low levels of the tration. These hearings ended incon m ost deadly chem ical known to clusively, and the EPA w ithdrew its m an--TCDD (dioxin)--during their proposed cancellation order. production. It is suspected th at dioxin Now, 10 years after A gent O range contamination, and not the principle was withdrawn as an accessory war ingredients in 2,4,5-trichlorophenox- weapon, 2,4,5-T and its close chemical yacetic acid (2,4,5-T) and 2-(2,4,5- cousin, Silvex, have, been suspended for trichlorophenoxy)propionic acid (S il most uses. This is only a tem porary vex), is the cause o f the spontaneous ban until a full review determines their abortions that prompted the agency to health effects and their benefits. take this " most drastic" step. Dow and the three other m anufac Long history of use turers claim that the herbicides are safe. To the contrary, the EPA in its Dow Chemical, the m ajor m anu suspension order and notice of intent facturer of both herbicides, introduced to cancel cites 40 anim al studies that 2.4.5- T to the m arketplace in 1948; it show th at the herbicides cause spon soon found its place in forestry m an taneous abortions, birth defects and agem ent, and clearance of rights-of- malignant tumors. But, until the Alsea, way, uses th at are now tem porarily O re., study, no tangible correlation banned. could be made between the dioxin- As a com ponent o f A gent O range, contam inated 2,4,5-T and its effect on 2.4.5- T was widely used by U.S. troops hum an health. carrying out defoliation operations TCDD, the suspected culprit, has during the V ietnam W ar. R eports never been found in hum an tissues, from V ietnam of m iscarriages and although dioxin has been found in a deformed babies forced the U.S. to few samples of beef fat and human withdraw Agent O range from use. The milk. T he unintended hum an experi culprit was the teratogen dioxin-- m ent at Seveso is m arred by u nder 2,3,7,8-tetrachlorodibenzo-p-dioxin reporting of pregnancies, legal and il (TCD D ), the same chemical that legal abortions, and probably even erupted from a chemical plant in 1976 spontaneous abortions. and showered the residents of Seveso, Italy. The Alsea studies In 1970, the EPA restricted the uses Pregnancy and abortion records of 2,4,5-T to forestry and pasture were b etter for the A lsea basin. In management and clearance of rights- 19/8, nine women wrote to the EPA of-way, banning its use in homes, stating that their 13 miscarriages could gardens and at recreational sites. The be correlated with peak forestry agency also directed m anufacturers to sprayings. S cientists who exam ined reduce the dioxin content to 0 .1 ppm. their m edical histories and the reasoning that this reduction would spraying periods could not pinpoint a 1lupv0' ri1 Q- DOW0966608 cause-and-effect relationship. How ever, the EPA pursued the m atter further. The agency called upon scientists from Colorado State University and the University of Miami Medical School to study rates Of m iscarriages over a six-year period. M iscarriages in the Alsea area where heavy spraying of 2,4,5-T occurs annually in M arch and April were compared to miscar riages in a sim ilarly rural are a in eastern Oregon where spraying does not occur. The findings: the miscar riage rate in the Alsea basin was sig nificantly higher than th a t in the con trol area, and the miscarriages in Alsea peaked in Ju n e and July, two to three m onths after the annual Spring sprayings. ably begin in July, are expected to take several years to complete. Safe or not? Dow claims that it is fighting E PA 's action, not because 2,4,5-T is such a money-m aker for the com pany-- it only accounts for 0.2% of Dow's an nual revenues-- but because " it's time to take a stand.'' Dow president David Rooke queried at a press conference: " If you can wipe a product with this much proven safety off the market, then w hat is safe?" T he safety o f 2,4,5-T is now being more carefully determined. The EPA is analyzing sam ples of soil, w ater, gam e m eat and hum an milk from the Alsea basin for TCDD. The University o f Illinois M edical C enter is planning AIR SAMPLING Study links miscarriages to herbicide sprayings : ^ Air Quality Sampler The EMI AQS-II. an economical alter native to continuous air monitoring, collects unattended samples tor up to 96 hours. Equipped with a low power electronic timer/controller. the AQS-II can be programmed to gather 4 to 24 sequential or random hourly sam ples. Leasing is available. EPA's Blum "alarm ing correlation" "This alarming correlation," deputy adm inistrator Barbara Blum stated at the press conference announcing the suspension, "comes at a time when 7 million pounds of 2,4,5-T are about to be used to control weeds on power line rights-of-w ay and in pastures, and to manage forest lands across the N a tion." The suspension, Blum said, would protect 4 million people from exposure to the Spring sprayings, and have minimal short-term economic impact. Blum cautioned that the epidemio logical study is highly suggestive, though "not proof of a cause and effect relationship." Dow and the 10-12 other plaintiffs seeking legal and ad ministrative relief agree. Dow calls the study "seriously flawed." The herbicide manufacturers with drew from an administrative hearing which was to decide on the correctness of the emergency suspension. The hearing was dissolved iast month. Dow then asked for immediate cancellation hearings before an EPA adm inistrative law judge. Such a judge was assigned to the cancellation hearings. He called for a prehearing conference early this month. The hearings themselves, which will prob- Dow's Rooke "seriously fla w ed " a computerized (medical) case history study of Vietnam veterans, and their spouses and offspring. A nother Illinois scientist, M. M. Yokoyama, is looking for alterations in levels o f hum an white blood cells and antibodies caused by exposure to Agent Orange. , Should dioxin show up in E P A 's collected samples, or should alterations in circulating levels of leukocytes and antibodies be traceable to dioxin ex posure, the environmental agency would have little difficulty in cancel ling the herbicides' registrations-- som ething it had attem pted to do wiLh 2.4.5- T in 1973. T he cancellation hearings wili d e termine whether the risks outweigh the benefits, or whether the risks are ac ceptable. However, the fact that fewer than a dozen of the several hundred potentially hazardous pesticides have been banned by the EPA in its nineyear existence speaks to the circuitous and ineffectual registration and can cellation procedures laid down by the pesticide law. M anufacturers can skillfully m anipulate these regulations to their advantage. And they have. The 2.4.5- T case is not unique: it is simply a microcosm of pesticide politics. LRE Radon Sampler Similar to the AOS. the Radon Sam pler collects a 12 to 46 hour sample in a single bag. Designed lo satisfy NRC requirements, the Radcn Sampler in cludes an electronic controller filter, regulated piston pump, and battery, all in an environmentally enclosed housing. Air Sampling Bags These pretested, chem ically inert, highly retentive duPont Tedlar" bags successfully collect many gases In stock sizes of 2, 5.10.15. 30. and 50 liters are supplied with fitting, tubing, and pinch clamp. Custom sizes and hardware are available. Peisonai Sampling Pumps These sm all and versatile Pulse Pumps have (low rates adjustable from 1 to 20 liters per hour and a high Mow rate of 90 liters per hour As both tiller sampler and bag filler they adapt to air sampling applications from industrial hygiene monitoring to ambient air collection. Rechargeable batteries optional. /IR O N M EN T A L ASU R EM EN TS. INC le id e s o o r ff st reet gr F-R ACN C IS4CifOi/'.1CQAfl.974R1R141 CIRCLE 1 ON READER SERVICE CARO V olu m e 13, N u m b er 6. June 1979 2,4-D FACT SHEET I % Background 2 , 4 - D is one of the m o s t w i d e l y used h e r b i c i d e s in the United States. There are approximately 1,500 products containing 2,4-D registered with EPA, and more than 70 million pounds of the active ingredient are distributed annually. The term "2,4-D" refers to the phenoxy h e r b i cide 2 , 4 - d i c h l o r o p h e n o x y a cetic acid and its 35 d e r i v a t i v e salt and e ster forms. 2 , 4 - D is used to cont r o l b r o a d l e a f w e e d s in a v a r i e t y of p l a c e s i n c l u d i n g h o m e l a w n s , c e r e a l and grain crops, commercial areas, commercial turf, rightsof-way, and forests. Public concern about the potential adverse health effects of 2,4-D has intensified since the emergency suspension of - 2 . 4 . 5 - T and Silvex in M a r c h 1979. This concern stems p r i m a r i l y f r o m 1) the c h e m i c a l s i m i l a r i t y of 2 , 4 - D and -- 2 . 4 . 5 - T as p h e n o x y h e r b i c i d e s , and 2) the q u e s t i o n o f 2 , 4 - D d i o x i n - c o n t a m i n a t i o n , e s p e c i a l l y c o n t a m i n a t i o n w i t h t e t r a c h l o r o - i" d i o x i n , a m a n u f a c t u r i n g c o n t a m i n a n t in 2,4,5-T, w h i c h c a u s e s DOW 357486 cancer and miscarriages. Due to the chemical s i m i l a r i t y of 2.4- D and 2,4,5-T, the public has expressed concern about the potential for cancer- and miscarriages from the use of 2 . 4 - D. T h e r e is a l s o c o n c e r n b e c a u s e the c o n t r o v e r s i a l m i l i t a r y d e f o l i a n t A g e n t O r a n g e , used in Vi e t Nam, was composed of 2,4,5-T and 2,4-D. Agent Orange was never r e g i s t e r e d b y - E P A for c i v i l i a n use In the U n i t e d S t a t e s . Its u se in V i e t N a m by the U .S .'m l 1 i t a r y has r e s u l t e d in c l a i m s of a d verse h ealth ef f e c t s to A m e r i c a n m i l i t a r y pers o n n e l . T h e V e t e r a n s A d m i n i s t r a t i o n is s t u d y i n g the s e c l a i m s . P r o m p t e d by these c o n c e r n s and E P A ' s need to r e s o l v e the questions surrounding the use of 2,4-D, the Agency initiated a review of.the available- information on the potential health ejffects of 2,4-D. This, r e v i e w w a s c o n d u c t e d in p a r t to * ^ -determine if the h e r b i c i d e s h o u l d -be r e v i e w e d u n d e r the RPA_R p r o c e s s ( R e b u t t a b l e P r e s u m p t i o n A g a i n s t R e g i s t r a t i o n ) or if .another regulatory action was appropriate. . I I . Agency Review and Conclusions Based on the results of this review, EPA has concluded that a) the p r e s e n t l y a y a i l a b l e i n f o r m a t i o n on the p o t e n t i a l adverse health effects of 2,4-D does not support a regulatory a c t i o n to r e m o v e 2 , 4 - D p r o d u c t s from the m a r k e t ; b) i n f o r m a t i o n f r o m s c i e n t i f i c a l l y v a l i d s t u d i e s d o e s not i n d i c a t e t h a t t.e continued use of 2,4-D poses an imminent hazard or unreasonable ad v e r s e effect when used a c c o r d i n g to label p r e c a u t i o n s and d i r e c t i o n for use; and c) the A g e n c y s h o u l d act q u i c k l y and v i g o r o u s l y to obtain b e t t e r t o x i c o l o g i c a l i n f o r m a t i o n on 2 . 4 - D. T h e s e c o n c l u s i o n s arc- b a s e d on t h e s e f o l l o w i n g considerations: .. . I 1. T h ere is no e v i d e n c e a v a i l a b l e at this t ime that indicates 2,4-D contains any form of dioxin. This includes the t e t r a c h l o r o - d i o x i n (TCDD), w h ich is a m a n u f a c t u r i n g contaminant of 2,4,5-T and causes cancer and miscarriages. T C D D is not t h e o r e t i c a l l y e x p e c t e d to be found in 2,4-D. The. m a n u f a c t u r i n g p r o c e s s e s and s t a r t i n g c h e m i c a l s from o which 2,4-D and 2,4,5-T are made are not the same. Although other much less toxic dioxins are theoretically p o s s i b l e in 2 , 4 - D , t h e y h a v e n o t b e e n f o u n d d e s p i t e thorough chemical analyses. 357487 2. B e c a u s e p r o d u c t s c o n t a i n i n g 2 , 4 - D h a v e b e e n registered for use since the 1940's, most of the scientific data submitted to support the product r e g i s t r a t i o n s now ; on the m a r k e t w e r e d e v e l o p e d m a n y y e a r s a go. W h i l e s o m e (..of t h e s e s t u d i e s a r e s c i e n t i f i c a l l y v a l i d , m a n y o t h e r s do ''not meet today's standards for scientific testing. As a result, there are s i g n i f i c a n t i n f o r m a t i o n g a p s in several CO areas including cancer-potential, reproductive effects, ^ n e u r o t o x i c i t y , and metabolism in-animals. 3. The s t u d i e s m o s t p e r t i n e n t to the q u e s t i o n o f .. -- tumor-causing potential (oncogenicity) of 2,4-D were considered inadequate and inconclusive. No valid conclusions could be drawn one way or another from the data. 4. A l m o s t all a n i m a l t e s t s c o n d u c t e d on the p o t e n t i a l reproductive effects of 2,4-D show that, unlike 2,4,5-T with its contaminant TCDD, there is a n o - e f f e c t level for injjjry. to the fetus' (feto.toxicly) fjrom 2 , 4 - D . A n o - e f f e c t _level in a n i m a l s t u d i e s is the d'o s l e vel b e l o w t.he jLoweg.f'dosage that-.produces observable -adverse effects. At c o m p a r a b l e d o s e l e v e l s , 2 , 4 - D i n d u c e s l e s s s e r i o u s fetotoxic effects than-2,4,5-T contaminated with TCDD. ^ In- t e s t s w i t h rats, 2 , 4 , 5 - T w i t h its T C D D c o n t a m i n a t i o n c a u s e d r e s o r p t i o n s of trfe f e t u s ( d i s s o l u t i o n of the u n b o r n , animal) at very low levels. This effect was virtually n o n - e x i s t e n t in rats fed 2 , 4 - D at the same d ose levels. Because of the significance of fetotoxic effects and because several of the r eproductive tests on 2 , 4 - D w e r e found to be scientifically deficient, new tests will be needed before sound conclusion can be made. -, ... 5. T h e s c i e n t i f i c e v i d e n c e a v a i l a b l e at t h i s t i m e d o e s n o t I n d i c a t e the p o t e n t i a l h u m a n e x p o s u r e is s u f f i c i e n t -- to r e s u l t in h u m a n h e a l t h e f f e c t s . ,,VJ-* /) ^ -. -C . The most vigorous authority available to EPA under ,, the pesticide law to fill information needs is a new section of FIFnA (Federal Insecticide Fungicide Rodenticide Act) passed in 197fl. This provision, known as 3(c)(2)(B), allows EPA to request any additional data from pesticide registrants that is considered necessary to maintain the registration of existing products. The Agency can immediately require the manufacturers to develop the data where gaps exist. The registrants have 90 days to show that they are complying. Their product registrations may be summarily suspended if they fail to meet the Agency's conditions. No other action could obtain this information any faster. EPA is putting the data requirements Into final form and they will be issued to the registrants after review by our Scientific Advisory Panel. These scientific experts will review and comment on the data requirements to assure that they will provide the information EPA needs to more definitively answer the questions on potential health effects of 2,4-D. 7. Based on a-'review of the toxicology data (see section IV below), and a review of the risks of other pesticide chemical now undergoing regulatory action, the Agency believes that the * risks of several other pesticides are higher and better document * 6 than those associated with 2,4-D. To put the review of these other higher priority chemicals aside in order to devote EPA iO resources to taking action against 2,4-D would not, in the Agency's opinion, best serve the public interest. CO cn III. Additional Actions In addition to requiring several Important studies of the manufacturers on 2.,4-D, EPA will also: 00 00 1. Conduct several tests on-reproductive effects;- fthrough L our. Office of Res'ea-rch and- Development) of seve-cal -de-rivaiives of 2,4-D in order to quickly get aew.inforraation a'nd have a good basis for comparison with the company-produced* dat a. . ' `2. Continue i*bs ondoing review of forest pest control' practices. This review will evaluate all chemical and nonchemical controls to identify the most environmentally protectivewavs to control forest pests. The Agency believes that a piecemeal approach-to forest chemical regulation only leads to confusion, both to the industry and to the public. Unless we review the whole range of possible controls, examining one chemical at a time only gives rise to questions about the chemicals which would be used to replace those examined and prohibited from use. 3. Review all new data as it comes in to determine if a chanqe in our regulatory posture Is warranted. This Includes evaluation the results of new animal tests as well as lookina Into reported incidents involving human exposure- to the chemical. - ' i!% ' .4. C o n t i n u e to s u p p o r t fie l d tests to m e a s u r e e x p o s u r e to 2 , 4 - D d u r i n g the p r e s e n t g r o w i n g s e a s o n . '7. '\ ` 5. EPA is i n f o r m i n g the I n t e r - A g e n c y W o r k G r o u p , `: e s t a b l i s h e d by the W h i t e H o u s e to s tudy the p o s s i b l e l o n g - t e r m effects of Agent Orange, of the actions being taken. EPA will also share its scientific findings with this committee. IV. T o x i c o l o g y B ac k g r o u n d The p o t e n t i a l h a z a r d of a c h e m i c a l is u s u a l l y m e a s u r e d in l a b o r a t o r y a n i m a l t e s t s . A n i m a l s are g i v e n d o s e s of a chemical over a specific time period. Scientists attempt to d e r i v e from most of these tests a "no o b s e r v a b l e e ffect level" (NOEL) -- the dose level below the dosage where effects are first o b s e r v e d . From the animal tests and NOEL's, the potential effects on humans and other animals c a n be e s t i m a t e d . A set of b r i e f d e f i n i t i o n s is p r o v i d e d b e l o w to p ermit b etter u n d e r s t a n d i n g of the s u b s e q u e n t v. discussion of toxicological findings. . 00^357489 A. Gene r a l terms 1. A c u t e o r a l t o x i c i t y (LD50) - t his t e s t d e t e r m i n e s the dose level which produces death in half the test animals after a single oral dose (short-term test). Used to p r edict the n e a r term toxicity of the chemical immediately upon contact with people or other non-target animals. 2. C h r o n i c f e e d i n g t e s t s - a n i m a l s a r e fed for :u m o s t their life span (usually g r e a t e r than 18 '-2 m o n t h s in r o d e n t s ) in o r d e r to d e t e r m i n e the d o s e level w h i c h s h o w s no toxic e f f e c t in test - a n i m a l s . Thi.s is the test from w h i c h the N O E L 7 is (usually) d e r i v e d . * 3. O n c o g e n i c i t y t e s t i n g - animals fed r e l a t i v e l y -'large d o s e s of the t est c h e m i c a l for t h eir l i f e . spa n ( u s u a l l y 1 8 - m o n t h s to 2 y e a r s in rodents) to try to i n d u c e t u m o r s . T h e s e t e s t s are. used. -J 7 to p r e d i c t w h e t h e r the c h e m i c a l m a y p o s e a 7" c a n c e r h a z a r d . 4. R e p r o d u c t i v e t e s t i n g - t h e s e t e s t s e v a l u a t e the effects of the chemical on the fe r t i l i t y of both- the male and female parents by exposing the animals for a period of time before breeding. s' The tests also measure the possible effects of the chemical on the pregnant female and the fetuses through several generations. (The test with rodents through 3 generations runs a p p r o x i m a t e l y 14 months. ) - ' - r> 'V"* DOV/357490 5. T e r a t o l o g y t e s t i n g - these t e s t s e v a l u a t e the e f f e c t s of t h e chemi c a 1 on f e t u s e s by e x p o s i n g pregnant females during the short period of time t h a t t he f e t u s is most s u s c e p t i b l e t o c o n g e n i t a l malformation. Teratogenic effects include c le ft p a l a t e , c e n t r a l nervous system d e f o r m i t i e s , eye and l imb d e f o r m i t i e s , and i n t e r n a l organ m a l f u n c t i o n . T h e s e a r e c o n s i d e r e d t o be l i f e - t h r e a t e n i n g e f f e c t s that put the animal at a disadvantage fo r s u rv iv in g in its environment. 6 . F e t o t o x i c i t y - f e t o t o x i c e f f e c t s can be seen i n e i t h e r the reproduction or t e r a t o lo g y t e s t s . T o x i c i t y may be s een i n t h e e x t r e m e f o r m as f e t a l d e a t h o r as l e s s s e v e r e p r o b l e m s , such u as d e l a y e d f o r m a t i o n o f b o n e s , r e d u c e d body 1 w e i g h t s a t b i r t h , o r edema ( a b n o r m a l f l u i d 1accumulation in the t i s s u e ) . Most f e t o t o x i c e f f e c t s a p p e a r t o be r e v e r s i b l e o n c e e x p o s u r e to the test chemical is c u r t a ile d . Therefore most f e t o t o x i c e f f e c t s are c o n s i d e r e d to be l e s s s e r i o u s t h a n t e r a t o g e n i c e f f e c t s , w i t h the exception of fetal death. B. Summary o f T o x i c o l o g y Review Mo s t o f t h e d a t a i n EPA f i l e s on t h e p o t e n t i a l h e a l t h e f f e c t s of 2 ,4 - 0 are centered_on the acid form, even though t h e r e a r e many d e r i v a t i v e s , such as s a l t s and e s t e r s . T h i s is b e cause t h e many forms o f 2 ; 4 - 0 m e t a b o l i z e to t h e a c i d form in the e nv i r o n m e n t and in t h e body. The d i s c u s s i o n of animal data below, t h e r e f o r e , concerns the acid form of 2 , 4-D.unless otherwise noted. 1. Acute t o x i c i t y - low to m od e ra te . The p o t e n t i a l 7 - f o r i mmed i a t e po i son i ngs f r o m c o n t a c t w i t h t h e - * c h e m i c a l i s un 1 i k e l y r- * 2 . N e u r o t o x i c i t y - T h e r e i s l i t t l e d e f i n i t i v e - i n / o r m a t i on . o n t h e p o s s i b l e n e u r o l og i . c a l e f f e c t s of 2 , 4-D. In .s e v e ra l reported cases of impaired n e r v e f u n c t i o n , i t was not known i f t h e i n d i v i d u a l s were p e c u l i a r l y s e n s itiv e to that type of e f f e c t or were exposed to other t o x i c m a t e r i a l s . , 3 . R e p r o d u c t i v e e f f e c t s ( e f f e c t s on t h e u n b o r n ) T e s t s have been c o n d u c t e d on r a t s , m i c e and hamsters to evaluate the possible reproductive e f f e c t s o f 2 , 4 - 0 . I n a l m o s t a l l t e s t s , a NOEL has been e s t a b l i s h e d . 2 , 4 - D c a u s e s some o f . t h e l e s s s e r i o u s f e t o t o x i c e f f e c t s , such as edema (s w e llin g of tissu es) at the lower dose levels t e s t e d , and causes I i f e - t h r e a t a n i n g b i r t h d e f e c t s ( s k e l e t a l ma1f o r m a t i o n s ) and c l e f t p a l a t e s only at the very high l e v e l s t e s t e d . 18079 B a s e d on t h e NOELs I n t h e an 1 <j 1 s t u d i e s , EPA e s t i m a t e s t h a t t h e l e v e l o f e x p o s u r e i n a "worst-case" s it u a t io n (eg. a person standing d i r e c t l y u n d e r a s p r a y p l a n e ) wo u l d be 500 to 1000 times less than the dose lev e l that ~ m i g h t c a u s e an e f f e c t . *; 1* Much of t h e d a t a a v a i l a b l e t o ju d g e t h e s e e f f e c t s was g e n e r a t e d by o l d s t u d y p r o t o c o l s , has d e f i c i e n c i e s in t h e t e s t met hods, and needs c l a r i f i c a t i o n by f u r t h e r s t u d y . i. EPA a l s o r e v i e w e d s u m m a r i e s o f t e s t s c o n d u c t e d ' i n R u s s i a w h i c h s t a t e t h a t some d e r i v a t i v e s o f 2 , 4 - D p r o d u c e d a d v e r s e e f f e c t s on u n b o r n a n i m a l f e t u s e s a t much l o w e r l e v e l s t h a n i n d i c a t e d by the data in EPA's f i l e s . These summaries c o u l d not be used in t he A g e n c y ' s r e v i e w b ecause t he i d e n t i t y o f t h e t e s t m a t e r i a l , and i t s i m p u r i t i e s , was u n c l e a r , and b e c a u s e t h e r e we r e no n u m e r i c a l d a t a t o back up t h e summary c o n c l u s i o n s . I n some c a s e s t e s t s need t o be done o n . s p e c i f i c d e r i v a t i v e s o f 2 , 4 - D . T 6 P ls e ^ o a 3. Oncogenicity -(p o te n tia l fo r causing tumors) Severa l rodent s tu d ies have been conducted to d a t e , but none of these s t u d i e s produced d a t a t h a t showed 2 , 4 - D was o n c o g e n i c in t e s t a n i m a l s . The t e s t s were c o nd u ct e d a decade ago and a r e c o n s i d e r e d t o be i n a d e q u t e by t o d a y ' s s c i e n t i f i c s t a n d a r d s . New s t u d i e s on r o d e n t s are needed. 4. M u ta g e n ic ity ( i n h e r i t a b l e e f f e c t s ) - The vast m a jo rity of the m utagenicity studies conducted on 2 , 4 - D _ a r e n e g a t i v e . H o w e v e r , t h e r e a r e t h r e e . . p o s i t i v e s t u d i e s . T a k e n as a g r o u p , t h e r e s u l t . s / o f t h e ; s t u d i es c a n - b e s t - be d e s c r i b e d as i n c o n s i s t e n t and . i n c o n c l u s i v e . A new s e r i e s ' o f t e s t s b e i n g . ' ' c o n d u c t e d by t h e D e p a r t m e n t o f H e a l t h , E d u c a t i o n , ^ ' a n d W e l f a r e wi 11 be r e v i e w e d by EPA when t h e y ' a r e -compl e te d '. 5. E p i d e m i o l o g y - No e p i d e m i o l o g i c a l s t u d i e s o f human h e a l t h e f f e c t s fro m 2 , 4 - 0 e x p o s u r e have b e e n c o m p l e t e d . H o w e v e r , EPA i s c u r r e n l t y 1 n v e s t i ga t'i ng r e p o r t s a b o u t a l l e g e d a d v e r s e e f f e c t s from p o t e n t i a l chemical exposure i n s e v e r a l p a r t s o f t h e c o u n t r y . EPA w i l l be l o o k i n g at t h e r e s u l t s o f t h o s e s t u d i e s and w i l l decide in the near fu tu re about a d d itio n a l f i e l d wo r k . V. Exposure to 2,4-D There are at least three ways that the average citizens might come into contact with 2,4-D - through the diet, during home use, and drift of the herbicide from nearby use. i a) Diet The EPA has set tolerances for residues of 2,4-D in various food crops. The Food and Drug Administration (FDA) routinely samples a variety of foods (the Market Basket Survey) which FDA considers to be representative of the average American diet. Samples are analyzed for pesticide residues. During the period of 1974 to 1977, no 2,-1-D residues were found in any of the products surveyed. However, during the 1965 to 1977 period, a variety of other food products were analyzed, of which about 1.1% were positive for 2,4-D in very minute quantities that were well below EP A's tolerance (allowable residue) levels. % b) Home use There are currently a number of registered home-use products which contain 2,4-D in a variety of formulations. Exposure to the herbicide in home-use situations will depend to some extent on the specific formulation used. If care is exercised by the homeowner in adhering to the directions for use and precautionary statement on the label, exposure to 2,4-D should be low. oo * c) Drift "Drift", the airborne transport of pesticide materials' to a non-target area,.is a common source of exposure. Sometimes, a pesticide will drift during application, depending on cl ima tic' cond itions- (tempo ra-ture , wind speed), type of formulation used, terrain (fores_fcs, moua-tai-cs) _ and type of _appl icat ion method used Jae.'ial, ground spr-ay) . Several States have imposed restrictions on 2,4rD use-in order to cut down on drift-potential . Once on the ground or target crop, the herbicide may become airborne again by the process of vaporization. This particular type of -drift has been the subject of intensive research by the producers of 2,4-D. Since the introduction of less volatile forms of the herbicide over the last few years, this kind of drift nas become much less extensive. 357492 U<\ : VI E n v i r o n m e n t a 1 P e r s i s t e n c e I t -vv ': 2 , 4 - 0 is not a p e r s i s t e n t p e s t i c i d e . Breakdown of t h e ' ' h e r b i c i d e begins almost immed ia tely a f t e r a p p l i c a t i o n at a r a t e d e p e n d e n t on s e v e r a l e n v i r o n m e n t a l f a c t o r s su ch as t e m p e r a t u r e , h u m i d i t y and medium ( a i r , s o i l , c r o p , w a t e r ) . The r a t e o f l o s s ( c ommonl y r e f e r r e d t o as t h e w h a l f - l i f e ) is a measure of the time req u ired fo r h a l f of the s u b s t a n c e t o be d e g r a d e d o r l o s t . % i' On s p r a y e d v e g e t a b l e s , t h e h a l f - l i f e v a r i e s f r o m 1 - 3 Ol w e e k s d e p e n d i n g on g e o g r a p h i c l o c a t i o n , c l i m a t i c c o n d i t i o n s , v e g e t a t i o n t y p e , a p p l i c a t i o n t e c h n i q u e and f o r m u l a t i o n u s e d . OX CD I n s o i l , t h e CO w e e k s , d e p e n d i ng h a lf - lif e varies on a c i d i t y , s o i l from type several days to 2 and amount o f r a i n . ' In w a t e r , the h a l f - l i f e v a r i e s from a few days to seve ra l m o n th s d e p e n d i n g on f a c t o r s su c h as o x y g e n c o n c e n t r a t i o n , a c i d i t y , l i g h t i n t e s l t y , w a t e r t e m p e r a t u r e and f o r m u l a t i o n used. . * A pril 22, 1980 '1* I- V I .9 HUNwf< TdcntiHcii*ion of Three Neulnil Conliimin.'inl.s in Production Crude 2,4-D 1.115 Beverley L. liuslon /~t< J * Tlic structure o f a nt:-.: -r neutral contam inant in production grade 2,4-D is derived by chemieal and spectroscopic data ami confirmed by synthesis. Two other neutral impurities are tentatively identified and shown to be isomeric with the major neutral o-.ntaminant by com bined glc mass spec troscopy. livid ace is presented to show that these im purities can interfere with the pic analysis of 2,4-D for 2,3,7,8-tctrachlorodiben/o-yj-dioxin DOW372303 MW0S3305 A llhough the toxicology o f pesticides is extensively studied, Z-&, little research has been devoted to the nature and -f- toxicological significance of trace am ounts of im purities in pesticide chemicals. However, the observation (Courtney c t a t., 1970) that samples o f 2,4,5-T (2,4,5-irichlorophcnoxyacctic acid) used in a teratogenic study contained significant am ounts of the highly toxic compound 2,3,7,8te(rachlorodihenzo-/>-dio\in (H igginbotham e l a !., 1968) has led to an increased interest in the nature of im purities in pesticides. Recently, we had occasion to analyze, by gas-liquid chro m atography,.the neutral extracts of some samples of produc tion grade 2,4,5-T and 2,4-D (2,4-dichlorophcnoxyacetic acid). The glc tracings of the extracts from both pcst: ides indicated the presence of several neutral contam inants an.I in the case of the 2,4-D extract three of these impurities had very sim ilar retention times to tlv.it of 2,3,7,S-ictrachlorodibenzo-/> dioxin. The present paper describes the isolation and char acterization of the major of these three impurities and the tentative identification of the other two. Furtherm ore, this work points out the need for camion on the part of the analyst in interpreting glc data alone when analyzing samples o f pesticides for impurities. EXPERIMENTAL M aterials. All solvents were pesticide grade and used as received. Production grade 2,4-D and the 2,3,7,S-tetrachlorodibenzo-/i-dioxin were generously supplied by Dow .Chemical Com pany, M idland, M ichigan. 2,6-Dichlorophcnol, 2,4-dichlorophcnol, and methylene iodide were ob tained from Aldrich Chemical Com pany, Eastman Organic Chemicals, and Fisher Scientific, respectively. Instruments. Mass spectra were obtained with a Hitachi Perkin-F.lmer (I? MS-4) mass spectrom eter coupled with a Perkin-Elmer (M odel 990) gas chrom atograph, fitted with flame ionization detectors. The stainless steel column, 6-ft X '/'m- o.d., was packed with 3% X 11-60 on 100 120 mesh C hrom osorb \V (HP), C hrom atographic Specialties Ltd. Operating conditions were: detector and injector temperatures, 220` C ; oven temperature, 200"C; carrier gas, helium at a flow rale of 34 ml/niin. N m r spectra were o b tained with a Varian A-60A spectrom eter in dcuieroehloroform solution with letramcthylsilane as internal stanilanl. Ultraviolet spectra were obtained with a Unieam model SP 18(H) spectrometer. Melting points were determined with an Elicit otlk-rmal capillary melting point apparatus an I arc unrorrcclcd. M itroaualyses were perform ed by A. II. (iygli, Miemanalysis I aboialories Limited, Toronto, Ontario. Research I a ho i atoi ies, I n o d and O i up D i n a l o i a l c , Mepai Il l i c i i l o f N a t i o n a l l l . a lth a nd W M l 'a ie , I u m i e y 's P a s til le , ( )l ( a w / u |< I A H I ( l u i a i u> Chrom atography. Baker analyzed reagent silica gel was used for column chromatography. Preparative thin-layer chrom atography was performed on plates coated with 0.5 mm silica gel (M N-Silica Gel G /u.v.:1,, M achenoy, Nagel & Co.). A mixture o f hexane-benzene (4:1) was used ns developing solvent. Glc analyses were obtained on several instruments using the colum ns and conditions listed in Table I. Extraction of 2,4-D Neutral Impurities. Production grade 2,4-D (I kg) was extracted by shaking with hexane (I I.) and filtering through a medium porosity sintered glass funnel. The extraction process was repeated xviih four additional (1 I.) portions of hexane. A total of 8 kg of 2,4-D was processed in this manner and the combined extracts were evaporated to dryness on a rotary evaporator at 40C. The residue was taken up in ether (100 ml) and extracted with two 50-c.:I portions of 10% sodium hydroxide solution, washc with water (50 ml), and the ether layer was dried with anhydrous sodium sulfate. Evaporation of the ether gave the neutral impurities as a pale yellow semi-solid. Purification of Impurity III. The neutral residue was dissolved in a m inim um am ount o f hexane and ch ro m ato graphed on a colum n o f silica gel (1.5 cm X 42 cm) Elution with hexane (200 mi) gave a fraction that contained impurity III as the m ajor com ponent as indicated by glc. P reparative tic o f this fraction gave a band (7?i 0.62) which was eluted with ether and shown by glc to contain impurity III with a trace of im purity II. Three recrystallizations of (his m aterial from hexane gave pure III as colorless crystals, mp 101 - 101.5'C : nmr 5 5.76(s,2 H), 7.25-7.41 (m, 6 H ); uv 228 nm (t 19,900), 281 (2740), 289 nm (2240); mass spectrum m / c 336 (parent ion). A n a l. C alcd. for C 13ILO .C L : C , 46.42; H , 2.38; Cl, 42.26. F ound: C ,4 6 .3 4 ;H ,2 .3 5 ;C I, 41.24. Synthesis of His(2,4-(lichloropheno.\y)mctliane and Bis(2,6-<liclilon>phenoxy)inellianc (3 and 5). The ap propriate dichlorophenol (16.3 g, 0.1 m ol), anhydrous potassium carbonate (14 g, 0.1 mol), methylene iodide (4 ml, 0.05 mol) anil dry acetone (100 ml) were com bined and refluxed for 8 hr. The m ixture was cooled, filtered, and the solvent removed in v a c u o . The residue was dissolved in methylene chloride (100 ml), washed with two 75-rnl portions of 10% sodium hydroxide solution, one 75-ml portion of water, anil dried over anhydrous sodium sulfate. Evaporation o f the solvent gave a pale yellow solid which was recrystalli/ed twice from 95% ethanol to give colorless crystals of 3 an d /o r 5. H is(2,4-dii.hloroplieno\y)m ethane, mp 100 1 0 2 'C [lit. mp 100.4- I00.K X (M iron and l.owy, 1951)]. M ixture melting point with III 101 102 "C ; n m ri 5.75 (s. 2 II), 7.25 7.41 (m, 6 I I); maw spectrum m / c 336 (parent ion). B is(2 .0 d iih ln ro - X^30 3phenosy)nielh:ine, mp 78.5 80"C; nun ^ 5.6X (s, 2 I :), 6.85 7-10 ^ (lit, 6 11); mass spi-etrum i/i/e 336(parent ion). i U l . N I t J ' J l . A 1 HI 1 n i u i i . h l . U i <\/U. L \ /M i Tabic 1. Glc Parameters Column Stationary phases Support Injector/ Oven defector 'temper- temperature, nture.v. Dlmcns'i :-s *C *C Detection Carrier gas flow rate, tiil/min Glass Glass Stainless steel Stainless steel Q IC'-9/l%//Sr*r.t/-t.*30 SE-52 2.5% SE-30 5% XE-60 3% 80 -100 mesh Chromosorb w (Hr) 80 100 mesh Gas Chrom Q 80-100 mesh Chromosorb W 100- 120 mesh Chromosorb W (HP) 5-ft X '/.-in. (o.d.) 3-fl X '/.-in. (o.d.) 6-fl X '/.-in. (o.d.) 6-ft X '/.-in. (o.d.) 225/210 220/220 230/250 220/220 190 Tritium foi!' electron 2* capture 185 Tritium foil* electron 60- capture 200 Flame ionization' 30* 200 Flame ionizatii - / 35 Carrier gas, nitrogen. *Carrier gas, h e liu m . Varan Aerograph Model 600-D. * Varan Aerograph Model 2100. 'Hewlett-Packard Model 700. ! Pcrlsin-lilmcr Model 9^0. Tabic II. Gas Chromatographie Comparison of Three 2,4-D Neutral Impuri lies and 2,3,7,8-Ti'.rachlorodibciizo-/J-dioxin Column QF-l/SE-30 SE-52 SE-30 XE-60 Retention lime, min 2,3.7,8-lclrachlorodioxin 11.5 7.4 7.9 5.8 2,4-D Impurities I II HI 9.2 10.4 11.6 5.6 6.5 7.4 6.0 6.7 7.7 5.7 6.3 7.6 A n a l. C a ic d .fo rC ,JH ,0 ,C I,: C, 46.42; H , 2.38. F ou n d : C, 46.56; H, 2.41. Synthesis of I.,2',4,6'-Tctrachlorodiphcnoxyir.-fhane (6). cr-2,4-Trichloroani.: 'c (2 g, 0.01 mol) [prepared according to the method of Lashua and Ranck (1966)] was stirred and re fluxed for 8 hr with a suspension of f.c potassium salt of 2,6dichlorophcnol in dry benzene. The cooled mixture was washed with water and the benzene layer dried over anhydrous sodium sulfate. Evaporation of the solvent gave a colorless solid which, upon four recrystallizations from 95% ethanol, gave colorless needles, mp 8 5 -S6C: nm r 5 5.75 (s, 2 H), 6.88-7.61 (m, 6 H) ; mass spectrum m j e 336 (parent ion). A n a l. C alcd.forC uH .O jC I,: C, 46.42; H ,2.38. Found: C, 46.53; H.2.48. RESULTS A N D DISCUSSION T he analysis of the neutral extracts of production grade 2,4-D was investigated by glc using the columns and conditions listed in Table I. It is apparent, from an exam ination of F igure 1, that several neutral im purities are present and, ns indicated in Table II, three of these components, labeled I, II, and lil in Figure I, have retention times similar to that of 2,3,7,8-tcirachlorodibenzo-/j-dioxin under the glc conditions stated in Table I. Isolation and Characterization of Component III. Since com ponent III appeared to be present in relatively sub stantial quantities, it was decided to isolate and identify this material. Hexane extraction of production grade 2,4-D followed by column chrom atography, on silica gel, gave a fraction enriched in com ponent III as estim ated by glc. Further purification of this fraction by preparative thin-layer chrom atography, on silica gel, gave a pale yellow solid which, after repealed recrystallizations from hexane, gave pure III as colorless crystals, mp 101 -101 ..S^C. The mass .spectrum o f III shows a parent ion at iu/e 336 uni an isotopic distribution pattern typical of four chlorine .atoms (M cl.afferty, 1967). C onsidcialinn o f this data together with the ele mental analysis (experimental) gives rise to CulUO.-CI, as a O O ro coc 4* Figure 1. Chromatogram of 2,4-D neutral extract. Column 5'SE30; temperature, 200'C; nitrogen flowrate 30 ml/min probable molecular formula for compound III. The ultiaviolct spectrum of III exhibits absorption bands at 228 nm (i = 19,900), 281 nm ( = 2740) and 2S9 nm ( = 2240), sug gesting a substituted phenol ether (Scott, 1964). The nuclear magnetic resonance spectrum of III consists of a complex m ultiplcl between 7.25 and 7.41 ppm (6 II) and a singlet at 5.76 ppm (2 H), indicating the presence of six arom atic protons and a substituted methylene group. These facts suggest two possible part structures 1 and 2 for com pound HI. o 1 ^ y ~ o-- ch4-- o-- ci, i s o s 4 2 A choice between part structures I and 2 was made when it was observed that an acetone solution of III did not react with aqueous silver nitrate. Since part structure I contains an ioni/nblc halide function it would he exported to give a pre cipitate with silver nitrate. I'url structure 2, on the other hand, contains only arom atic halogen anil would not Ik: Tabic III. Comparison of Physical Properties of 2,4-13 Impurities am) K.vnllicfic IPphcnoxymelliancs Compound His(2 ,6 -i- lilnrn- 2.2',4 ' Telrnehi.iro- phnioM ) diplienoxy- I methane II methane III Ills(2,4-dichliirn- phenoxv)methane Retention lime, min Mass speCrum (parent ion nnd main fragmentation ion 1 ;h i i u nm r, ppm 6.0,* 9.2* 336 175 ... Melting point, *C SE-30 column. * SE-30/QF-I column. 6.0,* 9.2* 336 175 ... 78.5-80 6.7 .* 10.4* 336 175 ... v 6.7 ,* 10.4* 336 175 ... . 85-86 7 . 7 / 11.6* 336 7 . 7 / 11.6* 336 175 5.76 (2 H ) 7.25-7.41 (6 H ) 101-101.5 175 5.75(2 II) 7.25-7.41 (6 H 100-102 5YMTHETIC COOOp TSOO- e &OOC - *500- ood i t, coupoundb DOW372305 Figure 3. Mass spectra of compounds I , IT, and I I I m odated by cleavage of one of the O -C H j bonds in 3 to give the ion 4, analogous to the loss of an alkoxyl group of a ketai (Budzikiewicz e t a t., 1967). Figure 2. Nm r spectra of compound I I I and synthetic liis(2,4dichloruphciin\y)mcthaiic expected to react with silver nitrate. Thus, part structure 2 was favored for compound III and furthermore, since the most reasonable precursor of III is 2,4-dichlorcphenol, strucluro3 could be written for com pound III. 3 The mass spectrum of compound III supports structure 3 since the main fragmentation ion at //< 175 reptesents the loss of C\l IOC'I.. ft mil (.',11,0 Cl, and t an !>e readily accom776 i a c i i l o o n n i t m . v n t.. ;n, n o . j. 1072 4 Structure 3 was proven to be correct by synthesis of bis(2,4-dichlor 7phcno\y)methni , and its identity with III con firmed by mixture melting point, glc retention time, and com parison of nm r spectra (Figure 2) and mass spectra (Table HI). The Identity of Impurities I and II. Since impurities Tand H arc present to a lesser extent than III, as indicated by glc, no attempt was made to isolate (hem; instead combined glc-mass spectroscopy was used to elucidate their structures. The mass spectra of impurities !, II, and III are virtually identical (Figure 3), suggesting that they are positional isomers. Furtherm ore, since it is know n that production grade 2,4-13 (Lot /092440) contains small am ounts of 2,6dichlorophciml , an impurity (W iden, 1970), it is reasonable to assume that com pounds I and II might have j ^ r ^ t Q i g S j DOW372306 OIL FROM 82 PEANUT GENOTYPES and 6. Structures S and 6 were synthesized and shown to be identical to Tnnd II, respectively, by com parison of glc reten tion time, on two colum ns and mass spectra (Table 111). Semiquantitntivc analysis of the commercial sample of 2,4-D used in this study indicated that com pounds I, II, and III were present at levels of 1, 10, and 30 ppm , respectively. The toxicological significance of these three compounds as impurities in production Grade 2,4-D is not known. However, from a study of the teratogenic effects of both production grade 2,4-D and purified 2,4-D (K hcra, 1972), it appears that com pounds I, II, and III have no adverse effects at the levels administer', d in his investigation. ACKNOWLEDGMENT \ T he author thanks Ray legs for technical assistance and W alter Miles for obtaining the mass spectra. LITERATURE CITED Budzikicwicz, H ., Djcrassi, G , Williams, D . H ., "Mass Spectrom etry o f Organic Compounds," H o ldcn-D ay, San Francisco, 1967, p 259. Courtney, K . D ., Gaylor, D . W., Hogan. M . D ., Falk, H . L ., Bakes, R. R ,, M itchell, 1., Science 16S, 864 (1970). Higginbotham . G . R ., Huang, A ., Firestone, D ., Verrclt, J., Rcss, J., Campbell, A. D ., Nature 220, 702 (1968). K hcra, K . S,, Toxicol. AppL Pharmacol, in press (1972). Lashua, S. G , Ranch, D . E,, to D o w Chemical Co., U.S. Patent 3,280,197 (Oct 18, 1966); Client. Abstr. 66, 23506(1967). M iron, S., Lowy, A .. J. Amer. Chem. Soc. 73, 1872(1951). M cLalfcny, F. W ., "Interpretation o f Mass Spectra," W. A. Ben jam in, New Y o rk , N .Y ., 1967, p 22. Scon, A . I., "Interpretation o f the Ultraviolet Spectra o f N atural Products," Pcrgatnon Press, 1964, pp 91-96. W iffen, E. F.,, D o w Chemical o f Canada, Lim ited, Sarnia, O ntario, private communication, 1970. Receiccd fo r renew October 29, 1971. Accepted January 24, 1972 4 18086 LOG RESPONSE I WORTHINGTON. HAMMONS. ALLISON . 34 1 2 7 5 ULa o O CO- -vl to . CO p EXPERIMENTAL PROCEDURES Genotypes. Selection o f the 82 genotypes was based either upon prior knowledge o f chemical composition (Holley an d H am m ons, 1968) or upon possible use in the develop m ent o f new commercial varieties. Virginia (A . hypogaea subsp. h yp o g a ca ) and Spanish (subi; . fa s tig ia t ) botanical types, varying w idely.in fatty acid com position, seed size, growth habit, duration, and other chara.-icristics, were included in the group, as were several representative com mer cial cultivars. AU genotypes were grow n at the Coastal Plain Station, T ifton, G eorgia, in 1965, 1967, and 1968, employing standard cultural practices (M cGill and Samples, 1969). At harvest the plants were dug, allowed to dry in the w indrow for 24 to 48 hr, stacked, and cured in the stack for 6 to 8 weeks prior to picking. Sample Preparation and Analysis. Samples were handshelled and stored at 4-5C until lime o f analysis. Since fatty acid com position is influenced by seed m aturity (W orth ington, 1969) only those seeds that appeared to be fully m ature, as indicated by seed size, abs-.-ncc o f wrinkles, and by pigmentation of seed coat and interior o f pericarp, were retained for analysis. Oil was expelled with a silverplated Carver press, filtered through W hatman paper, and the auloxidation induction period m easured by the method o( O lcotl and Einsel (1958) as modified by Young and Holley riQAM Oil s^mnli-.; (0.2 ml in 10-ml lieakers) w^re hnaip.-i in _ S' ) 6 s 18087 DOW 881002 me March 4, 1980 T H E D O W C H F " " " ' al C O M P A N Y 2,4,5-T AND SILVEX DEFENSE Cancellation hearings for suspended and i and silvex are scheduled to begin on Mart Law Judge in Washington, D.C. This will 10 years ago when Dow appealed EPA's proposed cancmui.iuu w*. for use in rice fields. _ The core team handling the defense includes a scientist from the Product Registrations group, along with Dow attorneys and outside counsel. Dr. Marguerite L. Leng, Senior Registration Specialist, will serve as Science Advisor to the legal team headed by John Scriven of the Dow Legal Department. The day-to-day operations will be handled by Mark Tucker, also a Dow attorney. Outside counsel includes Edward W. Warren, L. Mark Wine, Richard L. McConnell, and John A. Zackrison of the Washington D.C. law firm of Kirkland & Ellis, and Rudolph H. Schroeter of the Los Angeles law firm of LaFollette, Johnson, Schroeter, and DeHaas. Marguerite will provide a unique broad perspective on the science issues, including analytical methodology, environmental fate, metabolism, residues, and toxicology of 2,4,5-T, silvex and their TCDD contaminant. This in-depth knowledge was developed during 14 years in the registration group, mainly reviewing research reports and preparing interpretive summaries of data on Dow herbicides for submission to government agencies. Included were a total of 32 volumes on 2,4,5-T and silvex submitted in petitions for tolerances (safe residue limits) and in the Dow risk response to EPA's Rebuttable Presumption Against Registration (RPAR) for 2,4,5-T. Her responsibilities will be to review witness statements and documents in the vast repository of exhibits supporting both sides of the issue. The critical period will be during the time EPA presents its risk case, estimated to take about 6 months after the hearings get underway. We appreciate the considerable personal sacrifice Marguerite will make in spending most of this time in Washington away from her family. However, as she says "I have devoted at least 6 years of my career at Dow on this project, and want to see it through to a favorable conclusion based on science instead of politics." If you have any questions, please contact me or John Scriven. Manager, Product Registrations Regulatory and Legislative Issues Health and Environmental Sciences gt> < J. H. Ashley, Washington P" --- ce: C. A. I. Goring, 9008 F. D. Hoerger, 2030 M. L. Leng, 9008 J. G. Scriven, 2030 M. D. Tucker, 2030 Registration Group DOW 8S1003 n. a. uisnourger, 9001 J. . Donalds, 9008 B. C. Fischback, Pittsburg A. C. Fischer, 9008 R. F. Flannery, 9008 D. R. Frayer, 2030 P. J. Gehring, 1603 M. E. Getzendaner, 9008 C. Goodman, 9001 J. H. Haines, 2030 R. G. Hanson, Walnut Creek R. E. Hefner, 1776 J. C. Hansen, Washington B. B. Holder, 2030 R. C. Hunter, 9008 O. K. Jantz, 9008 D. J. Jensen, 9008 J. E. Johnson, 2030 J. Jones, Pittsburg E. E. Kenaga, 9008 R. J. Kociba, 1803 E. "V. Luoma, 574 D. D. McCollister, 9008 K.'R. McKennon, 2030 R. J. Moolenaar, 1702 R. D. Moss, 9008 W. W. Muelder, 9001 M. G. Norris, 9001 P. F. Oreffice, 2030 D. W. Osbome, 9001 J. K. Priddy, 9008 M. E. Pruitt, 2020 D. L. Rooke, 2020 B. A. Schwetz, 1803 L. A. Shadoff, 574 T. D. Sinclair, 2030 J. M. Theis, 9008 J. Venable, 1603 S. R. Vranish, 9008 A. J. Watson, 9001 C. S. Williams, 9008 C. D. Woods, 2030 * V*'v*o V- EG* 18089 V&jH/ t The Mortality Experience of Workers Exposed to Tetrachlorodibenzodioxin in a Trichlorophenol Process Accident Judith A. Zack, and Raymond R. Suskind, M.O. A standardized m ortality analysis was conducted on workers exposed to tetrachlorodibenzodioxin in a trichlo rophenol process accident at the Monsanto Company plant in Nitro, West Virginia. One hundred and twenty-one workers who developed chloracne resulting from this acci dent on March 8, 1949, were selected for study. Follow-up o f this group was 700% complete. The standardized mor tality ratio for a ll causes o f death was shown to be 0.69, with 32 deaths observed and 46.41 expected. For the categories o f malignant neoplasms and circulatory diseases, the standardized m ortality ratios were 1.00 and 0.68, respectively. Because o f the small size o f the cohort and the relatively small number o f deaths observed, the results o f this study cannot be considered conclusive. However, it is important that no apparent excess in total mortality or in deaths from malignant neoplasms or diseases o f the circulatory system was observed in a group o f workers with a high peak exposure to tetrachlorodiben zodioxin who were follow ed over a period o f nearly 30 years. The results o f this study w ill be incorporated with those o f a larger study which w ill include plant workers ex posed, in the course o f 2,4,5-trichlorophenoxyacetic acid production during the period 1948 to 1969. A wide variety of acute and sub-acute health effects has been reported in w orkers involved in the m an u factu re of 2,4,5-trichlorophenoxyacetic acid (2,4.5-T) from 2,4,5-trichlorophenol (TCP). The m ost consistent clinical finding is chloracne. a skin disease characterized by com- From the Department or Medicine and Environmental Health. M on^nto Company. 800 N Lindbergh B ivd, St Louis. MO 63166 (Ms Zdtk. Epidemiologist), and the Institute o! Environmental Health. Kemsmg Laboratory, the University ot Cincinnati Medical Center. Cincinnati. OH 45267 (Dr Suskind. Director) \ edones, cysts, pustules, and abscesses. Hepatic dysfunc tion, peripheral neuritis, disorders of fat metabolism , and porphyria cutanea tarda are other frequently reported findings in these w orkers.' C hloracne has been shown to be essentially due to 2,3.7,8-tetrachlorodibenzodioxm (TCDD),1 a b y p ro d u ct in the synthesis of 2.4,5-T The sub ject of this p ap er is the chronic health effects of exposurtto TCDD, as reflected in the m ortality experience of a cohort of M onsanto Company workers who developed sym ptom s of chloracne following a trichlorophenol pro cess ac cid en t at th e Nitro, W est Virginia, plant in 1949 Production of trichlorophenol began in the fall of 1948 at the Nitro plant. In this process, the reactants 1,2,4,5-tetrachlorobenzene, sodium hydroxide, and m ethanol were all added to the autoclave. Heat was ap plied and, when the pressure reached the desired point the au to clav e w as vented. O n M arch 8,1949, ab o u t six m onths after production start-up, a violent reaction and decom position occurred when tem perature and pressure within the autoclave becam e excessive. The relief valve opened and the fumes and tarry residues from the decom posed contents of the autoclave were discharged into the at m osphere and into the interior of the building. Employees w ho worked in the area of TCP production or w ere involved in the cle3n-up began to develop symp toms im m ediately following exposure to the material w hich w as discharged from the autoclave. Symptoms in cluded eye and respiratory tract irritation, headache, diz ziness and nausea, and a severe irritant reaction of the ex posed skin. After these initial sym ptom s subsided, the chloracne and other symptoms becam e evident. Ashe and Suskind** exam ined a total of 12 m ore severely affected workers on three occasions during the period of 1949 to 1953 A nother 26 persons with chloracne, apparently not related to th e accident, w ere also exam ined in 1953 The H aprintad from Journal o f O ccupational M adicm a January. >960, V olum a 22. No. 1 pp. 11*14 JOM I960 18090 clinical symptoms, in order of relative frequency,* included accidental industrial intoxication. acneform lesions; severe pains in muscles of upper and In 1976, a TCP process accid en t in M eda, Italy, resulted low er extremities, shoulders and thorax on exertion; in the co n tam ination of a large and densely p o p u lated fatigue; nervousness and irritability; decrease in libido; area.* A prelim inary m ortality study has been co n d u cted dyspnea; vertigo and intolerance to cold. O n examination, in two of the 11 towns affected. The overall m ortality rate all of the cases had chloracne. Several were severely hyper- did not differ from that expected, b ut increases in d eath s pigm ented, especially on the face. Of the six workers ex from liver cirrhosis and leukem ia w ere suggested. am ined in 1949 and 1950, four had liver enlargem ent and The chronic toxicity of TCDD exposure to animals has one had sensory loss in one foot. Liver im pairm ent as irv dicated by hepatomegaly, tenderness and soreness in the right upper quadrant and epigastrium and a delayed pro been more extensively studied. TCDD toxicity has been thoroughly review ed.' Chronic toxicity to TCDD is m anifested by liver necrosis, thymic atrophy, and deple jOW 239974 thrombin time, was observed.*5 * In 1953, four of the six workers exam ined in 1949 and tion of the lymphoid organs. Two studies indicate that chronic adm inistration of low levels of TCDD to rats is 1950 w ere re-examined and six additional workers involved in the accident were also examined. The findings in this later examination indicated a general regression of both the cutaneous and noncutaneous symptoms which had associated with an increased incidence of neoplasia. In one study, the oral adm inistration of TCDD produced an increase in h ep ato cellu lar carcinom as and squam ous cell carcinom as of the lung, hard palate/nasal turbinates, or been present earlier. All of th e w orkers show ed a m arked to ngue.11 In an o th er study, TCDD fed to rats p roduced im provem ent in their skin lesions -- there w ere residua of tum ors in 38% of the test an im als." N eoplastic nodules the acne and a few active lesions. In a few cases, workers and cholangiocarcinom as of the liver w ere observed continued to com plain of aches and pains of the lower ex The study rep o rted here will exam ine the m ortality ex trem ities and back, nervousness, excessive fatigue, and perience of a co h o rt of 121 em ployees involved in the dyspnea. No clinical explanation for these com plaints 1949 trichlorophenol process accident, with special em could be m ade based on the results of the physical ex phasis on cardiovascular disease and on neoplasms, par am ination.4 ticularly of the stom ach, liver, lung, and skin. The findings of the examinations by Ashe and Suskind are consistent with those w hich have been reported in Population and Methods other industrial episodes which occurred subsequently.1 In this study, the d ev elo p m en t of chloracne, a hallm ark The acute health effects of TCDD exposure are described in the literature,' but little is known of the chronic effects. Several reports describe the occurrence of cancer and other deaths in w orkers exposed to TCDD w hich suggests an association between exposure and the subsequent of TCDD exposure, was used to identify em ployees for study. The study population consists of all persons with chloracne w hich could be attrib u ted to the 1949 TCP pro cess accident O ne hundred and twenty-two em ployees w ho develo p ed chloracne following this incident w ere developm ent of a variety of neoplasms.*" These reports, identified from plant safety records dating to the time of t however, are generally of small groups of workers with the accident, and from w orkm en's com pensation and' relatively short periods of follow-up and are considered to plant m edical records. O ne hundred and tw enty-one be preliminary in nature. In a 25-year follow-up study, 17 w hite m ales w ere included in this study -- on e fem ale deaths were observed am ong a cohort of 75 C erm an w ho was living as of the en d p o in t of the study was n o t in w orkers w ho had been involved in a 1953 TCP process cluded in this m ortality analysis. It is assum ed that all of accident.* Of the 17 deaths observed (11-25 expected the skin disorders recorded in the p lan t records represent depen d in g on the choice of a control population), six true cases of chloracne and n o t oth er types of o cc u p a w ere from cancer (four or fewer expected), five from car tional or nonoccupational dermatitis. An analysis of the diovascular disease (as expected), two from suicide (fewer chloracne cases and exposures not associated with this than one expected), one from liver cirrhosis, o n e from a accid en t b u t rather with the norm al TCP/2.4.5-T p ro d u c urogenital tract disease, and tw o from external causes. Or tion processes will b e the su b ject of a future paper. the six can cer deaths, three w ere from stom ach can ce r in The data were analyzed by the modified life-table th e age group 60-69, a n um ber significantly higher than m eth o d using the u p d ated M onson program .'4 In this expected. Two other cancer deaths were from oat-cell m ethod of analysis, the age-, race-, time- and cause- carcinom a of the lung and o n e was from ad en o car specific m ortality rates for a standard population (in this cinoma of the colon. case, the population of the United States) are applied to A similar accident occurred in the N etherlands in 1963 the person-years lived classified by age, race, and tim e A in a factory producing 2,4,5-T * Eight deaths have been standardized m ortality ratio was calcu lated as the ratio of observed am ong 93 exposed workers. Five or six of these the observed d ea th s to the expected d eath s for 22 deaths were from cardiovascular disease. The proportion selected causes of death. The statistical significance of of deaths due to myocardial infarction was noted to be differences between observed and expected numbers was high. based on the Poisson distribution and statistical signifi jirasek et al* 10 and P azd e ro v a" follow ed 55 of the 78 ca n ce was d eterm in ed at the 5% level of significance. Czechoslovakian workers w ho were affected by chlor For the purpose of analysis, each m em ber of the study ac n e resulting from o ccupational exposure to 2,4,5-T and cohort was assum ed to have en tered the study on M arch pentachlorophenol. In this study, five death s w ere ob 8, 1949, the d a te of the accident. The vital status of each served. These included two deaths from bronchiogenic mem ber was determ ined using standard follow-up tech carcinom a (less than one expected), one from cardiovas niques and ascertained as of D ecem ber 31,1978 For each cular disease, one from liver cirrhosis, and one from an person found to be deceased, a death certificate was ob- DOW 239975 Tabte 1. -- Obstrvtd and Exported Deaths Among 121 Males Exposed to TetracNorodlbenzodloxin in a Trichloropheno! Process Accident. Cause All causes ol death All malignant neoplasms Buccal cavity and pharynx Digestive organs and pentoneum Siomacn Liver All other digestive organs Respiratory system Lung All other respiratory organs Skin Genitourinary organs Lymphatic and hematopoietic tissue Other sites Oiseases of the nervous system and sense organs Diseases of the circulatory system Arteriosclerotic heart disease, including coronary heart disease All other disease ot the circulatory system Diseases of the respiratory system Diseases of the digestive system All other diseases External causes of death - p < 0 05 tLess than 5 observed deaths ICO No. (Eighth Revision) 140-209 140-149 150-159 151 155-156 -- 160-163 162.163 - 172.173 185-189 200-209 -- 320-389 390-458 410-413 -- 460-519 520-577 -- 800-998 Observed 32 9 0 Q 0 0 0 5 5 0 1 0 3 0 0 17 13 4 1 0 2 3 Expected 46 41 9 04 0 30 2.53 0.50 0.18 1 91 3 02 2 85 0.17 0 15 1.16 0.88 0 94 0.36 25.01 17.74 7.27 2.78 2.26 3.18 3.78 SMfl 0.691.00 t f f f t 1 66 1.75 f t t t t f 0 68 0.73 t t t t t tained. The underlying cause of death was coded to the 8th Revision of the International Classification of Diseases, A dapted" by an experienced nosologist. Results All of the 121 m em bers of the study cohort w ere traced. Eighty-nine w ere verified living and 32 w ere verified deceased by death certificate. The results of the standardized mortality analysis of the 121-m em ber study cohort are shown in Table 1. The standardized m ortality ratio for all deaths is shown to be 0.69, with 32 observed d eath s and 46.41 expected. This is the only statistically significant difference shown in this table. There were nine deaths from malignant neoplasms with 9.04 expected. There were no deaths from stom ach or liver cancer. There w ere five lung cancer deaths versus 3.02 expected and one skin ca n ce r d eath with 0.15 ex pected. The malignant tum or was a fibrous histiocytoma presum ably of derm al origin, w hich is rare. There w ere three deaths from neoplasm s of lymphatic and hem atopoietic tissue with 0 88 expected. There w ere 17 observed d eath s from circulatory diseases with 25 01 expected. The stan d ard ized m ortality ratio for circulatory diseases was low at 0.68. Case sum m aries for the can cer death s are given in Table 2. Discussion Because the study cohort was small and only 32 death s were observed, the results cannot be considered con clusive. Nevertheless, the analysis of the mortality ex perience of these workers indicated no apparent excess of total m ortality or of deaths due to m alignant neoplasm s or circulatory diseases. fhe TCDD-exposed workers in the p resen t study repre sent the largest group ever investigated after long-term follow-up. The criteria fo< inclusion (presence of the workers at the 1949 accident and the subsequent occur rence of chloracne) limit the group to those with a signifi cant exposure at that time. The latency period of 29 years Tabl 2. -- Cancer Deaths Among a Cohort of 121 Males Exposed to Tetrachlorodibenzodioxin in a Trichloropheno! Process Accident. Year at Birth 1909 1910 1911 1922 1915 1920 1919 1907 1910 Year of Hire 1943 1927 1939 1945 1939 1346 1943 1943 1939 Year at Death 1962 1970 1964 1973 1970 1978 1973 1971 1978 Death Certificate Statement of Cause of Oeath Lung cancer (162.1) Pulmonary carcinoma (162.1) Broncniogenic carcinoma (162 1) Broncniogemc carcinoma 162 1) Lung cancer | 162 1) Malignant fibrous mstiocyloma ol suit tissue origin (173.9) Hodgkin s disease (201 0) Lymphatic leukemia (204 9) Acute myelogenous leukemia (205 0) ' Smoking history was ootained dy interviews wnn former co-workers of ine decedents Smoking History* Cigarettes Cigarettes Cigarettes Nonsmoker Cigarettes Cigarettes Cigarettes Pipe Cigarettes 18 is longer than that of any previous study, and the follow up is com plete Therefore, although the cohort is small, it represents the best opportunity so far to study the long term effects of TCDD on m ortality. By augm enting these data with the results of com parable mortality studies, the long-term effects of TCDD may be more definitely evaluated The dui<iOiS w ish io ih.m x M rs jan et Vun^. M r K a n d y Prcolet, dnd M rs P h y llit K o r te fo r th**ir a ssiW d ni f* w ilh |h#* d ata c o lle c t io n References 1 International Agency for Research on Cancer IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man Vol IS Some fumigants, the Herbicides 2.4-D and 2.4.5-T. Chlorinated Dibenzodioxws and Miscellaneous Industrial Chemicals Lyon IARC. 1977 2 Kimmig J and Schulz KH Occupational acne iso-called chloracne) due to the chlorinated aromatic cyclic esters Derm a to io R ic a 115 540-546. 1957 3 Ashe W f and Suskind RR Reports on chloracne cases. Monsanto Chemical Company. Nitro. West Virginia Reports of the Kettering Laboratory. December 1949 and April 1950 4 Suskind RR A clinical and environmental survey, Monsanto Chemical Company, Nitro. West Virginia. Report of the Kettering Laboratory. July 1953 5. Suskind RR Chloracne and associated problems Report to the Conference of the National Institute of Environmental Health Sciences on Chlorinated Dibenzodioxms and Dibenzofurans, April 3. 1973 6 Suskind RR Chloracne and associated health problems in the manufacture of 2.4 5-T Report to the Joint Conference. National In stitute of Environmental Health Sciences. International Agency for Research on Cancer. Lyon. France. January 11. 1970 7 Young AL. Calcagm JA. Thalken CE. et al The toxicology, en vironmental fate, and human risk associated with herbicide orange and its associated dioxin US Air Force Occupational and En vironmental Health Laboratory Report OEHL TR-70-92. Brooks Air Force Base. Texas. 1970 8 International Agency for Research on Cancer Long- term hazards of polychlorinated dibenzodioxms and polychlorinated diben zofurans (ARC Internal Technical Report No 78'001. Lyon. 1970 9 lirasek L. Kaiensky I. and Kubec K Acne chlorma and porphyria cutanea tarda during the manufacture of herbicides Cesk D e rm a to l 48 306-317. 1973 10 Jirasek L. Kaiensky I. Kubec K. et al Acne chlorma. porphyria cutanea tarda, and other manifestations of general poisoning during the manufacture of herbicides II Cesk D e rm a to l 49 145-157. 1974 11 Pazderova J. Lukas E. Nemcova M, et al Chronic poisoning bv chlorinated hydrocarbons formed in the production of sodium 2.4.5-trichlorophenoxyacetate P ra c L ek 26.332-339. 1974 12 Kociba RJ. Keyes DC. Beyer JE. et al. Results of a two-year Chrome toxicity and oncogenicity study of 2.3,7.0-tetrachlorodibenzop-dioxm in rats T o x ic o l A p p l P h a rm a c o l 46:279-303. 1978 13 Van M iller JP. Lalich )J. and Allen JR: Increased incidence of neoplasms m rats exposed to low levels of 2.3.7,8-tetrachlorodibenzop-dioxm C h e m o s p h e re 6 537-544. 1977 14 Monson RR Analysis of relative survival and proportional mor tality. Compuf B io m e d Res 7 325-332. 1974 .15 Eigth Revision. International Classification of Diseases. Adapted for Use in the United States US Department of Health. Education and Welfare. Public Health Service, PH$ Publication No 1693 Washington U S Government Printing Office. 1977 DOW 2 3 9 9 7 9 f)i :W 1"ii-sfeis-jC?. 1 |.irc.'-SrivV 1; . '-.- V: . . - - '. mi o O I o c h s or J-5. ACUTE AND SUB-ACUTE ORAL TOXICITY OF 2,4,5-THICHLOaOEHENOXYACETIC ACID FOR DOGS Victor A. Drill, Ph.D., M.D. Professor of Pharmacology Wayne University College of Medicine 1950 j j 1 > 7 i f :} ]i ] ] ] ] 1 T G ilo O 'MOO ACUI2 AKD S2-ACuI2 C U L OXICITY CS* 2f4-raicELcaa3ii2xaxYiC2rio a c i d f c r d c g s Viccr A. Drill, ?h.D., M.D, Profoacar of Phamacolo<3y Taysa Dnivoraity Collona of itodicia ITS2 * Investigations have shewa fact 2,4,j-trlchlcrophonoxyacotlc acid C? can. bo used as a ccrbicido (1)* in concentrations ranging from 0,1 Q ,to 0,2 per oent* Toxicological studies on the material era vory limitod* ^ , In the present study tho acuta and chronic oral toxicity, of 2 ,4 5"'? wa3 dotor-^> ' 'O - minaa in doga. In tho present- data h istciogical changes are not inaludod as rgi this phoso cf tho study is net yc't cor.pie tod.' V- ' . yethedn 'i O' 10- " Adult mongrel dogs of both saxes tore used*, tost of tho animals tore housed in the laboratory for a period of 2 to 3 souths before tho study . was started. During this oontrol period they were immunised against distemper with tho Croon vaccina.'The dogs -core fed a standard etock diet (Friokies} pd libitum. The 2 ,4 ,5 -" ussd was commercial material with a purity of ICO,,;.. . In the acuto studies tho calculated dose of 2 ,4*5 "'? as administered : as a single oral dose in eapsulos. The dogs ware ebsenvod for1a period of 14 days at which tins survivors ~oro autopsied. In tho'chronic study.the 2,45-T was administorad orally in capsules, $ days a wook ovor a 13 week'1 i period. Each capsule was imbedded in a 4 to 5 fivam pioce of commercial cannod dog food. The animals consumed this readily, thus avoiding tho 'con-./' tiausa trauma of .the stomach tubo. During tho study the animals wore weighed .v..: twico a v?cok* Control blood .counts were takoa bofero tho administration cf ' the drug, on tho 30th day and cn tho $0ih.day at.th'o completion of the study. Hemoglobin was determined with a photoelectric, colorimstor (2 ). upon the ':V death or at tho completion cf tho study* tho animals were autopsiod, tissues intestine . were taken from tho'lung, heart, livor, kidney, adrenal 3ploon, thyroid^and . evary or .testes and fixed in lO.t formalin. . 1 Symptoms produced. Tho survival at various dose levels of 245 is 1istad in Tablo 1. The LB50 is in tho range of 100 cgm* par kilo or slightly higher. . .. Tha.symptoms observod in tho dors were fairly mild and limited to a slirfit to moderate stiffness in tho hind'legs, with the development of atomin in the two dogs receiving .the highest doses of 2,45~T. No symptoms ware %> seen in tho one dog that diod following a single do3e of 100 ogm. per kilo (Tablo 1). . 2. Body Tioirht. Doses of 100 mgm. per kilo or grantor producod > a slight fall in body vraight during tho test period. Tho results are somewhat variable with the 0 mga. per kilo dpso.but indicate only a relatively slight ,offoot of the drug on body weight (Tablo 1). . 3. -Gross pathology. The changes observed vero limited to tho 1' * astro-intestinal tract and lungs. In dog 90 the ducdonal mucosa was a slight diffuse rad' in color and-ir* dog 74- small pin point hemorrhagic aroas wore observed in the mucosa together with sons blood in tho lumen. A small amount of blood cas also found in the small intestine' of dog 84. This, seme anirxil also presented a purple-black lower loft lobe of:the lung that had a* solid blood consistency*. IT. CTTBiTTIC CTlAL TOXICITY. . 1.' Mortality.' Doga that received donas of 2 , $ and 10 rgm. per kilo survived the 90 days. The dogs receiving 20 mgs. per kilo either in'a single or divided dcoo died during the study. '; 2. General svmotcms. No signs or symptoms were observod in tho -J - dcgs that survived tho study. In the degs that died tho prominent effects' sere weakness, slight stiffness in the hind legs, tad difficulty in tho swallowing of food, and bleeding from tho gums in cno 0 3 .(Table 2). o O ." 3 3rv reir-ht. The 4 0 3 a that died shored a loso of ever 2 ", kilos in body woight that started approximately 1 0 days 'beiera tho death of tho anir.nl. Tho surviving asinaio do not shew any significant chango in body weight during this parici, but tended to fluctuate ebout thoir. original weight* . / 1 4* Blood count. .Tho administration of 2,4*5-T id not have any significant effect on the hemoglobin cr rod call count (Table 3) Dogs IO3 , 7 0 , 57 find 1 0 5 showed sons decrcasa in total "white coll count during tho . study but the values did not fell lower than those soon in tho control dogs In tho differential counttikon before death, the per cost of lymphocytes,fall ' to very low le7 ol3 in deg3 5 7 find 1 0 5 . ^ : 5 * OrC3 3 pathology Animal Ivo. 93' showed a diffucoly reddened duodenum and jejunum. . Dog 1C5 autepsied cn the 59"* day showod a generalised jaundice as indicated by the sclera and tho finding of yellow fat'and yellow orsans throughout; the body. ' In deg 53' the'renal capsule was stripped with difficulty. ..... ., '; : 6 . Organ waipfcts, The animals that survived the .JQ day test por- . icd did not show any significant change in organ weights (Table 4) Various. Eiincr changes In organ weight can bo noticed in soma 1of the dogs fed the 2 0 E^m. per kilo do3o who died during tho study. These changes wore limited to possible increase in heart, liver cr kidney '`sight. , Discussicn. Single larga doses of 2,4,5-T can produce death in cogs. However, the central nervous system symptoms observed are quito mild, consisting chiefly of stiffness cf the hind legs with soma ataxia. These. symptoms seem to bo 'definitely less than these obtained with comparable doses of 2,4-D. However, further studies with dosoa of ljjO to 200 mg. por kilo migjit elicit a better sories of signs ana symptoms. The acutely intoxicated O ro animals lost only a slight amount of woight.. Cross pathological changes j-- * H-S o when ebsorvod wero confined to thegi'stro-intestinal mucoca and in ono deg `Jj to the lccvor left looo of tho lung. The decreased incidence of such changes as compered with.2,4-D paralleled tho mild contrul norvou3 offoots of the drug, In tho studios on. chronic oral toxicity of 2,4,5-? i was obssrvod, # that the animals survived doses of 2, 5 1 mg. par kilo without grossly . apparent ill effects, but succunbod to doses of 20 mg. por kilo. Thus the chronic toxic dcao seems to bo in tho same range as that obtained with 2,4-D. In those animals receiving tho highest dosage, 20 cg-kg of 2,4,5"? evidence of toxic effects consistently included slight muscle spasm, difficulty in swallowing food, and lcs3 in terminal body weight. A marked decrease in tho percentage of lymphocytes in the peripheral blood was noted in two instancoc, nhilo generalized icterus of the body organs and inflammation of duodenal mucosa wore each observed once. \ .. Summary 1. The acuta oral LD^q 2,45"? is ^ the o? 100 mg, per kilo cr higher. Acutely poisoned dogs show mild spastic signa accompanied by a loss in body weight. C-ross pathological changes wero uncom-. men and. wero confined to the small intestinos and lungs. 2. In chronic oral fcoding of 2,4,5"? ssos of 2, 5 and 10 per kilo allowed survival fcr 53 days and did not produce any symptoms or changes in body weight, organ woight or blood count. Dogs fed.20 mg. of -5- 2,4- per kilo pa? day died during tho study, edentng aild ojactic soap- toas,- a loss ia body T/oiht, and in 2 casos a decrease in tho por cont of o O lyaphocytoa in tho peripheral blood, liinor changos raro prosoat in tho duo denum in one dog, and jaundice ia anothor dog at autopay.- ,*> CD to |- ;;C5. S*i * >*-::''*^* ^ * ' il._; 1 v i v i , ' - ^w a:J*4;4<Li tVW^iri ?, ~ T T \ f 1. Hildebrand, 2. ZU, Scianca, 10^;ii.6S. 194 6 . . 2, . Evolya, K. A., J. Biol. Caes., 3* 19j. -O = *vl ; . ra l> .1 <1 i l '] -i>i-' ?31C 1 Effect of singlo eral cocos of 2,43-T la doga O O Dc3 J'-O. ' TV M ( OJJ 4 ? 90p 9-?g- 100 J 101 ICS? 100 21 Toro T Z 'r / V . .CO 290 130 ' 100 . 100 100 30 50 50 50 Died day o 3 s 7 s s s- wo s s T 'artslity N / . ) 'S - > 1/1 1/1 ' 1/ 4 ' 0/4 t 1 o33 - 1 .1 -U l -0 .5 -1.0 --1 .0 -o , h * o ,3 . -2.3 +0.1 -0 .6 * S v sn tcss* * 0 . . . 0 0 0 .0 0 0. 0 rc H"* V ,c oe , \ *. . ^ S - su rvival fe r 14 dey to s t period. ** colusa iadicatoc 3acral severity of sysptcsa es discussod la toxt.O - ao cssie fres aorsal. ' t \ .'V-.i . V- h e m. ` --*"c - ' \.fiW y~tl * .}. m*.*bio 2 O i;; Body weight' end survival of docs fed 2 ,4*3 I* for 90 days % I'; I; it ];;. 1 D 03 . TCo. ` * 77? 102'.! 8?? 99:i .7; &F 10313 937' 515 * ' 70? 3711. 69? v7 92l? '' lOja Dcnr> j k Initial ; IwHl nona .. . 7 . 3 aosa 7 12.1 2 11.3 2'. ' . 3.9 '5 '; 8.s -.5 10 * 12 .3 12 .3 10 : 13.0 10 /; 9 * 3 '*20 : 1 1 .s '->20 :' 1 0 .3 ' 20 20 : 14 .9 10 .9 . ?.V>iHit Day Diff. ' <Q . Diod . tw 1 .f-- * 7*7 ' -0.4 ` : 14*5' ; . 1 2 . 7 ` ` . 10.0 2.4-1 1.4 *1.1 s 3 3 s 0 ,<T. * ;`* 3.4 : 12.3 ii 0 ' 1 2 . 2 ' 9*2 ; 9*2 '7*9 . 12.7 8.2 * ' *0.2 -0.2 -0,3 -o.a . -0 .6 -2 .6 -2 .6 -2 .2 . -2 .7 1 *s S'' 1 i. s .. *c*> * .s 49' ` 75 11 ' ' 59.' <*. '\ ** ' '* * "fiaily doso divided and adiainiatsrad ' ia A.jj. # and $ in' P.a. ' * V <v . ' .# S - survived $ 0 day test 'period. ` T\ ) Ii if> l 181C3 *r*f '-.VV-,i -. " 'l .- t Ii i Table 3 D o E f f e c t c f 2 ,4 .3 ? on bene;;lcb in ,' t o t a l b lc o i and d if f o r o a t i a l eo u a ts ' . . v -\ - ' - JTOi D orn. rr/A 77? . nona * .. ' Day 0 30 1021 . UCCIO m 2 : 0 30 90 0 30 90 td> T T -r . X 9*60 1Q.75 12.74 12.29 13.31 19.95 11.9$ 14.49 l/*. 92 Tv7}3 n il. 3 .0 3 3 .6 3 5.3? 5 33 3 .7 3 6.47 5 .8 2 A ?0 6.93 me 16,100 13,300 14.700 14,500 14,9-30 6,400 12,450 16,700 1.6.900 "out 67 56 . 31 D iff e r e n t i a l co-.irvt T "an Ban Den ir. / * i 26 1 0 6 16 2 13 1 0 0 3 0 Death r " c:av .r-*.c i o .c5 55 39 2 0 4 , ` . 63 . ' 26. 48 51 4 l 0 0 7 .` 0 '70 , 24 62 23 89 13 2 12 2 0 4 .. 0 3 . : 00 57.'i 2 . 66? 3 a03:: 3 93-? . 1 0 . , . 5 5 * 1 0 . r- ' ' 70? 10 371 c 20 0 10.73 3.2S 30 90 13.35 14.J-.6 . 3O/ oOr6'*-) 0 11.90 6.21 30 12.93 . 6.17 90 19.32 7.09 0 10.94 3.50 .30 12.67* 3 .7 9 90 12.95 6.24 0 II.9 0 6.50 3 0 12.74 6.72 90 12.67 6*42 0 11.33 30 12.34 90 11.34 3 .9 1 . 3 .4 9 6.22 0 12.63 30 . 14.46 90 14. 34 6.37 6 .2 3 7.29 0 1 2 .3 4 3 0 3 33. 8,6 4 3 .9 3 49 8:93 9.07 17,100 l i , 300 17,203 12,600 17,400 19,200 13,100 16,600 - 7.-50 i+,Of .ij Or *. 13,000 13.600 13,250 13,650 ' f l r - l , j)X , * . i - ? J 15,400 16,400 3,100 3*1.0t ) /*V**V .16,450 6.900 75 22 0 56 ' 34 ' 9 ' 67 25 6 43 47 . 0 46 . 48 5 -55 44 l 47 . 5 4 . 71 20 3 21 8 29 0 69 24 4 66 28- 73-70/ 15 15 ' >. . . 6 56 70 73 . 94 . 67 72 24 27 12 23 93 12 4 3 / O 8 0 0/ 77 13 3 94. 4 2 0 '3 . 0. 1 : 02 0 .3. . . .0 - ` 1 1 0 0 ' 0 30. v 0- 17 1 00 . :<-. . 0 ,3 0. , . 3 . 0. 5 0 23 ' 0 16 00 0 . 4 0 10 00 . ' 0 . 10 . 0: 7 0 0 4.9 ' 69? 20 C 12.48 7 . 1 0 lb -.OjO 93 29 2 0 6 1r 94.? lOjii . / *. .... 30 14.66 20 0 12. .44 20 0 . 12.48 '3 0 . ' 12.93 6.82 5.79 6 .0 3 '5*81' 11,200 13.800 XOQOQ 14 ,6 5 0 71 63 51 73 : 28 .2 44 ;- 13 00 1 \ 0 1 11 2 0- 4 '.0 3 '0 1 ____ 59 11.37 6.14 7,176 '9 2 ` 9 1 0 0 69 (*' 1 < - daily dona divided and adriini ..4* --4 t ia'A.2i. and i in' ?.i^ ..18104 ; I- Dc\3 No. 77F 102M 8?F 99-M 86? 10 7 .1 93-" 55m 70? 57 69? 94F 105.-.1 Table 4 Organ weights of dogo fed 2,4,5 T for 90 dnya Baood on final body weight Dono n^/K Kidney . V(t cA'5 c nono nono 2 2 5 5 io 10 10 '20 c20 20 20 53 83 ...58 . 40 35 68 48 . 49 -37 51 62 . 73 68 . 4 94 5.72 ' 4*56 4.00 4.1? 5.44 4.17 4 .0 1 4*02 5 55 7.65 5*75 8 .3 0 I.ivor *;t fi/Kg B 268 405 557 as? 341 4.28 3 O2 412 292 335 . 390 3 55 284 34*8 ' 2 7 .9 43*1 35*7 ' 4 0 .6 3'**2 33*2 33*8 31*7 36.5 49*4 48*9 3 2 .2 Heart xib .cA's ' B 61 6 .1 0 95 6.55 70 6 . 1 5 75 7*50 7.50 86 6.88 74 6.44 73 5*93 70 7 . 6 0 65 7 . 0 7 67 8.49 79 6.22 67 0.17 Adrenal v/t e / t e B 1 .0 1.2 ia 1 .0 0 .8 1 .4 1 .2 1 .0 1 .2 1 .4 1 .5 1*5 1 .1 0 .13 0 0 .0 3 3 0 .0.37 0 ,10 0 0 .0 9 5 0 .112 oao.'j ` 0.032 0 .13 0 0 .19 2 0 .19 0 0.110 0 .13 4 Thyroid XI t cA'-C 'B 0.4 1.1 .. 1*3 .0,7 0 .5 0 .9 -*** 0 .7 ----- 0.8 O.O3 2 . O.0 7 6 0.102 0 ,0 70 O.OSO 0 .0 72 --- 0 .0 3 7 a-- -- -- 0 .0 90 t c = .daily do3o divided and adrrdniatorod $ in Ali.and in P.M. ' CSP ^ O cn . ` i c s i z o i 'M o a 1 123v 'i wiwniiii! nit~ o I W . THE ACUTE ORAL TOXICITY 0? 2,4,5-TRICHLG50PEEN0XYACETIC ACID TO EATS, MICE, GUINEA PIGS AND CHICKS , V . K. Rowe, D. D. McCollisier and H. C. Spencer Biochemical Research Laboratory The Dow Chemical Company Midland, Michigan su o ro ^ -* o cn 'I J .* J : i ? i t Rference III /3 THE ACUTE ORAL TOXICITY OP 2,^,5TRICLOROPKEHOXYACETIC ACID TO RATS, MICE, GiffiJEA PIGS A ED CHICKS o o w 0 V. K. Rov.-e D. D. I-ieCollister and H. C. Spancer CT 'C5 Elocheaical Research Laboratory The Dot; Chemical Ccrr.pany Midland, Michigan 1950 - 1- Single oral doses of 2 ,k,5-trichlorophenoxyacetic acid as a O O 10 percent suspension In olive oil were' administered by stomach tube r: to mixed sex groups of rats, mice and guinea pigs. Aliquots of a 3 percent solution in olive oil were administered to chicks. The white \ j -* -i' . : ' rats and the guinea pigs were selected from the stock colonies of this ' ' laboratory. White mice were obtained from the Carworth Farms and New o l\3 r;i <E Hampshire Red chicks were purchased from a commercial hatchery. Mature young adult animals were used in each Instance except for the chicks which were approximately three weeks of age. Table I summarizes the concentrations and volumes of olive 011 solutions containing 2,ii-,5" `:;*,ichlorophenoxyacetIc acid administered to each species. TABLE I SINGLE ORAL ABI-UNISTRAT ION OF 2 ,4,5-iHICHL0R0?KSN0Z?ACSTIC ACID Species Percent Material In olive oil Volume Given by Stomach Tube Minimum (cc.) Maximum (cc.) Rats Mice 10 10 0.7 0.05 2.9 0.3 Guinea Pigs 10 . 1.0 2.3 Chicks - > 3 0.3 5.8 ........-- i - ............. - * Mortalities resulting from the various single oral dosages t f. T k I j l I j of 2,^,5"trichlorophenoxyacefcie acid to the experimental animals are | given in Table II. All surviving animals were observed until it was v certain that they had fully recovered (usually about two weeks). j Symptoms of poisoning, particularly with the higher doses, were ataxia and myotonia. IBiCB DOW 0 H1^ O 0t H -2 - TA3LE II MORTALITIES RESULTING FROM THE ADMINISTRATION OF SINGLE ORAL DOSES OF 2 , 4 ,5-TRICKLOROPHEIIOXYACETIC ACID TO RATS, MICE, GUINEA PIGS AND CHICKS - Dosage Rats Mice' Guinea Pigs Chicks (K m ./tern.) No.fed No.died No. fed No'.died No.fed No.died No.fed No.died ; 0.1 a? 0.2 0.3 : 0.4 0.5 0.7 - ' 10 10 10 10 0 5 8 10 30 6 2 5 0 54 6 ` 5 4 66 53 66 60 60 64 6 5' 66 3 .3 The acute oral LD50 values wi th their 19/20 confidence limits were determined for' each species by use of the method described by Litchfield and Nilcoxon^. The results of the statistical treatment of the dosage-response data are presented- in Table III. TABLE III ACUTE ORAL L D So VALUES FOR 2 ,4 ,5-TRICKLOROPEENOXYACETIC ACID WHEN ADMINISTERED TO RATS, MICE, GUINEA PIGS AND CHICKS LDso (19/20 confidence limits) Species . _______ pm. A s m . ________________ Rats Mice Guinea Pigs Chicks O.5OO (O.39I *- 0.640) O.3S9 (0.2h 5 -- O.619) 0.381 (O.3O7 -- 0.472) (0 .2 11 -- 0.456) Slope . 'Function* 1.49 1.80 1 .2 1 1.40 A the fold change in dosage required to produce one unit standard deviation in response along the line. 1. Litchfield, J. T Jr., and Wileexon, F.: J. Pharm, and Expt. Ther. Qo: 99, 19^9- II gsmzof m o q -3 The Litchfield and Wllcoxon tests for parallelism of two lines and estimate of relative potency rev.ealed that the 2,^,5-tri- chlorophenoxyacetlc acid was significantly (19/20 probability) more toxic to the chicks than to the r a t s . There were no significant differences In either parallelism or relative potency as a result of the following comparisons: rats versus mice, rats versus guinea pigs, mice versus guinea pigs, mice versus chicks and guinea pigs versus chicks. I l I i I \ 18: to 112 1 VJ RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5--TRICHLOROPHENOMYACETIC ACID Biochemical Research Department The Dow Chernic al Company Midland, Michigan August, I960' t= J _F o A > S V* o AV*. ^ C*'1 ^ 1 ; I 1 i : ; ;{ t i i PROBLEM The widespread handling of 2,4,5-trichiorophenoxyacetic acid made it desirable for us to have a significant amount of toxico logical information.. Data on the oral toxicity was necessary for presentation-to the F.D.A. Hearing on residue tolerances. ! < MATERIAL '' Name : Formula: Structural: 2,4,5-Trichiorophenoxyacetic acid . 0 II C^ _ H-0-C-C-0-<^ Cl H N Cl Empirical: C oH cC Z> l oj0, EXPERIMENTAL RESULTS The acute oral toxicity of 2,4,5-trichIorophenoxyacetic acid for rats, mice, guinea pigs and chicks is given in the attached s u m m a r y w h i c h w a s presented in evidence at the F.D.A. Hearing. In .concluding our testimony on this material we stated, L1X 1 m 1123 i '3. H 2 S U L T S 0 ? T O X I C O L O G I C A L T ESTS ON 2,4,5-TRICHLOROPHEXGXYACETIC ACID Biochemical Research Department The Dow Chemical Company. Midland, Michigan August, 1950 mmmmm rc co ! i; ii 3 i r1 . i . :iir- i r. t. 9 JL C UCfc* & RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICHLOROPHENOXYACETIC ACID Biochemical Research Department The Dow Chemie al Company Midland, Michigan August, 1950 O O t--4 c~ PROBLEM The widespread handling of 2,4,5-trichlorophenoxyacetic acid made it desirable for us to have a significant amount of toxico logical inform a t i o n . . Data on the oral toxicity was necessary for presentation-to the F.D.A. Hearing on residue tolerances. r>.(yi (i ; ri a! Ti MATERIAL Name: Formula: Structural; 2,4,5-Trichlorophenoxyacetic acid 0 TI Cl___ / T \H - O - c i- O - Cl Cl Empirical: C H ,, C l o0 o EXPERIMENTAL RESULTS The acute oral toxicity of 2,4,5-trichlorophenoxyacetic acid for rats, nice, guinea pigs and chicks is given in the attached s u m m a r y w h i c h w a s presented in evidence at the F.D.A. Hearing. In concluding our testimony on this material we stated, c ." I <5 O O o I 1813 IlBREGISflftMtersE "Because of the similarity in chemical structure and in t o x i c i t y and physiological effect between 2,4-D and 2,4,5-T, it seems that a residue tolerance for the latter on fresh fruits and vegetable:: should be the same as that established for 2,4-D; it would seem that a residue of 20 p.p.m. would provide a wide margin of safety". ~Eye Irritation T.'hen 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 as a 10% solution in p r o p y l e n e glycol wa s introduced into the eye of a rabbit, it produced marked pain and slight conjunctival irritation immediately Jive hours later, severe conjunctivitis and moderate corneal damage was apparent. This picture remained about the same for two days before healing began. Jive days after the exposure mild conjunc tivitis and corneal damage was still apparent. a o So ct rc Skin Irritation Then 2 , 4 , 5 - t r i c h l o r o p h e n c x y a c e t i c acid as a 10% s o l ution in butyl carbitol acetate was applied repeatedly to the rabbit ear and bandaged repeatedly to the shaven abdomen, a slight simple irri tation developed in both instances. STmT'.; i 3 V The toxicological properties of 2,4,5-trichlorophenoxyacetic acid may be summarized as follows: 1. T h e m a t erial is m o d e r a t e in acute oral t o x i c i t y for the four species, rat, mouse, cavie, and chick. Of these species the rat seems to be the more resistant and the chick the most susceptible. 2. T h e material is markedly irritating to the eyes. 3. T h e m a t erial is slightly to m o derately irri t a t i n g to the skin upon prolonged exposure. 1 Pa HANDLING HAZARDS AND PR CAUTIONS FOR SAFE HANDLING " Swal l o w i n g It does not s e e m likely that 2 , 4 , 5 -trie 111or ophenox y a c e t i c acid can be swallowed accidentally in amounts dangerous to life, although this might be possible if concentrated solutions of the material w e r e available to children. If substantial quantities of the material were to be swallowed by anyone, serious ill effects could be anticipated. If the material should be swallowed, induce vomiting by giving a common emmetic such as 2 tablespoonfuls of table salt in a glass of warm water. Call a doctor. DOW <h: Eyes The 2,4 , 5-trichlorophenoxyacetic acid is capable of causing serious damage to the eyes. Precautions should be taken when, h a n d l i n g t h i s m a t e r i a l to prevent p o s s i b l e eye contacts. It is recommended when handling strong solutions of 2,4,5-trichlorophenqxyacetic acid that goggles, face shield, or other similar device be w o r n to p r e v e n t a ccidental splashes in the eye. It is belie v e d that the handling of the solid does not present a hazard simply because of its insolubility in aqueous medium. If the material in any form should get into the eyes, they s h ould be p romptly w a shed with flowing water for at least 15 minutes and medical attention obtained. Sxin " P r o l o n g e d and r e p e a t e d contact wit h the skin may re s u l t in t irritation. Occasional contacts for short periods are not expected to cause irritation. Strong solutions are no doubt more likely to cause d i f f i c u l t y than the dry solid. In case of contact wash the exposed area with soap and water. Do not wear clothing or shoes which have been contaminated with solu tions of the material. i Page 4 Inhalation hTo stud i e s of toxicity upon inhalation of 2 , 4 , 5-trichloro- phencrcyacetic a cid dust have been made. Y.'e believe, however, that in v i e w of the siird-larity w i t h this m a t erial to that of 2,4-dichlorop h e n c x y a c e t i c acid, that it is unlikely that toxic concentrations O of the dust would be inhaled, primarily because of the irritation O it will produce upon the upper respiratory passages. I I Reforonce II Ct T lC \S Fo o ! I \ I t" :< 18116 IN THE UNITED STATES COURT OP APPEAIS FOR THIS DISTRICT OP COLUMBIA CIRCUIT No. 2M3'I ! HARRISON WOLFORD, et al., .1.134 s! 'i HU t CO i HU 05 : Petitioners .v. CLIFFORD M. HARDIN, Secretary of Agriculture, Respondent pow 2 0086 on petition for review of an order . OP TnE SECRETARY OP AGRICULTURE Cl cn STATEMENT OP THE REASONS UHDERT.UIIG THE DECISION 0? THE SECRETARY WITH RESPECT .TO THE REGISTRATIONS OP PRODUCTS OUNTAINING 2,4,5-T iPursuant to this Court's order .of July 31, 1970, I have prepared, on behalf of the Secretary of the Halted.States Department of Agriculture, (1) an explanation of the standard used to determine when a particular hazard is sufficiently imminent to warrant immediate suspension of the registration of an economic poison under the Federal Insecticide, Fungicide, and Rodenticide Act (hereafter FIFRA), as amended, 7 U.S.C. 135-135h; * and (2) a description of the specific facts to which that standard has t been applied in order to reach the conclusion that suspension is not 18 i ^ warranted with respect to 2,*1,5-T products Tor (granular uses about the n. ' home, recreational areas and similar sites and for all uses cn rights of y -way, fd crop3, hay, pastlire, range lands and other farm uses. 9Si)800 1. The FIFRA authorizes the Secretary to suspend the registration of an economic poison inrredlately "when he finds that such action is neces- sary to prevent an imminent hazard to the public.11 7 U.S.C. 135b(c). . Congress did not define the term "imminent hazard" in the 195*1 amendment which created the suspension authority, and the legislative history of .-the amendment is silent with respect to deFinsmsnt of the .term. V.Mle -- the Government does not agree with the result reached in .Nor-Am Apj-icultural products. Inc, v. Hardin (No. 18,*178, decided July 15, 1970), where a divided Seventh Circuit panel concluded that there is judicial review in - 'b a district court of an order of the Secretary which suspends the registra tion .of an economic poisen, the Secretary does concur with the majority's .. .2 / -- exposition ..of the_term. ^imminent hazard.Specifically, the Seventh Circuit majority said: ~~l7 On April 20, 19/0, the Secretary suspended all uses .of 2,*l,5~T products in areas adjacent to lalces, ponds and ditch banks (the only uses "near aquatic sites ever approved); and all liquid formulations about the ,hone and recreational areas and similar sites (2*19). The introduction of 2,*1,5-T into water sources could threaten humans and aquatic life, and -- the use ;of liquid formulations around the hene, recreational areas and similar sites could involve direct exposure of pregnant women to undesir able concentrations of 2,3,7,8-tetrachlorodibes'Zojo-dioxin. It was not necessary to suspend granular uses about hemes, recreational areas and similar sites because they contain small amounts of 2,4,5-T (as lev; as one percent) in fertilizer mixtures, do not have to be mixed by the -- applicator and thus do not present the direct exposure hazards that are present in connection v/ith liquid formulations. On May 1, 1970, the Secretary issued notices of cancellation regarding all uses of 2,!1,5-T products on food crops intended for human consumption and all granular uses about the heme, and recreational areas and similar sites (250). 2/ I need not explore here the Government's disagreement with the t Nor-Am majority which/ since the Government's petition for rehearing in banc was granted on August 13, 1970, will now be considered by the full Court. - 2- Dow 2 0086 . c C; e rne. tern [viz., "inridnent hazard to the public"] appeared in federal legislation in "The Drug Amendments of 1962," an act amending the Federal Food, Drug and Cosmetic Act. Under that statute the "imminent hazard" finding is required for the :sun-navy suspension of a license to market drugs. if the suspen sion is to be effective prior to hearing. l.hlle the legislative history of the 196't amendment to the Federad. Insecticide, .Fungicide, and Rodenticid-e Act, giving the Secretary: of Apiculture similar suemary authority, does not seem specifically .to contain a discussion of the concept of "lir.nincnt hazard" it does refer to the fact that the new procedure is modeled after that contained in the Federal Food, Drug and Cosmetic Act. Viewing the similarity of the language utilized and the purposes for which it was intended, it seems clear that the language in .both acts should be similarly interpreted. The Senate report on the "Drug Amendments of 1962" reflected that an invvinsnt hazard to the public health would exist when the .evidence before the Secretary shoved that a drug v;es so unsafe as to create a public health situation "which must be corrected inrnsdilately, and cannot be permitted to continue while a hearing is .being hold." The Committee contemplated that the pov;or would be exercised only in tl:e exceptional case of an emergency which did not permit the Secretary to correct it by other means. S. Rep. Ho. 1 7 M (pt. 2), 87th Cong., 2d Soss. 7 (19-52). As Senator Eastland elaborated in his prepared report cn the Senate ...floor: "The committee believes that this authority, which .could have grave effects upon a manufacturer end upon .'the confidence of the public in a drug which might later be found appropriate for continued availability to physicians, should only be exercised under the most extrae conditions and with the utmost care." 108 Cong. Ree. 17366 (1962). Similarly the House of Representatives Report on the same provisions stated in part that it would be expected that in .'exercising the imminent hazard authority the Department would make.every effort, within the limits of its public responsi bility, to notify the applicant ar.d allow him to advance arguments why summary suspension was not required, or after suspension lias been invoked, why it might safely bo with drawn pending the hearing. H.R. Rep. Ho. 2^6^, 87th Ceng. : 2d Ssss. 8-9 (1962). ^'3 - 18119 Dow 2 008G53 While it is apparent that the Department has been concerned Yiith the environmental- impact of the use of mercury products, .It is certainly much less clear that the data available to the Department, at least insofar as our record is concerned, indi cated the necessity for emergency action ns opposed to continued and in-depth analysis study. Slip op., pp. 17-19 In a word, the Secretary agrees that an iieninent hazard exists when an economic poison presents a public health situation v;hich must be cor rected Immediately and cannot be permitted to continue while a hearing is being held and that the suspension authority must be exercised sparingly, i.e., only when a serious emergency exists. Effect and time, not time alone, are the determinatives of the Secretary's authority to suspend a registration, particularly in Dight .of the fact that the FUulA authorizes, suspension "to prevent an imminent hazard to the public [emphasis supplied]." In this connection the Secretary concurs with Surgeon General Jesse Steinfelcl's stata ;nt that "if an individual or an animal v;ere exposed to a carcinogen, and developed a cancer 20 years later, this is nonetheless as far as I am concerned, an inmient hazard and v;e.should .remove it." 'I .consulted with seven'Department scientists and we applied these criteria to the folio-ring facts regarding 2,*I,5-T and, after considera tion .of all the scientists* reccmmendations and opinions, I conclude that only the use .of 2,^,5-T products in areas adjacent to laJss, pends and ditch banks, and the use .of liquid formulations of 2,^,5-T around the home and recreational areas and similar sites present the tlrreat of an imminent hazard to the public. I novr turn to the scientific data , supportive of my conclusion. - H- -1 X 20 d o w 2 U8G5 2a* Ch-*cal Charactersties erri Synthesis of 2 jl,5~T The herbicide,' 2,1},5-T trichlorophenoxyacetic acid, commonly laiovm as 2^I5-T, consists of the.atoms of chlorine, oxygen, hydrogen and carbon. 3he 2,4,5~T acid has the following chemical fonnula: Carccer-cially, 2,^1,5-T is synthesized through a ccaplex series .of chemical ' reactions, beginning with benzene and chlorine, under a variety .of tempera.rture and pressure conditions. Proper control .of these conditions is . essential to minimize side reactions that can result in impurities in the * -..final product. Formulations containing acids, salts, esters and ester mixtures of 2,^,5-T, solvents and other ingredients, can be applied cither as liquids or solids, 2,il,5-T, when applied.at proper amounts, acts by disrupting normal metabolism in seme plants, but not others. The great v value .of 2,^,5-T jo that it acts selectively on plants, killing unwanted cjz brush and v/aeds, but not significantly injuring other forms of plant life. Considerable attention has been devoted to a trace impurity pro duced in step one of the reactive process. This is the formation of '2,3,7,8 tetrachlcrodibenzo jvdioxin (TDD), a highly toxic substance. TDD is one of about 75 theoretically possible "dioxins." Only six to . eight "dioxins" have been identified as trace contaminants in various chemical formulations, althougli TDD is one "dioxin" for which there is concrete evidence tint it is a teratogen. - 5 - 181 m w 2 OO8 6 6 O .Consideration .of the "dioxin" impurities originally arose from two' independent sources: (1) the chick cdmva factor; and (2) chloracne poisonings .of workers in chemical plants* In 1957> poultry farmers in the east and central United States suffered sporadic losses .of their .flocks (in seme cases up to 95 percent .of a single flock was afflicted). . Afflicted poultry exliibited "liydropercardium (abnormal ^accumulation of .serous fluid in the pericardial cavity), ascites (aecirnulaticn of serous : fluid) [and] liver and kidney pathology".(181 at p. 8). It was ascer tained almost immediately that the causative agent must be contained in : fatty acids or animal feed fats .of chlorinated aromatic contaminants (133-139, 146). However, it was not until 1957 that a scientist .at the research laboratories of the' Proctor and Gamble Ccmpary identified the' ` "chick edema factor" to be .of .the polychlorcdibe;"i30"p-d:loxin family, specifically l,2,3,7,8,9-h3xachlorcdibcn2o~p-clo:dn, a chemical "relative" i3/: - ..to TDD (138). Beginning in 1899, chamlcal workers were.reported to be .suffering a fora.of skin rash,' called chloracne (2*18). German doctors who studied . the .affliction determined it could be eliminated by the reduction .of "dioxin" impurities'in the reaction processes (148, 162; see also, 189 at pp. 241 and'336). The first reports of 2,4,5-T chemical workers in Germany :suffering chloracne v.'ere issued in 1957*- The cause'was identified as TDD. . ''.3/ Ihe data obtained in these investigations do not indicate that ; 2,4,5-T was involved with the chick edema problem. , -- 6 -- 18122 In 19^1, when the Dov/ Chemical Carpany in Midland, Michigan, responded - t o increased demand for 2,4,5-T for use in Viet Nam and attempted to speed up production by changing the reactive process, about 60 workers became afflicted withchloracne. Dov/, a major producer of 2,4,5-T, l-- ivodiately closed. cbv.-n its plant and undertook a study of the cause, v/hlch was identi~ "V : fled as TDD, Dov; then changed the temperature and pressure conditions of s the eariy reaction process to insure that not more than 1 ppm of TDD would be present in technical grade 2,1|,5~T. Dov/ reports that all 2,l|,5~T now being produced contains less than 1 ppm of TDD, in .many cases less than '0,5 ppm, and seme under'0,1 ppm. This has been confirmed by laboratory ..tests conducted by both the Departments of Agriculture and Health, Education, and Welfare. - T99800 s Moa 'b, ' Uses and'Benefits Of'2;*1.5~T 'Until relatively recent times the main weapon in weed control was physical removal (cultivation). Some chemicals like coman salt, copper sulfate, sulfuric acid, arsenates, and . heavy petroleum oils, M i l a n plant life and have been'used - : for many years to produce temporary soil sterility. Fire . has long been used to burn off weeds and other plants prior to replanting and along rights of way. Of these methods only -- -- .'-.cultivation [was] selective. Handler (ed.), Biology and the ;:Rituro Of Man, 82fr (1970). Although 2,4,5-T was first synthesized' in the 1930's, it did not receive .-extensive research consideration until World War II, when, as with so many other technological possibilities, it v/as subjected to accelerated research- for study of possible use in connection with the military .effort in South ' Pacific jungles. However, "the compound was never employed in connection v/ith any military operation ,during World War II. 'V 'Since 19!'l, Dow has monitored chemical effects at its plants by rabbit car tests (139 & PP 36l to 375) -'7 d o w 2 008GG2 2,4 >5-T was first registered for domestic use pursuant to the FIFRA in 1948. In the intervening 22 years, it has found increasing use for the control of brush and weeds In areas utilised for agriculture, forestry (primarily to clear deciduous hardwoods from conifer stands), industry (for rights of way and powerlinos) and home (for lawns and gardens). Between .i960 and 1968, production of 2,4,5-T in the United States exoanded from 7.9 million pounds to 42.5 million pounds3/-(180 at p. 34). In 1964, about 19 percent of domestic use was for agricultural purposes (approximately 7 percent on hay, pasture and range lands, and 12 percent for other farm uses), .7 percent 'was for. lawn and turf treatment, 7 percent on Federal lands, approximately 10 percent on private nonfarm forests, 2 percent for margins of aquatic areas and 6 percent for other uses, including by state and local governments. Almost half of use, 49 percent, was on non-Federally controlled rights of way (179 at p. 14). ihese usages are, of course, subject to fluctuation. Domestic use declined from 8.9 million pounds in 1964 to 4.6 million pounds in 1966, partly in response to shortage of supplies resulting frcm increased demand for military use abroad. In a society dedicated to production of high quality foods available to all'people at economic prices, all herbicides including 2,4,5-T play an important role by cutting losses to weeds that curtail food harvests. Similar considerations apply in the field of forestry. Much of the demand for herbicides has resulted from the cut-migration of workers who. formerly used hand cultivation methods in weed and brush control. / A substantial percent of the increased production was for military use in Viet Nam and for general export. . 8- 18:124 The benefits steaming from the domestic use of 2,*I,5-T have been summarized by Dr. Lee DuBridgo, Science Advisor to the President and Director of the Office of Science and Teclmology in the Executive Office of the President: We have here an example of a chemical substance intentionally placed in the environment by man for the betterment of his welfare. Where the aim has been to exchange capital for labor in land and waterway management, there can be no doubt . that this herbicide has proved its worth (189 at p. *J52, *15*0 These benefits have been translated in economic-terms by'reports of the Agricultural Research Service and the Economic Research Service of the Department of Agriculture: !,If- 2,*I,5-T was no longer used in this country it would cause an additional cost of 52 million dollars to achieve the sane result, by use of alternative herbicides and manual methods. If alternative herbicides (primarily 2,*!-D) were also banned, this cost would 6/ increase to 172 million dollars' (179, 180). c. Ecological Considerations and Persistence of Residues Involved in the Use of 2,*t,5-T_____________ . __________ 2,*I,5-T is not considered a persistent pesticide when compared with the chlorinated hydrocarbon insecticides. Degradation on plant tissue occurs rapidly and data available suggests that residues resulting from recommended rates of application are not of sufficient magnitude to Justify concern as potential health hazards. Generally, 2,*J,5-T persists in soils for not more than three months. It appears that within this 008663 r C < 6/ Both these figures are based upon the assumption that we will , maintain the present level of control. 9- - 18125 DOW 2 0 0 8 6 6 period the residues are broken down by coil microorganisnis tint use chemicals as sources of carbon and energy (88-9*1, 96, 99/ 100). Althqi.^ wind and water might carry the residues to non-target areas during the three-month period, this does not appear to constitute a significant hazard. A potential danger to humans and acquatic life could exists if substantial treatments are carried on near water' sources or by acci dental spills into v/ater'; therefore, the'Secretary has suspended all uses', of 2,4,5-T around lakes, ponds, and ditch banks. 4 Residues in forage crops decline rapidly, and may remain at low levels for eight weeks on forage grasses. (97, 98) Care must be taken to defer grazing by dairy cattle soon after spraying. Analysis by the Food and Drug Administration of dairy products (discussed more fully, infra, at p. HI ) reveals that not only is the finding of residues extremely rare, but, when found, residues are at very low levels. The available evidence suggests that animals eliminate residues even more quickly than plants. For instance, rats and pigs eliminate them very rapidly, one-half of the ingested dose being eliminated in 3 to 10 hours, mainly through the urine (33, 75). Dairy cattle also excrete 2,*J,5-T through urine rapidly (255). In terms of wildlife habitat, the application of 2,*I,5-T can have either beneficial or adverse effects depending on the circumstances and the species involved (^8). Thus, although an intense and unduly heavy application can adversely modity game habitat, judicious treatment of -10- 18126 selected areas of forests of low economic value can improve the habitat for the protection and production of wildlife such as game birds and deer. Although too high an application can reduce population of bees, proper applications on rights of way can create ecological "edge effects" very - v favorable as habitat for species such as grouse and wild turkeys (4). d. Public Health Considerations Involved in the Use of 2,4,5-T Recently there has evolved concern that 2,4,5-T may be a teratogen, i.e., an agent responsible for birth defects. However, the Secretary's orders of April 20,. 1 9 7 0 and May 1, 1970, suspending certain uses of 2,4,5-T, cancelling others, and not affecting other uses, were based on .exhaustive examination of available scientific evidence, which was analyzed and interpreted by experts in the Departments of Agriculture; Health,' Education and Welfare; Interior; and private sources. In 1963, the National Cancer Institute entered into a contract with Bianetics Research Laboratories, Inc., of Litton Industries (Bethesda, Maryland). Bionetics was to undertake! a study of the possible carcinogenic and teratogenic agents in a number of pesticides and related compounds. Eventually 86 compounds, including 2,4,5-T, were selected for study. After the study was completed by Bionetics in early 19^9, the National Institute of Environmental Health Sciences began a statistical analysis c c iC 7/ See generally, Odum, Fundamentals of Ecology, 278-281 (2d ed., 1959)s Hanson, Dictionary of Ecology, 123 (196*0 -11- ` 1 i1_o,47 Dow 2 00866G ..of the data accumulated by Bionctics. Because of .the finding that 2,'1,5-T appeared to be "causing significantly more deformities than ejected," particular attention was paid to this compound. The rav; data v;as also released to the Health, Education and Uelfarc..Secretary's Commission on Pesticides and Their Rolatj.cnship to Environmental Health (the tfrak Commission). * While the data ccnpilcd by Bionctics did not reveal 2,4,5-T to be a carcinogen (see .^3), it did indicate that the compound was teratogenic .to.strains of nice and rats injected doses suspended in DMSO, and fed dosages suspended in honey. Clef'c palate and cystic kidney were the predominant teratogenic effects.. However, the validity of the test results ' was almost immediately challenged. 'The Dow Chemical Company objected that the 2,^1,5-T used in the study was not a representative sr.-iiple of the presently being produced in this country. Dew pointed out that the 2,Jl,5~T used by Bicnetics contained the "dioxin" contaminant TDD at . the rate of 27*8 ppm, v-hile the 2,ii,5-T bejLng produced commercially containedsubstantially less .than 1 ppm. ..;Subsequent studies were undertaken by the Food and Drug Administration (FDA), the National Institutes of Environmental Health Sciences (NIEHS), % .the National Institute of Dental Research (on cleft palate defects), and by the Dow Chemical Company. DowTs preliminary studies indicated no teratogenic effects from relatively "pure" 2,^,5-T containing less than 8/ , 1 ppm at dosage levels under 2l\ rr.g/l:g per day. The PDA and NIEHS studies 8/ It does not appear chemically possible commercially to formulate 2,J|,fpT without traces of TDD, though such traces can be held as lev/ as 0.1 ppm. did show teratogenic effects when injected in D.'oO at 100 irg/lcg (though not at lower level's), even though relatively ''pure" lots of 2,4,5-T v;ere tested. Both the validity and the iiriplications to human health of the latter two stu-dj.es have also been subject to question. The HIEIIS study showed no teratogenic effect on rats, but did on mice. FDA studies of the "dioxin" contaminants have used the chick embryo (see 189 at pp. ISO-360). However, v;hile the sensitivity of the chick embryo to many factors makes it ideal for experimental screening study, tlds very factor makes questionable its relevance to public health. For jLnstance, an essential vitamin, vitamin A, is a teratogen under this test. c c As the World Health Or-ganigatlon has stated: K 0086G7 The chick embryo contributes greatly to basic er.bryological knowledge. However, for the screening of drugs for terato- . genicity, its use is not recommended. It is.too sensitive ...to a wide range of agents and affords no parallel with the anatomical- and physiological relationship existing betvreen pregiant mammals and her [sic] conceptus (189 at p. 402). - 2,4,5-T has been used in this country for more than two decades without one confirmed report ;of its having had a teratogenic effect on a human embryo or fetus, and the speculation that vddespread use of 2,4,5-T has caused birth defects in Viet Hem has not been confirmed. Reports of alleged widespread dosage caused by use of 2,4-D; 2,4,5-T and Silvex (a . chsmically-related compound to 2,4,5-T, but with a different selectivity) by the United States Forest Service near Globe, Arizona, "lest much of . their significance upon medical surveillance for alleged illnesses in 9/ humans and malformations in animals" (181 at; p. 37). Hot only is it ' 9/ Officials of this Department are currently preparing a definitive .report with respect to these allegations. . ~:13 9 difficult to extrapolate results obtained from tests on animals to hunans (omd vice-versa: vrltness thalidomide, to which rat strains showed no adverse reaction, see 27), but further there is a tremendous difference between the massive dosage purposefully fed tn test animals. and the insignificant amounts .of 2,*I,5--T v;hich reach the American people. The Food and Drug Administration has studied,'inter alia, residues'of 2,*1,5-1 s' on agricultural commodities between 196-4 and 1$6B, and found that there were none in the vast majority of cases. A few cases .of insignificant . .amounts were reported. I have previously mentioned that the BDA has set -no tolerance for 2,*1,5-T cn agricultural commodities, and if a commodity O -bore a residue it viould be subject to seizure.. Of the 5,300 food samples O _studied by the FDA,_o:ily two showed detectable residues' at -0.1 ppm limit ;of the detection mathc-ds. ''"It: must be emphasised that .teratogenicity can .be' induced in a wide variety, of the highly sensitive embryonic oi? fetal organisms by many <P o 00 an GO chemical agents, even vitamin A, if dosage is high enough. ' However, such a result on an animal dees not allow the inference that a similar result -would follow for a.human embryo or fetus. Hot oily are there significant differences in metabolic sensitivity which can only be correlated by comparable metabolic study, but, more importantly, potential teratogenicity -to humans end human embryos end fetuses must be gauged'.by analysis of the actual intalis mounts that can reasonably be expected to enter the .human body. t -lV- * 18130 On .the basis of the application of the standard I have described, supra, pp. 2 ~ , to the' presently .available scientific evidence hereto fore summarised, I conclude that (1) the Secretary properly suspended all uses of 2,J|,5-T in areas adjacent to lalrcs, pends and ditch banks, end the use of ail liquid fomr.ilations aromd the hcrr.e, recreaticnal areas and similar sites to prevent an Imminent hazard to the public; s (2) the Secretary appropriately issued notices of cancellation regarding all uses ef 2* ^ 5rT products crj food crops intended for human consump tion and all granular uses about the heme, recreational areas and similar sites since recent reports raise scr.'.e question about the safe use of 2,4,^-T products in these respects; and (3) the .ether uses of 2,il,5-T products do not present any foreseeable hazard to the public. Respectfully submitted, .Ned D. Eayley Director Science and Education 699800 s M o a * - 15 - i o jLO m 6 G 6 l MOO 1.07y 1135 Presence of chloracnegen and 2,3,7,8 -tetrachlorodibenzo-p-dioxin in 2,4,5-T from various manufacturers Hooker Compound TCP Animal Test * 1965 Assay (GLC) ** neg. <1 ppm Diamond 2,4,5-T active (mod-sever) 2 , 4 , 5 - T --- 5 ppm S 2 16 ppm 1969 Anima 1 Test Assay (GLC) O o 00 a o active <1 ppm Hercules 2,4,5-T neg. S^-Sg <1 ppm Sg 3 ppm*** Monsanto 2,4,5-T active Dow TCP neg-active S^-S^2 6.5-11 ppm '63 2 samples @ <lppm '64 6 samples, 1 @ 6 ppm 5 @ <3 ppm *65 3 samples @ <1 ppm <1 ppm * chloracnegen test by rabbit ear bioassay. ** chemical assay for 2 ,3,7,8 - tet.rachlorodibenzo-p-dioxin *** Dow was buying from Hercules in late 1965 and sample was from 3rd or 4th batch shipped and high dioxin thought due to "pushing" capacity of plant. 18132 REGISTRATION STATUS FESrRTPTTON:" ~ .. ., Use of silvex m pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT). U J Ji je fa<// .-VX g u j tj 'w '5 ,_ o a w tw VID (Veterinary Investigational Drug)-FDA IND (Human Investigational Drug)-FDA NDA (New Drug Application)-FDA DHuman OAnimal DOriginal OSupplement Antibiotic Form 5 or 6 - FDA Animal Human Original Amendment TEMPORARY PERMIT, PESTICIDE PESTICIDE REGISTRATION APPLICATION QOriginal Revision PESTICIDE TOLERANCE PETITION Temporary Permanent FOOD ADDITIVE PETITION - FDA Other : Agency: USDA 9-PP-1103 REPORT 1 1 3 f, product KURON (silvex) Date This Report No. ______ ______ ______ Date Fi led W i t h Gov 't 12/29/' Estimated 10/29/ Date of Approval Optimistic Pessimistic ______ ______ ______ Country: USA fi-i D o w C o n t a c t :(1) G. E. L y n n _________ (2) M. L. L e n g ________ (3) D . D . M c C o l l i s t e r ^Tot DATE ACTION 11/5/67 t/ ' M e e t i n g b e t w e e n C. L. D u n n o f I T F P H T a n d G. C. H o l s i n g o f H a z l e t o n L a b o r a t o r i e s I n c . , w i t h J. A l p e r t a n d L. L. R a m s e y of FDA to discuss tolerances for 2,4-D, 2,4,5-T, MCPA and silvex in range and/or pasture grass at the high levels that exist shortly after application. Mr. Alpert regarded 200 p p m as high and 700 p p m as unrealistic. R e m o v a l of animals from rangeland and adherence to pre-grazing intervals w e r e considered to be questionable as restrictions in l a b e l i n g . E x c l u s i o n o f d a i r y a n i m a l s w a s a c k n o w l e d g e d to. be practical. (FDA review of USDA petition for tolerances for 2,4-D in grain, PP 6F0459, concluded that 20 p p m in feed would result in less than 0.05 ppm in meat, but a two-week pregrazing interval was needed to avoid meat or milk contamination from animals grazing treated fields of small grain t r e a t e d at 1.5 lb a.e./A) c ( ( c c c 11/27/67 12/29/67 1/8/68 V 8/1/68 Request for extension of all currently registered uses of s i l v e x on p a s t u r e a n d r a n g e l a n d s u b m i t t e d b y C. L. D u n n o f Hercules on behalf of NACA ITFPHT. (Also 2,4-D, 2,4,5-T, MCPA) E x t e n s i o n g r a n t e d b y U S D A u n t i l Jan. 1, 1 969 ( l e t t e r to C. L. Dun n ) . (Also 2 , 4 - D , 2 , 4 , 5 - T , MCPA) Notification of extension published by USDA as PR Notice 68-1 for uses of silvex on pastures and ranges listed on page 698 of the Summary of Registered Agricultural Chemical Uses. (Also 2,4-D, 2,4,5-T, MCPA) PR Notice 68-11 issued by USDA informing all registrants that further limited extensions will be considered based o n a p r o g r e s s r e p o r t r e c e i v e d on o r b e f o r e N o v e m b e r 1, 1968, or by Submission of a petition for tolerances p r i o r to J a n u a r y 1, 1969- REGISTRATION STATUS DSCaiPTINTi '; Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT) 9-PP-110 3 REPORT Product SILVEX Date This Report DATE ACTION Tota. 10/29/68 R e q u e s t f o r f u r t h e r e x t e n s i o n r e q u e s t e d b y C. L. D u n n on behalf of NACA ITFPHT using tabulation of residue data p r e p a r e d b y M. Leng. 12/3/68 12/23/68 E x t e n s i o n g r a n t e d u n t i l J a n u a r y 1, 1 970 f o r a l l c u r r e n t l y registered uses of silvex on pastures and ranges found on pages 698 of the USDA Summary (2nd e d i t i o n ) . Assigned number 9-PP-1103. (Also 2,4-D, 2,4,5-T, MCPA) A letter from FDA reviewing PP 8F0675 for tolerances of silvex in various crops stipulated that if the present registration for silvex on pasture and rangeland grasses (extended) is to be retained, a tolerance for silvex on pasture and rangeland grasses should be proposed, and supporting residue data submitted. Depending upon the level needed, additional cattle feeding studies and proposals for meat and milk tolerances may be necessary. (Also others) t c * m. * 1/10/ 69 PR Notice 69-1 lists extension of silvex at 0.75 and 4.0 lb/A in p a s t u r e s and ra n g e l a n d as shown on page 698 of the USD A Summary (Also all uses of 2,4-D, 2,4,5-T and MCPA in pasture and rangeland). 1/15/69 M e e t i n g i n W a s h i n g t o n o f D. D. M c C o l l i s t e r a n d C. L. D u n n of Industry Task Force with Harry Hays and Harold Alford of P R D a n d L. L. D a n i e l s o n , D. L. K l i n g m a n a n d W. C. S h a w of ARS, USDA, to discuss Mr. Dunn's proposal of 10/29/68 to Dr. Hays that r e g i s t r a t i o n of the use of the four phenoxy herbicides on range and pasture grass be continued without the establishment of a tolerance on grass and that the regulatory tolerance be established only for the meat of animals feeding on treated grass. PRD concurred in the principle of exemption for qrass but pointed out that hay will require tolerances. 1/27/69 M e e t i n g in W a s h i n g t o n o f D. D. M c C o l l i s t e r a n d C. L. D u n n of Industry Task Force with Harry Hays, Harold Alford and D a v i d B o w e n o f PRD; L. L. D a n i e l s o n , W. B. E n n i s a n d D. L. K l i n g m a n o f ARS; a n d L. L. R a m s e y , F. J. M c F a r l a n d , V/. S t o k e s , J. G. C u m m i n g s , D. V. Ree d , H. B l u m e n t h a l , G. E. W h i t m o r e , H. R. G i t t e s a n d J. B. L a m b o f FDA. It was agreed that ARS and the Task Force would prepare a protocol for feeding studies for meat and milk which should specifically support the principle of exemption for grass. The protocol would be circulated to FDA for their evaluation. All four compounds should be studied in one comprehensive project and feeding studies should be done only with the acids. Continuation Paqe 2 18134 r \ L u r\H i i u n o i r t I U M T UK I DESCRIPTION: Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT). 9-PP-1103 Product SILVEX Date This Report xi DATE 2/26/69 ACTION M e e t i n g of T a s k F o r c e in w h i c h Mr. G. T. S c r i b a , of Regulatory Services reported that FDA legally duty or power to issue a tolerance regulation or exemption regulation in regard to range grass. NAC Director has no a tolerance iu ta . C H E M I C A L CO; 2/27/69 o Q 3 4/30/69 M e e t i n g o f C. L. D u n n a n d M. E. G e t z e n d a n e r w i t h p e r s o n n e l of the Crop Protection Division and ADP of ARS USDA to discuss tentative protocol for feeding studies to justify the use of all four phenoxy herbicide compounds on range and pasture grass. A n o t h e r m e e t i n g w i t h ARS (ADP) of U S D A to d i s cuss p r o t o c o l for feeding studies. 6 / 1 9 / 6 9 L e t t e r f r o m C. L. D u n n o f T a s k F o r c e to Dr. P a u l D e L a y , Director of Animal Disease and Parasite Division of USDA, enclosing proposed protocol for the animal feeding studies. He proposed that registrations of phenoxy herbicides on f ranges and pasture grass be continued without tolerances for range or.pasture grass, but with tolerances to be set for m eat at levels indicated by the data developed b y this feeding study. Included were fixed level feedings and a declining-rate feeding study approximating actual range e x p o s u r e as p r o p o s e d b y D. L. K l i n g m a n b a s e d o n d a t a o b t a i n e d by Texas A&M on residues of 2,4-D.and 2,4,5-T in range grass. The animal feeding, animal sacrifice, and taking of samples were to be conducted by USDA while the Task Force would be responsible for analyses of tissues and for submission of petitions, should tolerances be required. 1 X. 5' cA o 6/23/69 y L e t t e r f r o m C. L. D u n n t o L. L. R a m s e y o f F D A r e p e a t i n g proposal made 2/27/69 that registration for use of chlorophenoxy herbicides on range and pasture grass be continued without setting tolerances on the grass, provided that suitable tolerances for meat and milk are provided where needed. Enclosed was a copy of the proposed p r o t o c o l for fixed level.feeding for 4 weeks followed by immediate slaughter, for slaughter 7 days after discontinuing highest level, and for feeding at declining levels over an 8-week period. Arguments were presented that tolerances were not n e e d e d for g r ass (an o p i n i o n s u p p o r t e d by r e p r e s e n t a t i v e s from PRD and the Crops Research Division and Crops Protection Research Branch of U S D A ) , because grass does not move in interstate commerce and as a growing crop would represent a difficult policing problem. Although range grass is seldom cut for hay, appropriate label restrictions and precautionary Continuation Page 3 REGISTRATION STATUS DESCRIPTION:" Use of silvex ln pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT). 9-PP-1103 f?"P0RT P r o d u c t SILVEX Date This Report DATE D 6/23/69 j (cont'd < o ACTION statements would be required by PRD, USDA to prevent such harvest in the absence of a tolerance for hay. A separate project concerning milk is being undertaken (by Dow) and data from that study will be used in support of pasture grass registrations. Total 2?s. T H D O W C H Ldi 7/7/69 L e t t e r f r o m L. L. R a m s e y to C. L. D u n n s t a t i h g t h a t t h e y (FDA) do n o t agree w i t h his v i e w t h a t tole r a n c e s for these herbicides on range grass are not needed and that pasture grass is a separate situation. Reference was made to the Federal Register of 4/8/69 in which an order was published e s t a b l i s h i n g a t o l e r a n c e o f 40 p p m f o r d i c a m b a in or on grasses (pasture and rangeland) and grass hay. (See att a c h e d pages from EPA Summary for dicamba listing many restrictions^ e e 10/23/69 A r e q u e s t f o r e x t e n s i o n s u b m i t t e d b y C. L. D u n n on b e h a l f o f ITFPHT reported on progress in getting the necessary milk and meat residue data. Dairy cows are on hand in Midland and control pretreatment samples of milk are being collected. Feeding studies with 2,4-D and 2,4,5-T are scheduled to run c o n c u r r e n t l y at stepwise levels from 10 to 1000 p p m for 2 to 3 weeks at each level. Feeding studies with MCPA and silvex are scheduled to begin in February. The meat residue studies are to be conducted by USDA in Kerrville, Texas, in both cattle and sheep, at 300, 900 and 1800 ppm 2,4-D and 2,4,5-T and silvex, and at 250 and 500 ppm MCPA in the total diet for 4 weeks. The effect of a 7-day withdrawal and of declining levels in the diet will also be studied. Final details of the studies are still being discussed with USDA. 10/29/69 S t a t e m e n t a b o u t 2 , 4 , 5 - T i s s u e d b y Dr. Le e A. D u B r i d g e , S c i e n c e Adviser to the President and Executive Secretary of the President's Environmental Quality Control, listing actions to be taken because of Bionetics report that this herbicide caused teratogenic effects in rats and mice. c c c c c c 12/10/69 Dow initiated feeding study with 2,4,5-T in at levels of 100, 300, 900, and 1800 p p m in for 4 weeks with slaughter 1 day and 7 days feeding. cattle in the total after the Texas diet last 12/29/ 69 Word was received by ITFPHT that USDA has granted extensions for the use of 2 , 4 - D , MCPA and silvex in pasture and rangeland. No mention was made of 2,4,5-T although it was listed in the request,for extensions submitted to USDA on 10/23/69. Continuation page .8136 K t I KA rI ON STATUS DESCRIPTl'ClTT: Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT) * F "PORT Product SILVEX Date This Report DATE ACTION iota! 1/19/7 ) PR Notice 69-20 dated 12/22/69 received from USDA showing extensions granted for a number of pesticide registrations including 2,4-D on grass (hay crop), pasture (grass), and ranqeland (ranqegrass), ranqeland clearance (ranqes); MCPA on pastures (established); and silvex on pastures and ranges. 3/4/70 USDA notified ITFPHT that extensions were being granted 2,4,5-T in pastures (grasses), rangeland clearance and in crops pending completion of studies in progress. 3/19/70 ^ 4/2/70 Task Force notified that ARS was ready to proceed with the p r o p o s e d c o o p e r a t i v e m e a t r e s i d u e s t udy on 2,4-D, 2,4,.5-T, silvex, and MCPA to be done at Kerrville, Texas. Meeting of Industry Task Force to discuss progress in getting residue data on 2,4-D, 2,4,5-T, silvex and MCPA in various food crops, meat, and milk. Cost estimates are around $5,000 for most crops, $75,000 for sugarcane (Dow), $60,000 for m i l k (Dow) an d $3 8 , 0 0 0 for m e a t (Southwest R e s e a r c h Institute), plus cost of filing amendments and of filing petitions for tolerances to cover uses in range and pasture. Most companies are not willing to cost-share these large amounts. rfe 4/9/70 PR Notice 70-10 issued by USDA listing additional extensions including 2,4,5-T in crops and in pasture and rangeland. (Copy not received by regular channels; obtained by special r e q u e s t f r o m U S D A b y M. L e n g ) . 4/15/70 USDA announced immediate suspension of 2,4,5-T for some uses, and cancellation for other uses. Range and pasture, forests, and rights-of-way are not affected. 6/4/70 L e t t e r f r o m C. L. D u n n to R. D. R a d e l e f f o f U S D A a t K e r r v i l l e with revised protocol for feeding studies only at fixed levels of 2,4-D and silvex at 300, 1000 and 2000 ppm in the d i e t for c a t t l e a n d at 2 000 ppm' for shee p ; o f M C P A at 250 and 500 ppm for cattle and 500 ppm for sheep; and of 2,4,5-T at 2000 ppm for sheep. (Dow had conducted feeding study with 2,4,5-T at 100, 300, 900, and 1800 p p m in diet of cattle) Continuation Pago 5 7<3 O O1 1-KE D O W CHL'i'ilCAL (JOd f\ u u i >/ ' r\rt i i u n O T r t IVJO r U K I DESC f t W T n 1 --------------------------- Product SILVEX Use o f s i l v e x .in p a s t u r e and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT) Date This Report - 3 D A T E ________________________ To ta . A C T I O N ________________________ ________________ 6/17/70 Hearing before Senator Hart's Commerce Energy, Natural Resources and Environment subcommittee revealed that Ralph Nader's Center for the Study of Responsive Law, with some other organizations, has petitioned USDA to completely ban use of the herbicide 2,4,5-T and the related herbicide s ilvex. The petition asked the suspension immediately of all esters and formulations of 2,4,5-T and silvex "around the home, recreation areas, lakes, ponds, and on food crops, . w h ether o r n o t they are r e l a b e l e d . " It also a s k e d USDA. to make public the names of all suspended products, and to issue notices of cancellation of the registrations of 2,4,5-T and Silvex in all formulations for all other uses not yet mentioned. FDA tests have shown that Silvex causes birth defects in chicks but tests in mammals are incomplete. 2 . 4 - D w a s a l s o d i s c u s s e d a t l ength. Dr. T. C. B y e r l y , U S D A ' s t C Assistant Director for Science and Education said that " d i c h l o r o c o m p o u n d s s u c h as 2 , 4-D are u n l i k e l y to result...in__ ... ( \ the production of dioxins." DuBridge admitted that cases of substantial dangers have not been proven. Surgeon -3 -o General Steinfeld said that the 2,4-D data was uncertain. As of now, dat a is not suffi c i e n t to say that 2 , 4-D is oa t e r a t o g e n i c to h a m s t e r s . H. W e l l f o r d o f N a d e r ' s R a i d e r s s a i d that 2,4-D did not figure in their petition to USDA, but O ci) that it is "just as deserving of cancellation and probably suspension as Silvex," and that 2 , 4-D caused birth defects in chicks. The Surgeon General said that studies with 2.4- D in rats are underway. .... 12/5/70 N o t i c e p u b l i s h e d in F e d e r a l R e g i s t e r Vol. 35, No. 236, p. 185 5 0 , e n t i t l e d A m e n d m e n t o f S t a t e m e n t f o r I m p l e m e n t a t i o n w h i c h speci f i e d that registration should be continued in those instances in which a petition for tolerance or e x e m p t i o n t h e r e f r o m has bee n filed on or before D e c e m b e r 31, 1970, pending decision on such petition. 12/22/70 D o w s u b m i t t e d a m e n d m e n t s to T a s k F o r c e p e t i t i o n s (PP 8 F 0 6 7 0 for 2 , 4-D, PP 8F0675 for silvex, PP 9F0761 for MCPA) and resubmitted 2,4,5-T petition (originally PP 8F0669, refi as PP 1F1102). Included data on residues in grass from a pplication of 2 , 4-D, 2,4,5-T and silvex at industrial r a t e s (6 to 12 l b / A ) / c o m p l e t e r e p o r t s on r e s i d u e s i n m i l k and preliminary reports of Dow analyses for residues in muscle of cattle. (Analyses were to have been conducted by Southwest Research Institute in San Antonio but they had been unable to develop a satisfactory method.) The tolerance requests were not changed from those listed in the original Continuation Page ___ 6 8 K L l b I KA I I UN I A l Ub DES CH IP'1'1NI -( Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances (ITFPHT).` K t P UK I ( .roduct SILVEX Date This Report DATE 12/22/ 70 (Cont* d ACTION petitions submitted in December 1967. The 72-volume sub m i s s i o n w a s h a n d - c a r r i e d to W a s h i n g t o n b y E. R. L a n i n g to assure filing prior to the 12/30/70 deadline. To tal XIs 1/4/71 n 4 .j 1 / 5 / 7 1 / 1/6/71 is Letter dated 12/30/71 from Drew Baker of Pesticides Tolerances Division (formerly FDA) of the new EPA stating that tolerances are required for residues of phenoxy herbicides on pasture grass and rangeland grass. He referred to letter of 7 / 7 / 6 9 f r o m L. L. R a m s e y to C. L. D u n n o n this s u b j e c t . T e l e p h o n e c o n v e r s a t i o n s b e t w e e n D. D. M c C o l l i s t e r a n d L o w e l l E. M i l l e r , C. L. S m i t h and D r e w M. Baker, Jr. re n e e d for tolerances in grass. Concluded that a definite, numerical tolerance fiqure must be proposed for ranqeland or pasture grass to m e e t the requirements of the amendment published on 12/5/70. A m e n d m e n t to p e t i t i o n s s u b m i t t e d b y D. m o d i f y i n g Section F to.re q u e s t 300 p p m residues of 2,4-D, 2,4,5-T, silvex and land grasses or pasture grasses. D. M c C o l l i s t e r tolerance for MCPA in or on range- c , ^cm- -- - CO c1 O _> 2/5/71 Letter from EPA stating that additional fee of $2100 needed for each of 2,4-D, silvex and MCPA, and $1500 for 2,4,5-T. 2 / 2 2 / 7 1 D. D. M c C o l l i s t e r s u b m i t t e d a d d i t i o n a l fee o f $7800. 3/11/71 L e t t e r from E P A stat i n g p e tition amended as of 2/26/71. 5/3/71 PRD certified usefulness for silvex in pasture and rangeland (also for 2,4,5-T on 5/5/71, for 2,4-D on 5/14/71, and for MCPA on 4/21/71). 7/12/71 Letter from PTD stating that certain animal residue studies crucial to their review have not been reported. 7/21/71 Letter from Dow stating that residue studies had been c o m p l e t e d a n d an a m e n d m e n t w o u l d be s u b m i t t e d in 30 to 60 days. 7/29/71 P T D grants 60 days for r e ceipt of data. 10/13/71 A m e n d m e n t to petition submitted including complete report on residues of silvex and 2,4,5-trichiorophenol in muscle, fat, liver and kidney of cattle and s h e e p . . (Similar amend ments submitted for 2,4,5-T on 9/29/71, for 2,4-D on 10/1/71, and for MC P A on 10/21/71.) Tolerance p r o p o s a l m o d i f i e d to * Continuation Page REGISTRATION STATUS R'PORT D IST C irrPT T M l ' ~ 1 Product SILVEX Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force for Phenoxy Herbicide Tolerances ( I T F P H T )." Date This Report_ DATE -10/13/71 jcont'd. j j ACTION request 300 ppm for g r a s s , 0.5 ppm for rice straw and sugarcane bagasse, 0.1 ppm (negligible residue) in or on apples, plums, rice and sugarcane, and in meat and meat byproducts of cattle, goats, and sheep; and 0.05 ppm (negligible residue) i n mi lk.. :11/12/71 Receipt of amendment acknowledged by PTD and review begins. ^ 5/5/72 E P A p r o p o s e d i n t e r i m tolerances in Fede r a l R e g i s t e r Vol. 37, No. 89, p. 9 2 2 8 - 9 2 2 9 , i n c l u d i n g s i l v e x at 0.1 p p m for apples, rice, sugarcane, and plums (prunes). No interim tolerances proposed for silvex in grass, milk, or meat. (Interim tolerances also proposed for 2,4-D and MCPA in crops but not for 2,4,5-T.) 7/21/72 (' Letter from Pesticides Tolerances Division outlining ' deficiencies in petition for silvex tolerances, including need for data on bound residues in rice, sugarcane, grass, milk and meat. (Similar letters issued for 2,4,5-T on 5/4/72, for 2 , 4 - D on 7/19/72 and for M C P A on 11/10/72.) A l s o s t i p u l a t e d t h a t t o l e r a n c e o f 600 p p m s i l v e x w o u l d be' n e e d e d for grass (in c o n t r a s t to n e e d for 2000 p p m 2 , 4 - D and 1000 ppm 2,4,5-T). 8/30/72 Interim tolerances finalized by EPA including 2,4-D, silvex a n d M C P A i n t h e f o o d c r o p s c o v e r e d b y the p e t i t i o n s . ----(See F e d e r a l R e g i s t e r , Vol. 37, No. 169, p. 17554-17555). 9/6/72 Dow r e q u ested 90 days to submit substantive amendments to the phenoxy petitions. 9/13/72 Additional interim tolerances proposed by EPA including 2,4-D and MCPA at 300 ppm in grasses (pasture and rangeland) and grass hay, with statement that interim tolerances should not be set at that time for several pesticides including 2 , 4 , 5 - T (in anything) and s i l v e x (in grass and water). (See F e d e r a l R e g i s t e r Vol. 37, No. 178, p. 18565-18566) 9/15/72 Letter from P T D stating that petitions would be held in abeyance until 12/6/72. 9/29/72 Dow submitted comment to Hearing Clerk and requested that an interim tolerance of 300 ppm be established for silvex in grasses (pasture and rangeland) and grass hay, citing data in s u p p o r t a n d e n c l o s i n g p a p e r by M. L e n g e n t i t l e d " R e s i d u e s in C o n t i n u a t i o n P age ___ 18140 REG ! SCIATI ON STATUS RfORT DESCUiPtnJjn ;------------------------ " l P r o d u c t SILVEX Use of silvex in pasture and rangeland. Request for extension of all registrations submitted by NACA Industry Task Force( for Phenoxy Herbicide Tolerances (ITFPHT) .' Date This Report^ DATE ACTION Total Mis, 9/29/72 cont'd. Milk and Meat and Safety to Livestock from the Use of Phenoxy Herbicides in Pasture and Rangeland" reprinted from D O W N TO E A R T H Voi. 28, No. 1, p. 1 2 - 2 0 , S u m m e r 1972. 10/13/72 Letter from Drew Baker advising us that the letter to the Hearing Clerk and the two attachments were being considered by chemists and toxicologists in P T D . 12/2/72 Interim tolerances proposed by EPA on 9/13/72 were finalized t w i t h o u t m e n t i o n of silvex or 2,4,5-T. (See Federal R e g i s t e r Vol. 37, No. 233, p. 2 5 7 1 6 - 2 5 7 1 7 ) . 12/7/72 M e e t i n g o f D. D. M c C o l l i s t e r a n d M. L. L e n g o f D ow, a n d Lamar Brown of Rhodia with Drew Baker, now Acting Assistant D i r e c t o r o f t h e . n e w R e g i s t r a t i o n S e c t i o n o f EPA, a n d J o e _______ Cummings, Don Duffy, and Bob Quick of the chemistry review section, to discuss the status of work completed and underway at Dow to answer their questions of 5/14, 7/19, 7/21 and 11/10/72 on the phenoxy petitions. Mr. Baker stated that silvex was being cancelled for pasture and rangeland because the chemists could not agree on the tolerances needed, therefore the toxicologists could not determine that the levels proposed were safe. He also stated that "dioxin" was not the problem, although Joe Cummings indicated inadvertently that it was. Mr. Cummings also showed surprise that residues of the metabolite 2,4,5-trichlorophenol did not occur in significant amounts in tissues of animals fed high levels of silvex whereas residues of the trichlorophenol reached . 4.7 ppm in liver and 5.8 ppm in kidney of cattle given 1800 ppm 2,4,5-T in the total diet for 4 weeks prior to slaughter, and were about 0.3 ppm in milk of cows ingesting 1000 p p m 2,4,5-T in their feed. Discussion of problems related to cancellations under the new pesticide law (FEPCA) did not deter Mr. Baker from his commitment to cancel, apparently under orders from above him in EPA. He believed that all we had to do was appeal and ask for review by an Advisory Committee, during which time we could furnish the additional data needed to set the tolerances. However, under FEPCA, the Administrator can either issue a notice of intent to cancel allowing any interested party to request a public hearing, or he can request a public hearing himself. In any case, the reviewers would have to be able to provide evidence supporting their view that interim tolerances could n o t be s e t for s i l v e x as w e l l as for 2 , 4 - D a n d M C P A in grass in spite of the fact that considerable residue data was submitted on silvex in grass whereas no data was provided for MCPA in grass. ----ce c c c c b b Continuation Page ___q 18141 With a few e x c ep tio n s, the m od ern h e r b ic id e s a re of a r e la tiv e ly lo w o r d e r o f to x ic ity , , thus lim iting the hazard of th eir u se. A num ber o f r e se a r c h e r haw rep orted upon the tox icity of 2 .4 -D (2 ,4 -d ic h lo r o - phenoxyacctic acid), 2 ,4 .5 -T (2.4.5-trlch loro-p h en oxyacetic acid), and th eir e ste r s (38,53, 63,77,107,108)* The studies of M itchell, et a l, (1946) and of G rigsby and F arw ell (1950), in which they grazed liv esto ck on heavily treated p astu res, indicate that the practical hazard with th ese m aterials is v ery low , Rowe and Hymas (1954) bave studied 2 ,4 ,5 -T by adm inistering it to s te e r s . 'v /f*0 1:.3S Pei At K errville, we have studied sev era l herb icid es in sheep and ca ttle. (T able 4). (D- It is apparent that pentachlorophenol is one of the m ore toxic of th ese m aterials, producing poisoning in cattle and sheep in sin gle d o ses of 25 m g ./k g . Of th is group, d in itro -o -sec-b u ty l phenol is the next m ost toxic, proving lethal for sh eep after 4 daily doses of 25 m g ./k g . Oddly, the alkanolam ine sa lts of d in itr o -o -sec -b u ty l phenol are much le s s toxic, one sheep having tolerated 35 daily d oses of 25 m g ./k g . each without showing sym ptom s. oa Di: AT (0 i In g e n e r a l, the p h e n o x y -a c id grou p o f h e r b ic id e s w as o f v e r y lo w t o x ic ity fo r sh eep . The m ost toxic of this group w as 2-(2.4,5-trichlorop h en oxy) propionic a d d in the form of o o 2-` p rop ylen e g ly c o l butyl e th e r e s t e r s , 11 d a ily d o s e s o f 1 0 0 m g ./k g . ea c h p ro d u cin g death.' 00 This would still cla ss it as a rela tiv ely nontoxic compound. " 2-t cc 1II The fo llo w in g m a te r ia ls w e r e n o n to x ic w hen a d m in iste r e d d a ily in 100 m g ./k g . d o se s for 35 days: The alkan olam in e s a lts o f 2 ,4 -d ic h lo r o p h e n o x y a c e tlc a c id and o f 2 -m e th y l- 2/* I 4-chloropnenoxyacetic acid , the propylene glycol butyl ether e ste r s of 2,4-d lch lo ro - phenoxyacetlc acid and of 2 .4 .5-trich lorop h en oxvacetic acid, the trieth ylam in e sa lts of 2,4,5-trichlorophenoxyacetic acid and the sodium salt of 2,2-dlchlorop rop ionic acid. ! i 2,> 1 The ab sen ce of poison in g from th ese m a te r ia ls ad m in istered In su ch high d osages for 5 w eeks Indicates that th ere is little hazard to livestock to be expected from normal' 2,^ usage. Dehydroabietylam ine acetate (D elrad), an algaecide based upon tech n ical abietalam ines, is used at very low ra tes in re se r v o ir s and irrigation can als. C attle and sheep w ere unharmed by consum ing w ater treated with 100 p.p.m . D elrad, w hich is several tim e s the concentration u su a lly em p lo y ed . T h is com pound i s e x tr e m e ly d ista ste fu l to cattle and sheep and probably w ould n ev er be consum ed by them v o lu n ta rily even if the ch em ical w ere left ex p o sed . C attle w ere unharm ed by d o ses o f 200 m g ./k g ., sh eep by d oses of 250 m g./kg. Higher d o ses produced poisoning. I 2,* 2, B ecau se of the low to x icity and reduced hazard of h erb icid es, togeth er with co m paratively restricted u sage, the num ber of publications is sm a ll. Much o f the evaluation of to xicity of th ese m a te r ia ls h as b een done- w ith laboratory a n im a ls ra th er than with liv e sto c k . Reduction of Hazard by Control of T oxicity While very little can be done to change the sp e c ific to x icity of a p e s tic id e , sev e ra l things can be done to con trol it. T here a re certain com pounds w hich m ay be ad m in istered sim u lta n e o u sly w ith the p esticid e w hich antagonize it s a ctio n d ir e c tly or slow its action su fficien tly to enable the organism to deal with it. 146 i ex bu tu: sp sl< de tp1" L.''' * '"v I-'1;.-'.;-.'-. .;f r!Vi - 18142 TAHU 4.-- Summary of studies of the oral toxicity of herbicides to sheep and cattle at Kerrville, Tex. Chemical A n-tm aT Fre Dose quency Results SfitfiOO z m o o Pentachlorophenol Kg. tk e . Sheep 25 1 dose mild toxicity Cattle 25 1 dose mild toxicity (Delrad) Dehydroabietylamlne acetate Sheep Cattle 250 1 dose tolerated, higher doses toxic 200 1 dose tolerated, higher doses toxic Dinltro-o-sec-butyl phenol Sheep 25 daily death after 4 doses Altonolamine salts of Dinltro-o-sec-butyl Sheep phenol 25 daily tolerated 35 .doses* 2-(2,4,5-Trichloropbnoxy) propionic acid, Sheep propylene glycol butyl ethar esters 100 dally lethal after 11 doses 2-4-Dichlorophenoxyacetlc acid, 1 V nntV I n n -tn S a l t s Sheep 100 ' daily tolerated 35 doses* 2,4,5-Trichlorophenoxyacetio acid, alianolamine salts Sheep 100 daily tolerated 35 doses* 2,4-Dichlorophenoxyacetic acid, propylene Sheep glycol butyl ether esters. 100 daily tolerated 35 doses* - 2,4,5-Trlchlorophenoxyacetic acid, propylene glycol butyl ether esters Sheep 100 daily tolerated 35 doses* 2,4,5-Trichlorophenaxyacetio acid, triethylamine salts Sheep 100 . daily tolerated 35 doses* 2,2-Dichloropropionio acid, sodium salt Sheep 100 daily tolerated 35 doses* #At time 'of preparation of this table, these testa irere In progress. P esticides p rep ared in granular form for many nses accom plish th eir objective m ore effectively; y et, because of the weight of the granules, the am ount rem aining on foliage o r drifting aw ay fro m the a rea is g reatly reduced. ;| ' l! Biological C ycles To a c ertain ex ten t we have biological cycles involved in the use of p esticid es. F or exam ple, we m ay tr e a t a body of w ater for in se c t control and acco m p lish the objective, but organism s not k illed by the treatm en t m ay absorb and sto re the chem ical. They, in turn, according to th e ir biological position, m ay be consum ed as food by other species, and these in tu rn by o th ers, until there is ultim ately a rem oval fro m that w ater of a species serving a s food fo r m am m als o r b ird s, o r m an. The cycle is then continued out side the original environm ent* The accum ulation of pesticide m ay be attenuated or concentrated w ithin this cycle, depending upon the p a rt the anim al o r plant m ay provide of the to tal d iet of the consum - 14? i cow 2^08499 - nervous tissu e. Unfortunately, this Indicator often falls because anim als can die with high le v e ls of the en zym e In the blood w h ile oth ers liv e quite norm ally with none In the blood. C arbam ates.--In secticid es of this group are cholinesterase inhibitors but are g en er a lly not so a ctiv e In. this reg a rd . Sym ptom s o f poisoning g en erally p a r a lle l th o se produced by the organic phosphorus com pounds, with a few additions. U sually the ataxia is m ore pronounced. The poisoned anim al gen erally attem pts to move rapidly, but su c ceeds only in rolling en d -over-en d in a tum bling m anner, recovers rapidly, and repeats the p r o c e ss. T here is u sually a great deal of trem bling and shaking. The poisoning by carbam ates follow s the gen eral cou rse d escrib ed for the organic phosphorus com pounds. L esions of Poisoning Chlorinated H ydrocarbons.--A c u te poisonlnig by th ese compounds y ield s no pathog nom onic le sio n s. The le sio n s found are those exp ected in violent deaths fro m other c a u s e s -- c lo u d y sw e llin g , b lan ch ed v is c e r a , and h e m o r r h a g e s on v a r io u s o r g a n s . In addition, there m ay occa sio n a lly be con gestion of the lu n gs. The heart is g en era lly found to be in sy sto le. Subacute and chronic exp osu re lead to d egen erative changes in the liv e r and k id n eys. O rganic Phosphorus Compounds and C arbam ates.--T h is group u sually le a v e s the v iscera unm arked. C ertainly tnere are no diagnostic lesio n s. O ccasionally the lungs are edem atous or congested, and occasion ally there m ay be congestion with hem orrh ages in various viscera. H erb icid es.--T h e dinitro h erb icid es create sev e re damage in the kidneys and liv e r . The lungs are gray in co lo r. Often there is a yellow discoloration of a ll tissu e s and fr e quently the odor of the compound is e a sily detected. The 2 .-(2.4.5-trichlorophenoxy) propionic acid sa lts produce degenerative changes in the liv e r and kidneys and rather sp ectacu lar hem orrhages on the su rface of the h eart. The other phenoxy-acid com pounds are reported to produce kidney and liv e r d a m a g e .. D iagnosis of Poisoning Proper diagnosis of suspected poisoning of livestock has been a m ajor concern of ARS. To aid in this problem , we have developed film s describing and pointing out m ethods of arriving at correct diagnosis. D ifferential diagnosis w as not, and is not, easy in the ca se of chlorinated hydrocarbon p esticid es. The sym ptom s m im ic those of many d ise a se s in which the cen tra l or p erip h era l nervous structures a re a fflicted . In m any c a s e s , a d iagn osis can be reach ed only by elim inating all other p ossib le conditions. Proper diagnosis are reached only after carefiil evaluation o f the h isto ry o f exp osu re, sym p tom s, and the lesio n s (the la tte r being nonpathognom onic in acute chlorinated hydrocarbon poisoning, but may su ggest other d isea se p ro ce sses). D ifferential diagnosis of poisoniug by other p esticid es is not appreciably e a sie r . All m ust be approached .with caution and carefu lly studied. U se of C h olin esterase D eterm inations in D iagn osis.--T h e organic phosphorus in s e c ticid es and the new er carbam ates are recognized as inhibitors of ch o lin e stera se , eith er as the origin al compound or a s m etab olites of the o rig in a l. O rigin ally, we (92), and oth ers, felt that blood ch olin esterase activity would be an excellen t indicator of exposure and of poisoning. For single exposures we fe e l the method 149 AG 02318 dow 2 008293 1145 Some Salienc Aspeccs of 2,4,5-T Toxicology, Metabolism and Dissipation: Reaction of the Environment Total effect on selected ecosystems is the basis on which society will reject or accept a pesticide in the future. Man has used the herbi cide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and similar compounds to maintain synthetic ecosystems for the past 25 years.- Ecological benefits from the use of herbicides are discussed in a recent article by Barrons (9). This review summarizes research data on the effects of the chlorinated phenoxy aliphatic acids on the world ecosystem. Toxicological data are summarized by Rowe and Kymas (44) , Palmer and Radeleff (41) and by Seifres, et al.-- ^ Acute toxicity is usually expressed as the amount of chemical (mg/kg body weight) required to kill half a population of laboratory animals. This amount, known as the LD.q , is from 300 to 1000 mg/kg for rats, mice, guinea pigs and rabbits for the commonly used herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-T and [(4-chloro-o-toyl)oxy] acetic acid (MCPA). These values correlate well with levels found to be toxic to cattle. Dogs are some what more sensitive and chickens less sensitive than laboratory animals. The acute toxicity of 2,4-D and 2,4,5-T is not considered an important biological property in terms of the net effect on natural ecosystems. Compounds are administered in sublethal quantities over the entire life cycle to laboratory animals and its effects on tissues and organs -- Scifres, C. J., F. S. Davis and T. J. Allen. 1969. Unpublished com pilation of scientific publications related to chlorophenoxyacetic acid herbicides with special reference to 2,4,5-T. Available on request from Range Science Department, Texas A&M University, College Station, Texas. d o w a 008294 noted for the determination of chronic or subacute toxicity. These are so-called feeding studies. In general, the threshold for chronicity is about 10 to 25 percent of the value (100 mg/kg) (44) . Carcinogenic and tumorigenic properties are of interest principally from the standpoint of human consumption of herbicide treated foods, either directly or indirectly. The "standard" procedure is to feed small laboratory animals (usually rats or mice) the maximal dosage which can be tolerated without fatalities by stomach tube or by mixing with the ration, over the life span of the animal, then to examine the animal thoroughly for tumors. In one recent study (26) 2,4-D and 2,4,5-T were among 118 compounds tested. Eighty-nine of these, including 2,4-D and 2,4,5-T, gave no significant indication of tumorigenicity after oral administration. Chemicals causing malformations in the fetus, teratogens, have been publicized extensively in recent months. Research conducted by a commer- 2/ cial laboratory for the National Institute of Healthr- presented evidence that 2,4-D and 2,4,5-T, at oral dosages sufficient to cause death if fed over the entire life cycle, cause malformations of some fetuses when fed to pregnant mice and rats. There are three problems in interpreting findings of this type: First, there are undoubtedly species differences; that is some species or strains may be more susceptible to teratogenesis than others. Presently there is no way of knowing whether man is more or less susceptible than the mouse to induction of abnormal fetuses by --2 / Courtney, K. D., D. W. Gaylor, M. D. Kogan, H. L. Falk, R. R. Sates, and I. Mitchell. 1970. Teratogenic evaluation of 2,4,5-T. (Un published) . 1 8 46 d o w 2 008295 a given compound. Secondly, the dosage used in such studies is far in excess of that ever likely to be consumed by humans. Third, these lab oratory experiments are designed to test a single chemical. Man's environ ment contains a complex mixture of chemicals. These mixtures may interact with each other or with integral parts of the ecosystem to either enhance or inhibit teratogeniticity or other effects. Although no specific data are available, 2,4,5-T has been used ex tensively on rangeland grazed by pregnant cows and sheep for more than 20 years with no reports of resultant fetal malformations. The conditions under which 2,4,5-T is used (up to one pound per acre, followed by defer ment of grazing for at least 30 days) would seem to preclude any such effects. Rates and pathways of metabolism of 2,4-D and closely related herbi cides have been studied by many investigators since 1959 (5, 21, 25, 34, 42, 46) without clarifying certain details of metabolism. However, these compounds undergo a variety of reactions in plants from which the principal data have been obtained. Decarboxylation is a common degradation route for 2,4-D and related chlorophenoxy compounds. The percentage degraded varies from as little as 1 to as much as 57 in various species (39, 48, 53). Hydrolysis of the ester of 2,4,5-T (50) and probably the amide occurs readily in plants. Complexes with sugars, amino acids, proteins and other plant constituents also occur, but little quantitative data are available on this as a route of detoxification. Although the preponderance of data suggests that metabolism of 2,4-D 18147 and related compounds in plants is relatively rapid, no definitive data on the biological activity of metabolites are available. It is doubtful whether metabolism by plants is an important route of inactivation. However, it is possible that plants could store the herbicide in a slowly released form, resulting in long term residues at levels below 1 ppb, the present limit of resolution in water. Persistence is a relative term relating the length of life of a pesticide or its first metabolites to that of other pesticides as measur able by our present analytical methods. The insecticide DDT is considered relatively persistent because it does not decompose rapidly and reappears later in other parts of the ecosystem. Even though 2,4-D is one of the most rapidly decomposed of all herbicides (4, 27), and is at the opposite end of the persistence spectrum, it cannot be considered absolutely nonpersistent. The principal routes of detoxication in soil were summarized by Bovey (12). Soil microorganisms rapidly decompose 2,4-D. The rate of decomposition of 2,4-D in the soil is therefore increased by conditions favoring rapid microbial growth. Klingman (29) states that if 0.5 to 3.0 lb/A of 2,4,5-T is applied to a moist, loam soil, it can be expected to persist for 2 to 5 weeks . Of prime importance is the lack of accumu lation of 2,4-D in the soil from one year to the next at normal field rates (29). The related herbicides 2,4,5-T, silvex, MC?A and 4(2,4-DB) are thought to be similar to 2,4-D in soil persistence properties (46). However, there are few data substantiating this belief. 0082913 o o * fo ."Ajfc8 DOW2 008297 Current research at Texas A&M with the herbicide 4-amino-3 ,5,6-trichloropicolinic acid (picloram--3/) may suggest the fate of phenoxyacid herbicides in soils. If the herbicide is applied to trees and grass when in full foliage, no more than 10 percent of the applied herbicide reaches the soil surface initially ( ). Depending on soil type, about half this amount (5 percent) is leached out of the root zone (into the subsoil or the ground water). Approximately 2 percent.of picloram is adsorbed to soil colloids too tightly to be removed by plant roots. The remaining 3 percent comes into contact with the root system of the plant, and a small amount (less than 0.05 percent) is absorbed into the root system, and is retained or travels upward to aerial portions of the plant, some of which are eaten by animals. The remainder rejoins the herbicide not in contact with the root. Thus, approximately 7.5 percent or more of the original application moves into the subsoil or ground water. Thus, 0.018 lcg/acre from an 0.5 lb/A initial application may move into the lower soil strata. Thus, leaching of picloram is the major route of dissipation from soil. The principal difference between picloram and the phenoxyacids in the soil is the microbial degradation of the latter group. Aly (2) reported that 2,4-D persisted up to 120 days in lake waters aerobically incubated in the laboratory. It was decomposed biologically by lake muds, (83 percent within 24 hours) but only after extensive microbial adaptation techniques were used. The amounts of 2,4-D esters adsorbed on bentonite, illite and kaolinite ranged from 0.2 to 0.14 mg 3/ 18 9 DOW 2Q08298 per gram. Whether photolysis represents a significant route for the en vironmental degradation of phenoxy herbicides is undetermined. Decompo sition of 2,4-D to phenols occurred in artificial ponds under the influence of ultraviolet light (2) but was less extensive under natural conditions in the northeast (3). No information for 2,4,5-T persistence in water is available. Con sidering the relatively large acreages on which 2,4,5-T- is applied, this question seems to require additional study. Recent research has shown that some 2,4,5-T may be washed off treated areas. Trichell et al (51) found that a 2 lb/A application of 2,4,5-T caused 0.045 lb/A to appear in runoff water following 0.5 inches of simulated rainfall. The authors suggested that distance between the treated area and major water arteries is an important factor determining the amount of herbicide which will enter the water sources. When 4 lb/A of 2,4,5-T was sprayed directly over water to control riparian vegetation, maximum concentration in the stream was .04 ppm, and none could be detected downstream (43A). Formulation affects susceptibility to washing and leaching. Hauser and White ( ) found that as much as 27 percent of a foliage application of the ester of 2,4-D was washed away in the first 30 minutes of rainfall, while only 3 percent of the amine form was lost under the same conditions. It is not certain whether usual herbicidal applications can result in appearance of 2,4,5-T in the ground water. The rationale for this uncertainty is discussed in the section on persistence in soil. One instance of contamination of well water by 2,4-D has been reported (42). The persistence and mobility of chlorophenoxyacetic acids in air 18150 d o w ,2 008299 has not been extensively studied. The short chain esters of 2,4-D and 2,4,5-T are considered volatile and can cause biological damage to organisms physically removed from the source (42). The question has never been subjected to quantitative analysis. Significant photolytic degradation of phenoxyacids may occur in air; Crosby and Ming-Yu Li recently outlined the technical difficulties inherent in such studies (16). Although studies of pesticide persistence in plants are directly related to studies of plant metabolism, it seems appropriate to consider vegetation as a partitioning agent in the ecosystem as well as a metabolic agent. Most laboratory studies have demonstrated disappearance of 2,4-D in plants, with partial metabolism within a week (49). Hay and T'nimann (24) found that a 50 pg application of 2,4-D to bean (Phaseolus vulgaris L.) seedlings eventually disappeared almost completely, and about half the absorbed compound was degraded in 24 hours. Morton (38) found that 80 percent of the 2,4,5-T applied to fully expanded honey mesquite (Prosopis juliflora (Swartz) DC var. glandulosa (Torr.) Cockr.) leaves was altered in 25 hours. The maximum rate of change occurred between 70 and 85F. In this and many similar studies, however, 2,4,5-T may have been conjugated with sugars or proteins, and later released as parent molecule over time. Morton et al (37) also studied persistence of 2,4-D and 2,4,5-T in and on range grasses in the field (Table 1). Rainfall was the most important factor influencing disappearance in these natural ecosystems. Esters were completely hydrolyzed after one week to the acid and unknown Dow 2 008300 metabolites. In 1963, after 8 weeks and 3.1 inches of rainfall, concen tration of 2,4,5-T and unknown metabolites in and on green tissues of grasses such as Androuogon saccharoidas Swartz (silver beardgrass), A. scoparius Michx (little bluestem) and Pasnalum dilatatum Poir. (dallisgrass) was 25 and 7 ppm, respectively at 2.0 and 0.5 lb/A rates. Concen trations were 2.1 and 9.5 ppm, respectively in sideoats grama (3outeloua curtipendula (Michx) Torr) 8 weeks with 3.0 inches of rainfall after similar applications in north central Texas. Residue from 2,4,5-T amine at 1 lb/A was 9 and 2 ppm at 8 and 16 weeks after application, respec tively, from grasses treated in southeast Texas and was 19 and 5 ppm for the amine and acid formulations of 2,4,5-T respectively 8 and 16 weeks after treatment of grasses in north central Texas. Concentrations of the acid were 9.1 and 6 ppm, respectively, at 8 and 16 weeks after treat ment in north central Texas. Baur, 3ovey and Smith (10) applied 2 lb/A of 2,4,5-T ester and recovered 4 ppm from forage after a month, and 0.17 ppm after 6 months. In summary, trends from data available suggest that 2,4,5-T residues available to grazing animals on rangeland would be on the order of 300 ppm after one day and 0 . 1 to 0 .2 ppm after 6 months, depending upon rainfall. Merkle and Fertig (35) found 1 to 3.5 ppm of 2 ,4 ,5 -(trichloro)prop ionic acid (silvex) in trefoil 36 days after application of 1/2 ib/A. The residues were directly correlated to rate of application. These investigators showed that the amount of silvex residue remaining is more closely related to the time interval between applications and harvest d o w 2 005301 than to rate of application. For example, 0.25 lb/A of silvex left a residue of .23 ppm in seedling trefoil 2 weeks after application, but only 0.19 ppm remained after 6 weeks even when the rate of application was increased to 0.75 Ib/A. There was considerably less silvex in the plant regrowth than in the plant material originally sprayed. Never theless, silvex residues of 1.74 ppm were found in timothy regrowth originally treated with 3 lb/A. Silvex at 0.75 lb/A could be applied to trefoil in late October without leaving detectable residues in the plant growth the following spring and without serious damage to the trefoil stand. Linscott (33) found in greenhouse studies that about 90 percent 14 of C labelled 4(2,4-DB) applied to legumes disappeared within 30 days after treatment. Simulated rainfall accelerated herbicide removal. The herbicide was not found in legume regrowth after clipping. Linscott, et al (32) studied the decomposition of 4(2,4-DB), 2,4-D and silvex in silage, and found that ensiling reduced but did not eliminate 4(2,4-DB) and silvex. Cattle and sheep have been fed 2,4-D and 2,4,5-T on forages treated with these compounds, and pastured on 2,4-D treated fields. Elimination by excretion is apparently characteristic for the chlorophenoxyacetic acids (43). The following data are from studies in which 2,4-D or 2,4,5-T was fed directly to domestic livestock: When a lactating cow was fed 5.5 g daily for 106 days, no 2,4-D was found in the milk, liver, kidneys or fatty tissue, or in the blood serum of a calf given the milk (36) . 3 008302 Gutenmann et al (22) fed 5 ppm 2,4-D to a Jersey cow and could not detect the herbicide by gas chromatography in her millc the following morning. In sheep, about 96 percent of an oral dose of 2,4-BC14 was excreted unchanged in the urine within 72 hours (15). Slightly less than 1.4 percent was excreted in the feces. The 2,4-D in urine and feces was identified by electrophoresis and paper chromatography.. The edible sheep tissue contained 0.05 ppm or less. In another study, sheep given 2 grams of 2,4-D acid for ninety days were analyzed on the ninety first day arid residues in all tissues were between 0.05 and 1 ppm (43). Some data are also available for persistence in milk from animals fed 2,4-D treated forages. Klingman, et al (20) applied 2,4-D aster at 2 Ib/A (twice the normal rate) to forage, then analyzed the milk from cows grazing the treated forages. After two days, milk contained 0.01 to 0.09 ppm. After 4 days, residues were below 0.01 ppm, the limit of sensitivity of the method. The probability of 2,4,5-T being ingested by humans via the meat of domestic livestock seems remote, since most rangelands are sprayed in the spring, 4 to 6 months before the livestock are slaughtered. However, reports on 2,4,5-T persistence in meat of animals grazing 2,4,5-T treated pastures are not available. No information is available on persistence in wildlife. Mobility of herbicides in the environment is related to solubility in water, affinity for soil, and volatility. No comparative data exist O O iOl oow 2 008303 for the phenoxyacetics. Some formulations are volatile, but the tendency in agricultural practice has been to curtail usage of these "high-volatile" formulations. The ester formulations are generally less water soluble than the amine for many herbicides, but can be carried in irrigation water. If chemicals are ingested by successively higher links (animals, birds or fish) in the food chain, increases in tissue concentration may occur until injurious levels are reached. Mo instances of significant biological magnification of 2,4-D or its relatives have been recorded in some 25 years of extensive use. At present, no system of investigation which can evaluate this property in advance of large-scale usage in the environment has been developed. Limited research findings are available indicating that 2,4-D and 2,4,5-T interact with other chemicals in the environment. When 2,4,5-T is mixed with picloram, mobility of the picloram and its concentration in the lower parts of the plant increase in mesquite, while in live oak (Ouercus virginiana Mill.), 2,4,5-T mobility and concentration in the lower plant increase (10, 17). Recent studies by Allen at Texas ASM suggest that the activity of 2,4-D can be increased and decreased by certain natural plant constituents.--4/ Whenever biological activity of a herbicide is increased by interaction with other pesticides in the environment, the hazard from that herbicide may be increased, but the amount used may be reduced in view of the increased herbicidal activity. Very little specific data on the nature or frequency of pesticide interactions are available. This -4/ Allen, T. J. Personal communication. 18155 d o w 2 008304 question needs research attention. Sheets and Danielson (46) indicated that when 2,4-D is present in soil the number of microorganisms capable of inactivating the compound apparently increase. Repeat applications of 2,4-D were less persistent in soil, and therefore less toxic than the initial application. 3acteria capable of inactivating 2,4-D have been isolated from soil and grown in pure culture (6 , 8 , 20, 27). In one study 4(2,4-D3) was converted to 2,4-D by microorganisms in the soil and a raicroflora capable of quickly inactivating both 2,4-D and 4-(2,4-DB) was present in soils which had received 4-(2,4-D3) previously (52). Sollag, et al (1 1 ) reported that a phenoxyacetate-metabolizing Arthobacter spp. contained an enzyme that converts 2,4-dichlorophenol and other chlorophenols to catechol in the presence of MADPH and oxygen. Persistence of many herbicides in the soil is related to nalogenation of the benzene ring (IS, 19, 47). Soil perfusion studies of Audus (4, 7) established chat meta halogen substitution (2 or 5 position) renders a phenoxyacetic acid molecule more resistant to microbial degradation, but 4-substitution makes the molecule more labile. .Alexander and co-workers (1, 13) indicated that resistance of the phenoxyacetics or their derivatives to microbial degradation was governed by the position of the halogen rather than the number of halogens on the ring, (meta substitution decreased rata of degradation) and the linkage and type of aliphatic side chain also influenced susceptibility to microbial breakdown. The general conclusion is that the phenoxyacetics do not have per manent serious effects on soil microflora (46). However, in at least S0E800 M O a8 one scudy 2,4-D had a delayed lethal effect on 5 species of soil bacteria ac concentrations which did not inhibit growth completely (31). High rates of 2,4-D are required for inhibition of nitrification and ammonification, with considerable variation in susceptibility of microorganisms to 2,4-D (28) . Few data specifically for 2,4,5-T are available. The organisms which inhabit the top 3 to 5 inches of the ocean are extremely important to the well-being of man. Compounds which may in fluence the photosynthetic capability of these organisms, the phytoplank ton, are potentially dangerous to the world oxygen supply. For 2,4-D and its relatives, some evidence as to effects on growth of phytoplankton exists. Butler (14) found slight inhibition of phytoplankton (mostly diatoms and dinoflagellates) growing in water containing 1 ppm of the salts or acids 2,4-D or 2,4,5-T. No measurements of effects on the photosynthetic capability are available. Techniques have been for studying both acute and chronic toxicity of 2,4-D and some of the phenoxyacetics to fish have been studied exten sively. The general conclusion is that 2,4-D is relatively non-coxic to fish at customarily used rates, but materials used in formulating the phenoxyacetics (e.g. xylene, petroleum derivatives) are sometimes toxic ( ). Rowe and Hymas (44) found that ordinary field rates of 2,4-D and 2,4,5-T are not injurious to wildlife. Benefits in increase in forage availability from decreasing competition from woody, non-browse species through use of herbicides are summarized by Barrons (9). No data on .408800 5 M O a residues of 2,4-D or 2,4,5-T in meat of game species are available. Summary Man. has utilized herbicides such as 2,4-D and 2,4,5-T for the past 25 years to construct synthetic ecosystems which presumably serve his purposes better, '.Then natural ecological processes are disrupted, as after the use of herbicides, the chemical tools might have to be used repeatedly in order to maintain the "stability" of the synthetic eco systems. The chlorophenoxyacetic acid herbicides apparently serve man's purposes*gainst these beneficial effects. ,A1 1 possible ramifications of their use must be considered ar.d weighed. They are not toxic to mammals at the dosages required for maintaining the synthetic ecosystems. The bulk of the evidence gathered to-date suggests that the chlorophenoxy herbicides dissipate rapidly from soils where they serve as a carbon source for the microbial population. Plants metabolize or complex the compounds readily. If domestic animals or wildlife ingest forage con taining these herbicides, the chemicals are largely excreted via the urine. The possibilities of biological magnification seem remote. In general, the net effect on the ecosystem has been reflected in increased yields of desirable species for man's use. *1 -14- Alexa.nder, M. and M. J. H. Aieam. 1550. Effect of chemical structure on microbial decomposition of aromatic herbicides. Jour. Agric. and Food Cher.;. S: 146-150. Aly, 0. A. ar.d S. D. Faust. 1564. Studies on the fate of 2,4-D and ester derivatives in natural surface waters. Jour. Agr. and Food Chem. 12; 541-546. 3. New Jersey Agr. 46: 12 T/ Audus, L. J. 1964. In Physiology . 5. Audus, L. J. 1961. Metabolism, and ! ed., Encyclopedia of Plant'Phy Springer-Verlag. Berlin. Audus, L. J. 1955. Plant growth substances. Leonard Hill Limited, London. Jr' C .S 7 . .-.uaus, u. tj. 1960. In Herbicides and the Soil (E.X. Woodford and / d/v'v . ,J G.R. Sager, eds.) Blackwell, Oxford, pp. 1-15. " ..A rW , ti'' 8 . Audus, L. J. 1950. Biological detoxication of 2,4-D acid in soils: H - i r isolation of an effective organism. Nature 166: 556. 9. Barrons, K. C. 1969. Some ecological benefits of woody plant control with herbicides. Science 165 (3392). 10. R., R. D.B a u r , J . W. B o v e y a n d J . Smith. I969. H e r b i c i d e c o n centrations in live oak treated with militares of pido rati and 2,4,5-T. 7 11. Eollag, J . M., C. S. Helling, and M. Alexander. 195S. 2,4-D metabolism. Enzymatic hydroxylatier, of chlorinated phenols. J . Agr. Food Chen. 16(5); 626-G2S. i9 3ovey, R. W. I$6 6 ,, problems ir. residues of chemical herbicides ana thetr persistence m soiis and p..anc ana animal tissue. Unpublished presentation at a symposium on woody plant con trol, Texas A&M University. c < tc 008307 13. . c. :B u r g e r , I C . , I Sci. Am. 26: n i 7 14. B u t l e r , P . A . 1965 15. C l a r k , D. E., J. E McLaren. 195. Food Chem. 12; 43-45, 18159 IO 16. Crosby, D. G. and Ming-Yu Li. 1969 Herbieloo Phe tGCacor ir. Degrade tien. or Herb icicles (Kearney ar.d Kaufmann Marcel Dekker, New York. 17. Devis, F. S., F. N. Dovey er.d M. G. Merkie, 1963. paraquat end 2,4,5-T on the uptake and transpo in woody plants. Weed Science 15: 236-323. IS. DeRosa, H. R. and A. S. Newman. 1943. Comparison of the pe. of certain plant growth regulators when applied to soil. Sci. Soc. Amer. Free. 12: 222-226. 19. DeRosa, l-[. R. 1945. Persistence of come plant growth regulators when applied to the soil in herbieical treatments. 3ot. Gan. 107: 533-539. 20. Evans, K. C. and 2 . 5. N. Smith. 1954. The photochemical inactiva tion and microbial metabolism of the chiorophenoxy acetic acid herbicides. Diochem. Jour. 57. 1. Freed, V. K., and H. L. Monte;omet-/. 1963. The metabolism of herbicides by plants and toils. Residue Rev. 2: 1-13. 22. Gutenmar.n, N. H., D. D. Hardee, R. F. honia-a- and D. J. LiAs. 1963. Residua studies with 2,4-D in the dairy cow and in a natural and artificai rumen. J. Dairy Sci. 46: 1237. 23. Hauser, G. A. and E. A. Hauser. 7 Hays, I. R. and X. V. Tnimann. 1956. The fate of 2,4-D acid i bean seedlings, II. Translocation. Fiant Physiol. 31: 446-451. to, Hilton, J. Tj T^ T p t-> c u, and H. M. Hull. 1963. Mach1 s 4 X. i ..2 of herbicide action. .Ann Rev. FI. Physiol, 14: 353-334. 2 J e ^ . v--'4 j _ _ - _Bi_cassayj o_f_ p^est_i_c_ides and i--r.dustr-a' chemicals for tumoriganicity in mice: A preliminary note. Jour, of National Cancer Institute 42: 1101-liil. 27, Jensen, H. L. and H. I. Petersen. 1952. Detoxication of hormone herbicides by soil bacteria. Nature 17C: 39-40. Johnson, E. J. and A. R. Colmar. 1955. ' The effect of 2,4-D upon the nitrogen metabolism of some soi1 bacteria, proc. So. Need Conf. 8 : 305. 29. Klingman, G. G. 1961. Need Control; As a scie and Sons, Inc. New York. iohn Ni ley 3 0 . Klingman, D. L., C. H. Gordon, George Yip and H. ?. Burchfield. 1966, Residues in the forage and in milk from cows grazing forage treated with esters of 2,4-D. Needs d o w 2 008308 i i s - 3 1 Lamartiniere, C. A., L. T. Kart and A. D. Larson. 1969. Dalaycd lethal effect of 2,4-D acid on bacteria. Bull. Environ. Contain. Toxicol. 4(2): 113-119. 32. Linscotc, D. L., R. D. Hagin and A . J. Hright. 1965. Effect of ensiling on tine decomposition of several herbicides. Crop Science 5: 455-456. 33. Linscott, D. L. 1964. Degradation of 4-(2,4-DB) in plants. Jour. Agr. and food Chen. 17: 7-11. 34. Loos, M. A. 1969. Phenoxyalkanoic acids. In Degradation of Herbicides. (Kearney and Kaufman eds.) litreel Deklcer, New York. 35. Herkle, H. G. a: S. N. Fertig. 1953. The determination of residues of silvex in birdsfoot trefoil and grasses. Proc. Sf- XCKCC_. 265' / /? lu -o'. CVC-0- 36. Mitchcrl,r J.J W. ( /' (j , '4o<n. C'.'Cfck/i-rOs,'-GL C' U jT/r'- Gaetjens. 1946. p o o ix -lO i/t. ' ''< Tolerance of farm animals to feed containing 2,4-D. Joijr. Anim. Sci. 225-232. 37 Horton, H. L., D. E. Robison and R. D. Heyer, 1967. persistence of 2,4-D, 2,4,5-T and dicanba on range forage grasses. Heeds 15: 263-272. 38 Horton. H. L. 1966. Effect of temperature and humidity on foliar absorption, translocation, and metabolism of 2,4,5-T by mesquite seedlings. Heeds 14: 136-140. F'AexJ. 39 Norris, L. A. and V. N. Fred. 1966. Heed Res. 6 : 212. 40 N i c h o l s o n , H . ? . a n d T h o m a h ^ , J . R . 1965. Pesticide persistence in public water, their detection and removal. In Research in Pesticides. IS1-190. Academic press,New York. 41 Palmer, J . S. and R. D. Radeleff. 1969. The toxicity of some organic heroicides to cattle, sheep and chickens. USDA (AR(J)_ Production Research Report No. 106. 22 pp. 42. Potts, S. F. 1963. Spraying herbicides on woody plants near sensitive crops. Agr. Chem,. 18: 3-34. 43. Radeleff, R. 1964. Herbicides, plant growth regulators, and o fungicides. In Veterinary Toxicology. Lea and Febiger, Philadelphia. ., - , .. 4f 4/ . Rowe, V. 1C. and T.Afnymhs.<t - v . .\t) \ S * t i ' x L . vI'o- 'o-4/<*.'A -'-Sumn^ry or tox:i`c.r' -oAl* otgp y inform. ation on 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Amer. Jour, of Vet. Res. XV: 622-629. 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I O r j /) * TJ <"J r) .:i if.'-J"- f tJ u y r n O cj 41 u o : i r `< 4M O li 1-1 d i-t 9 i'. r i -, 4 o * '/ -u -i-i no r-4 n .O 1-4 4 r - l -y CJ l j r-4 a 11 !< H 1 O 1 O : 4 ;{ i to o -* i 4- 1 r <t- i ej 4 e l CJ ; i ** d co '-0 t i 01 CJ T j r - CJ r*:J. > CO vO r ; \ r,i r i *y vj rl * J l l . 1 y / i o 4 : y < j .Ci r* cj r 1o 01 M ; : o o rrJ 'M t 'M cj cj 4 C0 r-4 d *o *7 -rl t:0 44 O y .4 d O r4 <-! fn , o CJ 1i tr-*4 !i -J r o -, i u d -d d e . i-i i - o n A r ] y t) 4 CJ Ij O O y T :' ll v i 77 i n r-4 :j !.) 1 i CJ d d MI a i *: i >4 A c]( i 1 1 01 r' r) :0 O . i{ O O Hl 9 O ri 01 i s 0 9 "O CJ '1 i/) y *d A > p CJ i r< 'D 1 J d l 1 . 1 t i d O (J 'i i f < /I d d :i rj :o i-4 fj y O d e l d j r 1 11 r o (J rj o O i i d e l s r j O 4 y r l -, 4 P* r *fj P j4 tj d ji * rd A y e l 14 1-4 --/ i-, 'O I - , 01 44 CJ y O O TJ 1-1 d 1-1 r-l cj H <J 4 i-4 rl f i y .O T i iM : j J 4 O r\ 4 y >4 .o ;J CJ o !' p 4> r CJ t i y U 't r<^ rj \J r-4 yd- 9 n 'O 9 77 C J r-4 e l CO - ^ --J* v j - tr \ n O m 008310 CN2 CD H 05 rM 1165 ACUT2 AND SD3-ACUT2 COAL TOXICITY CF 2 ,45-5n i c h l cno h i d x o x y i c s t i c a c i d irca d c g s 63800 8 moq Victer A. Drill, rh.D., M.D, Profoaccr of Phcmncolo^y T/ayna University Collo^o of .'.io d ic in a ? .; j 18163 d o w 200783a Investigations have shown that 2 ,4 -trichlorophenoxyacotIc uoid (2 ,4 3 -T) can bo used as a herbicide (1 ), in concentrations ranging from 0 , 1 to 0,2 per cant. Toxicological studies ca tha material are vory Halted, In the present study tho acute and chronic oral toxicity of 2,i;,5-T v;as deter mined in dogs. Ia the present data histological changes uro not included as this phnso of the study is net yet comploted, .'.athcds Adult mongol dogs of both sosos were used, iiost of the animals 7/oro housed In tha laboratory for a period of 2 to 3 swaths before the study :a3 started. During this control period they were immunised against distemper with the Croon vaccina. Tha dogs wore fed a standard stock diet (Friskies) Ad llbitim. Tho 2,4,5-T 'wad wa3 commercial notorial with u purity of ICO., In tha acute studies the calculated dosa of 2,4,3-? was administered as a single oral dose in capsulos. The dogs ware observed for a period of 14 duys at v;hich tiaa survivors wore autopsiod. In the chronic 3 tudy tho 2,4,5-T was adrainistorad orally in capsules, 3 days a week over a 1 3 week poriod. Each oapsule was imbedded in a 4 to 3 grata piece of commercial cannod dog food. The animals consumed this readily, thus avoiding tha con tinued traurn of the stomach tube. During the study tha animals were woighod twico a wook. Control blood counts were takon bofere tho administration cf the drug, on tha 3 0 th day and on tho 9 0 th day at tho completion of the study. Hemoglobin was determined with a photoelectric colorimeter (2 ), Upon the death or at the completion of tho study, the animals were uutepsied, tissues intestine were taken from the lung, heart, livor, kidnoy, adronal, 3ploon, thyreid^aud ovary or te3tea and fixed in 10,i formalin. 1816 dow 2 007831 - ^ n 'T .'T rn n . `- i <*/ I. ACf7:~: r/uy. toxicity. 1. Symptoms produced Tho survival at various dcse levels of 2,4t.5"T io listed in Table 1. The IDjO is in tho range of 100 mga. par kilo or slightly higher. Tho symptoms observod in tha dogs v;orc fairly mild and limited to a slight to moderuta stiffness in the hind less, with the development of ataxia in tho two dogs receiving the highest d030 3 of 2 ,4 ,5 -T* hTo symptoms wore aeon in tho one dog that died following a single doao of 10 0 mga. per kilo (Tablo 1), 2. Body noif'ht. Doses of 100 mgm. per kilo or greater produced a alight fall in body weight during tho test period. Tho ra3 ult3 are sosowhat variable with the 0 mgm. per kilo dcsa but indicate only a relatively alight effect of the drug on body weight (Table 1). 3. Gross patholory. The chengo3 observed v:ero limited to the l gastro-intestinal tract and lungs. In dog 90 tha duodenal mucosa mis a slight diffuse red in color ard in 0 3 7 4 small pin point hemorrhagic uroaa were observed in the mucosa together with some blood in tho lumon. k small amount of blood was also found in tho small intoctino of dog 64. Thi3 Game aninul also presented a purple-black lower left lebo of the lung that had a solid blood consistency. it . crnrnic chat, t oxtoit y. 1. Mortality. Dogs that roceivod doses of 2, 5 a&d 10 mgm. per kilo survived the 90 days. The dog3 receiving 20 ngm. per kilo either in a single or divided dooa died during the study. . 2 General symptoms. Ko signs or symptoms ware observed in the i.8 i.'o5 dow a 0 0 7 8 3 2 -3- dogs that s u r v i v e d the study. In the dogs that died tho prominent effects "'ore wcaknoss, slight stiffness in the hind legs, and difficulty in tho swallowing of food, and bleeding from tho guns in cno dog.(Table 2), 3 Body wolr-ht. The 4 dogs that died shored a loss of ewer 2 kilos in body woight that started approximately 10 days before tho death of tho animal. Tho surviving animals do not shew any significant change in i body weight during this poriod, but tended to fluctuato ebout their original weight* 4. Blood count. .The administration of 2,4*5-f did not have any significant effect on the hemoglobin cr rod call count (Tahlo 3). Dog3 103, 70, 3 ? Qn<i 10 3 showed soma dccrcaca in total white coll count during tho study but the valuos did not fall lower than thoso soon in the control dogs. In the differential counttakcn b-eforo death, the per cont of lymphocytes fell to very lew levels in dogs 3 7 aad 1 0 5 3 Oc33 patholo-?' Animal ilo. 93 Avowed a diffusoly reddened duodenum and jejunum. Dog 105 autepaied on tho 59*h day showed a generalised jaundice as indicated by the cclera and the finding of yellow fat and yellow organs throughout the body. In dog 55 the renal capsule was stripped with difficulty. 6 . Organ weights. The animals that survived tho c;0 day test per iod did not show any significant chu'ngo in organ weights (fable 4) Various miner changes In organ weight can be noticed in some of tha dogs fed the 20 tugm. per kilo dose who died during tho study. These changes wore limited to possible increase in heart, liver or kidnoy weight. Discussion. Single large doses of 2,4*5"f can produce death in dogs. However, the central nervous system symptoms observed are quito mild, consisting chiefly of 3 tiffnos3 of the hind legs with some ataxia. These. d o w 2 .^07833 symptoms seem to bo definitely leas than thcsa obtained with comparable doses oT 2,4-D. However, further studios with dosoa of 150 to 200 rrg. por kilo might elicit a bettor sories of signs und symptoms. Tha ccutoly intoxicated animals lost only a slight amount of v;oight. Gross pathological changes when obsorvod wero confined to thagi'stro-intestinal mucosa and in ono dog to the lo'-vor loft lobo of tho lung. Tha decreased incidanca of such changes as compared with 2,4-D paralleled tho mild central nervous effects of tha drug. In tho studios on chronic oral toxicity of 2,4.5-T, it v;as observed that the animals survived dose3 of 2, 5 snd 10 as. par kilo without grossly apparent ill affects, but succumbed to doses of 20 rag. por kilo. Thus the chronic toxic dose aeoms to bo in tho same range as that obtained with 2,4-D. In those animals receiving tho highest doaago, 20 mg-kg of 2,45-T, evidence of toxic affects consistently included slight muscle spasm, difficulty in swallowing food, and loss in terminal body weight. A marked docreusa in tho percentage cf lymphocytes in the peripheral blood was noted in two instsncoa, whilo Generalized icterus of the body orceins and inflammation of duodenal mucosa wore each obsarvod once. Summary. 1 The acute oral LD^q for 2,4.5-T is in the range of 100 m3 , por kilo or higher. Acutely poisoned dogs show mild spastic signs aocampanied by a loss in body weight. Cross pathological changes v/ero uncom mon and were confined to the small intestines and lungs. 2. In chronic oral feeding of 2 , 4 doses of 2, 5 and 13 mg. per kilo allowed survival for S3 days and did not produce any symptoms or changes in body weight, organ weight or blood count. Dogs fed 20 mg. of 18 ^6? 2 ,4 -r por kilo por day died during tho study, sdorine mild spastic symp tom, a loss in body T/oight, and in 2 cace3 a dscrouoo in tha por cor.t of lymphocytes in tho peripheral blood. .Minor changes r.'oro precont in tho duodonum in ono dog, and Jaundica in another dog at autopsy. D O S to o o <E CO fs > I 1. nildobrand, E. .., Soiones, l%>. 2. Evlyn, K. A,, J . Diol, Chea, 115 6 3 * 1 9 3 6 . Dow o 007835 fa b le 1 Effect of ainglo oral doooa of 2,4.5-T in decs Deg j'-O. 7Mi .. . V'jM cijF C4f 90f 95 F 1 0 GM 101 M 10 6 ? 103 M noro r=-Vi-: on 3;;o 100 100 100 100 io 50 50 50 *Dicd d-y ? 1 Or* 7 0r* 0 w wC s s '"crtnlity 1/1 1/1 ) \ ' 1/4 ' J f o/4 J i:t lo 13 iry.u -1 . 1 -I./-. -0 . 5 -1 . 0 -1 . 0 -0,4 4 0 .3 -2 . 3 4.0.1 -0 . 6 Svantcas* 4 0 0 0 0 0 0 0 0 d o w 2 007833 * 3 s survival for 1 4 day toot period. * coluan indicates general savority of syrapterna 03 discussed in toxt.O a no change frea noraal. D0 3 r?o. 777 10?:'.: 377 99M 667 1031.1 937 35 707 3711 697 947 105.M 2T a b l e Body weight and survival of do^a fed 2 ,4 . 3 T for 9 ^ ays Do^t u^/iC nono nono 2 2 5 3 10 10 io *20 *20 20 20 Initial 1'j.a 7.3 12.1 11.3 8.9 8.6 12.3 12.3 13.0 9.3 1 1 .S 10.5 14.9 10.9 "oi-ht I *-a 7 .7 it}. 12.7 10.0 8.4 12.5 11.5 12.2 9.2 9.2 7 .9 12.7 8.2 Jill-# 10.4 2.4 1.4 *1.1 0.2 40.2 -0 .8 -0 .3 -0 .6 -2 .6 -2 .6 -2 .2 -2 .7 Day Mod s 0 OH 3 3 3 5 r* 5 49 73 11 39 cow 2Q07837 daily dosa divided and adrainistorod In s survived 9^ day test period. ' ( and i in P.M. Tibia 3 E f f a c t c f 2 , 4 , 5 T o n h e 202 l o b i n , t o t a l b l c o d a n d d i f i e r e n t i n i c o u n t s B 03 No* 7 7 - 1' Poca rrA> nona 1021 nona 877 2 99--I 2 667 3 103.1 3 937 10 55-'d 10 737 10 37iJ 20 ""57 " 20 9 /1.7 1 0 5 1 ;. 20 20 5dl ITb me :.-U . 0 9.60 5 .0 3 30 10 .73 5 .6 3 90 1 2 . 7/1 9 . 7 ? 0 12.29 3 53 3 13.31 3 .? 3 00 1 7 . 9 9 6 .4 7 0 1 1 . 9 5 * 5 2 30 I 4 .4 9 6 .1 3 00 1 A . 9 2 6 .9-7 0 1 0 .7 5 5 .2 8 30 13 .5 5 5 . 5 ^ 90 1 4 . 4 6 6 .0 9 0 I I . 9O 6 .2 1 30 1 2 . 9 3 00 1 7 . 8 ? 6 .17 7 .0 9 0 1 0 .9 4 30 1 2 . 6? 0 1 2 . 9 9 5 . 3 5 .7 9 6 .2 4 0 I I .90 6 .5 0 30 90 1 2 .7 4 1 2 .6 7 6 .7 2 6 .4 3 0 1 1 .5 3 30 1 2 . 3 4 90 1 1 . 3 4 5 . 9I 5 .4 9 L .o.**>) 12.65 30 14.40 -00 1 4 . 9 4 6 .3 7 6.23 7 .2 9 0 12 .34 5 .3 5 33 8 ,6 4 3. 9a 4? 8 .9 8 3 .0 7 0 1 2 .4 8 7 .IO 30 1 4 .6 6 6 , o 2 0 1 2 i 'A 0 1 2 .4 3 7.73 6 .0 3 30 1 2 . 9 3 5 . 3 1 _ _ 2_-- U t S L , 6 . 1 4 me 16,100 13,300 1 4 ,7 0 0 14,500 14,900 6 ,4 0 0 12 ,4 5 0 16 ,7 0 0 16,9 9 0 17 ,10 0 14,800 17,2 0 0 1 2 ,6O0 1 7 ,4 0 0 1 7 ,9 0 0 1 3 ,1 0 0 16,600 7 , "90 I6 .I 5 O 13,800 1 3 ,6 0 0 13.250 13,650 14.3 9 0 1 5 ,4 0 0 16,400 3 .3 0 0 16,400 1 6 ,4 5 0 6,900 16,050 111,20 0 1 7 ,3 9 0 1 6 ,3 0 0 1 4 ,6 5 0 7 .1 7 9 4'-: ir.; 7 56 ,9 -1 55 63 : 3 70 62 80 75 56 ' `>7 43 46 V' 47 54 71 69 66 7.7/ o -/ jo 70 73 54 6? 72 77 94 3 71 69 il 73 92 n i f i ornrtt i d l.y -x ) ".?n ` 1 16 0 13 1 3A9/ 2 O 4 91 i 2*--'r 2 23 12 17 0 22 0 C/ 9 29 6 47 O 43 5 4.4 1 20 3 21 8 29 0 24 4 23 A 19 I 15 6 24 4 27 3 12 6 23 3 33 0 12 6 13 4 29 2-3 ^2 4-4 18 9 3 2 2 0 4 2 4* i. c c m ,t f) 1^A ?. c :; i n * 06 c3 00 04 07 00 04 03 00 03 01 02 03 01 00 0 33 0 17 ' 00 0o 03 0 5 ..... 0 23 0 16 00 04 0 10 00 0 10 07 00 06 31 01 03 0 A 00 n -ith c: -'* a c fO - CC3 O -si OCG. 49 79 l1 90 * daily doaa diviod and adtnniatorod i in .il. and in P.L. Dog No. 77* 1 021.1 87F 99-M 60? IO3.M Dotf 55 M ?a? 5769? 94? .105.'.; Done ir<2/ii none none 2 2 5 5 10 10 10 *20 20 20 20 T able 4 Crfian noiehts of dcv^a fed 2 ,4 . 5 T for 90 days Ba3 od on final body woiht K idn ey vt e A s 33 4 .9 4 O3 5 . 7 2 58 4.56 43 4 .3 0 35 4 .1 7 68 5 .4 4 43 4 .1 7 49 4.01 37 4 .0 2 51 5*55 62 7.35 73 5 .7 5 68 8 .3 0 L iver r;t gA-G G 260 3-4.8 405 27.9 557 43.1 357 3 5 .7 341 40.6 2j28 3 4 .2 302 3 3.2 412 3 3.8 292 31.7 335 3^-5 390 49.4 395 43.9 262, 3 2 .2 Hniir t ut cA s G 61 6 .1 0 95 6.55 70 6.15 75 7.50 63 7-53 86 6 .8 3 74 6.2)4 73 5 .9 3 70 7.60 65 7.07 7 0.49 79 6.22 67 8 .1 7 Adron:Jl Y/t gA g G 1.0 1 .2 1 .1 1.0 0 .3 1 .4 1 .2 1.0 1 .2 1 .4 1 .5 1 .5 1 .1 0 .1 3 0 0 ,0 3 3 0 .0 3 7 0 .1 0 0 0.095 0 .1 1 2 0 .1 0 4 0 .0 3 2 0 .1 3 0 0 .1 5 2 0 .1 3 0 0 .1 1 8 0.13-4 T'!)v r o id ut G 0.4 0c032 1 .1 O.O76 1 .3 0.102 0,7 0.070 0 .5 O.Oj O o.9 0.072 ---- 0.7 0.057 ---- -- ---- ---- 0.0 0 .0 ;0 * = duJdy ocas divided and nd;rJLniatcred h in A.I,', and in l*.?j. CG :1 e m n o * M O O THE DOW CHEMICAL llG COMPANY M IDLAND. MICHIGAN A COMPILATION OF TOXICOLOGICAL INFORMATION REGARDING 2,4,5-TRICHLOROPIIENOXYACETIC ACID mu CONTENTS - Assembled July 1963 - References I RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICHLOROPHENOXYACETIC ACID II A C U T E A N D S U B - A C U T E O R A L T O X I C I T Y OF 2 , 4 , 5 - T R I C H L O R O PHENOXYACETIC ACID FOR DOGS III T H E ACUTE ORAL TOXICITY OF 2,4,5-TRICHLOROPHENOXYACETIC ACID TO RATS, MICE, GUINEA PIGS AND CHICKS IV SOME EFFECTS OF HERBICIDES ON PASTURE AND ON GRAZING LIVESTOCK V TOXICITY OF 2,4-DICHLOROPHENOXYACETIC ACID AND 2,4,5TRICHLORQPHENQXYACET IC ACID VI SUMMARY OF TOXICOLOGICAL INFORMATION O N 2,4-D AND 2,4,5-T TYPE HERBICIDES AND AN EVALUATION OF THE HAZARDS TO LIVESTOCK ASSOCIATED WITH THEIR USE ^ U G l/U c c G RESULTS 0 ? TOXICOLOGICAL TESTS ON 2 , 4 , 5 - t r ic k l o a o ? hexg>?;a c z t : c a c id Biochemical Research Department The Dow Chemical Company midland, Michigan August, 1S50 4^ 1 8 1K 6 007825 RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICHLOROPHENOXYACETIC ACID Biochemical Research Department The Dow Chemic al Company Midland, Michigan August, 1950 PROBLEM The widespread handling of 2, 4 , 5-trichlorophenoxyacetic acid made it desirable for us to have a significant amount of t o x i c o logical information. Data on the oral toxicity was necessary for presentation to the F.D.A. Hearing on residue tolerances. C O ^ MATERIAL Name : Formula: Structural 2,4,5-Trichlorophenoxyacetic acid O H Cl H-O-C-C-O- // I H \ Cl Cl Empirical: CgHgClgOg EXPERIMENTAL RESULTS The acute oral toxicity of 2,4,5-trichlorophenoxyacetic acid for rats, mice, guinea pigs and chicks is given in the attached s u m m a r y w h i c h w a s p r e s e n t e d in e v i d e n c e at the F.D.A. H e a r i n g . In concluding our testimony on this material we stated, 1$ "Becau.-e of the s i m i l a r i t y in c h e m i c a l s t r u c t u r e and in t o x i c i t y and p h y s i o l o g i c a l e f f e c t b e t w e e n 2 , 4 - D and 2,4,5-T, it seems that a residue tolerance for the latter on fresh fruits and vegetable., s h o u l d be the same as that e s t a b l i s h e d for 2,4-D; it would seem that a residue of 2Q p.p.n. would provide a wide margin of safety". Eye Irritation V.'hen 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 a c i d as a 10% s o l u t i o n in p r o p y l e n e g l y c o l w a s i n t r o d u c e d into the eye of a rabbit, it produced marked pain and slight conjunctival irritation immediate! dive hours later, severe conjunctivitis and moderate corneal daaag was apparent. This picture remained about the same for two days before healing began. - ive days after the exposure mild conjunc tivitis and corneal damage was still apparent. Skin Irritation then 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 a c i d as a 10% s o l u t i o n in butyl c a r b i t o l a c e t a t e w a s a p p l i e d r e p e a t e d l y to the r a b b i t ear ar. bandaged repeatedly to the shaven abdomen, a slight simple irri tation developed in both instances. SUMMARY The toxicological properties of 2,4,5-trichlorophenoxyacetic acid may be summarized as follows: 1. T h e m a t e r i a l is m o d e r a t e in a c u t e o r a l t o x i c i t y for the four species, rat, rxuse, cavie, and chick. Of these species the rat seems to be the more resistant and the chick the most susceptible. 2. T h e m a t e r i a l is m a r k e d l y i r r i t a t i n g to the eyes. 3. T h e m a t e r i a l is s l i g h t l y to m o d e r a t e l y _ r r i x a t i n g to the skin upon prolonged exposure. cow 2 0073 HANDLING HAZARDS AND PRECAUTIONS ACA SATE HANDLING Swallowing It does not seem likely that 2,4,5-trichlcrophenoxyacet ic acid can be swallowed accidentally in amounts dangerous to life, although this might be possible if concentrated solutions of the material were available to children. If substantial quantities of the material were to be swallowed by anyone, serious ill effects could be anticipated. If the material should be swallowed, induce vomiting by giving a common emmetic such as 2 tablespoonfuls of. table salt in a glass of warm water. Call a doctor. Eyes The 2,4,5-trichlorophenoxyacetic acid is capable of causing serious damage to the eyes. Precautions should be taken when handling th: s material to prevent possible eye contacts. It is recommended when handling strong solutions of 2,4,5-trichlorophenoxy acetic acid that goggles, face shield, or other similar device be worn to prevent accidental splashes in the eye. It is believed that the handling of the solid does not present a hazard simply because of its insolubility in aqueous medium. If the material in any form should get into the eyes, they should be promptly washed with flowing water for at least 15 minutes and medical attention obtained. Skin Prolonged and repeated contact with the,skin may result in irritation. Occasional contacts for short periods are not expected to cause irritation. Strong solutions arc no doubt more likely to cause difficulty than the dry solid. In case of contact wash the exposed area with soap and water. Do not wear clothing or shoes which have been contaminated with solu tions of the material. i *1 V s moq inhalation No studies of toxicity upon inhalation of 2, *1,5-trichloro- ; phencxyacetic acid dust have been made. \.'e believe, however, ehat in view of the similarity with this material to that of 2 ,4-dicl.lorophenoxyacetic acid, that it is unlikely that toxic concentrations of the dust would be inhaled, primarily because of the irritation it will produce upon the upper respiratory passages. ^9 l THE DOW C H EM IC AL 1166 COMPANY MIDLAND. MICHIGAN A COMPILATION OF TOXICOLOGICAL INFORMATION REGARDING 2,4,5-TRICHLOROPIIENOXYACETIC ACID CONTENTS - Assembled July 1963 - References I RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICHLOROPHENOXYACETIC ACID II A C U T E A N D S U B - A C U T E O R A L T O X I C I T Y O F 2 , 4 , 5 - T R I C H L O R O PHENOXYACETIC ACID FOR DOGS III TH E ACUTE ORAL TOXICITY OF 2,4,5-TRICHLOROPHENOXYACETIC ACID TO RATS, MICE, GUINEA PIGS AND CHICKS IV SOME EFFECTS OF HERBICIDES ON PASTURE AND ON GRAZING LIVESTOCK V TOXICITY OF 2,4-DICHLOROPHENOXYACETIC ACID AND 2,4,5TRICHLORQPHENOXYACETIC ACID VI SUMMARY OF TOXICOLOGICAL INFORMATION ON 2,4-D AND 2,4,5-T TYPE HERBICIDES AND AN EVALUATION OF THE HAZARDS TO LIVESTOCK ASSOCIATED WITH THEIR USE 130 u RESULTS OR TOXICOLOGICAL TESTS ON 2,4,5-TRICKLOROPHENOXYACETIC ACID Biochemical Research Department The Dow Chemical Company midland, August, 1950 o\ ft RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICHLOROPHENOXYACETIC ACID Biochemical Research Department The Dow Chemical Company Midland, Michigan August, 1950 PROBLEM The widespread handling of 2,4,5-trichlorophenoxyacetic acid made it desirable for us to have a significant amount of to xi co logical information. Data on the oral toxicity was necessary for presentation to the F.D.A. Hearing on residue tolerances. D O ^ MATERIAL Name : Formula: Structural 2,4,5-Trichlorophenoxyacetic acid O H ^1 H-O-C-C-O- ^ ^ Cl H N Cl Empirical: CgH^Cl^O^ 007825 EXPERIMENTAL RESULTS The acute oral toxicity of 2,4,5-trichlorophenoxyacetic acid for rats, mice, guinea pigs and chicks is given in the attached summary which was presented in evidence at the F.D.A. Hearing. In concluding our testimony on this material we stated, % \\ "Because of the similarity in chemical structure and in toxicity and physiological effect between 2,4-D and 2,4,5-T, it seems that a residue tolerance for the latter on fresh fruits and vegetable, should be the same as that established for 2,4-D; it would seem that a residue of 20 p.p.rn. would provide a wide margin of safet y" . Eye Irritation V.'hen 2,4,5-trichlorophenoxyacetic acid as a 10% solution in propylene glycol was introduced into the eye of a rabbit, it produced marked pain and slight conjunctival irritation immediate! -'ive hours later, severe conjunctivitis and moderate corneal damag was apparent. This picture remained about the same for two days before healing began. -ive days after the exposure mild conjunc tivitis and corneal damage was still apparent. Skin Irritation Then 2,4,5-trichlorophenoxyacetic acid as a 10% solution in butyl carbitol acetate was applied repeatedly to the rabbit ear ar. bandaged repeatedly to the shaven abdomen, a slight simple irri tation developed in both instances. SUMMARY The toxicological properties of 2,4,5-trichlorophenoxyacetic acid may be summarized as follows; 1. The material is moderate in acute oral toxicity for the four species, rat, mouse, cavie, and chick. Of these species the rat seems to be the more resistant and the chick the most susceptible. 2. The material is markedly irritating to the eyes. 3. The material is slightly to moderately -.rritating to the skin upon prolonged exposure. cow 2 0078; HANDLING HAZARDS AND PRECAUTIONS ECA SATE HANDLING Swallowing It does not seem likely that 2,4,5-trichlcrophenoxyacetic acid can be swallowed accidentally in amounts dangerous to life, although this might be possible if concentrated solutions of the material were available to children. If substantial quantities of the material were to bo swallowed by anyone, serious ill effects could be anticipated. If the material should be swallowed, induce vomiting by giving a common emmetic such as 2 tablespoonfuls of- table salt in a glass of warm water. Call a doctor. Eyes The 2,4,5-tri chlorophenoxyacetic acid is capable of causing serious damage to the eyes. Precautions should be taken when handling this material to prevent possible eye contacts. It is recommended when handling strong solutions of 2,4,5-trichlorophenoxyacetic acid that goggles, face shield, or other similar device be worn to prevent accidental splashes in the eye. It is believed that the handling of the solid does not present a hazard simply because of its insolubility in aqueous medium. If the material in any form should get into the eyes, they should be promptly washed with flowing water for at least 15 minutes and medical attention obtained. Skin Prolonged and repeated contact with the skin may result in irritation. Occasional contacts for short periods are not expected to cause irritation. Strong solutions are no doubt more likely to cause difficulty than the dry solid. In case of contact wash the exposed area with soap and water. Do not wear clothing or shoes which have been contaminated with solu tions of the material. A *On--a *1 'inhalation No studies of toxicity upon inhalation of 2,4,5-trichloro- ; phencxyacetic acid dust have been mado. \.'e believe, however, chat in view ox the similarity with this material to that ox 2,4-dicklorophenoxyacetic acid, that it is unlikely that toxic concentrations of the dust would be inhaled, primarily because of the irritation it will produce upon the upper respiratory passages. dow 2 007828 ll O a *H J C U A T A I. l i i ' p o i l N o . fX MC J-nccp-cc- 1975-1-1 ,n.l it \ f kv a u a ti o n of Cave in o g c n i c , c r a t o i i e m c 5 Mutagenic. Activities of Selectedd Pcessticides and Industrial Chemicals. Volume I: C a r c i n o g e n i c Study -1 r B 5 I v c p u f l Male Cngpletfen-AMH ion ft 17. A w c h a /( s ) S l\*rU iM !iin g No. on IW-nr. IV rio rn iii'y. U rj;ani.uon Name and A J J rc s s ! Bionetics Research Laboratories i 7300 Pearl Street j Bcthesda, Maryland 20014 12. >pnnsOfm$ Ofp,.\;ii*.ntion Naine arivi A d d re ss ! National Cancer Institute ! Division of Cancer Cause Prevention i Carcinogenesis Program 1 P.ethesda, Ma ryl an d 20014______________ J15. S upplem entary N o ;cs I Concept - Dr. ^aul Kotin and Dr. Hans ! Initial Planning - Dr. A J .' Pa ll o t ta Falk, NIH and Dr. E. 10. M rwjt c i / T -o is U nii No. 11. C o m r.iv l ` G ram No. PU 43-64-S7 PH 43-67-735 13. l'> pc m D e p u ri A; M enno Covurvti F I N A L 1963 - Aug. 1963 u. Ross Hart, BRL '.6.Absiracis Vol. I evaluates the c a r c i n o ge n i c p o t e n ti a l of 150 c o mm e r c i a l pesticides whic h have been used in part rs insecticides, herbi cid es, 6 fungicides. A total of over 20,000 mice were used in the ex pe rim en t. Eighteen virgin males 5 18 virgin females from hybrid strains CB6AKF1 and B6C5F1) were selected for a test on each compound for each route of admin ist rat ion used. An equal nu mb e r of mice served as u n t r e a t e d controls for both routes 5 another group of 36 served as ve h i c le con trols for each solvent. Two types of IS -month studies were c ar rie d out: . 1) a single s.c. injection in 0.0S ml of s u sp e n s io n in nape of nec k at ! 28th day of age; 2) d ail y oral s t o m a c h tube a d m i n is t r a ti o n from 7th to I 23th day of age, and thereafter mixe d in feed. MID c alcul ated on average i body weight per group was d e te r m i n e d in a p r e l i m i n a r y acute to x icity j study. No doses above 1,000 mg/kg were studied, except N-Propyl isome i r 7 Q D O r q q / i - f f . I l r p f h . i n w n <; s t u d i e d . ? f n t o t a l d o g - a o f 5 m p / m o u s e f e e n t ) 17. Key 'loco: a n d D o c u m e n t A n a l y s i s . 17o. D e s c r i p t o r : ; Bioassay > Carcinogens Pesticides Mice Toxicology Liver Neoplasms Herbicides Fungicides Reticulum Cell Sarcoma Pulmonary Neoplasms DDT Chlorohydrocarbcns Thioca rbama tes Environments Halohydrocarbons w 0 < to w (-- . O' o 17b. ld c n c iiie rs /O p c n -'.'r.J c d T erm s ! B6AKF1 Hybrid Mice B6C5F1 Hybrid Mice Dual clcan/dirty corridor Pesticide analysis Pesticide Residue system 18X8c i7c. c o s a t i lU c M /C c o u p IS. A v . u in 'u .iiy Nai**Rienr 06 -R io log ica 1 C M e dic al Release Unlimited f i > K M < * n Z' .** \h u *. J* t+1 Sciences 07-Chcmistry IV. u n iv )H .'p v .rl 1 11i "* 21. N o. u l 1Ni/je s 2U. l l l i i ! I ai c rwi \`>*irirn 22* u*ct**-uc Dow 2 007802 TAULE IV 32 LIST OF CHEMICAL COMPOUNDS STUDIED WITH CODE NUMBER, COMMON OR CHEMICAL NAME, DOSAGE AND VEHICLE OR SOLVENT Code No. 024 025 026 027 028 029 030 031 032 034 04 7 048 049 050 051 052 053 054 056 057 058 059 060 061 062 063 065 066 067 068 069 070 071 072 073 074 075 076 077 078 079 080 081 082 Common or Chemical Name Simazine Propazine Cap tan Piperonyl Bucoxide Piperonyl Sulfoxide N-Propyl Isome 2,4-D Isopropyl Ester 2,4-D Butyl Ester 2,4-D Isooctyl Ester Ethyl Carbamate Sevin IPC SDDC Dowcide-7 Zineb o ,p '-DDD Diuron Doaine Maneb Maleic Hydrazide Thiram Monuron PCNB 2,4,5-T Ferbam 2,4-D 2,4-D P >P'~DDT Atrazine p,p-- DDD Ethyl Tellurac Cap tax Ethyl Zimate Phenyl Isothiocyanate Pertnane Chloranil Altax Unads Dichlone Dicry1 Ethylene imine Nabam Agerite DPPD Vancide PB Bismate Subcutaneous Dosage mg/ka Solvent 1000 1000 1000 100 46.4 1000 100 21.5 21.5 25 100 215 464 46.4 1000 464 1000 1000 100 1000 46.4 100 1000 215 100 464 215 464 100 464 1000 215 464 100 215 464 1000 100 21.5 1000 4.64 10 1000 10 1000 0.5% gelatin 0.5% gelatin 0.5% gelatin Corn oil Corn oil DMSO Corn oil Corn oil Corn oil DMSO DMSO DMSO Dis. H->0 Corn oil 0.5% gelatin DMSO 0.5% gelatin 0.5% gelatin 0.5% gelatin 0.5% gelatin 0.5% gelatin 0.5% gelatin 0.5% gelatin DMSO 0. 5% gelacin DMSO DMSO 0.5% gelatin 0.5% gelatin Corn oil 0.5% gelatin 0.5% gelatin rU\ Ji-/vf gelatin Corn oil DMSO 0. 5% gelatin 0.5% gelatin DMSO 0. 5% gelatin DMSO Dis. H?0 Die. H2O DMSO DMSO 0.5% gela tin *Used during stomach tubing period only. Dosage mg/kg Oral Vehicle* DDm 215 46.4 215 100 46.4 2000 46.4 46.4 46.4 158 4.64 215 215 46.4 464 215 464 21.5 46.4 1000 10 215 464 21.5 10 100 46.4 46.4 21.5 100 46.4 100 100 46.4 215 215 464 100 iO 21.5 4.64 21.5 1000 46.4 10 0.5% gelatin 603 0.5% gelatin 102 0.5% gelatin 560 0.5% gelatin 300 0.5% gelatin 111 0.5% gelatin 6000 0.5% gelatin 111 0.5% gelatin 149 0.5% gelatin 130 0.5% gelatin 600 0.5% gelatin 14 0.5% gelatin 560 Dis. HiO 692 0.5% gelatin 130 0.5% gelatin 1298 0.5% gelatin 560 0.5% gelatin 1400 0.5% gelatin 82 0.5% gelatin 153 0.5% gelatin 3000 0.5% gelatin 26 0.5% gelatin 517 0.5% gelatin 1206 0.5% gelatin 60 0.5% gelatin 32 0.5% gelacin 323 0.5% gelacin 149 0.5% gelacin 140 0.5% gelacin 82 0.5% gelacin 300 0.5% gelacin 149 0.5% gelacin 323 0.5% gelatin 260 0.5% gelatin 153 0.5% gelacin 815 0. 5% gelatin 646 0.5% gelac in 1577 0.5% gelatin 377 0. 5% gelacin 30 G. 5% gelacin 73 0.5% gelacin 13 Dis. H2O 73 DMSO 3385 DMSO 121 181360.5% gelatin 34 51 dow 2 007803 TABLE VII NUMBER OF EXPECTED TUMORS AND MINIMUM TIGRIS II ClPENCE IN SUBCUTANEOUS STUDY MICE THAT CAN III; I'.CCAEDED AS SIGNIFICANT AT P=.Ub AND !'=. 01 LEVELS FOR SPECIFIED NUMBERS OF MICE NECROPS1ED I T1IF. STUDY CROUP* j . of mice ,,i.jpsieci in . iiudv prouD Expected total mice with tumor Total mice witli c-mcr (1) P-.05 Levo 1 Reciculum cell sarcoma Pulm. Ade noma (2) (3) hepa toma (4) Total mice with tumor (I) P-.0L Leve 1 Koticu Lum cell s a r c o s :i (2) Pu lm Ado-- noma n> Hepa toma () 72 71 70 69 68 67 66 65 64 63 62 , 61 \/ 60 59 58 57 56 55 54 53 52 51 50 7 13 7 13 7 13 7 13 7 13 7 12 7 12 6 12 6 12 6 12 6 12 6 '.12 6 11 6 11 6 11 6 11 6 11 5 11 5 11 5 10 5 10 5 10 5 10 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 8 4 15 7 10 6 8 4 15 7 10 6 8 4 15 6 10 5 8 4 14 6 10 5 8 4 14 6 10 5 8 4 14 6 9 5 8 4 14 6 9 5 7 4 14 6 9 7 4 14 6 9 5 7 4 13 6 9 5 7 4 13 6 9 5 7 4 13 6 9 5 7 4 13 6 9 5 74 13 6 J5 7 4 13 6 9 5 7 4 13 6 9 5 7 4 12 6 8 5 7 3 12 6 O 5 7 3 12 6 8 5 6 3 12 6 8 5 6 3 12 5 8 5 6 3 12 5 8 5 6 3 11 5 o 5 Using Chi Square Method '"Fourfold Table" - Yates Correction used for all hut total tumor . based on 613 Mice Nueropsied in the Total Nigative Control Croups: (1) of which 61 developed tumors. (2) of which 14 developed reticulum cell sarcoma. (3) of which 30 developed pulmonary adenoma (4) of which 10 developed hepatoma. ATTACHMENT PERTINENT DATA ABSTRACTED FROM TABLES XI AND XXV OF BIOXETICS STUDY (1968) Compound Name U ncreated Concrol-OA Subcucaneous Comp. No. S crain (1) A 8 U ncreaced Concrol-OB Subcucaneous A B U ncreaced Concrol-OC Subcucaneous A B Uncreaced Concrol I Subcucaneous A a Uncreaced Concrol II Subcucaneous A B Uncreaced Concrol I I I Subcucaneous A B C e la d n Concrol Subcucaneous A B Corn O il C oncrol Subcucaneous A B DMSO C o n c ro l Subcucaneous A B Tocal Negacive Concrol Subcucaneous A B 2 ,4 ,5 -T Subcucaneous A 061 B No. of Mice Ac S c a re MF 18 18 18 18 To c a l Mice X ecropsied >! F 16 17 17 13 Pulm onary Adenoma MF 11 11 18 18 17 18 2 0 18 18 IS 17 0 2 18 18 17 17 2 0 18 18 13 18 2 1 IS 13 13 13 1 0 13 18 18 18 0 1 12 18 12 13 1 1 IS 13 13 13 0 1 -- 4--4- 0 11 6 11 6 0 1 18 13 18 IS 24 1fc/4 24 24 16 18 13 13 21 21 19 20 10 00 -> 1 i. 1 24 24 2' 24 23 ^u 1.4/ c1 12 150 160 141 154 10 4 167 ln2 161 157 6 10 18 13 IS la 17 13 13 13 00 31 (1) S c r a in o t m ic e : A-B6C3F1 ( S c r a i n X in CAC r e p o r t ) B-BbAKFl ( S c r a i n Y in CAG r e p o r t ) o o N> cr o OC C JLB y Biochemical Research Laboratory 18.269 THE D O W CHEMICAL COMPANY - Subject TO XICITY OF 2, 4 , 5-TRICEL0R0PH2N0XY ACETIC ACID. ,'k i r Fil. T2l .l-g3-l Cbg. 1219A Rec'd 3-15-45 Fin'd 8-8-45 Work BJ C . G e i s e l ", ^ : ^ rJ M QZ-\ i ' XoTech. Service & Developcheck W. W. Allen W. R. Veazey c,'pl*Li^us<3 Rept. Br ' Z ' . / C . f ? -2 Problem - cs What are the handling hazards to be associated with 2,4,5-trichlorophenoxy acetic acid? O CO 05 Co Material - a O Name - 2,4,5-trichlorophenoxy acetic acid - K4568-2 3 Formula - Cl Structural - Cl O Cl 0. T ft* o ^ - 3 - o -h ^0 oo CO Empirical - CgH^OgClg OG Source - Organic Research Lab. - L. Begin Ref. - #410-5-258 Experimental Results - Acute Oral - Weighed doses of the acid were emulsified in 5-10% gum arabic solution and fed to cavies in single oral doses by means of a stomach tube. The material as the sodium salt,ad justed to pH = 10,was also fed. The results are given in Table I Table I The Acute Oral Toxicity of 2,4,5-Trichlorophenoxy Acetic acid and it3 Sodium Salt for Cavies. Material 100% Survival Dose 100% . Lethal Dose gAg- Acid 0.3 1.0 ; r. Salt - pH = 10 0.3 0.5 I F s r F p ORT IS THE PR0 PERT OF ' THE DOW CHEMICAL COMPANY 1219 25 25C 1144 1819 n irnon * on Page 2 These figures indicate that the subject material is moderately toxic when given in single oral doses. No information upon the effect of repeated administrations was obtained. It was noted in this acute oral work that the animals did not exhibit the nervous symptoms caused by 2,4-dichlorophenoxy acetio acid, sodium salt. The animals fed large doses of this latter material exhibited paralysis of the posterior portion of their bodies. Skin Irritation - The acid as a 10$ solution in butyl carbitol acetate was applied 10 tinges in 11 days to the ear and shaven belly of a rabbit. A very slight irritation developed on the ear and a slight to moderate irritation appeared on the belly. A 1$ aqueous s o lu tio n o f th e sodium s a l t f a i l e d to p ro duce any s i g n i f i c a n t i r r i t a t i o n on th e e a r even a f t e r 20 ex p o su re s in 27 days; s im ila r exposure on th e b e lly r e s u lte d in a v e ry s lig h t sim ple i r r i t a t i o n . Conclusions 2 ,4 ,5-Trichlorophenoxy a c e tic acid has a m oderate acute o ra l to x ic ity and is s lig h tly ir r i t a t i n g to the skin of ra b b its . Y/e conclude that excessive exposure to the dust of either the free acid or the salt v/ould be likely to cause skin irritation as would exposure to concentrated solutions for prolonged periods of time. Exposure to dilute solutions such as those used in practical application would not be expected to cause trouble except among those who persist in spraying themselves or who do not take the necessary precautions to prevent prolonged contact with the material. R easonable h andling p re c a u tio n s which w ill p rev en t i n g e stio n and prolonged skin co n tact w ith th e m ate ria l w ill s u ffic e * to prevent serious trouble. jf-5 copies. 1 X BIOCHEM ICAL RESEARCH LABORATORY T est on /{ Acute oral toxicity, Bsix, Cavy Prob. T 2 ^ . / / ' a j - / 1219-U3-5C-10-^3 BIOCHEMICAL RESEARCH LABORATORY J P j ZjLs T- a-x. Vo. f**' ^ Test on (J - Ac ute oral toxicity. Rt* Cavy 4/'/ u & U i,) JidiAjWi (7 Prob. T 73./y - 2 3 -/ Date sdo-^sr Animal Weight # kg. ? 2.4 - o 7 ^ 2. / Jr /TjLO 2.2.S' S'2./ SS^? el<3 <r" S /7o /^ .. ^ 0 3 . -S'-j s~ Dose Dose gm/kg gnu f C-C, Remarks %| <? / .3 S'!O 7/ 5 'I 1/ 3- / .05 o 72-0 /P.J" 5"^0 K 4-^OflM. W*:T 3. _ ./Osix. /:5.y^H 7 ^ ~ l ,, n J .^!.5rf. A l. 3 .a 1 Jh-lIum, 7 .* .// / ii.tL J 4/> <3Ao- l \ -. - . .17 7 0 17.70 <5 /Oi.c. 7,7a Stlkv/7 : ( * * .J o. 7 J.f 2. iZ.i. .. #V2- Ws'ifcV' A o. J" .z(c7 r 2.7s1 1 _('-AAVIg1rv^I ^ Itf/>. 10.*.< C.7S' 2-o V . c / o. A .la%0 J /0X toc.c.rsa ^9^/> 7 > S f2^7 N -,' v* .`r,? A 0 .<bi S\Z ?-> s~Y , . J lind3-10' */>" \ ____ ___V***~J%*.**_ V _ _ _ _ _ 1_ LS~7/ /. 0 .57/ S 7 J * * 1 tocc. }0.-1/3)joJ_dk+ \ 3-3**1 ^JL^ H w > < a L-? -^Laju 6Li X ^ 3 - /8 00 C Cj /Le^t?jU-^JL ^ - u rTL .4/*^--- 1 S % F :y ^ f -A-V-L - f n *^jZa~<s.V- ; P _ ?.'i* ui.i. 1219-- 43-- 5C-- 642 l` ^ ,--v Q i 8 TA'd'IM BIOCHEMICAL RESEARCH LABORATORY a 6 'c Test on /f </S ' ? ' ^ Acute oral toxicity. c J M n ^ j U L ^ ^ UX*. U * J ) nC a.v..y. (<K\s Prob. T 2 3 //-.z 3 -/ Date A n im al U TT W eigh t kg. tj 4 ( /c- g- ^ <7.a / X/l.2 )_________ _ Dose * Dose gm /kg ' gnu tt / . Remarks rH fn % . o V o .3 i * -i i IX. ___ 7 . 1 ? ____ i 1 3 c V 3 3 > 2 L7 35 7 33o 3^"o 3 0 .3 o -A" AO" .o M . / / . / 7 jT ^7/ tf* - -----r^T p! ^ 7 3?0 . / / l/*.0 # , / o 3 / ________ 1_________________ / z r - * *7. .So ^7/ i J 4 -U ^ 3-V 6 7 , 3 f f 0 . j" . / m " / f j T l / . ^ _____ 2 1 b t M l \ t i,A _ // 1219--43--5C-642 - ;: i / 181 BIOCHEMICAL RESEARCH LABORATORY Test on / { 7 j> Skin I r r i t a t i o n %' ~ -____ & ' j s c L ^j U ^ X / 77^ (/ No. o f A p p l i c a tl o n s _ L aT ~ S o ^ Qu arii-i^ertirvs ftt: Ear AbAomrxt -- SZ , O i^ J u u A . . p 7 p n n nr ^ o n Rabbi ^ D ate 3 - 2 - */dT~ 3 - Z l ' l/ s * 3-12- i S' 3- 2 3- V i" 3-^k-4S 3 -Z 7-V S0 s-^ v -4 'S ' v -v x V w -V 5T i ~ l *2- &~f Cage # Prob. T Day E x?UpT. R eaction 0 f 1 7 f / 6 r. .7 / Cl- 7 <? 7 /<?_ /f A 3 ... v <r" C, 7 r f /0 /It. ' " /V if u _/ ^ 3 aJTt X . jlJ J s^ U j j^ a . V'-T-- _-- --n -- r?T * w ^ - ^ __ _ i <i> i ^ ..< - / Jd - X/ V Ao -u /4 /- T L > ~ A ', >1 V * .. * /v< & ^ i c NC *U 9^. . . n (/ - 1 2 1 9 -2 0 --1M--1^5 1 " *. : .." * A ^ r.: . v.'A^V-i "^*V; ..* t/- /?*V'. - ;:s 1813^ Test on BIOCHEMICAL RESEARCH LARORATORY A Y~Z> ? - . _____ / Ofo ^ Skin Irritation No. of Applications /O QOTTrtlta^irve -JSas Abdomen Rabbit Date 3 - if -*^5" Cv*~tLM- ' 77 / 72. ^~~7 Caffe .7 Prob. T <2 3 --------------------------------------- -- -- DIa.xTiy Exp. TMTm Reaction t c c 0 1 / 7 ? K i - .... .7^-- --- r / 4L <5- * ~; ~ "/yiyod^ -- -- V* l.aJ' mJ + -- X . --. -AA/jO^r *_ ^ -.2 , 3 -y c y S~ 7C 7 ? *T /fo J ^ o ^ x ^ i p - ' X Ti . ....... 3 / * T 'T'dyf^sAs-r?^L-^ArL-A-iT/7'^*-.-. -<r/. ^rr X3 - 3 6~tJ-5 // / / 2 -XT' /.`f 7 /^- D-^O _.. I'Mvc,) '/ - 4 - *tz>" X. ^ <v- a- A */-- <-- 4^5 -- <- , ... ... (SA A -- . . ....`':'*:";/-.*i\r;;V' 1219-20-1 M-l 4-p -f .-.\41 ''"i(V*:'- .L . -- **/ !..*.>v:M<**Ki*',.'7N;* BIOCHEMICAL RESEIARCH LABORATORY ' /> ^ / a~<' - -SL. Tes Skin Irritation Q ^ a r-tVtq-t ive No. of A ppl icati ons^ - Ear -M ad cnnen , 0 ~ " Z to p iM * O U < U * -u P . / Rabbit ff 7 - / ^ 9 O 7 -- Cage # J3 5* Prob. T-i. J . - / ^ Date Day t*xp, # ? / 3 " 3 o - A 5 ^ z <+ 1 -3 -4 r~ 4 y ,-/.y.r.y^- jj2l . Z zZ l Z ' -Y s^ ^ C r J jL `/ /1Z . r . Y * JZ. X .Z z Z . <r _k_ X -PZ- / o zz Reaction ^3 "J i/ // // // .. -Z_ // feZnt. </-/->- `/ s ' i-zr-Yf y *^/",T,im----------*- 'Vr --*mr. Ji* vz: z Zz=, /JOH/ 2~d 2vM _X _ZI # 0 / J -b - f n 7 jXaiZU. // Z ..r P iP x . ..y-.* -iZZzZZ Zfl zz 2Z, A^wcO < Z Jf c L Z J #> jL tf = o . i r . f A t/ - x 6 >y,>; .Z . i>* -=*-* f, ;-.... ** 1219-20-1M -1U 5 X "B BIOCHEMICAL R LARCH LABORATORY y/6 <=*~<s -^sr. a . Test on / C r x * - z- a * z z . z j) > Skin Irritation C-fe^-ti-&a<Wrv e No. of Applications i.tsT 11 Es t Abdomen Rabbit >T Z U tQ L , L C ^ - J l <j ; ? ~ / f ^ ~ h Cage 3 T Prob. T 2 3 . / / ~ 3 - / Date Day Exp. 3^rW-T" or >3Vr / V- ~-- ^ a-- y3 2- 3 i _ / < ^ 23 3 /^ Reaction . ./.3~ /=-/0 /. 6 2- -------- j6 - x) L C c .. c c <J~ 3--VS- y* y- /- a" - y s " 7 /- f> -/-r- y- 7- -/-r / / /^ _ / - if - /5'" / 5 /,, /-/ 3 - / ^ // /-T / 7 -*<7 -7SJy*-- /..<-/^ v ,7 ^-<27727^ y y . 7 <? f >^. wA<y-oX-<^i -7 o--- V __s/\.lA<P-JL< *-C-K /> J Z * (!! // / 3 a.fv'/r'V. ^ < 2 ^ . -j . /f / / y y r - A-# / c T J,/ / - y -s 77 j/. a * - y r -L, ST" / r v. </.*/<- 7? y - x r ^ y y ^ ,A7 O-o y rsfrt-, , T -if_ __ ^e j ^ `y . ^ ; T ' : ^ r .......- ........ - 3-0/ " \ ~~ " ~; . y-z..c.-f 1219-20-1M -14-5 _ .. , / r] .'."i , t''V. ~'vi ?' 'U /, #.-.iV- # . 18137 of S W 'i Q 11 ( O THE DOW CHEMICAL COMPANY MIDLAND DIVISION April 25# 1967 K. E. Coulter Midland Divisin Research & Development 566 Building CHLORACNE RESEARCH PROGRAM History of Chloracne Incidences at Dow; Historically, Dow Chemical has been involved in chloracne incidences ever since Dow began the production of chlorophenols. At first, the commercial production of chlorophenols was conducted a.t 206 Building. During the period 1934-36 there was a severe outbreak among the employees which resulted in an unsuccessful lawsuit. The chloracne incidents were traced bacJtJMtqnfOTSworking conditions and the manufacture of I^Swicide P. JEhe manufacture of Dowicide P has since been terttlMped. New and improved working facilities for-the Dowicide group were constructed at 265 Building in 1940. In the late 1930*s, Wes Stoesser in 20A lab got a serious chloracne attack from ^ohffcorm a t e d diphenylene oxideT^ Drastic treatments were used tJT" cure Lilia incident and no further work was done on this series of compounds. It is suspected that many of the other incidences of chloracogare caused by chlorinated diphenylene oxides or^a'naioguejfrthereof. In the next ten years, another unfortunate situation occurred in the chloracne situation. Some of the Dow customers complained aborrtTDcbeaemikiitis and/or chloracne from the use of Dow's - Q i understand financial adjustments were made and the production of Dowicide 3 terminated. A purified material of. related structure is now sold as Dowicide 31 and 32. During the period 1940-65, the product 6x)( diphenyl oxide chlorinated to the hexa level) wasSaanufactured at 206 Building and at least one severe case of chloracne occurred because of this product. The production of 6x has been terminated. In the research lab at 172 Building, there were some cases of chloracne from research exposures. In one case, a severe case resulted from the recycling of residues from the manufacture of 2,4,5-trichlorophenol using glycol as a sojaewfc-.-- -In_other case, several mild cases occurred h.vdrolvziiig nolvchlorobenzenes using aqueous caustic at high temperature. Thediemical -lyslcs Lab had several incidences of chloracne from the recycling of caustic insolubles in the alcoholic caustic hydrolysis of tetrachlorobenzene 18198 DOW 1516484 - 2- In 19^4, the 2,4,5-trichlorophenol hydrolysis step was modified for economy and safety reasons from 10($ caustic to 23$ caustic, but this change caused an unforeseen increase in the chloracnigens. This increase in chloracnigens was sufficient to cause many mild incidences of chloracne and two severe cases (lost timers). In 1966 a new process was put into operation using the Bcehringer procedure involving a batch reaction at low temperaturesj and, so far, has operated satisfactorily, ^ tj After they started up their(Dowici^explant, the Canadians began to experience chloracne incidence-a^jn their employees. In Midland, more than half of the (ffowicide^employees have chloracne of varying intensity and it is impossible to say when or where these incidences occurred. Research Program in Progress for Chloracne Reduction: Because of the prevailing existence of chloracne in the Dowicide plants and a sincere desire to reduce or eliminate this, research has been initiated in 1966 on this problem. Progress has been slow due to the complexity of the problem. The problem involves isolating- and determination of the identity of the chloracnigensAfter being properly identified, work can progress on its reduc tion or elimination in the process. In commenting on chloracne, we must keep in mind that chloracne is a cosmetic evidence of the attack and serious liver damage is. an invisible effect of the attack. Rabbit ear tests are a positive sensitive method of determining the chloracne activity of chlorophenol impur-i t.ip a---- ---------------- --- -- P re se n t Knowledge o f C hloracnigens: Compound Activity 2,3,7*8 Tetrachloro - Very positive Cli-3 - Not active . Clg-r - Possibly some cpds. active Cle - Not active Very a c tiv e 0 Very active C l, - Unknown a c t i v i t y CL ,-Ci . -- OCT CL. Compound 0' -3Activity Clx-a - Unknown activity Cl3-a - Some very reactive Cl4-a - Some activity? Materials possessing unexplained, chloracne activity: Some Midland batches are mildl reactive Many Canadian batches are mild reactive Dowiclde G All sludges are active All decompositions are active Some have activity DOW 1 5 1 6 4 8 G I 18200 4 -4- Midland Research Program on Chloracne: The following chloracne activity fractions are being examined for isolation, identification, and minimization of chloracne activity. Unfortunately, due to shortage of technical help the program is proceeding quite slowly. 1. Diehloropnenoi still residue. 2. Pentachlorophenol process samples. a. Dowicide 6 and 7 "active" batches b. Dowicide G sludge. c. Dowicide G scrubber sludge. The method of research is to first concentrate the sample (remove chlorophenols), then fractionate by chromatography, test fraction on rabbits, then further fractionate by chromatography, then test fraction on rabbits, etc., then determine structure by micro analysis, then determine method of analysis in original sample, then investigate process changes which will minimize the chloracnigens in the process. The Benzene Research Lab and the Biochem Research Lab are collabor^ ting in this project. The Benzene Research Lab does the chemical research and the Biochem Research Lab does the testing on the rabbits on their charge. Alex Widiger Benzene Research Laboratory W Building cc: Ww.. HH.. HHaabbeerrssttrroohh.. 447744 BBldd<g R. C. Sauers, 474 Bldg. S. L. Bender, 172 Bldg. E. C. Staehling, 258 Bldg 18201 DOW 1 5 1 6 4 8 7 / Quik Print IHSTANT PRINTING & PHOTO COPYING 98 Prince William Street, Saint John, N.B. E2L 2B3 (506) 634-8840 can "Distom Print" Post-ItTM Notes R O A R O A R . . . ask our sales rep. for details U J t ' O O t i / I82C2- i <:vj-0 Mi-: N T OK H E A L T H . E D U C A T I O N . A N D W E L F A R E PL' U U C H E A L T H SLR V ICE *5 VcfT'i.T 1 >m <\ MN 0 ]? 33? i n ou r t 1cp ho.oo cc-ovorsr.c io n on N -ocow r . * t, '* \ , t r ;c hi v.-oohonoy.y n o e t i c a c i d . it is \ 'f'r: i- ' s r h.: :t e m >.nat io n of th' . .' 7 :-.-i hi 2 ,'. , l tr ichi oroph- :.oy." LIV, - .v... ' 1 . -( r . A s i m e n Li one*.:, 'li'-.rip, r o >.:. V.'.-ll w h i c h p a v e ovii'o: ; c ' = '* ' -- 0 : 'i'.iO cr. r. ' tr ichlovnno* .'r - - _ r e f e c t whi e n in " r e s e n t in ve \\n i ' *i; 1 is t h i v 'ht to be. r e s n o n s i h l o is 2 ->.*! -..in: .vSN V |< ih!1onee a n n,''r in L `J (. ^ ^ -l m t v \ ".j p r>>.-. col o r 3 0 v- a n d l i v e r ' vrat-u ; :.-v. *. : .i is pn c--.ncK:slve evihenc. i:o- . f.-,-;- T.i.ii' h', nine.- n o o n e ha* v p r s. ;ir. ; i ('.'v.'< r oa 1 2 ,4 , '> T oul . t h t T ' j f o r e , a r e the fn 1 1 o w i.ny,: r. r.v.:& o ' chc o u r e s t p r e p a r a t i o n o f 2 ,4 , 1 (o.-'i M e . .ra - o f t he c o v-'orcial 2 ,4 , 5 t r i c h l o r n p h o r .1 re it is e .-*Ceri fieri, in p o w d e r form. 'r.T's of the e o T w r c ial 2,4,5 trichloronhenox.vnearic acid iso-octyl r, 'cfop} the r ;l Is ifiern have* been ad-ien. C3 C"l C2; Oa c?iO ( jic 'i ) 247 AU - TOCER J ; MATSON H ; BENSON HH - LYMPHOCYTE CHROMOSOME ANALYSIS OF AGRICULTURAL WORKERS DURING EXTENSIVE OCCUPATIONAL EXPOSURE TO PESTICIDES - EMIC/73/016741 - MUTAT RES; 21:335-340.1973 - EMIC/ORNL DOH2I5736I 1820 249 AU - ANDERSEN KJ ; LEIGHTY EG ; TAKAHASHI HT TI - EVALUATION OF HERBICIDES FOR POSSIBLE MUTAGENIC PROPERTIES '4 f i? SI - EMIC/72/0I3029 SO - J AGRIC FOOD CHEM! 20:649-656,1972 AB - EMIC/ORNL 370 AU AO TI SI SO AB rv > ^ -a'-n A ARNOLD HR SANTELHANN PH LYND JR OKUHOHA AGR. EXPT. STA., STILLWATER. PICLORAM AND 2,4-D EFFECTS HITH ASPERGILLUS NIGER PROLIFERATION CA/003/003379C HEEDS; VOL 14, ISS 1, 1966,09-90 CBAC COPYRIGHT: CHEM ABS THE EFFECTS AND RELATIVE DEGRADATION OF C94-75-71 2,4-0ICHL0R0PHEN0XYACETIC ACID 2,4-0 AND 11916-02-1] 4-AHIN0-5.5.6-TRICHL0R0PIC0LINIC ACID PICLORAM ON ASPERGILLUS NIGER, STRAIN ATC6275, HERE DETERMINED BY BIOASSAY WITH TOPCROP VARIETY OF SNAP BEANS. FUNGAL PROLIFERATION WAS REDUCED BY 2,4-0 AT 10 AND 50 PPM., WHILE PICLORAM AT UP TO 50 PPM. DIO NOT REDUCE FUNGAL GROWTH. ASPER6ILLUS NIGER DEGRADED 2,4-0 MORE EFFECTIVELY THAN PICLORAM . FUNGAL MYCELLIUM CONTAINED BOTH HERBICIDES AFTER EXPOSURE. BOTH HERBICIDES CAUSED PROFUSE MERISTEMATIC CELL PROLIFERATION HITH ABNORMAL INTERNOOE ELONGATION AND SEVERE LEAF AND STEM CURLING IN THE SNAP BEANS. 15 AU - Aullcino F ; Bignami M I Carere^A ; Ccnti G J Morpurgo 6 AU - Valeich A TI - tkitational studies with soma pesticides in Aspergillus nidulens. SI - PESTAB/76/2257 SO - Nutet. Res. 3B(2): 13; 1976. AB - PESTAB. Induction of mutation and somatic recombination by the A 'AI!'"1 following pesticides has been studied utilizing Aspergillus nidulans as test organism: aminotriazole (araitrola), captan, dalapon, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, \ neburon, picloram, and tordon (pleloram as the triisopropanotamtne salt). Point mutation has been studied as induction of 8-azaguanine resistants utilizing a spot test at three different pHs. Only captan turned out to be mutagenic. Induction of non-disjunction and mitotic crossing--over were studied with a diploid strain of the mold by: e) utilizing a spot test previously described at three different pH s , b) plating conidi a of the mold on a medium supplemented with the maximum nonlethal dose of the pesticide and screening for the appearance of recombinant sectors in the colonies. Captan, aminotriazole, and dichlorvos Induce somatic recombination. Especially noteworthy is the action of dichlorvos in inducing crossing-over. (Author abstract by permission, abridged) 174 AU - Bignemi M j Aulicino F ; VelcichA > Carerc A i Morpurgo 6 - 1st. Superiors di Sanita, Rome, Italy - Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. - PESTAB/77/2780 - Mutat. Res. 46(6): 395-402 1977 (27 References) - PESTAB. Thirteen pesticides, aminotriazole, benomyl, captafol, captan, dalapon-Na, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, neburon, picloram and tordon were tested for ability to induce (1) point mutations to S-azaguanine resistance, (2) mitotic crossing-over, and (3) mitotic non-dis jm e t Ion and v hoplotdization in Asperiglllus nidulans. Tests were performed at three different pHs, i.e. 4.5, 7, .2. Three of the pesticides, captan, captafol and dichlorvos Induced point mutations; dichlorvos also Induced a high frequency of mitotic crossing-over and non-disjunction; benomyl Induced a very high frequency of non-disjunction whereas aminotriazole Induced weekly both types of somatic segregation. (Author abstract by permission) 104 O o rvj CD ^>4 CO cn J f02 D 0 W2 1 5 7 3 5 6 133 AU - Palmar JS J Radeleff RO TI - Tha toxicity of some organic herbicides to cattle, ahaap rtd chickens. SI = KAPAB/7S/01212 SO - U.S. Dapt. Agr. Prod. Res.', Rapt. No. 106: 1-26, 1969; (REF:21) A8 - HAPAB Tha rasults of studies of tha toxicity of 29 organic harbicidas to cattla, shaap and chickans hava baan prasantad. Thasa included chlorophanoxy compounds ( 2,6-0, 2,4,5-T, silvax and HCPA ), amide compounds ( diphenamid, COAA and TCBC ), phenyl urea compounds ( monuron, linuron, fanuron and diva-on ), thiocarbamate compounds ( diallate and triallata ), triazine compounds ( atrazina, simazina, promatona, propazina, ate. ). and benzoic acid compounds ( dicainba and 2,3,6-TBA ). Miscallanaous compounds investigated included bromacil, isocil, pholychlorobicyclopentadiana isomers, propanil, dichlobenil, picloram and dalapon. A total of 126 yearling cattla, 190 1- to 2-year- old shaap and 700 6-waek-old chickans ware studied. Repeated doses of tha herbicides ware administered in gelation capsules or as water- diluted solutions by drench or in pipettes. Tha usual period of study was 10 days or until toxicilogic affects appeared. However, in a number of instances, longer and shorter term studies ware mada. Tha signs of posioning by most of tha herbicides included anorexia and reduced weight gains. In many instances, idgastion in tha rumen seamed to have been imparled. Hacroscopic lesions were somewhat variable and nonspecific. The liver and the kidneys were most frequently Involved. An arbitrary yield of forage and rate of comsumptlon were selected to evaluate the hazard for cattle, sheep and chickens likely to exist under most severe conditions of use. The most common rates of application of many of thasa herbicides formulations are not a hazard to cattle, sheep or chickens. The maximum, rates of application of some approach or surpass a hazardous level In one or more of the tests species. ( Author abstract modified i Toxicology and Pharmacology 70/00/00, 360 1969 -a (,(|E1S I ZHOO D,, 126 AU - World Health Organizatio Ti - Health aspects of chemical and biological weapons; raport of a MHO Group of Consul{at*. SI - HAPAB/70/01829 SC - MHO, Geneva, i960 31 p . 1970 AB - HAPAB This raport is an attampt to analyza tha health affacts of possible chew!cal and biological warfare on different civilian population groups-the purely military aspects ora largely not considered qualitative and quantitative estimates of the health affects of selected chemical and biological agents employed under specified hypothetical conditions are made. Among the agents discussed are the anti-plant agents 2,4-0 2,4,5-T, piclorana cacodyltc acid monuron brcmacil agent white, agent blue and agent purple as wall as tabunt sarin and soman. References are given at the ends of sections. GENERAL 70/11/00 997 1970 ....^ \ 133 13S 1I AU TI SI SO Ji- /-. - Galson AM - Plants people and politics. - HAPAB/70/00844 - BioScience; 20(7): 405-10, 2970; (REF:17> - HAPAB Contemporary man is confronted with many problems concerning social urw-ast, pollution and overpopulation. Some of the chemicals that man depends so much on to produce high levels of agricultural products are having deleterious effects on man and his environment. Chlorinated hydrocarbons like DOT, dieldrin and aldrin, once considered very useful, have now become menaces because of their persistence in the biosphere and their potential poisoning of various kinds of creatures. Present and future pesticides must be more thoroughly test4d and then chosen and used with greater selectivity. Some of tha more recently introduced herbicides have been shown to be potential herbicidal analogs of DOT. (Vie of the suspected chemicals is 4-amino-3>56trichloropicolinic acid t picloram or Tordon ) . Uhder optimum conditions is some soils, 20 to 50X of applied picloram disappeared after 467 days ( Youngson at al., 1967 ) . On other soils low in moisture and poor in inorganic matter and air, only 3.3A of the applied picloram disappeared in a similar period. It takes between 10,000 and 100,000 parts of exogenous carbon to oxidize one part of Tordon t Tschirley, 1969 ) . The dangers of further use of this herbicide are evident. Another chemical 2.4- 0, can also become a menace under certain conditions by causing a massive increase in the nitrate content of pasture plants as to sicken animals eating these plans ( Stabler and Mhitehead, 1950 ) and must be used very carefully. Pesticides fabricated around heavy metals, such as lead and mercury, or other elements such as arsenic ( trivalent ) do not become completely detoxified once applied. The presence of rising quantities of lead and mercury in the environment has caused growing concern in public health. Science must not be misapplied. Thoughtless and indiscriminate use of chemicals such 2.4- 0 picloram, 2,4,5-T and arsenical cacodylic acid by industry, the military and other groups and organixations has and may continue to cause ecological havoc. The legal structure of our government as well as the influence of internajttonal committees and commissions offer many ways to promote rational use and limited control of our environment and must be amployed before it Is too late. GENERAL 70/07/00, 263 1970 133 i -j JL 2C7 6^ r' J 78 AU TI /^7l SsIo - ANON - Degree of hazard when using herbicides. - KAPAS/73/82233 - S. Afr. Sugar J.; 57(2): 71; 1973 ; (REFsl) - HAPAB Tha aoat useful factor for assessing tha potantlal hazard of a pasticida to husans and domaatic animals although not completely reliable la tha acuta toxicity of tha chemical. Oral and dermal toxicitiaa according to tha L050 valuaa> ara quita high for paraquat ioxynil, diquat and aoma othar harbicidas while picloram bromacil asulan simazine dalapon and diuron have ralativaly low toxicitiaa. Inhalation toxicity of aatariala ia ora difficult to aaaaaa than dansal or oral toxicity and ia ganarally laaa iportant axcapt in tha caaa of apacific fuafgwits and othar volatile atarials. 13G o CD no c_n 75 AU - Nailanda JB Tl - Survey of chemical and ralatad uaapona of uar. SI - HAPAB /73/2870 l SO - Naturuiaaanachaftan60: 177-183; 1973(PEF:29) AB - HAPAB. Tha uaa of chanleal paaticidas in warfare probably began in 1961 when herbicides ware employed to defoliate areas of S. Vietnam. Agent Orange the primary defoliant is a mixture of 29-0 and 265-T and was used unt 11 1970 whan it was. shown highly teratogenic. A 5-fold increase in tha incidanca of primary nepatoma in heavily defoliated areas of S. Vietnam was believed caused by residues of tetrachlorodioxin in tha mixture. Agent White a mixture of 26-D and picloram and substituted for Agent Orange retains its phytotoxicity in soil for decades. Organophosphates have also been produced for warfare although their use has not often been documented. These compounds attack the active center of AChE causing an accumulation of ACh and a consequent paralysis of voluntary and involintary muscles; concentrations of as little as 0. 01 mg per m(SUP)3 of air are almost instantly fatal. They are rapidly hydrolyzed in alkaline solution and poisoning can be treated by simultaneous administration of atropine and pralidoxime. CD 1 3 5 ;CO 85 AU - Westing A H TI - Herbicides in war: current status and future doubt. n t SI - KAPAB/73/01016 SO - Biol. Conserv.; 6(5): 322-327; 1972 ; (REF:i3) AB - HAPAB Approximately 26 million kg of 26-0> 22 millton kg of 265-T 1.5 million kg of picloram and 3 million kg of dimethylarsinic acid were used by the U.S. to clear forests and damage crops in South Vietnam between 1961 and 1971. Single applications were made on approximately 152 of the forest area and multiple ones on four percent. Alterations included reduced diversity of plant and animal communities loss of mineral nutrients accelerated erosion and reduced product!vity. Flights over affected areas showed almost complete recovery of agricultural areas but feu signs of regeneration in mangrove and forest areas It is estimated that one decade will be necessary to restore singly-sprayed areas and several decades for the multiply-sprayed areas. Timber losses probably totaled 67 million cubic meters and rice destroyed would have fed about 896000 people. Farmers and woodsmen have experienced mild temporary ailments attributed to herbicides. Death and illness of livestock reported by civilians were probably caused by the anti-personnel chemical 'CS' (ortho-chlorobenzalmalononitrila) or the malathion used in malaria control programs. 134 182C8 U. S. D EPA RTM EN T O P HEALTH, EDUCATION, AND VyELFARE FOR RELEASE IN A.M. PAPERS National Institutes of Health Tuesday, February 7, 1978 Office of Cancer Communications (301) 496-6641 ! DOH 215 7 2 5 0 Availability of a report on animal tests of picloram for cancercausing activity (carcinogenicity) was announced by HEW's National Cancer Institute in today's Federal Register. Technical-grade picloram, a systemic herbicide, was given in feed to rats and mice for 80 weeks. According to a summary of the report included in the announcement, under these test conditions the incidence of neo plastic nodules of the liver was suggestive of the ability of the compound to cause benign liver tumors in female Osbome-Mendel rats. In mice, picloram was not found to cause tumors. The tests are part of the Institute's Carcinogenesis Testing Program. Copies of the report, Bioassay of Picloram for Possible Carcinogenicity, are available from the Office of Cancer Communications, National Cancer Institute, Bethesda, Maryland 20014. ,167 210 6 2 8 S IZMOO i vt K ' 'k-bl'X - Ctcc) y & K- - Cicc) ^ ; D0W2 I 5 8 4 3 0 Scand j work environ health 8 (1982) 37--42 a> Mortality of 2,4-dichlorophenoxyacetic acid and^2,4,5-trichlorophenoxyacetic acid herbicide applicators in Finland First report of an ongoing prospective cohort study by Vesa Riihimaki, MD, MSc, Sisko Asp, MPoISc, Sven Hernberg, MD ' RIIHIM AKI V, ASP S, HERNBERG S. M ortality of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid h erb icid e applicators in F inland: F irst rep o rt oi an ongoing p ro sp e c tiv e c o h o rt s tu d y . S ca n d j w o rk environ h ea lth 8 (1982) 37-- 2. S o m e recent epidem iologic studies have suggested th at chlorinated phenoxy acid herbicides are h u m a n c a rc in o g e n s. T h e m o rta lity e x p e rie n c e in a c o h o rt of 1,926 m en w h o h a d s p ra y e d 2,4-dichlorophenoxyacetic acid (2,4-D) an d 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) d u rin g 1955-- 1971 h a s b e en fo llo w e d p ro s p e c tiv e ly fro m 1972 to 1980. T h e to ta l p h e n o x y acid exposure w as generally ra th e r low because the duration of w ork had m ostly been less th an tw o m onths. In 1972-- 1976 m o rta lity from all n a tu ra l causes in th e co h o rt w as only 54 / of th e e x p e c te d v a lu e (b a se d o n a g e-sp e c ific ra te s fo r th e g e n e ra l p o p u latio n ), a n d in th e su c c e e d in g 4 -a p e rio d 81 */ of th e e x p ec te d v a lu e . In th e assessm ent of cancer, m o rtality allow ance w as m ade for 10- and 15-a periods of latency betw een the first exposure and the sta rt of the recording of vital status during the follow -up. No increase in cancer m ortality w as detected, and the distribution of cancer types was unrem arkable. No cases of death from lym phom as or soft tissue sarcom as w ere found. The study resu lts m ust, how ever, be view ed w ith great caution ow ing to the sm all size of the cohort, the low past exposure, and the brief follow -up period. K ey terms: ch lorinated p h enoxy acids. The herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) have been used in F inland since about 1955 to destroy brushwood vegetation. Their peak con sumption occurred during the latter part of the 1960s w hen 50-- 70 t of active ingredients of both compounds were sprayed p er year. Since 1972 the use of these chemicals has declined. Four governmental organizations or stateowned companies (the Finnish State Rail ways, the Highway Authority, the Forestry Authority and an electric power company) have been responsible for the m ajority of herbicidal use to control brushwood, and during the years of intensive appli- 1 Institute of O ccupational H ealth, H elsinki, Finland. R eprint requests to: D r V R iihim aki. In stitu te o f O c cu p a tio n a l H e a lth . H a a r tm a n in k a tu 1, SF-00290 H elsinki 29. F in lan d . cation about 500 w orkers, alm ost exclu sively males, were employed annually in this w ork (20). The chemical control of brushwood m ainly involved the spraying of foliage in rights-of-w ay (railroads, highways, electric transmission lines) and in forests. The spraying season was restricted to about two to eight weeks during the sum mer. Spraying was, for the most part, perform ed with portable, manually oper ated or m otor-driven knapsack rigs, w ith the exception of that done by the Highway A uthority which mainly used moving ve hicles. T hroughout the 1950s and 1960s th e methods of application and the work habits did not change appreciably. Be cause the workers were not informed of .any hazards connected w ith the spraying, no special precautions w ere taken to avoid inhalation of the aerosols or skin contact. 0355-3140/82/010037-06USD2.50 0 H215843 1 The most extensively used commercial herbicide preparations were 2 : 1 m ixtures of emulsified esters of 2,4-D and 2,4,5-T dissolved in water. Even pure 2,4,5-T prep aratio n s were used. It is now well appreciated that the eventual hazards from the use of 2,4,5-T m ay be associated with a toxic dioxin im purity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It is not known w ith'~certainty how extensive the exposure to TCDD may have been in the early years of 2,4,5-T application. A nalysis of some old herbicide preparations used in F inland in 1962-- 1967 suggested th a t the TCDD content ranged betw een 0.1 and 0.9 mg/kg; in one of the samples it was below the detection lim it (18). Public concern about alleged adverse health effects from brushwood-control herbicides precipitated an extensive survey of the situation in 1972 by an ad hoc w orking group (20), w hich, how ever, did not find m aterial evidence to support such claims w ith regard to m orbidity. However in a nationwide search for suspected phenoxy acid-related symptoms and signs, several reports of skin disorders were received. These mostly pointetfe.to ob vious toxic reactions, and, in at feast one case, the skin disease, which was also later seen by a dermatologist, suggested a mild form of chloracne. A m ortality study incorporated into the survey did not allow any conclusions to be draw n regard ing m ortality rates in general and cancer m ortality rates in particular owing to the doubtful validity of the cohort and the short period, of latency. It was intended to follow this cohort of herbicide sprayers in the yeacM o come. New siflpicions of a possible carcino genic- effect -of exposures to chlorinated phenoxy acids w ere raised in Sw eden (1, 8), and they prompted us to begin a 5-a pro spective m ortality study in 1977. An in terim report was prepared at the request of the dtatem ational Agency for Research on C ancer (19), but it was not refined into a scientific report because we had reser vations as to its power to provide signifi cant evidence with respect to the problem of the cancer risks associated with phen oxy acid exposures. More recently further epidemiologic studies (2, 5, 11, 12) and a case rep o rt (9) have shown or suggested an association betw een exposure to chlorinated phenoxy acids 'and increased cancer risk. In res ponse to the renew ed public concern. Finnish health authorities established a new w orking group in 1979 to evaluate risks associated with the use of chlorinated phenoxy acids (and some other herbicides). The working group supplemented earlier m ortality studies by assessing the cancer m orbidity in the cohort in 1967-- 1974 through the files of the Finnish Cancer R egister. The observed num bers of different types of cancer did not signifi cantly differ from those expected on the basis of the cancer incidence in the general population, and no cases of soft tissue sarcom a w ere found (15). Subsequently, a further 4-a prospective follow-up of m ortality in the original cohort was instituted. -J- The debate on the hazards of chlorinat ed phenoxy acids still continues, and only a limited num ber of studies have been made available for the assessment of risks. O ur observations, while not formally published, have been referenced iry^bis context. We therefore felt it was ing^ftt- ant to present the results of this 9-a pro spective follow-up on the m ortality ex perience of the Finnish 2,4-D and 2,4,5-T herbicide applicators in detail although we acknowledge the prelim inary nature of these results. M aterial and methods The cohort of herbicide applicators was collected in 1972 from th e personnel records of the four main Finnish em ployers involved in chemical brushwood control. The cohort included 1,971 m ale workers who had been exposed for at least tw o w eeks d u rin g 1955-- 1971 to 2,4-D and 2,4,5-T. T he d ata w ere collected by office personnel in the respective com panies from different sources including old payrolls, because the files did not al ways provide inform ation on specific assignments. Therefore, in some cases the files of exposed w orkers were based on the memory of the forem en and clerks, and the reported exposure characteristics, eg, the duration of exposure, w ere likely to be imprecise. Special instructions were given to guarantee that pensioners and those 38 0 0 H 2 I 58 4 32 who had changed jobs would not be om itted. The duration of exposure to the herb i cides had been quite brief. Threefourths of the study population had a sum m ed exposure d u ratio n of two to seven workweeks, and only one-fourth of eight w ek s or more, as of 1071. divided over one or more sum m er seasons. For many persons in the cohort further exposure m ay have taken place after 1971. About tw o-thirds of the study population had had their first exposure rather recently, in 1966-- 1971. At the onset of exposure one-third of the individuals in the cohort had been less than 25 a of age, and the study popu lation is still relativ ely young. In the beginning of 1972 the m edian age of the F o restry A u th o rity em ployees was 33.5 a, and the corresponding figures for the Highway A uthority employees, power company employees and State Railways em ployees w ere 38.0, 38.3 and 47.8 a, res pectively. D uring 1955-- 1971, 45 individuals in the cohort of 1,971 persons died. Thus in the beginning of 1972 th ere w ere 1,926 persons alive who w ere follow ed through 1972-- 1980. The lists of nam es and identification numbers were checked against the popula tion data register at the Social Insurance In stitu tio n , w here ev ery citizen is regis tered. In all, 15 individuals could not be traced, and they were removed from the cohort. For those who had died, the underlying causes of death were obtained from death dertificates registered at the C entral Statistical Office. The expected num bers of deaths from different causes were calculated by m ultiplying the age- specific person-years of observation by the cause and age-specific national death rates for 1975. The fractional age group values w ere sum m ed to yield correspond ing figures for the whole cohort. The person-years and expected numbers of deaths w ere assessed in a conventional way. as well as by making allowance for the m inim um of 10- and 15-a periods (of latency) between the first exposure and the start of the recording of vital status during the follow-up. Results During the 9-a prospective follow-up period the cohort accum ulated 16,694 p e r son-years of observation, and 105 persons had died from n a tu ra l causes versus 155 expected. The follow-up was divided into two tim e periods, 1972-- 1976 and 1977-- 1980, and the observed, as well as ex pected, d eath s are presented in table 1. In 1972-- 1976 the n um ber of deaths from all n a tu ra l causes was only 54 /e of the ex pected value, whereas, during the succeed ing 4-a period, the observed deficit in deaths w as less th an 20 9/o (observed/expected = 0.81). A t variance to the pre viously published m ortality data over 1972-- 1976 (19) the present investigation found one m ore death from cancer and six additional deaths from other natural causes. One case had been om itted due to a technical erro r. T he loss of three cases may be attrib u ted to a delay in reporting since the deaths had occurred at the end of 1976, w hereas the inability to find the th ree others is unexplained. In the present investigation an e rro r of this kind is un- Table 1. Observed (O) and expected * (E) deaths among 2.4-dichlorophenoxyacetic acid (2.4-0) and 2.4.5-trichlorophenoxyacetic acid (2,4.5-T) applicators during the prospective follow-up period of 1972-- 1980. In the calculations no allowance was made for latency period. Follow-up period Number of person- years All deaths OE All cancers OE All natural causes of death OE 1972-- 1976 1977-- 1980 1972-- 1980 9,442 7,252 16.694 62 92.9 82 91.1 144 184.0 9 17 26 a Based on age-specific national death rates for males in 1975. 18.1 18.4 36.5 41 76.7 64 78.6 105 155.3 39 i Table 2. Observed (O) and expected a (E) deaths among 2.4-dichlorophenoxyacetic acid (2.4-0) and 2.4,5-trichlorophenoxyacetic acid (2,4,5-T) applicators during the prospective follow-up period of 1972-- 1980. Allowance was made for 10- and 15-a latency periods in the calculations. Cause of death 10-a period ol latency OE 15-a period ol latency OE All causes All cancers Lung cancer All natural causes 88 118 6 20 24.3 12 11.1 71 103.6 a Based on age-specific national death rates for males in 1975. 35 5 4 33 53.6 11.3 4.7 48.1 m 8 SIZMOGi Table 3. Localization of malignant tumors found among 2.4-dichlorophenoxyacetic acid (2.4-0) and 2,4,5-trichlorophenoxyacetic acid (2.4,5-T) applicators, and expected values.1 during the prospective follow-up period 1972-- 1980. Allow ance was made for a 10-a latency period in the calculations. Site or type of tumor Observed number of cases Expected number of cases Lung Stomach + esophagus Prostata Multiple myeloma Metastatic, unknown origin Other sites and types Total 12 4 2 1 1 -- 20 11.1 3.7 1.1 0.2 0.3 7.9 24.3 * Based on age-specific national death rates lor males in 1975. 171) w ere found (table 3). Follow -up for m ortality in the four em ployer groups w ith the 10-a latency period included (table 4) showed a m arked and unex plained underm ortality among the High way A uthority employees. Although there were uncertainties with regard to data on the actual duration of exposure, m ortality figures were assessed separately for the reportedly more heav ily exposed populations. M ortality fig ures (all natural causes and all cancers) for those exposed for six weeks or more or during 5 a and for those exposed for eight w eeks or more or during 5 a (table 5) did not uncover any indications of an in creased m ortality in these subgroups. likely since the search for individuals in the cohort was performed by the register officials themselves. W hen allow ance was made for 10- and 15-a latency periods, the num bers of per son-years w ere restricted to 8,445 and 2,888, respectively, and the num bers of d eaths from n a tu ra l causes w ere 71 and 33, respectively. E ven in these populations there w ere considerably few er natural d eaths th a n expected (m ortality rates 69 /o of th e expected values) (table 2). The total num ber of cancer deaths was also lower than that expected, while the occurrence of certain cancer types approximated the expected values. The distribution of different types of m alignant tum or in the population w ith a 10-a latency period was rather unrem arkable, and no cases of death from lymphomas [International C lassification of Diseases (ICD) 200-- 202, 8th revision] or soft tissue sarcomas (ICD Discussion We have followed the m ortality of a cohort of 1,971 men exposed over a period of at least two weeks to 2,4-D and 2,4,5-T herbicides during 1955-- 1971. Because of the methods used in collecting individuals for the cohort in 1972, selection bias could not be totally excluded, and thus the validity of the retrospective follow -up is questionable. For the prospective follow up of all those alive in 1972 (totaling 1,926 men) similar problems do not exist. The results of this investigation show that the general population was not an appropriate comparison group for the herbicide applicators, probably because of work requirements which selected ablebodied and healthy individuals. This un derm ortality seems to decrease w ith time, a phenomenon typical of the "healthy w orker effect" (16), although even 10-- 15 a after the first exposure, at a relatively 40 O m 1o e !8 s I ZMOt n young age, n atural m ortality was still slightly iess than 70 "/o of th a t for the general population. With regard to cancer m ortality the h e a lth y w o rk er effect is know n to play a less significant role (16), and the present observations show the same tendency. Chem ically-induced cancer has a long inducation-latency period, and therefore it was necessary to assess the m ortality ex perience of the herbicide applicators after a relativ ely long tim e, at least 10 a, had elapsed since the first exposure. This was even more im portant because the estim at ed total exposure had been quite low and of sh o rt du ratio n . W hen allow ance was m ade for 10- and 15-a periods of latency, which of course restricted the small ma terial even more, no increase of cancer m ortality was uncovered. Furtherm ore, the distribution of the cancer types was un rem arkable. No cases of death from lymphomas or from soft tissue sarcomas w ere found in th e population w ith the 10-a latency period, the expected values being 0.8 fo r lym phom as and 0.1 for soft tissue sarcomas. No inform ation was available on the sm oking habits of th e individuals in the cohort. The question arises of whether the pres ent findings can be used in support of argum ents that exposures to 2,4-D and 2,4,5-T, and to im purities in their com m er cial preparations, do not cause increased long-term risks of fatal diseases, particu larly cancer. This investigation cannot be regarded as a conclusive negative study for a num ber of reasons: (i) the cohort was relativ ely sm all, (ii) the exposures to h er bicides w ere low, p articu larly in term s of duration, and (iii) the follow -up period was still too brief to dem onstrate in full the possible cancer risks from herbicide exposures. Obviously, further observations on the m ortality of this cohort with the passage of time will perm it the drawing of more valid conclusions. Yet, it needs to be pointed out th at the present results do not confirm the pre viously shown association between mixed herbicide exposures and increased overall cancer risks (2) as fa r as specific exposures Table 4. Observed (O) and expected * (E) deaths among 2.4-dichlorophenoxyacetic acid (2,4-0) and 2.4.5-trichlorophenoxyacetic acid (2,4,5-T) applicators of different employers during 1972-- 1980. Allowance was made tor a 10-a latency period in the calculations. Cause of death State Railways (2.209 person-years) Highway Authority (2.302 person-years) E Forestry Authority (3.343 person-years) OE Power company (591 person-years) 0E Alt causes All cancers All natural causes 39 49.0 12 27.4 10 10.6 3 5.4 34 44.7 10 23.3 * Based on age-specific national death rates lor males in 1975. 31 6 22 35.1 6 6.9 1 29.5 5 7.1 1.4 6.1 Table 5. Observed (O) and expected a (E) deaths among the more heavily exposed 2.4-dichlorophenoxyacetic acid (2,4-0) and 2.4,5-trichlorophenoxyacetic acid (2.4.5-T) applicators. Cause of death No latency period Person- _ year _ E 10-a period of latency Personyears o 15-a period of latency Personyears o E Exposure lor six weeks or more during 5 a All natural causes All cancers 6,300 34 66.0 6,300 8 15.4 Exposure lor eight weeks or more or during 5 a All natural causes All cancers 4,338 23 50.9 4,338 5 12.0 3.588 29 3.588 a 2.717 2.717 21 5 * Based on age-specific national death rates tor males in 1975. 48.0 11.3 40.1 9.5 1.378 15 23.9 1.378 1 5.7 1,151 1.151 13 21.5 1 5.1 41 ^^H* I to 2,4-D and 2,4,5-T are concerned. N either did our observations support the finding that a m arkedly increased risk of lympho ma is associated w ith exposures to ch lor inated phenoxy acids (11). T he association between an increased risk of soft tissue sarcoma and exposures to chlorinated phenoxy acids, chlorinated phenols, and th e ir chlo rin ated dibenzodioxin (TCDD in particular) and dibenzofuran im purities has been strengthened by recent epidemi ologic (6, 10) and clinical (3, 13, 17) ob servations. Unfortunately, no certain con clusions on the risk of soft tissue sarcoma can be draw n in the present study because the m aterial is sm all. TCDD is a recognized anim al carcinogen, whereas the chlorinat ed phenoxy acids, including 2,4,5-T, are not (4, 7, 14). H ow g reat a poten tial risk of cancer for m an is associated w ith ex posures to 2,4,5-T containing sm all am ounts of TCDD is not known. The results of this investigation can only sug gest that m inor exposures to 2,4,5-T do not pose great risks of cancer. Acknowledgment The authors wish to thank D r L Teppo for his valuable comments on the m anuscript. References 1. Axelson O. Sundell L. Fenoxisyror och cancer. Lakartidningen 74 (1977) 2887-- 2888. 2. Axelson O, Sundell L. Andersson K, Edling C, Hogstedt C. Kling H. Herbicide expo sure and tumor mortality: An updated epidemiologic investigation on Swedish railroad workers. Scand j work environ health 7 (1980) 73-- 79. 3. Cook RR. Dioxin, chlorance, and soft tissue sarcoma. Lancet 1 (1981) 619. 4. Environmental Protection Agency. Decision and emergency order suspending registra tion for the forest, rights-of-way, and pasture uses of 2,4,5-T: A. Findings relat ing to adverse effects in test animals. Fed regist 44 (1979) 15878-- 15880. 5. Eriksson. M, Berg N, Harden L, Moller T, Axelson O. Casecor.trolstudie over maligna mesenkymala mjukdelstumorer och ex position for kemiska substanser. Lakartidningen 76 (1979) 3872-- 3875. 6. Eriksson M, Hardell I, Berg NO, Moller T. Axelson O. Soft tissue sarcomas and ex posure to chemical substances: A case- referent study. Br j ind med 38 (1981) 27-- 33. 7. FIFRA Scientific Advisory Panel. Report of the FIFRA Scientific Advisory Panel on proposed section 6 (b) (2) Notices for 2,4,5-T and Silvex. Washington, DC 1979. 20 p. 8. Hardell L. Malignant mesenchymal tumours and exposure to phenoxy acids -- A clinical observation. Lakartidmngen 74 (1977) 2753-- 2754. 9. Hardell L. Malignant lymphoma of histocytic type and exposure to phenoxyacetic acids or chlorophenols. Lancet 1 (1979) 55-- 56. 10. Hardell L. Relation of soft-tissue sarcoma, malignant lymphoma and colon cancer to phenoxy acids, chlorophenols and other agents. Scand j work environ health 7 (1981) 119-- 130. 11. Hardell L, Eriksson M, Lenner P. Maligna lymfom och exposition for kemiska sub stanser, sarskilt organiska losningsmedel, klorfenoler och fenoxisyror. Lakartidning en 77 (1980) 208-- 210. 12. Hardell L. Sandstrom A. Case-control study: Soft-tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. Br j cancer 39 (1979) 711-- 715. 13. Honchar PA, Halperin WE. 2,4,5-T, trichlorophenol, and soft tissue sarcomas. Lancet 1 (1981) 268. 14. International Agency for Research on -Cancer. IARC monographs on the evalua tion of the carcinogenic risk of chemicals to man. Number 15. Lyon 1977. 354 p. 15. Laakintohallituksen tyoryhma. Laakintohallituksen tyoryhman mietinto no 4. Vesakontorjunta-aineet. Helsinki 1980. 40 p. 16. McMichael AJ. Standardized mortality ratios and the "healthy worker effect" : Scratching beneath the surface. J occup med 18 (1976) 165-- 168. 17. Moses M, Selikoff IJ. Soft tissue sarcomas, phenoxy herbicides, and chlorinated phenols. Lancet 1 (1981) 1370. 18. Rappe C, Buser HR. Bosshardt H-P. Identification and quantitation of poly chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in 2,4.5-T ester formulations and Herbicide Orange. Chemosphere 7 (1978) 431-- 438. 19. Riihimaki V, Asp S, Seppaiainen AM. Hernberg S. Symptomatology, morbidity and mortality experience of chlorinated phenoxyacid herbicide (2,4-D, 2.4.5-T) sprayers in Finland: A clinical and ex perimental study. Working paper for an IARC working group meeting on "Co ordination of epidemiological studies on the long-term hazards of chlorinated dibcnzodioxins and chlorinated dibenzo furans." Lyon 10-- 11 January 1978. 10 p. 20. Vesakontorjunta-aineiden terveydellisia vaikutuksia tutkineen tyoryhma. Vesakon torjunta-aineiden terveydellisia vaikutuk sia tutkineen tyoryhman mietinto. Helsinki 1973. 69 p. Rcceived (or publication: 16 December 1981 42 169 O O rv> en co co 2,4,-D (2,4-dichlorophcnoxyacetic add) !. 2.4-D, n-butyl ter 1. 2.4-D, isooctyl ester (Ded-Weed LV-69) 1. Dibenzofuran ' 2.7-Dichlorodbenzo-p-doxin (DCDD) . DMA-4 (2.4-D/dimethylamine salt) . 2,4.3-T (2,4,5-trichlorophenoxyacetic i---------------------------- - h - ' - ! k - s-yj'Witf) >' 1h i ve- i V . q S - L l c d ) ? - 1 k - z o o e i-C ic o )* > ' k_- W b - L i c o \ V r , K- ' m S i - C i e o Y i ve- e e s - Cioi add) . 2 .4 ,5 -T , /-buyl ester ; k - l ' i s t e - L i e o ) ? . 2 ,4 .) /. iv ib u iY l e tte r ; W ~ O itS - 5 H - n i'- ( ic o ) P 2.4.3-T. isooctyl ester (Ded-Weed LV-4T) yK ~ l a g n i - ( j c o ) p 2.3.7.8-Tclrachlorodibenzo-/>-dioxin (TCDD) i K- *-^ 8 4 1. . ...------ ---- . ^ ::'ro:feY-I &W&S&3 Mutagenicity Testing of Agent Orange Components and Related Chemicals Kristihn M o r t e l m a n s ,* St e v e Ha w o r t h T W illia m Sp e c k ,i a n d Er r o l Z e ig e r j ' M icrobiol G enetics D epartment. S R I International. 333 Ravensw ood Avenue. M enlo Park, California 94025: tG enelic Toxicology Division. M icrobiological Associates. 5221 R iver Road. Bethesda. M aryland 20816: XDepartment o f Pediatrics. Case W estern Reserve University. School o f M edicine. Cleveland. O hio 44106; a n d C e llu la r a n d G enetic T o x ic o lo g y Branch, n a tio n a l In s titu te o f E n v iro n m e n ta l H e a lth Sciences. P.O. B o x 12233. R esearch Triangle Park. N orth C arolina 27709 R eceived M arch 26. 1984; accepted M a y 18. 1984 Mutagenicity Testing of Agent Orange Components and Related Chemicals. M ortelmans, K,, H a w o rth , S., Speck, W., a n d Zeicer, E. (1984). Toxicol. Appl. P harm acol. 75, 137-146. Components ofthe herbicide Agent Orange--2,4-dichlorophenoxyaceiic acid (2,4,-D) and 2,4,5- irichlorophenoxyacetic acid (2,4,3-T) and fheir esters, and the contaminant 2.3.7,8-leirachlo- > s todibenzo-p-dioxin (TCDD)-- and related chemicals were tested for mutagenicity using Salm onella ty p h im u riu m strains TA98, TA100, TA1535. and TA1537. No mutagenic activity was observed for any of the chemicals tested. The herbicide Agent Orange, used as a de foliant in Vietnam during the 1960s, was for mulated as a 50:50 mixture o f butyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyaceticacid (2,4,5-T). It also contained free 2,4-D and 2,4,5-T, as well as a number o f other esters, di- and trichlorophenol, and 2,3,7,8-tetrachiorodibenzo-pdioxin (T C D D ) (Hughes el a l.. 1975; Young e t al.. 1978). D ioxins, including T C D D , are produced during the manufacture o f 2,4,5-T and other pentachlorophenols, and were pres ent as contaminants o f the commercially used products (IA R C , 1977). Although there is no new exposure to Agent Orange, 2,4-D is currently used by home gar deners, and both 2,4-D and 2,4,5-T are used for industrial control o f rangeland, forest land, and in the cultivation o f rice (R. Mountfort, E P A , January 1984, personal com m unica- I 1Towhom all correspondence andrequests for reprints should be addressed. tion). D ioxins, specifically T C D D , are a by product o f 2,4,5-T manufacture (IA R C, 1977), have been reported as possible products o f combustion (Rawls, 1979), and are biologi cally available in contaminated soil (M c Connell e t al.. 1984). Additionally, a number o f accidents have occurred that resulted in humans being exposed to polychlorinated dibenzodioxins and polychlorinated dibenzofurans (H u ff e t a l., 1980). Because o f the wide spread exposure o f Vietnamese and Am erican military personnel and civilians to Agent O r ange in Vietnam, and the widespread exposure o f farm workers and home gardeners to 2,4D and 2,4,5-T preparations, these substances were tested for mutagenicity in the Am es S a l m o n e lla assay. T h is report presents the S a lm o n e lla test re sults o f 2,4-D, 2,4-D/dimethylamine salt, 2,4D n-butyl ester, 2,4,5-T, 2,4,5-T n-butyl and isobutyl esters, T C D D , 2,7-dichlorobenzo-pdioxin, and dibenzofuran, in addition to com mercially available (1979) home gardening 137 004I-008X/84 $3.00 CovyritJH I9M by A adcm ie f i a t . 1ml Ai s h a of riproducilo* io toy form racrm d. o o ro cn CO CO O'! 7 h iA. 1 -m . IW J.'HJW . ' I OO TABLE I Agent O range-Related Chemicals T ested in S a lm o n e lla Name 1. 2,4,-0 (2,4-dichlorophenoxyacelic acid) 2. 2,4-0, n-butyl ester 3. 2,4-D, isooctyl ester (Ded-Weed LV-69) 4. Dibenzofuran 5. 2,7-Dichlorodibenzo-p-dioxin (DCDD) 6. DMA-4 (2,4-D/dimethylamine salt) 7. 2.4,3-T (2,4,3-trichlorophenoxyaceiic acid) 8. 2.4,5-T, n-butyl ter 9. 2,4,3-1, iu ih u lyl 1er 10. 2,4.5-T, isooctyl ester (Ded-Weed LV-4T) 11. 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCDD) CAS No. 94-75-7 94-80-4 25168-26-7 132-64-9 33857-26-0 2008-39-1 93-76-5 93-79-8 49.18-72-I 25168-15-4 1746-01-6 Supplier* Dow Radianr Thompson-Hayward Fisher IITRI Dow Dow Radian Radian Thompson-Hayward IITRI Loi AGRI7III4 NS F80553 B4X NS M924024 AGRI33711 NS NS 9057 NS Label purity (%) NS* NS 67 Technical NS NS NS NS NS 64 NS Analyzed purity W 99 99.6 _ _ _ -- 99+ 99.4 99.5 -- 97.6 ' Dow Chemical Co.. 202 Dow Cenlcr. Midlaml. Mich -IKM0; Radian ( orp., R.O. Hot A u s im i. T i; v. 7 X 7 1 hoinpsnn-l l.ivu.m l ( Iu ' u i k '.i I P.O. Box M s - K.nis.i\ Cits. Kam. 6 M I0 ; liih c r Sncnuiu. 1* i > Hi)\ |,M)7. Houston. Tex 77?M. W in.ms InsMtulc of Ki*st\jrs`h Insinui' t lue.uhi . Ill hllhlh. 'ixvificd (V>rp TOO o: oi ae ro ilj &8P `i, fS 0|-!iai ae i' ca 33 'h 3 3 ?-> a P 8. S 3 f I' a " 5 5 S ?, * K o fttS8lg**S E-SS. f 3 & " | : ik S:s b 3'iS.h'.<JS rii il? n ' N i h t n r i ^ r nI h g. "Hr g ' H " 2? * fN S Me M S-8 MUTAGENICITY OF AGENT ORANGE COMPONENTS 139 preparations o f 2,4-D (isoocytl ester) and 2,4,5- T (isooctyl ester) (Table 1). M ETH OD S All ch em ica ls w ere tested in a p re in cu b a tio n m o d ifi cation of the Ames Salm o n ella plate incorporation assay (Am et at.. 1975: Maron and Ames, 1983); details of theprotocol aredescribedelsewhere(Haworthet at.. 1983). Briefly, fresh overnight cultures of Salm o n ella typhim urium strains TA1535, TA1537, TA98, and TA100 (ob tained from Professor B, N. Ames, Berkeley. Calif.) were used with the 90Q0g supernatant fraction of liver ho mogenate (S9 fraction) fromAroclor 1254-induced male Sprague-Dawley rats or male Syrian hamsters. About 1 X 10* bacteria were mixed with 0.5 ml of either 0.1 m (pH 7.4) sodium phosphate buffer or S9 mix (containing 10%S9), and 50 or 100>dof the test chemical or solvent, in each of three tubes. This mixture was incubated at 37*C for 20 min, after which time 2 ml of molten top agar containing a trace amount of biotin and a growthlimiting amount of histidine was added. The mixture was then poured onto minimal agar plat and incubated at 37*C for 48 hr, after which time histidine-revertant colonies were counted. Table I lists the chemicals tested, source, purity, and grade. The testing laboratories received and tested all chemicalsascoded aliquots. Solubilityinformation, storage conditions, and safe handling procedures accompanied each chemical. All chemicals initially were tested in triplicate plat at five dose concentrations, separated by half-log intervals. Dimethyl sulfoxide (DMSO) was the solvent. The high dose was 10 mg/plate unless limited by solubility and/or toxicity. The high dose was selected based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S9. Because of the limited amount of TCDD available, it was rested up to 100 ng/plate in the first experiment and 1000ig/plate in the second. All tests were repeated at least once, at least 1 week following completion of the original test The repeat test was also performed in triplicate. The following directacting positive control chemicals dissolved in DMSO were used: for TA1535 and TA100, sodium azide; for TA98, 4-nitro-o-phenylenediamine; and for TA1537, 9-aminoacridine. 2-Aminoanthracene was used as the positive control for all strains in the presence of the hamster and ratliverS9 mixes. Concurrent solvent andpositivecontrols were included in all experiments. A mutagenic response was defined asa reproducible, dose-related increase in the number of histidine-independent colonia over the spon taneous incidence; there was no requirement fora specific magnitude of increase. Decoding took placeafterail testing was completed and the data were evaluated. RESULTS N ine chemicals were tested in only one lab oratory each, while 2,4-D and 2,4,5-T were each tested in two laboratories. N o mutagenic responses to the test chemicals were observed under any o f the assay conditions either in the original or the repeat tests; however, vary ing degrees o f toxicity were observed with the different chemicals. A ll positive controls gave the expected responses. Differences observed in levels o f toxic responses by the different bacterial strains can be explained by the fact that the strains are not isogenic. There is al ways the possibility that weak mutagenic re sponses could be obscured by cell toxicity. W hile this possibility has been considered, there is no available evidence which dem onstrates that some chemicals are mutagenic on the plate test only in the presence o f cell toxicity, and that this toxicity is sufficient to mask the mutagenic response. Table 2 presents the test results for each chemical. Since each chemical was tested at least twice, with the dose range adjusted for the second test based on the results o f the first test. Table 2 presents only the data from the final test o f each chem ical. DISCUSSION. Epidem iological studies have been per formed in an attempt to establish a correlation between exposure to chlorinated phenoxy herbicides and carcinogenesis in humans. Special attention was given to 2,4,5-T and 2,4D , m ajor constituents as n-butyl esters in Agent Orange, and contaminants o f these products. T C D D , the most biologically active and most widely studied dioxin, is o f particular interest because o f its carcinogenicity. In gen eral, the epidemiological data on the phenoxy herbicides are considered inconclusive because o f small sample sizes, limited exposure levels, and/or design o f the study (Coggon and Acheson, 1982). zimizHoo 1 O U '.9 {>?" r if: TABLE 2 T esting R esults Dote* NA* TAIOO RLI 0 i) 33.0 too.o 3330 t.000.0 3.3330 10.000.0 POS' 113 1 I4.J* 9} I M 1 107 1 1.0 102 1 4.7 111 l 11.3 79 l 4.9 136 i 6.1 143 i 127 1 13) t 143 1 53 6.7 3.2 4.1 6.3 hV 646 l 114 1433 1 2 7 7 J H U NA MU 131 i 6.9 129 t 124 1 in * 113 l 41 1 6.2 3.3 6.0 9.9 9.JP IS9I i 61.1 2.4-D (2.4-dirhlorophcnoayacciic acid) IDMSO J* | C W R ) ' 7 1 12 6 1 3.0 3 1 0.9 1 l 3.9 4 1 1.5 4 1 2.0 I x 00 I X 1.7 7 X 2.6 7 X 1.0 7 l 0.9 9 x 2.0 IIX 2.1 9 1 0.7 7 0.9 3 1 1.2 3 X 0.9 4 X 09 3 1 0 .) 4 l 0.7 3 X 1.2 I X 09 I1 0.7 6 X 2.0 7 X 1.7 9 x 2.2 6X 0 9 I.Op 1011 1 18.5 123 1 6.1 1274 i 234.2 6 l 0.9 61 1.3 6 1 0.3 3 i 0.9 I 1 0.7p 137 i 23.1 16 X 15 x 15 l 17 X 17 1 13 X 1.2 1.5 2.7 2.1 1.5 1.5 331 1 9.0 T a 98 21 x 3.2 21 x 3.3 17 1 2.3 21 X 4.1 21l 21 22 x 30 3 x 2 Sp II x 20 i 13 x 13 x 31 2.6 19 I9 20 2.6p 2637 i 261 2 0.0 100.0 333.3 1000.0 3J33.3 10.0000 POS I07 II 1 71 i 10 1 23 1 1l 4.2 6.2 3.) 3.2 3.1 0.3s 330 i 23.3 94 l 96 1 91 i II 1 91 i 01 37 9.6 4.1 3.6 3.3 0.0s 107 i 30.1 71 1 71 1 71 1 61 1 II 1 01 3.1 1.2 4.1 2.3 30 0 0* 1991 1 73.1 2.4-D (2.4<dichlorophcnoftycctic acid) |t)M S O | (SRI) 13 1 71 51 91 31 0i 3.0 2.6 1.3 1.7 0.9 0.0s 6 l 07 6l 0 7 3 l 0.7 4 1 00 01 00 7 2.7 3 l 0.9 7 0.6 6 1 07 3 1 0.7 I 1 0.7 3 i 0.9 3 1 0.3 4 x 0.6 3X 03 4 X 1.5 0 X 00 6 1 1.3 4 1 0.0 7 0.3 7 1 1.0 3 i 1.2 I 1 0 7 333 1 11.9 211 1 2 1 .2 413 1 24.4 64 1 426 189 1 13 9 3 l 1.2 7 X 31 3 1 1.0 3 l 0.3 3l 01 00.06 397 1 37.6 20 i 2) X 23 x 21 1 11 3X 2,7 3.1 4.6 20 09 2.3 784 X 31 8 30 X 1.7 29 t 3 4 28 X I I 24i 3 7 28 X 4.6 3 x 30 334 x 14.: 31 1 23 x 27 22 x 28 x 7 09 40 09 22 09 17 1664 x 901 0.0 0.3 1.0 3.3 10.0 3 ) JO 100.0 331.0 t.000.0 3.333.0 10.000.0 POS 14) i n it 111 1 141 i 131 1 121 1 7.7 3.9 1.3 7.0 1.9 3.3 1239 1 37.3 139 1 1.2 1)3 t 132 1 133 1 136 l 130 1 6.1 3.2 12.6 1.0 3.5 1032 1 14 6 132 1 14.9 165 i 149 1 161 i no i 161 1 3.1 13.1 6.4 21 2.9 1001 1 24.1 30 1 32 1 33 1 31 1 35 x 23 1 2.1 1.7 5.0 3.3 1.3 l.2 i 937 i 31.1 2.4-D. fl'hutyl (tie r |D M S O ) JILCG G JI II 1 2.0 17 i 0.3 14 1 0.6 13 1 2.0 13 1 0.9 16 1 0.9 II 1 0.3 111 2.3 19 i 1.3 13 1 2.9 12i 1.3 14 i 4.0 7 x 0.9 3 x 09 1 1 2,3 6X 07 7 X 1.2 7 1 18 60 1 3.1 127 i 43.3 3 1 22 3 l 1. 7 1 1.7 1 0 3 6 1 to 4 1 06 7i IX II II1 li1 14 1 22 X 17 x 21 1 16 X 16 X 1.9 2.0 2.0 2.9 ).i 1.7 1367 l 77.3 32 l 18 3 4 i 1.7 30 x 37 x 29 x 21 x 26 X 1.8 1.7 06 30 12 33 X 29 X 30 X 29 X 32 x 23 1.0 3.2 31 27 890 X 20.3 o o H rm" s: > ni -I > r rnwr7wuu7 92 i 12.6 1 0 3 i 3.1 10 i 13.3 90 i 4.2 12 i 11.4 61 i 10.1s 123 1 4.9 94 i 91 1 94 i 80 z n .) 28 3.0 3.1 91 1 6.7 107 x 11.0 86l 11.3 Il1 5.2 13 1 6.4 32 x 6 2 20 i 16 i 23 x 19 i 20 i 24 x 2.4-D. isooclyl exicr IDcd-wccd. LV-69 ) |D M S O | JSRIJ I 1 1.7 17 x 0.9 121 l.l 13 1 3 0 |A 10i 1.7 151 1.3 13 x 0.3 10 x I1 02.02 3 x 1.7 3i 09 6X 06 4 l 1.5 3 x 10 6 1 1.2 6 x 0.3 6 X 1.3 7 x 1.2 f1 7x 11..23 6 X 0.9 7 X 0.3 6 t 06 34 x 4.3 3J X 2.3 26 x 2 0 27 * 6.4 24 x 6 3 43 X 10 43 x 44 i 3 1 64 24 4A 33 x 2 3 I9 X 2.9 I3 i 0.3 12i 0.3 0OH2 I 5O*i I `t -- -- p 0 3 00-0 A +1 H H H 44 4 i 44 44 3 Q 5D 2 Cp --r* <-}"i Npi OC i M ti H H M t 2222"i s p p * p < Q H tttiH tt 1 p>& * ti -- -- 44 44 t i 44 f* > p * e i i r4141****'4O41 < 4 44 44 44 H 44 H 41 H H 44 *1 4 44 * i #^ ^^ V "^4 --^ ^*^--pi p-> -- o " O44 41 41 44 44 O O ** --44 44 H H 44 44 44 41 44 41 44 H f p* o --^ 44 44 41 41 44 41 p* -- p* *r 44 44 +4 H H 44 44 41 44 t i t i N N <e 41 41 41 41 P* O 44 41 N a0 41 44 M--0n 04444 4 44 41 H 44 41 41 41 44 44 44 m O p-t p r- -- Vp* r. ~'w-- *"i Ow41t Pr4**1 --41 >4-1i -A1 *444 44 44 44 44 44 44 oo32 142 MORTELMANS et AL. r9. d 0* 5X? >9 0*4"" _ n _ 5 d to-X>0 ~90 _ I <-' i 41 H M rt *0 09> 44M444| H44 *"* r* ~ 1 44444444H44 44 i rt r 10 o _ * -- o0 a 20 4 M*1 H04144 44 X-- -- 4400 /--**-- - -- 0 44(44i 0HX4444.1. *4 0H -- ri r i -- 040d9 <00 r4*4--44444T440*\4*04 H * __ n9 9 as rT d 10 r- g X 9o -y <0 O (> < 4 44 M 44 44 44 44 44 H 44 44 H 44 4 44 +4 44 44 44 41 H -- as 0 v 0 *T 0 -- 9 --0 0 0 0 --0 9 0 01 ** d as -- 99-- -- (N 9 n O5 --9 o ----9 r- ,, ,,O 9 <0 r* r i o -- o -- 9 0 4* 44 44 44 44 - 44 44 44 44 44 44 44 44 44 44 0 44 44 44 44 " -- 0 +\ 0 0 0 0 O *0 r - o 0 0 > - " TABLE 2-- C o n tin u ed II X 0 ' -- * < Z n * Ij \r- < as 0 _ 9 *> 9 #* n 4 4 1 44 44 -- 0 --. 0 0 44 r* ac 3 i d2T 1- 9* 0 --l 2 r> d 9 9 9 -- -- H t4 44 44 4 M s 0 O B 4 44 44 44 44 44 X 0 as -- <** * 2 44 O ' >4 44 >4 44 O r-> O 0 4; 44 44 Oa 0 ** r* 0 6 *1 -A r o 0 30 44 44 44 44 44 H 0 4 0 0 44 44 r0 r*i 5 41 44 44 44 44 44 0 r> O -- X 'L 9 r-t 5 599 d 2 44 44 44 44 * i ! 00 ri 9 e 44 44 44 44 H 44 f r* n 0 0 0 -- 2 9o w 44 44 44 44 44 44 r*. 0 0 a <-) _ .j O - a V* o M r ? 44 44 44 44 44 H 0 0 0 O -- 44 0 44 0 H *-> 0 <0 ri 44 N 9> 44 <n % --<0 a 44 44 44 44 44 44 <0 0 ---- 44 r>. d0 > ri O 90 ** <0 rr 4 4 44 H 41 0 00 s 7 O M ri (0 _ c 44 44 41 H 44 44 >* m m rx a 0 -- X * m 10 0 o 10 44 44 44 44 44 44 I! r> <0 a " 0V <0 44 w 44 9- 44 m H f0 O 0 <0 0\ 44 44 44 44 44 44 m-- *0 -- r as 44 *0 0 0 <9 )<144H ij --PI o " 4 ?4 44 44 44 44 44 tO 9 0 -- OS10 44 0 4 O o'> rv'a-- O 44 44 H 44 44 M 44 :- s-- :-- s_ 2-- j 430 ^r>'o d MsO9> a^?5o --g( 8 4 44 44 *4 44 44 44 < 9> O*O*"1 O0 0>--*. O >*> MK OM MM AM H m <a4< 444 i t CD Cj O <? 2E cn CD * cn 44 M * a ;? t f) 4 I. MUTAGENICITY OF AGENT ORANGE com ponents 0.0 100.0 333.0 1.000.0 3.3330 10.0000 POS 161 X 7.1 125 X 11.6 136 s 5.0 145 1 11.9 144 X 5.7 131 x 9.0i 1311 X 17.2. 149 7.8 144 X 7.1 141 X 2.6 107 X 12.0 100 X 4.3 102 X 5.0 1011 X 219 175 X 3.5 .164 X 2.7 162 X 4.9 152 X 6.0 144 X 10.7 115 X .: 1440 X 101.1 37 x 2.1 34 x 1.3 28 x 1.7 31 x 5.2 31 x 4.0 27 x 2.1 966 X 19.1 .O-T. isohutylester|UMSO| |IG(i| 17 x 09 16 x 0.7 (I X 2.6 10 x 0.9 14 X 1.9 10 X 0.6 17 X 20 13 X 21 12 X 2 1 II X 2.4 14 x 12 10 i 0 9 6 X 1.7 5x 06 5 X OU 6 t 1.0 3 07 4 1 06 II X 1.3 90 X 1.2 440 X 9 H 7 X 1.7 7 X 09 6 X 0.9 6 X 10 8 X 0.3 3 X 10 94 X 6 1 8 X 3.0 1 X 2.7 9X 03 9 X 1.8 7 X 2.0 3 x 1.8 134 x 1.2 21 X 32 19 X 2.3 19 X 3.5 17 i 15 21 X 3.1 II X 1.7 1480 X 28.6 34 x 10 27 x 4.9 27 x 1.9 26 x 2.0 25 x 09 14 X 00 1021 x 150 31 x 5.2 31 x 55 40 x 24 34 x 19 25 x 4 5 25 x 24 1405 x 549 00 3.3 100 330 1000 3330 POS 81 x 12.6 93 X 4.9 114 X 7.2 W) X 5.2 100 X 1.3 102 X 1.5 621 X 46.4 140 7.8 156 X 8.0 152 1 14.0 166 X 6.5 139 X ll.l 135 X 14.2 714 X 29.8 154 x 4.2 130 X 10.4 134 x 9.1 128 x 10.7 122 x 14.9 158 x 7.0 1093 x 28.0 .4.3-T.isooctykster(Ded-weed LV-4T) ID M SO) |CWR) 9 X 1.2 10 x 20 13 X 1.2 II X 1.5 9 X 1.2 II X 1.2 10 X 2.5 13 x 0.3 14 1 1.5 15 X l.l 15 X 1.2 15 x 2.2 10 X 1.0 II X 1.9 II X 2.0 13 i 3.2 10 2 0.7 12 x 2.0 5 X 0.3 6 X 03 5 x 0.6 5 x 09 6 X 09 5 X 0.6 6 X 1.0 12 1 09 9 x 22 9 x 1.3 10 x 2 1 10 X 2.3 616 x 93.4 92 x 20.3 120 x 9.3 137 t 39.5 44 i 0.3 II X 1.7 1 X 1.9 10 X 05 10 X 0.3 10 X 17 1 X 1.5 90 x 6.4 15 x 1.2 12 x 1.5 13 x 0.9 16 x 3 2 12 X 1.7 13 x 0.9 343 x 32 6 24 x 12 21 X 3.0 21 x 2.9 23 x 2 5 24 x 10 23 x 0.7 572 x 1214 22 x 1.3 23 x 35 22 x 33 21 x 31 21 x 1.) 22 i 23 803 x 1070 00 10.0 330 100.0 333.0 1.0000 POS 141 10.1 170 x 14.9 178 1 8.6 162 X 5.7 116 X 0.9 149 X 7.8 374 x 21.2 154 X 188 X 172 x 193 X 154 X 177 X 9.0 7.3 2.5 3.0 5.5 6.8 153 x 430 :55 117 i 117 i 111 X 124 116 x 12.8 12.3 7.5 0.6 12.4 9.4 1527 66.3 TCDD (2.3.7.1'Trirachlorodibcni<>>/>-dioain)(DMSO) (SRI| 13 X 1.3 15 x 3.5 17 1 0.6 17 0.7 11 X 0.6 18 X 3.9 8 X 1.0 11 X 25 II X 37 1 X 3.4 7 1 12 8 X 20 5 x 1.0 S X 0.7 II1 1.2 8 x 34 9 1 3.2 1 X 2.3 5 x 1.3 5 X 0.3 6 1 00 7 x 06 3 x 0.7 7 2.U 9 X 1.7 7 X 0.9 II X 1.0 4 i 1.2 8 1 10 7 1 1.2 301 x 17.0 121 2 144 242 x 12.2 122 1 III 164 X 4 2 3 x 00 5 X 1.5 4 X 0.6 6 X 3.0 8 x 0.3 7 X 1.5 41) 1 22 8 19 X 3.5 22 X 3.3 20 X 46 II X 3.7 Ut n 18 X 3.1 723 X 32-2 24 t 2.1 27 i 35 29 x 2.7 26 i 31 2) x 13 26 1 06 384 x 22 1 21 x 32 2) x 36 21 x 20 24 t 3.3 2) x 2 2 II i 28 1204 X 101 *ig/plate. *NA. not activated;RLI. Aroclor |254*induced nl liver$9; HLI. Aroclor 1254-induced Syrian hamster liverS9. *DMSO. dimethyl sulfoiidc (solvent). 'CWR. Cue Western Rcicrvc University;EGG. EGAG Maun Research Institute;SRI. SRI International. 'His* rcvtrunu: mean 1 SEM (threeplates). /p.precipitatepresent on plates;a.slightclcarin|ofbackground lawn; t.complete clearingofbackground lawn,colonics not counted. 1POS. positivecontrol.TAI00 and TA1333. NA: sodium uidt (CWR; 3.2pg/platc:EGG: 2.5ig/plate;SRI: 1.0jii/pluic);TA9K. NA; 4-nitro-r*-phcnylcncdiaminc(CWR: 1.5pg/piaic:EGG: 12-0ug/platc:SRI: 5.0ug/plate); TAI537. NA: 9-aminoacndine (CWR: 33 pg/pUie: EGG: 80 ug/platc;SRI: 50 pgZplaic). TAI00. TAI335. TAI537. and TA98 with S9: l-aminiMnthraeene (CWR. R U and IILI; 1.0. 20. 20.and 1.0 g/pljic:F.GG. RLI. I5 ug/plaie(allstrains):EGG. HU: 0.75 ut/pialc (allstrains):SRI. RLI and HLI: 1.0.2.5.2 5.and 1.0ug/platcl. GJ I; KGO *>.*v- N> . N co 9 1 <i8 S I ZHOG 144 m o r t e l m a n s e t a l . T C D D was carcinogenic for male and fe male O sbom e-M endel rats and B6C3F1 mice following administration in the diet for 2 years at dose levels o f 0.01, 0.05, and 0.5 n g /k g j week for rats and male mice, and 0.04, 0.2, and 2.0 jig/kg/week for female mice (National Toxicology Program, 1982a). These findings confirmed and extended an earlier 2-year chronic study which produced tum ors in male and female Sprague-Dawley rats fed 0.01 and 0 .1 n g TCDD/kg/day for up to 2 years (Kociba e t a i , 1978). T C D D also was carcinogenic for female, but not for male, mice in a skin-paint ing study (National Toxicology Program, 1982b). D C D D , however, was not carcino genic for O sbom e-M endel rats o f either sex, or female B6C3F1 mice, but produced mar ginal increases in a variety o f tum or types in male mice, which was considered as " sugges tive o f a carcinogenic effect" (National Cancer Institute, 1979). T C D D has also been reported to act as a promoter o f diethylnitrosamine carcinogenesis in the livers o f female rats (Pitot e t a i , 1980), and o f //-methyl-iV'-nitro-A'-nitrosoguanidine carcinogenesis o f the skin o f female, hairless mice (Poland e t a i . 1982). There appears to be disagreement on the bacterial mutagenicity o f T C D D . Hussain et a i. (1972) and Seiler (1973) reported T C D D to be mutagenic for S a lm o n e lla strain T A 1532 without metabolic activation. G ilbert e t al. (1980), using S a lm o n e lla strains T A 9 8 , T A 1 0 0 , T A 1530, T a 1532, T A 1535, TA 15 37 , TA1538, TA1950, TA1975, TA1978, and G 46 , and Geiger and Neal (1981), using S a l m o n e lla strains T A 9 8 , TA 1 0 0 , TA 15 35 , TA 15 37 , and TA 15 38 , reported T C D D nonmutagenic both with and without metabolic activation. The report by Hussain e t a l. (1972) is difficult to evaluate since the data arc pre sented only as mutation frequency against survival, and the positive response by Seiler (1973) was obtained in a spot test, which re quires that the test chemical diffuse into the agar. Although the dose and solvent are not indicated, the reported solubility o f T C D D in water (0.2 X 10~7 g/100 ml; 0.2 ppb), and in chloroform (0.037 g/100 ml) (Crummet and Stehl, 1973) makes it unlikely that T C D D would diffuse into the agar. T he studies by Gilbert e t a l. (1980) did use T A 1532, although Geiger and Neal (1981), as well as our study, did not use TA 1532, but instead used TA1537 which is isogenic to T A 1 5 3 2 and contains the rfa mutation (Am es e t a l.. 1975), which ren ders the cells more able to take up large m ol ecules. In all probability, the doses in our and all other reported studies were beyond the range o f solubility o f the T C D D . T his lack o f solubility m ay be the reason for the lack o f toxicity at doses up to 1000 #ig/plate in our study (Table 2) and at the maximum doses tested (2000 and 20 zg/plate) in the other two negative studies; Hussain e t a l. (1972), how ever, reported a mutagenic response at survival rates between approximately 30 and 1%. T h e lack o f agreement between the bacterial mutagenicity and carcinogenicity o f T C D D could be the result o f a num ber o f factors: the chemical was too insoluble to be taken up by the S a lm o n e lla (addressed above), the S9 preparations used were not capable o f metab olizing T C D D to a D N A -b in d in g moiety, D N A -b o u n d T C D D does not induce mutation in these S a lm o n e lla strains, or T C D D cannot form D N A -b in d in g moieties under the con ditions o f the S a lm o n e lla assay. There is evidence that T C D D is metabolized by rodents, in v iv o and in vitro , via an N A D P H -dep en den t cytochrome P-450-dependent mixed-function oxygenase (Gasiewicz e t a l.. 1983), albeit at a slow rate, but no me tabolites have been identified. T his inform a tion suggests that either the S9 mix used in S a lm o n e lla mutagenicity experiments is not the correct com position to support T C D D metabolism, or is not used under the optimum conditions (i.e., T C D D concentration, liver enzyme activity, incubation time, etc.) for metabolizing T C D D . However, there is no ev idence as to whether the metabolites o f T C D D represent toxic or detoxified products (Gas iewicz e t a i , 1983). There are re s onstrate the pxas If T C D D car! it probably inifec indirect m e ch a r 1 catalyze the in i* idases ( P o la n d mediate other'll! The lack o f a m o n e lla with 2.-4C is consistent wii* literature (Shiraa a i . 1977; Probit 1983; Schoeny, 1 published mutagt 2,4-D, 2,4,5-T, a o f 2,4-D. RE Ames, B. N,, McCab Methods for deteeb the S a lm o n e lla /n u c test. M u t t. R e s S X j Cocoon, D., andAq herbicides causes 1059. ; Crummet, W. B..ws nation of chloriiuae furans in variousraa ! } 5, 15-25. Gasiewicz, t . a ., Ota Neal, R. a . (1981) taboiism of 2,3,218 in experimental ubn R isks o f C hlorinates (R. E Tucker, A.U.' f 495-525. Plenum..1* Geiger, L E., andNs testing of 2,3.7,8to dine auxotrophsoAS Appt. Pharm acol. S tt Gilbert, P., SaintSU cter, M. (19SO).2ao< and azooenzenes tan E n viro n . C o n ta m . 2ft Haworth, S,, Law W., and Zeiger, X_( test results for 250dtae ! 3-142. Huff, J. E , Moore, J . E (1980). Long-torn- benzodioxins and pd! viron. H ealth Perspae MUTAGENICITY OF AGENT ORANGE COMPONENTS 145 T h ere are n o studies rep o rted w hich d e m H ughes, B. M,, Fee, D. C., Taylor, M. L.. Tiernan, wwitwMSpp p onstrate the presence of D N A-bound TCDD. If T C D D cannot interact with DNA directly, it probably induces cancer in rodents by an T. O.. Hill, C. E.. J r., and Wu, R. L C. (1975). A n a ly tic a l M e th o d o lo g y f o r H erbicide Orange. Vol. I. D eterm in a tio n o f C h e m ic a l C om position. Technical Report 75-0110, pp. 31-66, Air Force Systems Com indirect m ech an ism . T C D D receptors which mand. Aerospace Research Laboratories. Wright Pat catalyze th e in d u c tio n o f m ixed-function ox terson Air Force Base, Ohio. idases (Poland and K nutson, 1982) also may mediate o th e r T C D D -related effects. The lack o f m utagenic responses in S a l Hussain, S., Ehrenberg, L , Lofroth. G., and Gejvall, T. (1972). Mutagenic effects of TCDD on bacterial systems. A m b io 1, 32-33. IARC (1977). Some fumigants, the herbicides 2.4-D and d m o n e lla w ith 2,4-D , 2,4,5-T, an d dibenzofuran 2,4,5-T, chlorinated diberzodioxins and miscellaneous ,e is consistent w ith the results rep o rted in the literature (Shirasu e l a i , 1976; Zetterberg et industrial chemicals. A R C M onographs on th e E val ua tio n o f C arcinogenic R is k o f C h em ica ls to M an. 15. >f a i . 1977; Probst e l a l., 1981; M oriya e t a i , International Agency for Research on Cancer. Lyon. Kociba, R. J., ICeyes, D. G., Beyer, J. E,, Carreon, ir 1983; Schoeny, 1982). T h ere are no prior R. M.. Wade, C. E., Dittenser, D. a .. Kalnins, :S published m u ta g en icity d a ta on th e esters o f R. P,, Frauson, L. E., Park, C. N., Barnard, S. D., O 2,4-D, 2,4,5-T, or the dim ethylam ine salt of 2,4-D. H ummel. R. A., and Humiston, C. G. (1978). Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. REFERENCES Appl. P harm acol. 46, 279-303. Maron, D. M., and Ames, B. N. (1983). Revised methods Ames. B. N., McC a nn. J,, and Yamasaki, E. (1975). for the Salmonella mutagenicity tesL M utat. R es. 113, Methods for detecting carcinogens and mutagens with 173-215. the S a lm o n e lla /m a m m a lia n m ic r o so m c m u ta g e n ic ity McC onnell, E E., Lucier, G. w ., Rumdaugh. R. C,, test. M u ta t. R es. 31, 347-364. Albro, P. W,, Harvan, D. J., Hass, J. R., and Harris, Cocoon, D., and Acheson, E. D. (1982). Do phenoxy M. W. (1984). Dioxin in soil: Bioavailability after inges herbicides cause cancer in man? T h e L a n c e t I, 1057- tion by rats and guinea pigs. S cien ce 223, 1077-1079. 1059. Moriya, M., O hta, T., Watanabe, K,, MiyaZawa, T., CrummET, W. B.. and STEHL, R. H. (1973). Determi Ka to , K., and Shirasu, Y. (1983). Further mutage nation of chlorinated dibenzo-p-dioxins and dibenzo- nicity studies on pesticides in bacterial reversion assay furans in various materials. E nviron. H ealth Perspect. systems. M u ta t. Res. 116, 185-216. 5, 15-25. National Cancer Institute (1979). Bioassay of 2,7-dichlo- Gasiewicz, T. a ., Olson, J. R., G eiger. L. H., and rodibenzo-p-dioxin (DCDD) for possiblecarcinogenicity Neal, R. A. (1983). Absorption, distribution and me (CAS No. 33857-26-0), Technical Report No. 123. Be- tabolism of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) thesda, Md. in experimental animals. In H u m a n a n d E nvironm ental National Toxicology Program (1982a). Carcinogenesis R isks o f C hlorinated D ioxins and R elated Com pounds bioassay of 2,3,7,8-tetrachlorodibenzo-p-dioxin (CAS (R. E. Tucker, A. L. Young, and A. P. Gray, eds.), pp. No. 1746-01-6) in Osbome-Mende! rats and B6C3F1 495-525. Plenum, New York. mice (gavage study). Technical Report No. 209. Re G eiger, L E., and N eal, R. A. (1981). Mutagenicity search Triangle Park, N.C. testing of 2.3,7,8-tetrachlorodibenzo-p-dioxin in histi National Toxicology Program (1982b). Carcinogenesis dine auxotrophs of S a lm o n e lla ty p h im u r iu m . T oxicol. bioassay of 2,3,7,8-tetrachlorodibenzo-p-dioxin (CAS A ppl. P harm acol. 59, 125-129. No. 1746-01-6) in Swiss-Websler mice (dermal study). G ilbert, P., Sa in t-Ru f, G., Poncelet, F,, a nd Mer Technical Report No. 201. ResearchTriangle Park. N.C. g e . M. (1980). Genetic effects of chlorinated anilines Pitot, H. G , Goldsworthy, T ,, Campbell, H. a ., and and azobenzenes on S a lm o n ella ty p h im u r iu m . Arch. Poland, A. (1980). Quantitative evaluation of the pro Environ. C o n ta m . T o xico l. 9 , 533-541. motion by 2,3,7,8-teirachlorodibenzo-p-dioxin of hc- Haworth, S., Law lor, T.. Mortelmans, K., Speck, palocarcinogencsis from diethylnitrosamine. Cancer W., and 2^IGER, E. (1983). Salmonella mutagenicity R es. 40, 3616-3620. test resultsfor 250chemicals. Environ. M utag. S(Suppl.), Poland, a ., and Knutson, J. C. (1982). 2.3,7.8-Te- 3-142. trachlorodibenzo-p-dioxin and related haksgenated ar Huff. J. E , Moore, J. a ., Saracci, R., and Tomatis, omatic hydrocarbons: Examination of the mechanism L. (1980). Long-term hazards of polychlorinated di- of toxicity. A n n u . R ev. P harm acol. Toxicol. 22, 517- benzodioxins and polychlorinated dibenzofurans. E n 554. viron. H ealth P erspect. 36, 221-240. Poland, A., Palen, D.. and G lover, E (1982). Tum or x< , 2 2 5 , v>,-.v. vX CU O *nr rvs c_n co -p - co 3? V V ei 2D O I f 5 8 3 7 3 "v'-'-v < `f y Ii, . 1.^\fit*r;- '.' '. 1'}:: .4 f jV tV`'..*; `f f ; ; i fu., < 1 . v:i-tv'. *.... ; l--`oi,r. ;:7vv;i'` ;:' *'<n: . ., f f v */ . iJ'- ` i, . -f: U. ',.i.-H"i'T.K.. r- ...... . i.'t ;//,`'S'- 1'.' ; r)0; 1 `pi * Jf ,y.- ^' V.M Y '- 't-ty *)/&: .:*3 r 4 :'V .f>/-' ^' V s: *" 3-OXA-5,10-DIAZABICYCL0(5.2^DECAE-ifoNE:' 5-(p-CHLOROBENZOYL)-10-METHYL-, i, fc 1.. f- <'i.. .i; 3C15H17C1N23 c 15h 17c i n 2o 3 k -9 2 ,6 6 6 3-OXA-5,10-DIAZABICYCL0(5.2.l)DECAN-U-ONE: 5-(p-CHLOROBENZOYL)-10-METHYL-, ALC-LP 1791 ~ \ L# 72^7 ----- 1---- CH2 N-CO-r)-Cl gensi DCR H N-CH_ CO DLSO ----- 1--- CH -- 0 chim. II 3/ 22/68 2 g. Midland Lit. Ref. New MP: 95-96C. White crystals Est. Sol.<l$ water;^25$ ethanol '.,'iTif.''f `V. :f,' 885^ C04T1 p n u n c n in u I umizMoa 4 , V __ * N0 i CHIX/i'.O:'::" N-'XY *!*UK1C7Ti/.J D i'is'"- i r;t; i-.'iit-. : I . i n i t s O.l rrv/1 -{/ 100M2I 58375 There is an urgent need to control aquatic weeds in certain - ` *s lakes, streams') and irrigation_d'-tct'.i:s, especially in tropical or sen:tropical areas. Waters to which such applications are m a\de . *\ generally are nat used directiy~as sources of drinking water. t However, there is a possibility that some herbicide will find its i k way to finished water and, therefore, a need for a standard. This section deals with three closely related compounds, 2,4-D i v (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorouhenoxyacetic Q f~ ^ ^ --- --------- ;------- " *=--------^---- ^ ---------'ay,v-2. acid), and 2,4,5-TP or silvex,2,(2,4,5-trichlorophenoxyptopionic acid). ------ --- ^----- 7 v Each of these compounds is available in a variety of salts and esters /tT ej- s\ that may have marked differences in their practical herbicidal - ./2 3 t ' f properties. However, in the body, the salts or esters are rapidly hydrolyzed to the corresponding acid. / - '-x' Studies of the acute toxicity of the chlorinated phcnexyalkyl ~ * - acids are summarized in Table 1. There is considerable variation ^ .. ' " .. ' (up to about three-fold) between the various species studied with regard to oral toxicity. It appears that the ac:uutte oral toxicity vx-. of the three compounds is of about the same magntiitude. That of 7 silvex may be somewhat leSsT /W'k'l ^ :[ V"' '-'v, ''' ^ vai ^ Dosages of 10 mg/kg/day for about 90 days frequently lead to mild..i ^ 4, ,r N#-. iiwig ................... i UUHZI 58376 2 In a two-year feeding study of silvex in both male and female rats, the no-adverse-effect level was found to be 53 ppm (about 2.6 mg/kg/day) of the acid equivalent. In a similar two-year study in I dogs, the no-adverse-effect level jwas 100 ppm (about 2.6 mg/kg/day) of the-acid equivalent for female beagles and 30 ppm (about 0.9 mg/kg/day) for male beagles. - , j Available data on the toxicity of chlorinated phenoxy-acids to man is summarized in Table 3. The entries all relate to 2,4-D. t A daily oral dosage of 500 mg (about 7 mg/kg) of 2,4-D produced no apparent ill effects in a volunteer over a 21-day period (Kraus, as cited by Mitchell _et_ aJL.-> 1946). When 2,4-*D was investiga'ted as a possible treatment for disseminated coccidiojlomycosis, the patient __ _ /i had no side effects from 18 intravenous doses during 33 days; each of the last 12 doses in this series was 800 mg (about 15 mg/kg) or more, the last being 2000 mg (about 37 mg/kg) (Seabury, 1963). //.' A nineteenth and final dose of 3600 mg (67 mg/kg) produced mild `N symptoms. . ..... ... .. In addition to these specific data, the favorable record of use experience of 2,4-D is also pertinent. Sixty-three million pounds of 2,4-D was produced in 1965. In association with this very large production and use of 2,4-D, there have been no confirmed cases of occupational poisoning and few Instances of any illness even duetto ./ yi '' r- -.-.`T 2,4-D required to produce symptoms in man is probably 3000 to4Q00 mg :(or about 45 to 60 mg/kg). '-A comparison of other..toxicity ~vslueaj for 2,4-D, 2,4,5-T, and silvex as listed in Table 1 and ?. indicates 3 D0 H2 1 5 8 3 7 7 that the toxicity of these three compounds is of the same order of magnitude. Therefore, in the absence of any specific toxicologic data for 2,4,5-T and silvex in man, it might be estimated that the acute oral dose of 2,4,5-T and silvex required to produce symptoms in man would also be about 3000 to 4003mg (or about 45 to 60 mg/kg). i ! Although the chlorinated phenoxy-acids act as growth hormones i in plants, they have no hormonal action in animals. Howrver, the mechanism of their toxic action in animals is poorly understood. Parenteral and oral administration of toxic doses of 2,4-D to experimental animals brings about a characteristic complex of signs and symptoms which has been studied particularly in dogs (Dalgaard- Mikkelsen and Poulsen, 1962; Bucher, 1946; Drill and Hiratzka, 1953; and Hill and Carlisle, 1947). After a few hours the animals display a disinclination to move. This passiveness is gradually aggravated., and a picture of myotonia develops with rigidity of the skeletal muscles and ataxia. The condition may improve transitorily with movement. In severe cases the animals show progressive apathy, depression, and muscular weakness, especially of the hindlegs, with periodic, clonic spasms, and, finally, coma. Animals killed quickly by large doses of 2,4-D are thought to die of ventricular fibrillation. It may be significant that the myotonia which is so characteristic of 2,4-D poisoning in animals has not been seen in man. The muscular signs in animals are accompanied by marked anorexia; frequently, irritation of the nose and eyes is indicated by scratching reactions. Further, bleeding from the nose and mouth may occur, as well as diarrhea with blood-stained stools. Local irritation of the alimentary 4 D0H2I58378 tract often causes vomiting. However, this sign may be absent, even after oral administration to dogs. Autopsy may reveal necrotic ulcers of the oral mucosa and signs of irritation with histologically i demonstrable inflammatory changes.and necrosis of the small intestinal mucosa, as well as focal necrosis in the liver and degeneration of j the renal tubules. Cattle given repeated oral doses of silvex have developed swellings in the parotid or submaxillary region, apparently j from a local irritating effect (Palmer t l., 1964). When given by mouth to dogs, even in fatal cases^2,4,5-T has produced only weak signs in the forms of ataxia and stiff movements of the hindlegs. Nonspecific hyperemia, of the lungs, liver, and brain was found in the case of a man who committed suicide by ingesting a large amount of 2,4-D. Repeated doses in animals, which may or may not eventually lead to death, produce loss of appetite, loss of weight, vomiting, depression, roughness of coat, and general tenseness and muscular weakness. The action of ingested phenoxy-acid herbicides on muscular function, including their production of myotonia and ventricular fibrillation, which is reminiscent of that following administration of halogenated acetic acid compounds, suggests an interference with carbohydrate metabolism (Dalgaard-Mikkelsen and Poulsen, 1962). Two cases of a transitory diabetiform condition observed in spraying personnel following work with chlorinated phenoxy-acid herbicides point in the same direction. However, hyperglycemia and glycosuria i ft9 3 1 could not be reproduced with certainty in rabbits; only one out of five animals responded in this way in exploratory experiments, where 5 the doses ranged from 125 to 500 mg/kg and the period of administration from 6 to 50 days (Lorenzcn and Lyngstfe, 1957). No effect of silvex on blood sugar levels was noted in cattle fed quantities of the herbicide sufficient to cause significant symptoms in all test animals and death in one animal (Palmer t a_l., 1964). Following feeding to dairy cows, silvex was almost quantitatively ! r excreted in urine (St. John et a_l., 1964). No residue of this compound j was detected in milk. , The current recommendation for control of alligatorweed by silvex ! ,, i is application of the propylene glycol butyl ether ester at the rate of 8 pounds acid-equivalent per acre. A second application is usually made during the same summer' to kill plants missed in the first application. Assuming that the water was 45 cm (about 1.5 ft) deep, appli cation of silvex at the rate of 8 pounds per acre would result in a concentration of 2 ppm. Since the average depth of rivers, lakes, reservoirs, and other potable water supply sources is probably considerably greater than 45 cm, the average concentration would actually be less than 2 ppm. Furthermore, although it has been shown (Cochrane t a_l., 1965) that the free acid persists in water for many weeks, silvex applied to a stream would be carried along by the current and would be diluted as tributaries entered the stream. Thus, anyone who received his regular drinking v;ater from a treated stream would receive the compound in a significant concentration only for a few hours in connection with any single application. Furthermore, since only two water treatments are planned per year, the total duration of dosage would be short. : 0 0 M2 158379 c D0H2158380 I 6 In studies of the persistence of silvex in closed artificial aquatic environment in the laboratory, mean concentrations ranged from 0.820 ppm immediatedly after application to 0.046 ppm after nineteen weeks (Cochrane e_t a_l., 1965). The ester of silvex was rapidly hydrolyzed to the free acid. The authors speculated that, in addition to loss through adsorption or absorption of hydrosoil or aquatic vegetation, degradation also occurred. A field study of silvex persistence was carried out in a creek having very little water movement that had been treated at a rate of 8 pounds acid equivalent per acre (Cochrane e a_l., 1965 and Nicholson, unpublished data). After the first application, concentrations ranged from 0.083 ppm one day after spraying to 0.0011 ppm after 21 days. Silvex was not detectable after 35 days. After the second application, concentrations ranged from 0.019 ppm one day after spraying to 0.048 ppm after 70 days. Following the third application, concentrations decreased from 73 ppb immediately after spraying to 2 ppb after 48 days, and to 0.1 ppb after 6 months. Silvex could not be detected one year after application. Another field test was carried out in a fast-flowing stream which provided maximal opportunity for dilution and interchange of water. Residues of silvex were not detected except during the first few hours following treatment at the rate of 4 pounds of silvex acid equivalent. The maximal level found in this study was 0.00005 ppm (Nicholson, unpublished data). These studies indicate that the 2 ppm value cited above as a maximum for silvex concentration immediately after spraying is reasonable, D0 H2158381 I 7 but that the compound would persist in a given location for only a short period, particularly in flowing water sources. As calculated above, the maximal concentration of silvex in the water supply following use for aquatic weed control would be approximately j 2 ppm. Based on an ingestion of 2 liters of water, the maximal daily intake per person of silvex from this source would be 4 mg per.treat ment (0.06 mg/kg for a 70-kg man). The toxicity studies discussed above for silvex in experimental animals, and the much more extensive data in experimental animals and/or in man for the closely related compounds 2,4-D and 2,4,5-T, indicate that this level should be safe. However, there need be no situation in which a phenoxy herbicide is applied to water that is used immediately for drinking. Dilution and decomposition will account for significant loss. Therefore the maximal acceptable level may be set safely and practically at 0.1 mg/1. =DOM215 83 82 I REFERENCES Bucher, N. L. R.: Effects of 2,4-dichlorophenoxyacetic acid on experimental animals. Froc. Soc. Exp. Biol. Med. >3:204-205, 1946. Cochrane, D. R., Pope, J. R. and Nicholson, H. P. : Progress report on silvex persistence. Unpublished report from the Southeast Water Laboratory, PHS, DHEW, Athens, Georgia, 1965. Dalgaard-Mikkelsen, S. V. and Poulsen, E.: Toxicology of herbicides. Pharmacol. Rev. 14:225-250, 1962. Dow Chemical Company: Unpublished data, 1964. Drill, V. A. and Hiratzka, T.: Toxicity of 2,4-dichlorophenoxyactic acid and 2,4,5-trichlorophenoxyacetic acid. A report of :their acute and chronic toxicity in dogs. Arch. Industr. Hyg. Occup. Med. 7:61-67, 1953. Goldstein, N. P., Jones, P. H. and Brown, J. R.: Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic acid. J. Amer. Med. Assoc. 171:1306-1309, 1959. Hayes, W. J., Jr.: Clinical Handbook on Economic Poisons. PHS Pub. No. 476, U. S. Government Printing Office, Washington, D. C., Revised 1963. Hill, E. C. and Carlisle, H.: Toxicity of 2,4-dichlorophenoxyacetic acid for experimental animals. J. Industr. Hyg. Toxicol. 29:85-95, 1947. Lehman, A. J.: Chemicals in foods: A report to the Association of Food and Drug Officials on current developments. Part II. Pesticides. Assoc. Food Drug Off. U.S., Quart. Bull. ljj:122-133, 1951. Lorenzen, I. and Lyngs^e, J. : En unders$gelse over virkningen af et kloreret fenoxyacetat pa kulhydratstofskiftet hos kaniner. Nord. Hyg. Tidskr. 38:153-157, 1957. Mitchell, J. W., Hodgson, R. E. and Gaetjens, C. F.: Tolerance of farm animals to feed containing 2,4-dichlorophenoxyacetic acid. J. Anim.' Sci. :226-232, 1946. ______ Nicholson, H. P.: Unpublished data. Nielsen, K., Kaerape, B. and Jensen-Holm, J.: Fatal poisoning in man by 2,4-dichlorophenoxyacetic acid (2,4-D): Determination of the agent in forensic materials. Acta Pharmacol. Toxicol. 22:224-234. 1965. Palmer, J. S. and Radeleff, R. D.: The toxicologic effects of certain fungicides and herbicides on sheep and cattle. Ann. NY Acad. Sci. 111:729-736, 1964. e 8 E8SI2f)00 2 Palmer, J. S., Clark, D. E. and Hunt, L. M: Toxicologic effects of silvex on yearling cattle. J. Amer. Vet. Med. Assoc. 144:750-755, 1964. Rowe, V. K. and Hymns, T. A.: Summary of toxicological information on 2,4-D 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, 1954. Seabury, J. H.: Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. Arch. Environ. Hlth. 7^:202-209, 1963. St. John, L. E., Jr., Wagner, D. G. and Lisle, D. J. : Fate of atrazine, Kuron, silvex, and 2,4,5-T in the dairy cow. J. Dairy Sci. 47: 1267-1270, 1964. Table 1: Compound cw\ The Acute/Toxicity of Some Chlorinated Phenoxy,alkyl Acids to Experimental Animals. / LD50 Value Species Sex Rclite (mn/kg) Reference 2,4-D Guinea pig Mouse Rabbit Rat Rat Rat Dog $ - - * 7 M M&F oi^i Orfil Oral Oril Oril Orli Oral 1000 375 800 666 500 375 100 Hill and Carlislej 1947 Hill and Carlisle, 1947 Hill and Carlisle, 1947 Hill and Carlisle, 1947 Lehman, 1951 Rowe and Hymas, 1954 Drill and Hiratzka, 1953 2,4,5-T Ra t Rat Dog M - M&F Oral Oral Oral 500 Rowe and Hymas, 1954 300 Lehman, 1951 10 0 Drill and Hiratzka, 1953 Silvex Chick Guinea pig Mouse Rabbit Rat Rat M&F M F F M&F M&F Oral Oral Oral OrajL Oral Oral 1190 1200 1410 819 650 500 Rowe and Hymas, 1954 Rowe and llymas, 1954 Rowe and llymas, 1954 Rowe and llymas, 1954 Rowe and Hymas, 1954 Dow Chemical Co., 1964 I l I OD zo V 8 8 S I 2 H 0 0 < c*- Table 2: f Toxicity of Repeated Doses of Chlorinated Phenoxyalkyl Acids to Experimental Anima1s / Compound 2,4-D 2,4,5-T Species Dosage Dog 10 mg/kg/day Dog 20 mg/kg/day Rat Rat Rat Sheep about 15 mg/kg/day (300 ppm in diet) about 50 mg/kg/day (1000 ppm in diet) about 150 mg/kg/day (3000 ppm in diet) 100 mg/kg/day Cow 250 mg/kg/day Dog Dog Sheep Sheep 10 mg/kg/day 20 mg/kg/day i , 100 mg/kg/day 250 mg/kg/day Duration 90 days 90 days 112 days 112 days "--* 481 days 7 days 90 days ""* 369 days 7 days Ef fee t none detected 3/4 died 18th to 49th i.day none detected 4/5 died 5/5 died in 12 days none detected lethal, sick after 4 days none detected , 4 of 4 animals died 11th to 75th day lethal lethal, 6ick after 3 days Reference Drill & Hiratzka, 1953 Drill 6c Hiratzka, 1953 Hill 6. Carlisle, 1947 Hill 6c Carlisle, 1947 Hill 6. Carlisle, 1947 Palmer 6c Radeleff, 1964 Palmer 6c Radeleff, 1964 j Drill 6c Hiratzka, 1 9 5 3 iDrill & Hiratzka,9 1953 l : Palmer 6c Radeleff, 1964 Palmer 6c Radeleff, 1 9 6 4 Silvex Cow (yearling) Cow (yearling) 25 mg/kg/day 50 mg/kg/day I Cow 100 mg/kg/day (yearling) /' / , 73 days 73 days / 29 days anorexia, dyspnea, and parotid enlargement after 28th dose, all animals developed anorexia, dyspnea, and parotid enlargement one animal died death Palmer 6c Radeleff, 1 9 6 4 Palmer 6c Radeleff, 1 9 6 4 Palmer 6c Radeleff, 1964 y .bo - SfleOSIZHOO; Table 2: (Continued) Compound Silvex Species____ Dosage Dog (female) Dog (male) Dog Dog 0.9 mg/kg/day (30 ppm in diet) 2.6 mg/kg/day (100 ppm in diet) 13 mg/kg/day (300 ppm in diet) A0 mg/kg/day (1000 ppm in diet) Duration 2 years 2 years 89 days 89 days Rat about 2.6 mg/kg/day 2 years j(53 ppm in diet) Rat !about 5 mg/kg/day 90 days (100 ppm in diet) i Rat 10 mg/kg/day 90 days (about 200 ppm in diet) Rat about 15 mg/kg/day 90 days (300 ppm in diet) Rat ! 30 mg/kg/day 90 days (about 600 ppm in diet) Rat about 50 mg/kg/day 90 days (1000 ppm in diet) Rat i100 mg/kg/day 90 days (about 2000 ppm in diet) (Continued) . Effect Reference No effect Dow Chemical Co., 1965 No effect Dow Chemical Co., 1965 none detected Palmer & Radeleff, 1964 growth depression and liver pathology, possible anemia and increased alkaline phosphatase No effect Dow Chemical Co., 1964 Dow Chemical Co., 1965 liver and kidney weights increased - no other evidence of pathology none detected, although liver and kidney weights apparently not determined slight growth depression - pathological changes I in heart, liver, and kidney liver weight increased Dow Chemical Co., 1964 Dow Chemical Co., 1964 Dow Chemical Co., 1964 Dow Chemical Co., 1964 some growth depression - Dow Chemical Co., 1964 pathological changes in heart, liver, and kidney liver weight increased Dow Chemical Co., 1964 988SIZM00i: Table 2: (Continued) Compound____ Species____ Dosage Silvex Rat about 150 mg/kg/day (3000 ppm in diet) Duration 90 days Rat 300 mg/kg/day 90 days (ab)ut 6000 ppm in diet) Rat about 500 mg/kg/day 90 days (10,000 ppm in diet) Rat 600 mg/kg/day 90 days i (about 12,000 ppm in diet) Sheep 100 mg/kg 11 doses \,~- CP -, t3 ' Effect Re ference 7/20 died before 90 days Dow Chemical Co., 1964 - growth retardation and pathological changes in heart, liver, and kidney liver weight increased Dow Chemical Co., 1964 and growth retarded 8/20 dead at 32 days - Dow Chemical Co., 1964 anorexia and weight loss - pathological changes in heart, liver, kidney, and testes 13/20 animals died - Dow Chemical Co., 1964 liver weight increased and growth retarded in survivors death Palmer & Radeleff 1964 i I 8E8SIZMOQ : Table 3: Summary of Available Information on Toxicity of the Chlorinated Phenoxyalkyl Acid Herbicides to Man No. of Compound persons Exposure____ Route Dosage Symptomatology , ______ Outcome_______ Reference 2,4-D r volunteer oral 500 mg/day for 21 days none no illness 3 occupational--^ dermal very small 1/ peripheral neuritis" illness & resp. persisted 1 therapeutic I.V. 18 doses dur no side effects ing 33 days; each of last 12 doses was 800 mg (about 15 mg/kg) or more; 18th was 2000 mg (about 37 mg/kg) 1 therapeutic I.V. 19th and final coma, muscle fibril recovered dose of 3600 mg lation, hyporeflexia, (67 mg/kg) urinary incontinence, and profound muscle weakness 1 suicid oral unknown prolonged stupor recovered attempt 1 suicide oral >6500 mg violent convulsions--2/ death Kraus, as cited by Mitchell et al., 1946 Goldstein et al. t 1959 Seabury, 1963 Seabury, 1963 Hayes, 1963 Nielsen e t _al., 1965 These three cases of "peripheral neuritis" among men very recently exposed to 2,4-D were seen at one clinic in a single month. No valid toxicological or epidemiological evidence was given to support a causal relationship. The symptom picture was unlike that seen in experimental animals or in man in 2,4-D poisoning. Although very large amounts of 2,4-D and related compounds have been manufactured and used, no other Bimilar cases have been reported. Thus, epidemiologic considerations and an inconsistent symptom picture suggest that other etiologies should be sought for these illnesses. 2/ Possibly of anoxic origin._____________ V'1 C3 ID 98E8SIZHOO; VO LU M E 33 N U M BER 2 F ILE - 15 J A N U A R Y 1984 Biochemical Pharmacology ( F o u n d e d hy S ir R u d o l p h A . P f t e r s ) -'7K' ' 2 ' 0 ' i a - U c c K ' 1! * b ' C l O C C m bd] 166 i DOM 2-1 5 8 't 3 b CONTENTS Commeniary Michelle W. Kloss. Gerald M. Rosen and Elmer J. Rauckman Research Papers Stuart R. Stone. J. A. Montgomery and John F. Morrison D. Mc Ewen Nicolls and Maciej J. K uliszewski Jacqueline F. Sinclair Philip Wiebki.n. Linda M. Zaitlin. E. Lucile Smith and Peter R. Sinclair W. Mark Lafranconi. Raymond C. Duhamel. Klaus Brendel and Ryan J. Huxtable Ronald J. G erson and Zahir A. Shaikh Peter Wollenberg and Walter Rummel R. Fears. W. R. Rush. P. Walker and H. Ferres R. Fears. A. Esmail. P. Walker. W. R. Rush and H. Ferres I69 Cocaine-mediated hepatoioxicity. A critical review 175 Inhibition of dihydrofolate reductase from bacterial and vertebrate sources by folate, aminopterin. methotrexate and their 5-deaza analogues 18 1 Kidney urokinase activity following acute exposure to lead 187 Aminopyrine and biphenyl metabolism in cultured hpa tocytes. Induction by alcohols I9I Differentiation of the cardiac and pulmonary toxicity of monocrotaline. a pyrrolizidinc alkaloid I99 Differences in the uptake of cadmium and mercury by rat hepatocyte primary cultures. Role of a sulfhydryl carrier 203 Vectorial release of sulfoconjugales in the vascularly per fused mouse small intestine 209 Hyperalphalipoproteinaemic activity of BRL 2631-1-- I. Enhanced cholesterol turnover in rats 219 Hyperalphalipoproteinaemic aclivity of BRL 26314-- IIInhibition of atherosclerosis in rabbits Continuiti on outtide httek fo u r This journal is now listed in Currant Auurcncxs in Biological Scirncrx. Send to your nearest Pergamon office lor a free specimen copy. ISSN 0006-2952 BCPCA6 33(2) 169-342 (1984) Pergamon Press Oxford Now York Frankfurt ! / . n i l . <MH. W Pmiu-J in Un;..:. V V . W I i - '4 > |V<U K ' 'l'b'i' - C lc c ) 4 5fc- Ct^c:-) >4 ^ I D 1jvi Pcr.ni.<nl'rcvlui { ?.. F EFFECTS OF 2.4-DICHLOROPHENOXY ACETIC ACID AND 2.4.5-TRICHLOROPHENOXYACETIC ACID ON PEROXISOMAL ENZYMES IN RAT LIVER * t- Y oichi K a w a s h i m a .* H a r u y o K a t o h . ' S e t s u k o N a k a j i m a .* H iroshi K o z u k a * and i- M i t s l r u U c h i y a n i a * Faculty of Pharmaceutical Sciences. Toyama Medical and Pharmaceutical University. - -h.'t* Suiiani. Toyama 930-01. Japan; * National Institute of Hygienic Sciences. }{ i-18-1 Kamiyoga. Setagaya. Tokyo 158. Japan i y {Received 20 May 1983; accepted 18 August 1983) Abstract-- The effects of feeding 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.4.3-irichlorophenoxy- acetic acid (2.4.5-T) on the level of peroxisomal enzymes in rat liver were studied. The concentration of triglyceride in serum was decreased and the activity of cyanide-insensitive palmiloyl-CoA oxidation, catalase and carnitine aceivltransfcrase increased. However, the extent of the increase in the activity of these enzymes by treatment with 2.4-D was less pronounced than that by 2.4.5-T treatment. The * administration of 2.4-D or 2.4.5-T increased the concentration of polypeptide with a mol. wi of 80.000 in the light mitochondrial fractions of the liver from the rats. y.. Several hypolipidemic agents are known to cause with ice-cold 0.9% NaCl and rinsed in cold 0.25 M proliferation of peroxisomes in rat liver [1-6]. Ethyl sucrose. Part of the liver was homogenized with cold i! 4-chlorophenoxyisobutyrate (clofibrate) is one of the 0.25 M sucrose and fractionated into nuclear, heavy ts most common inducers of peroxisomes and is used mitochondrial, light mitochondrial, microsomal and clinically as a drug. Lazarow and de Duve [7] pre cytosolic fractions by the method of de Duve e t at. sented evidence suggesting that hypolipidemic action [15]. The other part of the liver was frozen at -3 0 " of clofibrate is attributable to the induction of until th e peroxisomal enzymes were assayed. The & *- j; cyanide-insensitive fatty acyl-CoA oxidizing activity and that the activity is localized in the peroxisomes. Recent studies on several peroxisome proliferators frozen livers were used within 1 month as described previously [16]. E n z y m e a ssa ys. Cyanide-insensitive palmitoyl- including clofibrate have shown that these com CoA oxidation was assayed by the method of Laza pounds are carcinogenic in rats and mice [8-14], It row and de Duve [7] with minor modification [16]. is im portant therefore to study the effects of other One unit of activity was defined as the amount of aryloxy acids on peroxisom al enzymes. 2,4-D and enzyme required to reduce 1/tmole of NAD per min. 2.4.5-T are well known as herbicides. In spite of Catalaserctivity was determined by the method of their structural similarity to clofibrate, little infor Aebi [17]. Carnitine acetyltransferase was assayed mation is available about their effects on lipid metab according to the method of Kahonen [18]. Urate olism. particularly their effects on peroxisomal oxidase and glutamate dehydrogenase were assayed enzymes. The present study was undertaken to according to Leighton e t al. [19]. investigate the effects of 2.4-D and 2,4,5-T on the S o d iu m d o d ecyl su lfate-polyacrylam ide g e l electro induction of peroxisomal enzymes involved'in lipid p h o r e s is . Sodium dodecyl sulfate-polyacrylamide gel metabolism. electrophoresis was performed essentially according to the method reported by Reddy and Kumar [20|. MATERIALS AND METHODS except for the use of slab gel of 2 mm thickness. Light mitochondrial protein (20 ug) was loaded onto C h e m ic a ls . Palm itoyl-CoA , acetyl-CoA, L-carni- a stacking gel. Electrophoresis was carried out at tine. clofibric acid and bovine serum albumin were 25 mA for c a 5 hr until the marker dye reached the purchased from Sigma Chemical Co. (St. Louis, bottom o f the gel. The proteins were fixed in the gel M O ); N AD and CoA from Oriental Yeast Co. with 20% trichloroacetic acid for 30 min and then (Tokyo. Japan): and 2.4-D and 2,4.5-T from Tokyo stained with 0.25% Coomassie brilliant blue in Chemical Industry Co. (Tokyo, Japan). All other m cthanol-aceti acid-w ater (5 :1 :5 . v/v) for 2 hr. chemicals were of analytical grade. Destaining w ajjjo n e by repeated washing in 20% A n im a ls . Male rats of Wistar strain, weighing methanol containing 7% acetic add. 150-200 g. were used. Rats were fed a d lib . a com O th e r a n a lytica l p ro c e d u re s . The concentration of mercial diet or a ground diet containing 0.25% cholesterol in the serum was measured by the method t (w/w) 2.4-D. 2.4.5-T or clofibric acid for 3, 7 or 14 of Zurkowski (211. To measure the concentration of days. triglyceride in the serum , lipid was extracted from S u b c e llu la r f r a c t i o n a t i o n s . The rats w ere decapi serum by the method of Bligh and Dyer [22] and tated and the livers isolated. The livers were perfused triglyceride was isolated by TLC on silica gel G using 241 CD O r-o Ol GO co h ,!l s i ' 'i l( i!l i f i ; ;i Y. Kav\ asmim \ </ ni. Tiihle I- Elice-.* ni 2.4-D and 2.4.5-T on the concentration of triglyceride :nul cholesterol in rat serum Conditions Triglyceride (.umoles/ml serum) Cholesterol (<imolcs/ml serum) Control 2.4-D 2.4.5-T (3) (3) (3) 1.15 = 0.14 0.37 = 0.03 0.36 = 0.10 2.5o = 0.25 1.65 =0.11 1.35 = 0.54 Each value represents mean = S.D. The amount of triglyceride and cholesterol was determined as described in the text. Numbers in parentheses are the numbers of animals used. The rats were fed a control diet or a diet containing 2.4-D or 2.4.5-T for 7 days. petroleum ether-diethyl ether-acetic acid (80:30:1. v/v) as developing solvent. After adding pentadecanoic acid as internal standard, triglyceride was recovered from the silica gel by extraction with chloroform -m ethanol-w ater (2 :2 :0 .5 , v/v) accord ing to Stern and Pullmann (23). The amount of triglyceride was determined by measuring the amount of fatty acid in trigylceride by GLC. The conditions of gas chromatographic analysis have been reported previously (24). The concentration of protein in the enzyme preparations was determined by the method of Lowry e t al. [25], with bovine serum albumin as standard. RESULTS Table 1 shows the effects of 2,4-D and 2,4,5-T on the concentration of triglyceride and cholesterol in the serum. On administration of 2,4-D or 2,4,5-T for 7 days, the concentration of serum triglyceride decreased to approximately one-third of the control. The concentration of cholesterol was also decreased by this treatm ent, although the extent of the decrease was less considerable. The effects of 2.4-D, 2,4,5-T and dofibric acid on relative liver weight are com pared in Fig. 1. Marked hepatomegaly was produced following the administration of dofibric acid to rats, in good agreement with earlier repons [16,26). The relative liver weight increased 1.6 times compared Fig. 1. Effects of 2.4-D and 2.4.5-Ton relative liver weight. 2.4-D (). 2.4.5-T (O) and dofibric acid (). Each value was obtained from three to ten animals. Vertical bars represent the S.D. to that of control rats after treatment with dofibric acid for 14 days. Significant hepatomegaly was pro duced by the administration of 2.4.5-T. although the effect was less marked than that observed with clofibric acid. 2,4-D did not cause hepntomeealv. Figure 2 shows the time-course of changes in activity of cyanide-insensitive palmitoyl-CoA oxi dation. catalase and carnitine acetyltransferase when rats were fed a diet containing 2.4-D. 2.4.5-T or dofibric acid. The activity of cyanide-insensitive palmitoyl-CoA oxidation was increased by the adm inistration of 2.4.5-T and reached its maximum value 7 days after the initiation of the administration. The maximum activity was approximately 10 times that of the control and approximately 60% of that induced by dofibric acid, when compared on a g liver basis. However, the total activity of cyanide-insensitive palmiiovl-CoA oxidation in the whole liver was 34% of that induced by dofibric acid feeding owing to the less hpatomgalie effect of 2.4.5- T com pared with that of dofibric acid. The adm inistration of 2.4-D also increased the activity of cyanide-insensitive palmitoyl-CoA oxidation, although the extent of the increase was less pro nounced than that obtained by treatm ent with 2.4.5- T. The activity of catalase per g liver was slightly increased by treatm ent with either 2.4.5-T or dofibric acid, whereas 2.4.5-T did not change total activity of catalase in the whole liver. 2.4-D did not affect catalase activity. The activity of carnitine acetyltransferase. as well as cyanide-insensitive palmitoyl-CoA oxidation, was induced by treatment with either 2.4.5-T or dofibric acid. Treatm ent with 2,4-D caused a slight, but significant, increase in the activity of carnitine acetyltransferase. Figure 3 shows the data on subcellular distribution of cyanide-insensitive palmitoyl-CoA oxidizing activity and m arker enzymes for both mitochondria and peroxisomes. The highest activity of cyanideinsensitive paimitoyi-CoA oxidation was localized in the light mitochondrial fraction. The distribution pattern of the activity is similar to that of urate oxidase, but not to that of glutamate dehydrogenase. Figure 4 shows the electrophoretic profiles of pro tein from the light mitochondrial fractions from livers of rats which had been fed 2.4-D. 2.4.5-T or dofibric acid. In accordance with the findings reported by Reddy and Kumar [20). the concentration of poly peptide with a mol. wt of approximately 80.000 in light mitochondria was increased by feeding with dofibric acid. 2.4-D and 2.4.5-T also increased effec- 18244 DOH2 I 581(38 E lk v i' i>l 2.4-D ;i(ul 2.4..''I tm poru\i'om;il cnzwtu^ 24.' D0W2158439 Fig. 2. Effects of 2.4-D and 2.4.5-T on peroxisomal enzymes. Rais were fed a diet containing 0.25ci (w " ) 2.4-D. 2.4.3-T or clofibric acid. Enzyme activity in the post-nuclear supernatant of the livers was assayed as described in the text. Values represent the mean - S.D. from three or four experiments. A and D. cyanide-insensitive palmitoyl-CoA oxidation: B and E. catalase: C and F. carnitine acetyl- transferase. 2.4-D (G ). 2.4.j - T (O ) and clofibric acid ( ) . lively, but somewhat less m arkedly than clofibric acid, the concentration o f the polypeptide in the light m itochondrial fractions. DISCISSION Clofibrate is a hypolipidemic drug and a potent inducer of peroxisomal /-oxidation (7j. Since many other peroxisome proliferators increase the activity of peroxisomal /-oxidation [27--29] and decrease the serum lipid level [4.30), it is considered that the hypolipidemic action may be due to the increased activity of peroxisomal /-oxidation. The peroxisomal /-oxidation system is distinct from the mitochondrial one in its response to KCN [7], In the present study, we showed that the admin istration of cither 2.4-D or 2,4.5-T causes a decrease in the serum triglyceride concentration and an increase in the cyanide-insensitive palmitoyl-CoA oxidizing activity. Not only the oxidation activity, but also catalase and carnitine acetvltransferase were induced by 2.4.3-T. Catalase is a peroxisomal enzyme and. in rats, is induced slightly by treatment with clofibrate |7. 16|. Carnitine acetyltransfcrase has been reported to be localized in both mitochon dria and peroxisomes (18) and to be greatly induced by treatm ent with peroxisome proliferators [6. 18). O u r subcellular fractionation studies showed that the distribution of cyanide-insensitive palm itoyl-C oA oxidizing activity induced by 2 .4 .5 -T is consistent with that of urate oxidase, a m arker enzyme for peroxisomes. These results suggest that the increase in the activity o f p alm ito y l-C o A oxidation in the liver is due to the increase in peroxisome activity. In spite o f the fact that catalase is a peroxisomal enzym e, a large am ount o f catalase was distributed in the cytosolic fraction. This m ay result from leakage o f the enzyme from the peroxisomes into cytosol, because it has been suggested that peroxisomes grow and then burst [31). S im ilar leakage of catalase into cytosol was observed in the livers o f rats which had been treated with other peroxisome proliferators [29,30|. The present study showed that the concentration of polypeptide with a mol. wt of approximately 80,000 in the light mitochondrial fractions was effec tively increased by either 2.4-D or 2.4.5-T. The polypeptide has been reported to be increased mark edly in peroxisomes of livers from rats treated with peroxisome proliferators [20|. Osumi and Hashimoto [32) showed that a peroxisomal protein having activi ties of both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase corresponds to the peroxisome-specihc polypeptide with a mol. wt of 80.000. Accordingly, our results show that both 'l. KX'A1 0 n 8 S IZ M 0 a -T, N MlP S NMUPS Fig. 3. Subcellular distribution of cyanide-insensitive pulmitoyl-CoA oxidizing activity induced hv 2,-l.j-T. The livers were homogenized and fractionated inio nuclear (N ). heavy mitochondrial (M ). light mitochondrial (L ). microsomal (P) and cytosolic (S) fractions as described in the text. Enzyme activity was determined as described in the text. Each value represents the mean of three separate *\ experiments. Rats were fed a diet containing 0.25r c ( / | 2.4.5-T for 7 days. A . Cvanidc-inscnsiuve palmitoyl-CoA oxidation: B. urate oxidase: C. glutamate dehydrogenase: D . catalase. 2.4-D and 2.4.5-T. like clofibric acid, are inducers of peroxisomal /-oxidation enzymes. The. ability of 2,4.5-T to induce peroxisomal fioxidation was less pronounced than that of clolibric acid, whereas the effect of 2.4.5-T on peroxisomal enzymes was greater than that of 2.4-D. Most of the peroxisome proliferators are known to cause hepa tomegaly and induction of catalase as well as of peroxisomal /-oxidation and carnitine acetyltransferase. In fact. 2.4.5-T produced hepatomegaly and induced catalase, peroxisomal /5-oxidation and car nitine acetvltransferase. However. 2.4-D differed from 2.4.5-T in not causing hepatomegaly or catalase induction. Lazarow ct til. (33) pointed out that an increase in activity of peroxisomal /-oxidation is not always accompanied by an increase in catalase activity or liver size. O ur previous study also showed that the increase in peroxisomal /-oxidation activity by clolibric acid is not always correlated to the pro duction of hepatomagalv and an increase in catalase activity in rats of a hormonally altered state (16). The potency of 2.4-D and 2.4.5-T to induce peroxisomal enzymes may be due to their structural similarity to clofibric acid. However, n should be noted that phenoxyncetic acid. 2-chlorophenoxvacetic acid and 4-chlorophcnoxyacetic acid hardly caused hepatomegaly or an increase in the activitv of peroxisomal /-oxidation, catalase and carnitine acetyltransferase (data not shown). Several inducers of peroxisome, including c a librate and its derivatives |8 - I4 |. have been found to produce tumours when administered to rats for prolonged periods and a relatively low dose of clolibrate promotes hcpatoearcinogenesis induced by dtmethvlniirosaminc |34|. A case-control study showed that exposure to 2.4-D and 2.4.5-T causes a 6 times increase in the risk of soft-tissue sarcomas (35). It may therefore be necessary to take the potency of these herbicides to proliferate peroxi somes into consideration in the study of tumourigenesis of these aryloxv acids. I ^ ***** JH8SIZM00S si EMect1ol 2.4-1) and '.J..1' - 1 on |icro\iv>in:il enzyme-- 1`^ r Mar -V- >rr M r M X **mtd m +vmr*m -- * MW Fie. J. Sodium dodecyl sulfate-polyacrylamide eel electro phoresis of the light mitochondrial fractions of the livers. Rats were fed a diet containing 0.25r'r (w/w) clofibric acid. 2.4-D or 2.4.5-T for 7 days. Approximately 20,tie of light mitochondrial protein was analyzed by electrophoresis as described in the text. A . Control: B. clofibric acid fed rats: C. 2.4.5-T fed rats: D . 2.4-D fed rats. The arrow indicates the position of the polypeptide with a mol. wt of 80.000. REFERENCES 1. D . J. Svoboda and D. L. Azarnoff. J. Ceil. Biol. 30. 442 ( 1966). 2. Z . Hruban. M . Gotoh. A. Slesers and S.-F. Chou. Lab. Invest. 30. 64 (1974). 3. J. K. Reddv and T. P. Krtshnakantha. Science 190. 787 (1975). 4. J. K. Reddv. D. E. Moodv. D. L. Azarnoff and M . S. Rao. Life Set. 18. 941 (19761. 5. D . E. Moodv and J. K. Reddy. Toxic, appl. Pharmac. 4 5 .4 9 7 (1978). 6. D . E. Moodv and J. K. Reddv. .4m. J. Path. 90. 435 (1978). 7. P. B. Lazarow and C. de Duvc. Proc. ntttn. Acutl. Sci. U .S.A. 73. 21H3 (1976). 8. J. K. Reddv. M. S. Kao and H C Moody. Cancer Res. 36.121111976). 9 J. K. Reddv and M . S. Rao. ] natn. Cancer Inst. 59. 1645 ( 1977). Ill D. J. Svoboda and D. L. A/arnoll. Cancer Res. 39. 5419(1979). 11. J. K. Reddv and S. A. Oureshi. Hr. J. Cancer 40 . 476 (1979). 12. J. K. Reddy. M . S. Rao. D. L. Azarnoff and S. Sell. Cancer Res. 39. 152 (1979). 15. J. K. Reddv. D. L. Azarnoff and C. E. Hicnile. Sature. Land. 283. 397 ( 19S0). 14. J. K. Reddy. N. D. Laluani. M. K. Reddy and S. A. Oureshi. Cancer Res. 42. 259 (I9S2). 15. C. de Duvc. B. C. Pressman. R. Gianctto. R. Wattiaux and F. Applemans. Riochem. J. 60. 604 ( 1955). 16. Y. Kawashima. H. Katoh and H. Kozuka. Biochim. biophys. Acta 750. 365 (1983). 17. H. Aebi. in Methods o f Enzymatic Analysis (Ed. H. L \ Beremever). p. 673. Academic Press. New York (1974).' '18. M . T. Kahonen. Biochim. biophss. Acta 428. 690 (1976). 19. F. Leighton. B. Poole. H. Beaufay. P. Baudhuin. J. W. Coffev. S. Fowler and C. de Duve. J. Cell. Biol. 37. 482 ( 968). 20. J. K. Reddy and N. S. Kumar. Biochem. biophys. Res. Commun. 77. 824 (1977). 21. P. Zurkowski. Clin. Chem. 104. 451 (1964). 22. E. G. Blieh and W. J. Dver. Can. J. Biochem. Physiol. 37. 911 (959). 23. W. Stern and M . E. Pullman. J. biol. Chem. 253. 8047 (1978). 24. Y. Kawashima and H. Kozuka. Biochim. biophys. Acta 713.622 (1982). 25. O. H. Lowry. N. J. Rosebrough. A. L. Farr and R. J. Randall. 7. biol. Chem. 193. 265 (1951). 26. R. Hess. W. Straubli and W. Riess. Suture. Lond. 208. 856 ( 1965). 27. P. B. Lazarow, Science 197. 580 (1977). 28. T. Osumi and T. Hashimoto. J . Biochem. 83. 1316 (1978) . 29. T. Sakurai. S. Miyazawa. T . Osumi. S. Furuta and T. Hashimoto. Toxic, appl. Pharmac. 59. 8 (1981). 30. H. Ishii and T . Suga. Biochem. Pharmac. 28 . 2829 (1979) . 31. C. de Duvc and P. Baudhuin. Physiol. Rev. 46. 323 (1966). 32. T. Osumi and T. Hashimoto. Biochem. biophys. Res. Commun. 89. 580 (1979). 33. P. B. Lazarow. H. Shio and M. A . Leroy-Houvei. 3. Lipid Res. 23. 317 (1932). 34. Y. Mochizuki. K. Furukawa and N. Sawada. Carcino genesis 13. 11)27(1982). 35. L. Hardell and A . Sandsirm. Br. J . Cancer 39. 711 (1979). 1 ; .v .-X-'S THE DOW CHEM ICAL CO M PANY M ID L A N D M IC H IG A N REQUEST FOR PRELIMINARY TOXICOLOGICAL STUDIES - 1803 BLDG. MOLECULAR FORMULA f'ty * MOLECULAR eight SYNONYMS ^7 J A/ structural formula or COMPOSITION 9 0 ^3 0 * # 90 Q 7 3 ** G d o '/ / ,/ A oS* ,,r . 0 yo j ? < /{* /? 174 TOR. OR K NO. k l J 7-2- FILE T s .`A- 11 - 6 2 . '` r -A , *F *J& *' -rZ jL G Ji {FPOn ^AJnO*NUO P'ATdki'COLOGICAt ^ESTS: * : D0 M2,15 83 72 STAGE O f DEVELOPMENT: RESEARCH BENCH PROPOSEO AMOPOTENTIAL USE: Q - PILOT PLANT--------- SEMI PLANT ! " PLANT r CD REGISTRATION " --- **cTr*CDC?ANGLING: AMOUNTS TO ae hanOl EO: HUMAN E X P E R I E N C E IN H A N O L 1N C: NOTE.. " iNTfC -H ;.S.A. o 18 9 (BaGO THE DOW CH EM IC AL ?<t 7 " ^ 55 /</- / / V j t ' ) /trV ?7>- COMPANY MIDLAND A p r i l 13, 1970 6 9 8 S IZMOO: G . E . Lynn Ag. Products 9008 Building RESULTS OF CHLORACNE STUDIES CONDUCTED ON ASSORTED 2,4-D, 2 , 4 , 5-T, SIL V E X SAMPLES A N D THEIR E S T E R S > ORANGE AND DIAMOND'S CROP RIDER AMINE 4T-2. As p e r our t e l e p h o n e c o n v e r s a t i o n o n A p r i l 1, 1 970 h e r e is a summary of results of all chloracne studies recently com pleted on assorted 2,4-D, 2,4,5-T, Silvex Samples and their Esters, Orange and Diamond's Crop Rider Amine 4T-2. Chloracne T--Number 2578 2586 2587 2588 2589 2590 2601 2609 Name 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T 2611 2,4,5-T 2612 2,4,5-T 2602 2603 2604 * 2605 2606 2613 2669 2670 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D Reference Number 120449 120349 120369 120419 120459 120479 120070 Butyl Ester 60/40 PPI-93-3 Iso Octyl Ester PPI-93-5 D o w a n o l E s t e r (PiB) PPI-93-6 Lot # 130667 lot # 90530 .. L o t # 9 0 5 4 0 Lot # 90670 Let # 90970 Lot # 94847 Butyl Ester 60/40 AS I Iso Octyl Ester AS-7 2/5/70 Iso Octyl Ester DE 20 A 2/6/70 Chloracne Response Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative -- Negative Negative Negative Negative Negative Negative Negative !D0H2158370 G . E = Lynn 2- - A p r i l 13, 1970 Chloracne Number 2671 2607 2608 2610 2668 2667 Name Reference Number 2,4-D -- ------- --Dowanol E s t e r (PiB) A S -2 2/5/70 2,4-D D o w a n o l E s t e r (PiB) PPI-93-1 Silvex D o w a n o l E s t e r (PiB) PPI-93-2 Silvex Iso Octyl Ester 60/40 PPI-93-4 Orange M-3151 Lot # IRS 7 Crop Rider Amine 4T-2 Diamond Sample Chloracne Response Negative Negative Negative Negative Negative Positive Duplicates and triplicates were run on the majority of the a b o v e samples. O n l y D i a m o n d ' s C r o p R i d e r A m i n e 4T--2 e x h i b i t e d a positive chloracne response. F r e d L. D u n n Toxicologist Biochemical Research Laboratory 1803 Building Phone: 636-3165 wkw cc: V. K. Rowe, 1803 J. M. Nor r i s , 1803 J. F e r n a n d e z , 1710 File IDOH2158368 i 176 Todd, R. L. , A Case of 2,4-D Intoxication. J. Iowa Med. Soc. 52, 663-664 (1962) 18252 Dsherbants slectifs et substances de croissance. Aperu technique. H iiet pathologique su r l 'hom m e au cou rs de !a fab rication de l'ester du 2 .4 D. (1) 'Per K. M. ASSOULY i (Parta). Le dsir d'obtenir de belles rcoltes a. de tous temps, t le souci permanent dm agriculteurs. Pour y parvenir, ils ont essay divers moyens : enrichissement da sot par des engrais; accroissement dn rendement-par l'emploi de substances de croissance; enfin, destruction des mauvaises herbes qui, en absorbant les engrais. Peau et les matires minrales, entravent le dveloppement des plantes utQea. On est parvenu aujourd'hui, dans lo domaine des produits de croissance et des herbicides, des rsultats tout--falt remarquables. Mais leur fabrication sasdte des Inquitudes dont le Conseil Suprieur `Hygine de Franc* s'est fait l'cho dans sa sanco du 13 fvrier dernier. On a d'abord pens que ces produits pouvaient prsenter' une action cancrigne, et cette apprhension s'explique fort bien quand an voit Umanire dont.ccs substances agissent sur les plantes, od elles provoquent des tumeurs, des hyperplasies. Mais finalement, cette aetion cancrigne a t carte par le prefesseur Simonnet. Par contre, le Conseil Suprieur d"Hygine de Franc* a soulign le danger d'intoxication, que pouvaient prsenter ces produits pour les ouvriers qui les fabriquent. Avant de noos attacher i l'tude de la toxicit des substances en cause, noos t i oyossa utile de rappeler quelques notions techniques leur sujet. COMPOSITION ET ACTION DES HORMONES VGTALES . : Nous avons employ les mots d'hormones vgtales, parce que c'est ainsi qua l'on dsigne couramment ces produits destius accrotre le rendement. Il conviendrait peut-tre d'employer plutt (comme le remarque Pierre Chouard) le nom de substances de croissance qui dsigne, sans prjuger de leur nature Intime, les agents chimiques de tous ordres, capables d'exercer, de trs petites doses, une stimulation puissantesur l'longation des cellules vgtales Jeunes. Primitivement, ces substances ont t appeles auzines ou hormones de croissance vgtales ou phyto-kormones. En ralit, les auzines sont seulement les substances fabriques par la planta cQomme et qui exercent ce phnomne de stimulation, d'longation cellulaire par un mcanisme hormonal. On s'est aperu, ces 10 dernires annes, qu'une multitude d'autres substances avaient le mme pouvoir que les auxines; on les s dnommes hormones vgtales. Ces hormones ont une action qui varie suivant le moment d'utilisation et le but recherch : elles exaltent la croissance des plantes ou, au contraire, la ralentissent; elles acclrent la germination des graines, excitent la formation de racines, de germes, de boutons, de fleurs; ou, nu contraire, ralentissent ces fonctions; clics permettent la formation artificielle de fruits sans graines, sans fcondation par le pollen; elles aident (1) Co M i t e n i c a t i o n la Socit d e Mdecine du T r a t t t U , le 30 novembre 1054. .. /Lf-'L .fH-f 0 0 H215 83 42 18253 . D S H E R B A N T S E S U B S T A N C E S DE. CROISSANCE' 27,, 4 la rparation des dgts causs par U gele, facilitent U transplantation, le bouturage C'est dire que leur champ d'application est .vaste. Qut sont cet hormones T -- Ce sont des drivs oxy-actiqus'du noyas bnne et du noyau naphtaline ; des acides phnyl, nnphtyl et Indol-actique, preplontqne et. butyrique, ainsi que certains de leurs drivs, comme par exemple de nUrOee, des esters alkyllques, des sels d'alcali et d'ammonium, etc... L'un des plus connus est l'acide 2-4 dlchiorophnoxyactique, qu'on appelle, per abrviation, le 2-4 D. Chacun de ces produits a une action partiealiire t leur emploi varie-donc suivant le rsultat obtenir : par exemple, pour le bouturage, on utilise l'acide inrinl rtlrpis. l'acide lndol-butyrique ou l'acide naphtaline-actique ptutt que le 2-4 D. qui a une action trop dformante dans ce eus; contre la chute des fruits, on es sert-de l'acide naphtalne-actlque ou de ses sels, ou bien encore du 2-4 D. mais pende dilution. Signalons aussi que le 2-4 D. appliqu en pulvrisations ou arosols, stimule j-- - ment la maturation des fruits. ;. .v Un nombre considrable de corps ont t ainsi tudis et mis au point et certains (comme le 2-4 O. dont nous parlons plus haut) se sont rvle tre de l'ordre d'ans centaine de fols plus actifs que l'auxine proprement dite; si bleu qu'aux doses ordinaires ce n'est plus le pouvoir normal de croissance qui s'exerce, mais l'excs de ce pouvoir,, aboutissant la toxicit. De cette toxidt a driv un emploi nouveau : l'ntnisatlMt comme herbicide. . .. - - Cette utilisation des hormones comme herblcidet slectifs cet venue aussi de la cou- naissance de leur action stimulante sur les diverses fonctions de la plante* y compris la formation de tumeurs. L'usage des hormones, en tant que substances ds croissance, provoque souvent, aprs l'application, des hyperplasies, des cois cicatriciels qui e forment sur la surface de section d'un organe, tige ou ptiole en gnraL Les hormones,- 4 doses herbicides, ont une eetion Intensifie; elles provoquent une exaltation foOe des manifestations de croissance par hypertrophie et hyperplasie, une activit dsor donne qui entraine l'apparition de monstruosits les plus diverses, la consommation intense des rserves et finalement la mort, au stade herbac. Il s'agit donc d'une destruction radicale, puisqu'il n'y a pue formation de graines. Cette action est, en outre, slective; les hormones s'attaquent aux mauvalsea herbes" mais respectent les plantes utiles. E t ceci s'explique facilement : la rsistance dm plantes verte suivant les espces; celle des crales, par exemple, eet beaucoup plus forte que celle des ravenelles, coquelicots ou bleuets. C'est pourquoi le traitement est sans effet sur les crales, h condition cependant qu'elles aient dpass le stade 3 feuilles- - II devient ais de composer un produit de telle manire que seules les mauvaises herbes soient touches, tandis que les autres plantes resteront Insensibles son action. II convient, en outre, d'observer, lors de l'emploi, certaines prcautions pour obtenir un bon rsultat. En gnral, on opre 4 la Un du printemps, lorsque les tiges des plantes ont atteint leur plein dveloppement et ont puis les rserves de leurs racines. On vaporise le produit, en solution, sur le feuillage; Il adhre fortement, pntre rapide ment dans les feuilles; la sve le vhicule jusqu'aux racines; 11provoque alors, eu mme temps qu'un arrt de croissance au stade herbac, une prolifration dsordonne dee cellules qui aboutit 4 la mort, ainsi que nous l'avons vu. La destruction est obtenue eu 15 Jours ou i mois. Ces produits prsentent un avantage considrable sur les dsherbants habituellement employs : chlorate de soude, qui dtruit toute vgtation sans distinction; adde sulfurique, qui acidlAe le terrain, ou sulfate de cuivre, susceptible d'une lgre slec tivit, mais dont la manipulation prsente de graves dangers ou des inconvnients et dont l'action n'est pas aussi totale. .. FABRICATION DE L'ESTER DU 2-1 D I.a fabrication de ces hormones vgtales s'effectue seulement pendant quelques mois, en gnral ile fvrier 4 mai, c'cst-4-dlre pendant la priode o l'agriculteur e besoin de ces produits. Il existe sur le march deux sortes de produits dsherbants; l'un est base d'ester de l'acide 2-4 n . que l'on mulsionne dans de l'huile minrale avec un mouillant. L'utili sateur procde 4 une seconde mulsion du produit dans l'eau et le projette ensuite D0W2I583U fiMirrw j 1.'.Ci i -.-C 28 . . _i ' -* sur les surfaces dsherber au moyen d'un pulvrisateur. En cas de pluie, le produit, ' fix par le mouillant, adhre nanmoins. Le second qui sembla avoir la faveur des ' agriculteurs, ne contient ni mulsifiant, ni mouillant. A basa de sel de souda du 2*4 D. Il est directement soluble dans l'eau. Mais 11 risque d'tre lav par la pluie, puisqu'il' ne contient pas de mouillant. : iI De nouveaux produits basa d'ester sont actuellement en fabrication, comprenant une plus grande quantit d'mulsifiants. On pourra alors verser la produit directe . ment dans l'eau. Le mlange sa fera seul, sans qu'on ait mme besoin de l'agiter. D a n ^ usine que nous avons visite, on procdait seulement la raction d'estri- . Acatiun de l'acide 2-4 D. qui arrive tout prt d'une usine da province. Ouvrons une parenthse pour signaler que dans cette mina qui fabriqua l'acide lui-mme, les ouvriers sont imprgns d'une trs forte odeur de phnol, caractristique de ce produit. Cette odeur est extrmement tenace; elle persiste, mima lorsqu'ils quittent leurs vtements de travail. Ce qui leur a occasionn quelques dsagrments ; t I on considre, par exemple, leur prsence indsirable dans les lieux publics, cafs, tramways, etc.... Fermons ici notre parenthse et revenons notre nain parisienne. 1i La raction d'estrification a lieu partir du sel da soude de l'acide 2-4 D. sur lequel on fait agir de l'alcool thylique i 95*, port une temprature de 70*. A ce mlange, on ajoute ensuite de l'acide sulfurique k 98*, ce qui entrane une lvation considrable i iI de temprature. La cuve est alors refroidie et maintenue 70*. Cette opration ayant lieu en vase clos, l'alcool distille et retombe dans la cuve. Le produit est ensuite lov k l'eau, toujours en vase clos et on procde k une dcan i tation. II reste alors dans la cuve un liquide sirupeux qui contient environ 1 p. 100 d'nlcool et qui dgage encore une forte odeur die phnol. I 1 PATHOLOGI* L'Interrogatoire des ouvriers affects k cette opration rvle qu'ils prsentent les troubles suivants : somnolence avec sensation de jambes lourdes, Irritation des voles ariennes suprieures, gastralgie avec perte d'apptit, got sucr dans la boucha avec hypcrsallvation, sensation d'brit et d'hypersensibilit de l'oue, le moindre brait faisant sursauter les ouvriers. Les ouvriers employs au conditionnement te plaignent d'asthnie avec jambes lourdes, sensation d'brit et de tte vide. A quoi pourraient lre'dus les symptmes signals cl-dessus TOn pent imaginer qu'ils sont causs par le produit fini lul-mme ou par les manations des diffrents consti tuants, au cours de la fabrication. C'est ainsi que l'Irrltation des voles ariennes suprieures est bien connue dans l'Intoxication par les phnols, qui produit des catarrhes (laryngites, trachites). D'autre part, Il est signal par les auteurs que l'Intoxication chronique par le phnol -- d'ailleurs discute -- peut se manifester entre autres par des troubles digestifs, anorexie, ptyalisme, cphales, vertiges. Or, les ouvriers nous ont signal tous ces signes, parmi les troubles qu'ils rattachent l'exercice de leur profession. On pourrait rattacher galement quelques-uns des symptmes relavs aux manations probables des vapeurs d'alcool en cours de fabrication. Cette opinion n'est toutefoU pas partage par l'Ingnieur que nous avons vu ]>cndant notre visite, car, d'aprs lui, d'autres fabrications mettant en Jeu des quantits importantes d'alcool ne prsen teraient pas les Inconvnients signals par le personneL En rsum, on peut donc dire que la plupart des signes pathologiques peuvent tre dus au dgagement de vapeurs de phnol. Toutefois, des signes plus particuliers, tels que le got sucr, In sensation d'hypersensibilit de l'oue paraissent originaux et lus au produit fini lul-mme. Les troubles rapports cl-dcssus pourraient constituer un syndrome caractristique du travail au contact des produits en question, si d'autres auteurs les avaient tignala. Nous avons prord i des recherches bibliographiques, mais nous n'avons pu trouver de rfrence, du moins dans les sources que nous avons utilises, concernant leur net Ion sur le prrsonnrl employ il la fabrication. k * cfsr TT v-4 # & & & & :.-; ' - j - ;l1 D Q U 2 I 58315 IfclllJ. D S H E R B A N T S E T S U B S T A N C E S D E C R O IS S A N C E 29 Par contre, diverses publications signalent les travaux effectus sur tenr toxicologie. Il nous parait Intressant de rsumer ci-dessous les recherches effectues dans les diffrents pays du monde. TOXICOLOGIE f.e professeur R. Fabre, dons un rapport prsent aux Journes Mdicales Interna tionales de Paris eu 1937, signale les tumeurs qui se forment chez la plante autour d'une piqilrc d'htro-auxlnc, acide indol 3-nctlque. Il termine son rapport sur quelque questions pleines d'Intrt : <Les auxlnes vgtales ont-ellea une action sur le mta bolisme ou la croissance d'un organisme animal 7 Aucune exprience dfinitive n'a t tente dans ce sens Jusqu'Ici. Mais il serait fort intressant d'tablir un parallle entre l'action encore bien discute des hormones sexuelles sur le dveloppement des plantes et celle des auxlnes sur le dveloppement des animaux. Et s'il y a action, sur quelle cellule de l'organisme porte-t-elle 7 Autant de question sans rponses, qui doivent tenter physiologistes et mdecins. De mme, cette question de la toxicit du 2-4 D. pour les hnmains fut pose lors de la deuxime runion du Norlh Central States IVeed Contrl Confrence. Quelques membres relatrent alors des cas d'ingestions accidentelles de quantits variables, mais toujours sans altration apparente de la sant des accidents. Kraus fit savoir l'assemble qu'il avait lui-mme absorb quotidiennement 1/2 g de 2-4 D. pur pendant 3 semaines, sans ressentir de dsagrmenL Des expriences furent tentes Bcltsvllle, afin d'prouver la toxicit du 2-4 D. sur les animaux. La premire consista i faire patre, pendant 1S Jours, des vaches et des moutons dans un pturage pralablement trait avec l'herbicide. On avait mme doubl la dose habituellement ncessaire &la destruction des mauvaises herbes. Les' animaux furent ensuite examins par des vtrinaires, et trouvs en excellente sant. Dans la seconde exprience, on fit absorber une vache 5,5 g de 2-4 D. per jour, et ce pendant 3 mois. I.'animal consomma facilement le grain d'alimentation avec lequel la substance chimique tait mlange. Sa production laitire ne subit aucune baisse et un veau, nourri entirement de son lait pendant 1 mois, ne montra aucun symptAme de maladie. Une autopsie n'a pas rvl d'effet apparent du 2-4 D. sur les diffrents organes de cette vache. M. K. Bjom et H. T. Northen donnrent pendant 4 semaines une dose quotidienne de 200 mg/kg des poulets. Cela fut sans effet. Mais la dose de 7S5 mg/kg fut mortelle. E. V. llill, et H. Carliste, entreprirent des expriences sur 450 souris, 150 rats blancs, 125 cobayes, 70 lapins et 3 singes. Le 2-4 D. fut administr avec les aliments, en Injec tions Intra-veineuses, k des doses croissantes pour atteindre et dterminer la dote mortelle. A ce stade, les animaux meurent soudainement de flbrilatlon ventriculaire aigue. S'ils chappent k 1a mort Instantane, on voit apparatre la raideur des extrmits, avec de l'Incoordination musculaire, la lthargie, la stupeur et finalement, le coma et la mort. Ces symptmes sont constants, quelle que soit la faon d'administrer le 2-4 D. et quel que soit l'animal utilis pour l'exprience. L'investigation montre que le 2-4 D. est un compos relativement peu toxique dont la dose mortelle est de : 375 mg/kg d* sourit, 1 000 -- de cobaye, ' . 606 -- de rit, 800 -- de lapin. quand ces doses sont administres en solution aqueuse par la boucha. En supposant que la tolrance pour l'homme soit similaire k celle des animaux, la plus forte dose que pourrait supporter un homme de 75 kg serait da 15 g. Ces expriences ont t conduites avec du 2-1 D. utilis u>mme herbicide, sans que sa toxicit soit augmente par celle de ses solvants. Aucune exprience n'a t entreprise pour mettre en vidence l'Intoxication chro nique avec les effets de l'absorption par la vole respiratoire. Ue leur ct, Klng et Prnfound firent des es.iis en aquarium sur la brme et le bar. Ils trouvrent que dans ce milieu clos, le 2-4 D. A I p. 100 n'est pas toxique pour ces O Si, <0O - r jniv 9il 8 S I Z Mo a l .m . i i 30 poisons, mais qu'il le devient lgrement 4 100 p.'lOO, concentration qui ne peut tre , . que temporaire et locale en eau courante. .- . . t- ' . i iI I ] / 00 .. V .* F.n vrit, on se trouve l devant un produit nouveau dont le mode d'action lui-mme sur les vgtaux n'est pas encore tris dfini en agronomie et reste encore un peu mys- , , trieux. En ce qui concerne l'organisme humain, le problme est entier, matant do moini que nos recherches nous permettent de l'aOftnner. . nsuu On a donn le nom d'hormones vgtales i certaines substances chimiques capables d'exercer, A de trs petites doses, une stimulation puissante sur l'longation dot cel lules vgtales jeunes, action exerce habituellement par une substance labore pmr la plante elle-mme. Ces hormones sont des drivs oxy-acllqucs du noyau benzne et du noyau naphtaline, des acides phnyi. naphtyi et indol-actlqne, propianlque ' cl butyrique, ainsi que certains de leurs drivs : nltriles, esters alVyllques, sels d'alcali, d'auirnumum, etc... L'un des plus connus est l'dcida 2-1 dicblorophnoxyactique, appel couramment 2-4 O. L'action intensive de ces hormones les a fait utiliser comme herbicides slectifs. Elles agissent en provoquant une exaltation folle des manifestations de croissance, une activit dsordonne qui entrane l'apparition des monstruosits les plus diverses, la consommation Intense des rserves, et finalement la mort an stade herbac. Les ouvriers employs Ala fabrication de l'ester du 2-4 O. se plaignent de somno lence avec sensation de Jambes lourdes, d'irritation des voies ariennes suprieures, de gastralgie avec perte d'upptil, de got sucr dans la bouche avec hypersalivatlon, de sensation d'brit et d'hypersensibilit de l'oue, le moindre bcolt les faisant sursauter. Des expriences faites sur les animaux ont montr que le 2-4 D. absorb avec tes aliments ou Inocul en Injections intra-veineuses, n'talt toxique qu' dose* assez leves. . .: snuoounu v-. '> > , .- ' Bjo h n , M. K . e t N o r t iif x . H : T . -- RITeets o f 2-1 D leh lo ro p h en o x y -a r tic add o n ciiiek t S rte n te . 2'J. 11M8, p. 170-480. nKASSK-rtnosSAin. L. -- Le maurohei herbes. C.iioi a r i>, P. -- l,,e pro S rs rcen t <i:in la co n n n lssn o ce e t l'e m p lo i d e s su b s ta n c e s d e cro is- aiu-r. Jiecne inlernntinnale de Uolantque applique rt d 'Agriculture troplook, a** 307,308, 303. .110. l i t , 1 1 2 , 1018; n 311. 311. 317, 313, 319. 320, 1019. r.*vot.-KTTT>, H i.-htrU . -- Ce m e rv e ille u x 2-1 D . M inistre d e l'A g r ic u ltu r e , i O s b e e . 1 -A O n R . 13. -- l.es h o rm o n e s v g ta le s. R a p p o rt jir sc n U a u x Jo u rn e s .Mdicales In tem a tta n a ie * 1937, P a ru . M irrcintLL , J . \V\. H o r.r.io v , R. E. et Oa st ic x s , C. P. -- Toiersusce o f f a n a a n im ais to feed ro iita in iiig 2-4 U . ucjii. J . .-Irii-n.ii .Science. 5 , 1948. p. 228. , lI'Alui.nui.iAl-L'_l..V.! .e. tT.Ca--m ,11.--2 - 1 I ) . I w t n isu s . liq g . S T oA irlty ew ot 2-1 .i l e t l e r , 8 , 1 for e3 x4 p8 e, rpi. m9e.ntalanimais. Indu*. //|rf. et T m i.-ot. 29, n* 2 , 1!H7, p. 88-96. J akot. -- Pliyloliorm i-nr. C onfrence faite 8 la Maison d e la C h im ie, W 15 d cem b re 1911. - ' It I tI tj i 7 t 'l I 1 !t in K-4568K-2372- >1983 Chamost'here,Vol. 12,No.4/5,pp 749-759,1983 Printed in Great Britain 0045--6535/83/050749-11J03.00/0 01983 Pergaaon Press Ltd. a B n n op oh- oodc m s o n a u x a c n s a u c s a n s ohubd sdubs CN the Brjaium and anatnaim omcm annicawtrs A .L . tu x tj mad B.M. Shepa id 022ioe of Bwironwental Medicine, Vatarans Administration Keshington, O.C. 20420, O.SJL. //o Msuacr " Pv anvizoneantal or occupat ional health 1senes have received the sustained International attention that has bean fro mart an the pbenoxy herbicides and th eir associated chlorinated dlbenao-p-dioodna contaminants. The Veterans Adtainistrmticn in cooperation with other federal agencies in the United States Government has Initiated extensive health studies of veterans erpoasi to Agent Orange and its dioxin canbmrijiant during the Vistnm Conflict. The basis for resolving phanoxy berbiddes/dioxin controversies oust in large Measure stem from the results of scien tific inquiry. nmcoociicN The oae of herbicides to control vegetation has bean one of the m et persistent controversial subjects srlslng from the Vietnam Conflict. The Obited States Air Force applied re s t of these herbicides In dense jungle erase to ixicover id expoee hidden enemy staging areas, d e a r vegetation from the vicinity a t m ilitary bases and along linen of aoneunieation, end to destroy enemy crops. The objectives were bo provide defoliated nones that would reduce eobuehee and disrupt army attacks. Sis re st connonly used "defoliant* was 'Agent Orange,* a mixt ure of the two cemmericsl herbicides, 2,4-0 (2,4-dichloro* phenoxyacetic ad d ) id 2,4,5-T (2,4,5-tridUocophanoxyacetic a d d ), widely asployad for any years for brush control in forestry puajusus throughout the United States and for weed control in agriculture. During a S-yaar period fron 1965 to 1970, the O.S. Air Force applied note than 40 million lite rs of Agent Orange in South Vietnam. Some two million American m ilitary personnel served 1-year tours during the m m per i od. Recently, many veterans of that era have reported medical problem that possibly stem froa eapneure to Agent Orange during th eir m ilitary easigneents. Their oonplaints have ranged from tingling in the extremities to rare forms of cancer. Some veterans have fathered children with birth defects a d have suggested that Agent Orange is the culprit. The besis for resolving the Agent Orange controversy must in large measure stem froa the resu lts of s d e n tific inquiry. Accordingly, the Veterans Administration (VA) in cooperation with other Federal- agencies in the United States Government has in itiated extensive health studies of veterans exposed to Agent Orange and its dioxin oontmninant, 2,3,7,S-tetrachlorodibeno-p-dicocin (TODD) during the Vietnam Conflict. In addition, nunerous studies of non-veteran papulations exposed to the phenoxy herbicides and TOD have been encouraged. This artic le will briefly review the various federally sponsored health studies. 749 *-- .-v a>-w_ ,,3-82 5 8 a> hi!; CISCDSSICH 1b conduct pideniologic stu d ies o f hum n population erp-eed bo the phenaxy h erb icid es and th e d io x in co n tam in an t* , i t ix n e c e e s e ty to f i r s t know w hat h a a lth outcom e* should b e a aiae tad fo r study. Secondly, ana n u t ba ab le to id e n tify a stu d y co h o rt(s) w ith a high lik e lih o o d o f exposure and s c o n tro l groqp w ith a low o r no p ro b a b ility o f exposure. L a s tly , a su f f ic ie n t nusher o f in d iv id u a ls in each h a r t o u st be lo c a te d , re c ru ite d n d eraninari in cedar bo g iv e s t a tis tic s ] v a lid ity to th e r e s u lts . Uhfoctunafeely, th ese th re e requirem ents, so ia p a rta n t foe tb s aonduct of v a lid stu d ie s , are in ttiesaalvaa not c le a rly d e fin e d w ith r e s p e c t t o e i t h e r th e rh ae I r s l e used i n V le tim a as t h e p o p u la tio n a t r i n k . I t i s apparent th a t h e alth stu d ies o f the e ffe c ts o f phanoxy h erbicide w d /o r th e dio x in c o n ta a in a n t a re v a ry d i f f i c u l t under c o n d itio n s o f normal a g r ic u ltu r a l uaa a ltu a tio n x , b u t b e c o se im an a t l y o r e c o e p le x * e n c o n d u cted w ith o aho r t * b r i e f l y exposed a decad e ago d u rin g th e con d i t i ons o f w ar i n a t r o p i c a l e n v ix o n a e n t. pw ar B r ie f l i t e r a t u r e Beview Vary l i t t l e la reported in th e s c ie n tif ic lite r a tu r e concerning th e lo n g - ta n adverse e f f e c t s o f 2 , 4 - 0 , 2 ,4 ,5 - T , and TGDD o n h c a a n s . A cuta and s u b a c u te e f f e c t s a r e re p o r t ed q u i t e d f ot a l y fo llc w in g a cc i d e n t a l e x p o s u re s , s u i c i d a l g e s t u r e s , and i n d u s t r i a l a c c i d e n t s , b u t th e re is a g re e t d eal a t confusion concerning th e n a tu re o f lo n g -te s t e ffe c ts i f any. Much o f th e r a d i c a l know ledge aoncar u in g th e e f f e c t s a f 2 ,4 - 0 end 2 ,4 ,5 - T i s d e riv e d from case repo r ts . Since a e ry o f the p a tie n ts d escrib ed in th ese re p o rts were p h to w l t l p l e e h a n io l a g en ts, i t i s d if f ic u lt to determ ine the ch aeicels th a t produced s p e c ific symptoms. Of th e v e s t a rra y o f synptoae a ttr ib u te d to 2,4-D , th e n o e t c o n s is te n tly r epo rte d p ro b lem involve psychological behavior, the nervous system , th e liv e r end th e in test i n t s . \ M edical d a ta asso c iate d w ith erpm iire to 2 ,4 -0 cose p rim a rily from spraying in c id e n ts , but d a te fo r 2 ,4 ,5 -T and TQX cam ftem in d u s tria l exposures. Since th e f i r s t oonraerical p ro d u c tio n o f 2 ,4 ,5 -T , numerous in d u s tr ia l ep iso d es have involved exposure to t r id U o r c p h e n o l , 2 ,4 ,5 - T a n d TGDO. F i f t e e n o f t h e 23 e p is o d e s re c o rd e d in th e l i t e r a t u r e were ap p aren tly the re s u lt o f occupational exposures during in d u s tria l production of c h lo rin a te d phenols. B ut, on s ig h t occasions, p e rso n rel were exposed during cleanup f o llo w in g e x p lo s io n s o r t o Imprope r l y d e co n tam in ate d w orkshops ( 1 5 ) . unf o r t u n a t e l y , t h e e f f e c ts o f 2 ,4 ,5 -T in th ese episodes could no t ba d e a r l y d istin g u ish e d from the p o ss ib le e f f e c t s o f TCCO. Symptoms a t t r i b u t a b l e t o 2 ,4 ,5 - T a n d TCDO e x p o su re in c lu d e a l l o f t h e sy m p to m o f 2 ,4 - 0 e x p o s u re , i n a d d it i o n t o a s k in d i a e a s e , c h l o t a o i e . Many s c i e n t i s t s b e lie v e th a t chloracne is th e 'hallm ark* o f exposure to th e d ib en x o -p -d in x in s, e s p e c ia lly 2,3,7,8-T OE O . QU oracne i s a akin re a c tio n c h a ra cte riz ed by a aeniform d e rm a titis w ith comedo n e s (b la c k h e a d s) and i n c l u s i o n c y s t s o r p a p u le s and f r e q u e n t l y p u s t u l e s eo s e v e re t h a t th ey cause permanent sc a rrin g . M orphologically, i t is sim ila r to ad o lescen t earn , but i t i s u s u a lly so re sev ere, p a rtic u la rly on the upper face, e a rs , and neck. A ctive chloracne l e s io n s bave been repor t ed many y e a rs a f t e r ex posure .to TOED, b u t th e c o n d itio n u s u a lly c le a r s up spo n tan eo u sly in s few m onths. P rs s s tu te a g irg o f in volved a k in a re a s has been rep o rted in sane instances as w ell as th e ^p earan o e o f hyperpigm entation. 18259 751 S everal cbm ao n tzo l epidem iological s tu d ie s aonductsd by Swedish s c i e n t i s t s have rep o rted evidence o f a s t a t i s t i c a l r e la tio n s h ip between * s o ft tis s u e can cers" and exposure to th e phanoxy h e r b ic id e s and TQX3 (4 ) w h ile s i m i l a r s t u d i e s by F in n is h (9 ) and Mew Z ealand s c i e n t i s t s (11) dhow no r e la ti o n s h ip to s o f t t i s s u e sarcom a. The d a ta by Tung a t a l . (13) f r a * Vietnam h a s re ce iv e d w idespread a tte n tio n , b u t th e s c i e n t i f i c o c a a m ity has viewed th ese s tu d ie s w ith c aut i o n . Except fo r angiosarcom a, a ra re type a t c m c e r, which Is know to be caused by v in y l d ilo rld e exposure, th e re a re v i r tu a l ly no oanoers o f connec t iv e o r . o t h e r " s o f t t i s s u e " w hich have a known e t i o l o g y r e l a t e d t o c h e m ic a l e x p o e u re . The f i r s t re p o rts o f huaan b i r t h d e fe c ts a ttr ib u te d to Agent Orange ^ p e e re d in V ietnam ese newepspe r a i n Ju n e 196 9 . As a r e s u l t o f t h e p u b l i c and s c i e n t i f i c f u r o r couaod by t h e s e r e p o r t s . C u ttin g e t a l . ' (3) and H eselaon e t a l . (7) conducted two In dependent m irveys o f South Vletnemese h o s p ita l re c o rd s. Although n e ith s r repo r t reached d e f in ite co nclusions on th e v a lid ity o f th e a c c u sa tio n s, both re p o rts acknowledged th a t search es o f th e reco rd s p n b a b ly would have re v ea le d any marked in c re a s e in b ir th d e fe c ts o r in tro d uc tio n o f a s t r i k i n g d e f e c t , su c h a s t h e d e f e c t s produced b y th a lid o m id e . S u b seq u e n t r e p o r t s by TUng e t a l . (13) in 1971 vd Rose and Rose (10) In 1972 c en tere d c n c li n i c a l o b se rv a tio n s and -in te rv ie w s s n d u c ta d in Hanoi w ith refugees t t o claim ed th a t th ey were re p ea te d ly sprayed w ith de f o lia nt s in South Vietnam . The author s r epo rte d u n u su ally high ra te s o f a b o rtio n s and save r s b ir th d e f e c ts in both b tn an s and <- a n im a ls . In announcing th e r e s u lts o f stu d ie s conducted in South V ic tim in 1972 and 1973 (2 ), a c o m i t t e e a p p o in te d by t h e N a tio n a l Academy o f S c ie n c e s r e p o r t e d t h a t i t c o u ld f i n d to c o n clu siv e re la tio n s h ip between exposure to h e rb ic id e s and b ir th d e fe c ts In humans, but th e io m l t t e s recognized th a t a v a ila b le b ir th re co rd s were not adequate fo r d e f in ite c o n c lu s io n s . The o e m o itte e a l s o c o u ld n o t c o n firm o r deny r epo r t s t h a t some h im a n s, e s p e c ia lly th e H ontagnards, and dom estic anim als became i l l o r d ie d a f t e r th e y were exposed to h e rb ic id e sprays o r a f te r th ey consizmd tr e a te d p la n ts o r contam inated w a ter. In th e c o v e r l e t t e r t o th e r e p o r t , th e p r e s i d e n t o f t h e N a tio n a l Academy o f S c ie n c e s s t a t e d ! "On b alan ce, th e untoward e ffe c ts o f th e h e rb ic id e ptogrma on th e h e a lth o f th e South Vietnamese people appear to have been a n a lle r than one m ight have feezed". In 1982, Townsend e t a l . (12) r e le a s e d a re p ort, an t h e i r stu d y o f th e o f f s p r in g o f p ro d u c tio n w o rk e rs expose d t o 2 ,4 ,S - T and TCEO. The s tu d y was b a se d a n an I n te r v ie w q u e s t io n n a i r e a d m in is te re d t o 370 w ives o f men w ho-had worked i n a r e a s w here th e y c o u ld have been exposed t o TCCO and t o a c o n tr o l g roup o f 345 w ives o f men i n th e s u e aanpany tb o had nev er to rk a d in such a r e a s . The stu d y found no s t a t i s t i c a l l y s i g n i f ic a n t d i f f e r e nc es in the in stan ces o f m isca rria g e s, s t i ll b i r th s , In fan t d eath s, o r congenital m alform ations in the t*o groups. Early Federal E ffo rts The U nited S ta te s Environm ental P ro te c tio n Agency, th e U nited S ta te s D epartm ent o f A g rira ltu ra and th e N ational I n s titu te o f O ccupational S afety and H ealth have a l l conducted 752 lim ite d atudie* on th e p o te n tia l a sso c iatio n s o f phenoxy h erb icid es w ith hunan spontaneous a b o rtio n s and b irth d e fe c ts . Two s t u d i e s were c o n d u c te d b y th e E n v iro n m e n ta l P r o t e c t i o n Agency t o e v a l u a t e th e r e l a t i o n s h i p be t we e n h e r b i c i d e s s p ra y in g and re p ro d u c tiv e e v e n t s . John so n (6 ) r e p o rte d on th e d a ta used in e a r ly 1979 by th e E nvironm ental P ro te c tio n Agency in th e d e c isio n to o rd e r th e suspension o f re g is tra tio n s o f 2 ,4 ,5 -T fo r uses in fo re s t, rlg h te-o f-w ay , end p a stu re s . He r e p o r te d t h a t t h e in c id e n c e o f ^ o n t a n e o u s a b o r ti o n s o v e r a 9 - y e a r p e r io d in A l t a i , Oregon, was found to ba h ig h e r th an tb a r s te a in two o th a r re g io n s o f Oregon t h a t had low er ra te s o f 2 ,4 ,5-T usage. A lthough, a c y c lic p a tte rn in th e Incidence o f ab o rtio n s in the A lias g r o ^ c o rre la te d p o s itiv e w ith th e p a tte rn o f annual 2 ,4 ,5-T sp ray usage, th e Agency adonw ledged i t was n o t p ro o f o f a aause and e f f e c t re la tio n s h ip . In a n o th e r study spons o r e d by th e EPA, howeve r , t h e l n d d e n o e o f c l e f t p e A t e o v e r a p e r io d o f 32 y e a r s i n A rkansas was shown n o t t o be r e la te d t o th e usag e o f 2 , 4 , 5 -T . The in c id e n c e o f c l e f t p a la te f a r re g io n s w ith a low p ro p o rtio n o f r i o t a cre ag e (in d ic a tin g low exposure t o 2 ,4 ,5-T) and f o r re g io n s w ith h ig h 2 ,4 ,5 -T exposure in cre ased o v er th e y ears s tu d ia d . T his tre n d was a ttrib u ta d to b e tte r d e te ctio n o f c le f t p a la te and not to h erb icid e usage (8 ). The U n ite d S t a t e s De p artm e n t o f A g r i c u lt u r e sp o n s o re d a s tu d y d a s ig n a d t o e v a lu a te t h e r e la ti o n s h ip batween hixeen s a l e ex p o su re t o 2 ,4 -D and th e ln d d e n o e o f sp o ntaneous a b o rtio n s in th e i r w ives. In t h i s stu d y re p o rte d by Cana111 e t e l. (1 ), q u e stio n n a ire s p e rta in in g to t h e s e two f a c t o r s w ere s a i l e d t o a lm o s t 1 5 ,0 0 0 p e o p le who were e o p lo y e i i n o c a q a t i c n s in v o lv in g th e m an u factu re o r u se o f h e r b ic id e s . From th e re sp o n d e n ts , e group o f 134 e a s e s o f M isc a rria g e s were s e le c te d and aoapazad to a c o n tr o l groiq> o f 311 c a s e s o f l i v e b i r t h s . The c ases were analysed to determ ine th e incidence p e re n ta l exposure to 2 ,4 -0 p r io r to co n cep tio n , and th e ln d d e n o e o f o th e r re le v a n t confounding f a c to r s , such a s enoking illn e s s e s , end drug u se . The d a ta were o btained o r v e rifie d by telephone In terv iew s, lb p o s itiv e a s s o c ia tio n was e sta b lis h e d betw een 2,4-D exposure end a b o rtio n s, f a r th e e n tir e s tu d y p o p u la tio n , toe a g ro u p o f 21 c a s e s o f w iv es o f young f o r e s t and o a m a r i e a l w o rk e rs , a n a s s o c i a t i o n as s u g g e s te d , b u t was n o t found f o r any o t h e r s u b g ro u p . F u r t h e r in v e s tig a tio n s were su g g ested to c l a r i f y w hether t h is r e la tio n s h ip re s u lte d from b ia s e s in s tu d y d e s ig n o r r e f l e c t e d a r e a l i n c r e a s e among t h i s n a i l s u b s e t . Q IS op <=$ -f if i tB * I it k m % I* iIfeF.-* i I n November 1979, th e N a tio n a l I n s t i t u t e fo r O ccu p atio n al S a fe ty and H ealth receiv ed a r e q u e s t t o d e te rm in e w h e th e r an e x c e s s r u d e r o f b i r t h d e f e c t s o c c ur r e d anting th e c h il d r e n o f th e m ain ten an ce enployees w orking f o r th e Long I s l a n d R a il r o a d , New Y ork, and w h e th er th e s e d e f e c t s may h av e been cau sed by e x p o su re t o 2 ,4 ,5 - T . Hnnchar (5 ) i n v e s ti g a te d 170 liv e b ir th s observed from saong th e stu d y p o p u latio n o f approxim ately 1,400 em ployees. S ie re p o rte d th a t 42 o f th e l iv e b ir th s had a t l e a s t one n o n in fec tio u s h e a lth problem d u ring th e f i r s t y e ar o f l i f e . I t wes found th a t a l l m ajor b ir th d e fe c ts aanbinad and in g u in al h e rn ia were underrepre sen ted in th e study p o p u latio n (3 observed v ersu s 3.81 expseted fo r m ajor b i r th d e fe c ts and 2 obeerved v e rsu s 2 .3 expected fo r in g u in a l h e r n ia ) . However, 1 8 61 s? 1 l f n* cl -^ ?r ^ t l r t?f1- *a l i irt 753 m e ta ta r s u s adductu* (S o b se rv e d v e rs u s 3 .4 7 e x p ec te d ) and t e a r d u c t o b s tr u ct io n (3 o ts s r v s d v ersu s 0.22 expectsd) w n both s ig n ific a n tly over t ep rseen tsd in th e stu d y p o p u latio n . Hbnehar concluded th a t th a sa l a t t e r d a fa c ts were probably rw a n a p re sin ta d in tb s study population dua to diagnostic b ia s. ------ Previous stu d iaa there fo re suggest th a t th a n is very l i t t l a epidam ialogic d a ta associating TOO o r th a pbenaxy herbicide w ith a iy lo n g -ta n h a alth e ffe c ts in fan am o th a r than c h io ra c n e . However, as n o ted by tta lf e (1 4 ), n e ith e r la t h e n s t r ong ev id en ce to -- i m . th a aheanoa o f such e f fe c ts , (tost sta d ia s have not included s u ffic ie n t m ak e rs at a ttr a c ts t o d e t e c t an increased r i s k a t an issarewon a a n d itlo n . A lso th e r - " ^ o f o b s e rv a tio n in sany s ta d ia s h a s bean in ad eq u ate t o d e te c t r illn a s a w ith a long lafeancy p e rio d between exposure and illn a s a . T h e n i s c u rre n tly no r e lia b le evidence th a t lin k s d io x in exposure to cancer o r b irth defects in h iaan s. H ealth S ta tu s o f Vietnam V e tera n s As o f August 1982, n o n th a n 15,000 Vietnam v e te ra n s have su b m itted to th a V eterans A dm inistration fee d is a b ilitie s * iic h they believe a re tha re s u lt at exposure to h erb icid es. These claim* d e sc rib e ace th a n 130 d if f e r e n t e f fe c ts in fiv e m ajor c a te g o rie s s p a y to ia trie , d e rm a to lo g ic a l, re p ro d u c tiv e , n e u ro lo g ic , and a n o e r . The a r i a n da t a v a lid a ta l i n k s b e o man e x p o su re t o A g e n t O range and TOO i n t h a s e n s e t h a t aywp te a s r e p o r ta d by th a v e te r a n s have a ls o bean d o c u aa n ta d i n o t h e r r a s a s a t e x p o su re t o th e h e r b ic id e s o r t o ICED. But m ost o f thasa sysptzne, a .g . , peripher a l neuropathy, fa tig u e , w eight lo s s , and acee p sy ch o lo g ical d istu rb an ce , a r e a cu te syxptcnB th a t s a n if a e t th aaselv aa a h o rtly a f te r exposire . Such ayaptaaa a r is in g y e a rs a f t e r th e l a s t Iraoun exposure a re s o e t l i k e l y not cens arl by 2 ,4 -0 and 2 ,4 ,5 -T o r T O O . A la r g e nunbar o f th e v e te ra n s have d a is ie d d e rm a to lo g ic a l p eo h le n e, and many b e lie v e d th ey had e h lo ra c n a . 1b d a ta , how ever, no o ases o f ehloracna have bean confirm ed by p h y sical exam ination in th is grexp o f v e te ra n s . D espite th a lack o f so lid s c ie n tif ic evidence to eupport th e v e te ra n s' a lle g a tio n s th a t A gent o ra n g e i s th a cau se o f t h e i r d i f f i c u l t i e s , a n a r e c o g n iz e s t h a t warn o f t h e v e te r a n s hava d e f i n i t e l y exp e rie n ce d s e r io u s h e a lth problem s. T h e re fo re , th e pu rp o se o f many o f th e gove r nmen t s t u d i e s i s t o d e te rm in e w h e th e r A gent G range i s r e s p o n s i b l e f o r th e s e h e a l t h p ro b le m s . I f A gent Orange i s n o t t h e c u l p r i t , o t h e r f a c t o r s as s o c i a te d w ith t h e V ietnm n war sny be re sp o n sib le . Consequently, th e goal o f o th e r resea rc h e ffo rts is to determ ine i f V ietnam v e ta ta n s as a group a r e ex p erien cin g so re o r d if f e r e n t h e a lth problmns th an th e ir c o u n te r p a r t who d id n o t s e r v e i n t h a t p a r t o f tb a w o rld . I n su ch a a o n p ls x s i t u a t i o n no s in g le stu d y can provide a l l o f tb a answers . Thus, th e re i s a need fo r a n u tb e r o f d if f e r e n t approaches to exam ining th a h e a lth o f th e Vietnam v e te ra n . c s r a a r e idvestigaticks Hhat has been th e response o f th e V eterans A dm inistration and o th e r governm ental agencies t o t h e c r i t i c a l i s s u e s r a is e d b y A gent O range? E a rly a c t i o n by t h e VA in c lu d e d t h e develognent o f a cansm ic a tio n p ro cess fo r g ath erin g s c ie n tif ic and a d ic al in fo r m ation gli09'ZHOO 754 ^ 2 Z`0 `9 I Z M 0 (I lUSfi -V*<. illi.I:<n-i>rCi>T-V.'Ji f which would b u r d i r e c tl y on th e problem , in a d d itio n , e a r ly in 1978, th e V eterans A dm inistration e sta b lish e d th e Agent Orange R eg istry in o rd e r to acoonpllsh four o b je c tiv e s: (1) lb id e n tify a ll. V letn m v e te ra n s expressing a concern about th e p o ssib le adverse h e alth e ffe c ts o f th e ir exposure to Agent Orange. (2) l b p ro v id e a e a c h a n i t fee V letnm a v e t e r a n s t o v o ic e t h e i r i r w i m t o a p h y s i c ia n , re ce iv e a p h y sic a l exam ination end o b ta in re sp o n sib le n rra n to aam e.cf q u e stio n s . (3) l b se rv e e s a aactianima f o r follow -up o f th e s e in d iv id u a ls i f , a t a l a t e r d a te , new in fo c se tlo n develops as a r e s u lt o f th e v a rio u s re sea rc h e f f o rt s underway o r being planned. (4) 10 o b t a i n , a s a b y -p ro d u c t, acne p r e li m in a r y in fo rm a tio n o n t h e c u r r e n t h e a l t h s t a t u s o f t h s ve t e r ans *ho have p a r t i c i p a t e d i n th e r e g i s t r y . th e Agent Orange R e g istry provides us e s s e n tia lly w ith a l is ti n g o f nanas and a d d re sse s, together w ith sons bsdegrorod d ata a t a se lf-re p o rte d group o f v e terans w ith serv ice in Vietnam. A c a o p ie te p h y sic a l e x a e in a tio n and a group o f b a se lin e la b o ra to ry t e s t s a re provided to each v e te ra n . Iba inform ation iro n th is e f fo r t is c u rre n tly being placed in to a im p u ta r d a ta b u * . Ib e ra a re alm ost 100,000 Vietnam v e te ra n s th o have been examinad by th e V e te ra n s A d s d n ls ta t io n . He a r e in th e pr o c e s s o f c o l l a t i n g th e d a t a t o d e te rm in e w h e th e r o r n o t t h e r e a r e any s i g n i f i c a n t h e a l t h t r e n d s i n t h e s e i n d iv i d u a ls <4vlch m ig h t s u g g e s t a r e a s for fu rth er investigation. t. 1CTO A ssay o f Hunan A d ip o se T is s u e S in c e TCDD i s known t o a c c in u l a te p r e f e r e n t i a l l y i n th e a d ip o s e t i s s u e o f c e r t a i n s p e c ie s o f la b o r a to ry a n m ala, i t was su g g e ste d , e a r ly in th e h i s t o r y o f th e A gent .Orange is s u e , t h a t t h e n a l y s i a o f tem an f a t f o r TCDD m ig h t p ro v id e a way o f d e te rm in in g A gent O range e x p o s u re . In a d d itio n , i f d e te c ta b le le v e ls o f t h i s tu b s ta n ae w ere p r e s e n t, th e r e m ight be some re la tio n s h ip bo h e a lth problem s. Although m ethods fo r TOO a n a ly sis have lnptuvcd in re c e n t y e a r s , no such stu d y had bean c a r r ie d c u t in Inm ans w ith known ex p o su re to h e rb ic id e s c o n ta in in g t h i s t o x i c o o n tm rin a n t. c o n s e q u e n tly t h e VA em barked on a s n a i l f e a s i b i l i t y s tu d y t o t e s t th e me th o d o lo g y a d s e e w hat c o n c lu s io n s m ig h t be draw n re g a rd in g th e s ig n if ic a n c e o f th e r e s u l t s . The stu d y was c a r r ie d o u t in th r e e g ro u p s o f a d u lt m ales as follow s: (1) tw e n ty V ietn am v e te r a n s who c la im e d h e a l t h p ro b lem s r e l a t e d t o A gent O range e x p o su re a n d who v o lu n te e re d f o r t h e f a t b io p s y . (2) T h ree D .S . A ir Force o f f i c e r s w ith known heavy and r e l a t i v e l y re c e n t exposure in conne c t i o n w ith h e r b ic id e d is p o s a l o p e r a t io n s b u t who d i d n o t s e r v e i n V ietnam . (3) Ven v e t e r a n s w ith no s e r v i c e in V ietnam and no known e x p o su re t o h e r b ic id e s who ' 755 spectrom etry. lh * re s u lts o f till try im p le x and te c h n ic a lly d if f ic u lt an aly sis indicated th a t secy la # le v e ls o f TOD, b eliev ed to be 2,3,7,8-TCDO , could be d e te c te d in hunan a d ip o se tis s u e in th e range 3 to 99 p a r t s - p a r - t r i l l i o n . D ie le v e ls , how ever, d id n o t . c o r r e l a t e w e ll w ith known e x p o su re mt5 n o n -e x p o su re , and t h e r e was no o o r r e l a t i o n w ith h e a l t h s t a t u s . The s tu d y r e s u l t s , howev e r , in d ic a te d t h a t th e a ssa y method was f e a s i b l e , b u t would se rv e no c lin ic a lly o r a d m in is tra tiv e ly u s e fu l purpose w t i l * ta were a v a i l a b l e a i background l e v e l s o f TCCD i n t h e g e n e r a l U n ite d S t a t e s p o p u la tio n . A ccordingly, th e V eterans A dm inistration, in coopera t i e r w ith th e rniilinm aiH il P ro te ctio n A gency (ERA) h a s i n i t i a t e d an I n te r a g en c y a g ra e e e n t t o s tu d y l e v e l s o f 2 , 3 , 7 , S - t O S i n edlpoee tis s u e fra a aelectad group o f O .S. s a le s . The B nvironeental P ro te ctio n Agency has bean dam ographically c o lle c tin g adipose tis s u e fra a th e O .S. g e n era l populatio n ( i . e . , by re g io n , age, sex and ra c e ). T h is N atio n al Adipose T is s u e Bank v e s i n i t i a t e d in 1968 and now c o n ta i n s i r e c i s i e n t from o v e r 1 2 ,0 0 0 i n d i v i d u a l s . R epresented w ith in th is bank i s a v ailab le adipose t is a m f r a a approxim ately 500 melee bom betw e en 1937 and 1952. Many o f th e s e in d iv i d u a ls w i l l h ave s e rv e d i n th e m i l i t a r y d u rin g th e V la tn a a e ra and i t la raaso n ab le to eatme th a t sane a c tu a lly serv ed in Vletnma d u rin g th e p e rio d o f A gent O n n g e u se . A r e t r o spect iv e stu d y o f s e le c te d c h lo rin a te d d io x in s md fu ran s nay e s ta b lis h d a ta an background le v e ls o f 2,3,7,8-T Q 30 in th e O .S. male p o p u latio n a s w ell a s w hether se rv ic e in th e m ilita ry and e s p e c ia lly in Vletnme h as had an e f f e c t cn the le v e ls o f TOD in fllp T Il tis s u e . The s t u d y w i l l be c o n d u cted i n t h r e e { b a s e s . H ia se I w i l l b e t o o b t a i n th e name and s o c i a l s e c u rity m n b er fo r the approxim ately 500 m ales noted above. T his inform ation w ill ba used to d e te a d n e m ilita r y serv ice s ta tu s . Phase I I w ill be th e developm ent o f a n a ly tic mathods f o r t h e d e te r m in a tio n o f a e l e c t a d d io x in s ( e s p e c i a l l y t h e 2 ,3 ,7 ,8 - JTQX)) and f u r a n s i n huaan a d ip o s e tis s u e . The method w i l l be su b je cted to rig o ro u s in te rla b o ra to ry v a lid a tio n by an independent A nalytic Referee, e .g ., the A ssociation o f O ffic ia l A nalytical Q iam ists. R u se I I I w ill ba th e a n a ly sis o f th e adipose tis s u e and th e p re p a ra tio n o f a f in a l rep o r t. Phases I and I I ahould ba coupleta d w ith in f is c a l year 1983, and the repo r t from Phase I I I should be a v a ila b le in e arly 1985. Vietnam v e te ra n M o rta lity Study The va h a s i n i t i a t e d two la rg e m o r ta lity s tu d ie s which w i l l a n a ly se and e rn p a re d e a th r a te s and c a u s e -o f-d e a th p r o f ile s between v e te ra n s w ith s e r v le t i n Vletnma and com parable v e te ra n s w ith no s e rv ic e in Vietnam . I t is e stim ated th a t approxim ately 300,000 Vietnam and V ietnam -E ra v e te ra n s have died sin c e the' s t a r t o f th e V ietnam C o n flic t. T his number in c lu d e s ap proxim ately 52,000 ocnPa t d e a th s. The s tu d ie s w ill use e x is tin g a x p u te r record* to id e n tify e grxxp o f approxim ately 60,000 deceased v eteran s and determ ine cause o f death and Vietnam s e rv ic e s ta tu s . The stu d ie s a ls o include independent v a lid a tio n o f th e computer d a ta bases used in the stu d ie s. I t is iap o rtan t to note th a t the m o rtality stu d ie s w ill not p rovide inform ation s p c d f ie ia lly re la te d to {barney hartoieide exposure, b u t ra th e r to s e rv ic e i n Vietnam . 18264 756 Vietnam B cperlanoe tw in Study "tarn V ietnam E x p e rie n c e Twin S tu d ? w i n i t i a t e d by a g ro u p o f r a s t s r c h a t a f f m a tte r s a t th e W etenna A d a in istratlo n M ed lcal C enter, S a in t In u la , H laaourl. The atudy w ill id e n tify p a ir s o f tw in v e te ran s here ona tw in served In V latnaai during th e p a rlo d o f Agent Grange e p re y in g and tb a o t h e r s e r ve d In m a r e a o t h e r t h a n S o u th e a s t A s ia . A p p ro x im ately <00 p a i r s o f tw ins w ill be esaainad taring a a e rie s o f p e y d io lo g ic, p h y sio lo g ic and him tiam tial ta a t a . lh a d rv e lo p e a n t o f a p ro to a o l f o r th e stu d y and th e a sta b lla ia a a n t o f a Twin R e g istry iro n which t o s o l i c i t p a r t i c i p a n t s ace now underw ay. The a tu d y w i l l fo c u s p r i m a r i l y a n t h e t o t a l Vietnam ex p erien ce r a th e r th an any s in g le fa c to r such a s Agent O range. I f s u f f ic ie n t m a t e r s o f i d e n tic a l tw in p a ir s a re i d e n t i f ie d , th e r e s a y be an oppor t u n ity bo draw some aoneluaions regarding the e ffe c ts o f sp e c ific asp ects o f th e V letn ai experience sudi a s ooabat, herbicide axpoaure o r uaa o f in a e c tid d c a . B irth D efects and M ilita ry Servloa jn Vietnam Study I n 1981 t h e C e n te rs f o r D laei a s C o n tro l (CDC) i n A t l a n t a , G e o rg ia l n i t l a t a d a s tu d y d a a ig n a d to determ ine I f V latn aa v ete ran s are a t an ineraaaad r is k o f having c h ild re n w ith b ir th d e f e c t s . S in c e 1968 CTC had m a in ta in e d a r e g i s t r y o f a l l b a b ie s b o m w ith d e f e c t s i n th e g r e a t e r a e t r o p o li t a n A tla n ta a r e a . Of th e s o r e th a n 15,000 c h ild r e n i n t h i s r e g i s t r y , approxim ately 7,500 had s ig n if ic a n t anatom ical d e fe c ts a t b i r th . The in v e s tig a to rs w ill a t t e s t bo lo c a te and in terv iew both p a re n ts o f a l l 7,500 o f th e c h ild re n in th is group. In a d d itio n , th e p a re n ts o f 3,000 matched co n tro l norm al bo m d u rin g the sane tim e period w ill be interview ed. Since the m ajor o b je c tiv e o f th is study w ill be to determ ine dietber an unusually high pro p o rtio n o f fa th e rs o f babies bom w ith d e fe c ts aarvad in V ie tn a a , in fo r m atio n w i l l be ga th e re d about Vietnam a e rv ie a a s w e ll a s o th e r f a c to rs rich say be asso c ia ted w ith the occu rrence o f b irth d e fe c ts . I f the study dem onstrates th a t a V ietnaa v eteran has an ln o e a sa d ris k of fath erin g a c h ild w ith a d e fe c t i t aey be d e s in b le to a ttm ip t to determ ine i f th e in c re a se is a sso c ia te d w ith Agent Orange exposure o r w ith same o th e r f e c t o r ( s ) . The stu d y i s scheduled to be a m p le te d by th e err! o f 1983. The A ir fo rc e H ealth Study I n 1979 t h e A ir f o r c e i n i t i a t e d th e p r o to c o l f o r a oa ip c e h e n siv e e p id e m io lo g ic stu d y o f th e RANCH HAND p e r s o n n e l, e group o f a p p ro x im a te ly 1 ,2 5 0 men who a c t u a l l y co n d u cted th e a e r i a l , fixed-w ing h e rb ic id e spceying m issions in Vietnam from 1962 through 1971. The study i s designed around th e ^ ic s tio n ,,* B e v e th e re been, a re th e re now, o r w ill th e r e be in th e re a s o n a b ly f o r e s e e a b le f u t u r e , any a d v e rs e h e a l t h e f f e c t s eaong RANC3 BAND p e rs o n n e l c au sed by repe ated expoeure t o H erbicide Orange?* The in v e s tig a tio n is o p p o sed o f threw in te g ra te d elem ents -- a m o rta lity study o f those in d iv id u a ls who have d ie d s in c e t h e i r e x p o su re , a m o rb id ity study t o exam ine th e c u r re n t h e alth s ta tu s o f th e s tu ly su b je c ts, and a follow -up study to look fo r delayed e ffe c ts ewer th e n ex t 20 y e a rs . The m s rta lity and m o rb id ity study e l ev en ts see being conducted s im u lta n e o u s ly on t h e RANCH HMO p e rs o n n e l and a v e ry a a r e f u l l y m atched c o n tr o l g roup by Ho 4cn f J r fjs * | f jt f ; ~ & -4 * i *1* | * ? t r *P t 00H2I60729 757 th e o m ot personnel trac k in g procedures coupled w ith an e x te n siv e review o f m ilita ry R adical and personne l reco rd j d e ta ile d fa c e -to -fa c e q u e stio n n a ire to c ertain c u rre n t and p a st h e a lth event as 11 as o ccu p atio n al d fam ily d a ta ; and oaspeeharsive p h y sic al exam inations, psychological ta s tin g and d iag n o stic lab o r a to ry stu d ie s to determ ine e x a c t h e a lth s ta tu s . A dditional q u e stio n n a ire s and p h y sic al ex an in atio n s are to be adm inistered p e r io d ic a lly A iring th e follow-Mp p h a se . R a su lts from th e m o rta lity a n a ly s is a re to be rep o rted in s a r ly 1983 and th s r e s u lts o f the i n i t i a l q u e stio n n a ire and cur re n t h e a lth sta tu s a n a ly sis see to be released in eid-1983. E p id e m io lo g ic S tudy o f Around T ro o p s Eiqnos s d t o A gent O range P u b lic Law 96-151 d ta r g e d t b s V e te r a n s A t o r i n is tr a t io n t o 'd e s i g n a {SAkouul t o r and c o n d u ct an a p id w rin lg iesl study o f parsons 8 w hile serv in g in th e Arsed fecees o f th e United S ta te s d uring th e period o f the V le tn sn c o n f lic t, were saptsail to any o f th e c la s s o f to e s ic s ls tanotn a s 'th e d io x in s ' produced d uring th e aen u fa ctu re o f th e v a rio u s phanoxy h e r b ic id e s ( in c lu d in g th e h e r b ic id e known a s 'A g e n t O ra n g e ') t o d e te rm in e i f t h e r e n a y be l o n g - te a s a d v e rs e h e a l t h e f f e c t s i n s u d i p e rs o n s f r e n such e x p o s u r e ." Ef f o r t s a r e s t i l l underway to s u c c e s s fu lly c o l l a t e a protocol and to id e n tif y s u ita b le c o h o rts . T his w ill be a huge r e s a so d i e f f o r t which w ill in clu d e in te r v ie w and e x a a d n stie n s in v o lv in g s e v e ra l -thousands o f v e te ra n s . A co n tra c t to r th s d e sig n o f th s stu d y was s e r f ad bo th s School o f P ublic B ealth , u n iv e rsity o f C a lifo rn ia , Los A ngelas. A a a jo r p re b ia s in the d e si^ s o f sudi - a study r e la te d to th e fa c t th a t th e re were v ery tow re co rd s a n ln ta ln e d th a t te u ld lin k s p e c if ic ground t roops to her b ic id e exposure. A nother problem encountered was th a t l i t t l e i s known c o n c e rn in g l o n g - t a r s o r d e la y e d lum en h e a l t h e f f e c t s r e s u l t i n g f r e n e x p o su re bo th e p h e rax y h e r b ic id e s o r th e c o n ta m in a n t, TCDO. C o n se q u e n tly i t i s d i f f i c u l t t o d e s ig n d a t a c o lle c tin g in stn x sa n ts and ted m iq u ea hen th e outcome v a ria b le s ace so ill- d e f in e d . Howe v e r , s u c h d e s ig n work h a s b e en a c c o n p U s h e d , and i t i s hoped t h a t th e a c t u a l s tu d y w i l l g e t inderwey e a rly in 1983. (XNCUS3CN N eith er th e government nor th e s c i e n t i f ic txxenunity has re so lv ed th e naaezous con t r ov e rsie s (environm ental, s a d ic a l, o r p o l it i c a l ) inv o lv in g th e use o f Agent Orange in Vietnam from 1962 to 1970 n o r th e uae o f 2 ,4 ,5 - ? w ith in th e a g r ic u ltu r a l s e ttin g o f th e U nited S ta te s . A re p o rt by th e N a tio n a l Acadasy o f Sciences in 1974 docianented aces o f th e environm ental inpaets o f Agent Orange, but fo rtu n a te ly , th e arrangsaenta th a t tenainatad th e c o n flic t precluded a d d itio n a l s c ie n tif ic s tu d ie s in th a t sz e a . Such stu d ie s s ig h t have M inimised c u rre n t n a d ic a l cones m i about h e rb ic id e e x p o su re. M otsearer, to d a te F ed eral agency p o sitio n s an environm ental hazards asso ciated w ith T030 and 2 ,4 ,5 -T are e ith e r not w ell defined o r no t ifo n n ly accepted, thus perpetuating the controversy. The s c i e n t i f i c aaom unity o u st o o n tin u e to conduct v a lid re se a rc h cn c o n tro v e rs ia l e n v ito n a en tal and h e a lth -re la te d is s u e s in o rd e r to provide a r e lia b le b a sis to r a p p ro p ria ta d ecisionm aking. The V e tera n s A d m in istra tio n sta n d s firm ly ccnm ittad to working c lo s a ly w ith o th e r agencies of the F ederal G ovarm er.t as w ell as w ith th e p riv a te acto r to 1!i OQ w9 9v QO 758 a. o b ta in a a auuiy answ ers a s q u ic k ly a s p o s s ib le o a n s is ta n t w ith sound s c i e n t i f i c p r i n c i p i a s In o edsr to re so lv e th is p a rp la rin q is s u e . , the rs s o lu tio n o f the cont r ov ersy , however, w ill be achieved on ly follow ing th e p u b lic 's acceptance o f th e outoone o f s c ie n tif ic in v e s tig a tio n s. To th a t and s c i a n t i s t s a u s t accept th e r espon s ib i l i t y fo e n o t o n ly conducting q u a lity research, but also fo r tran slatin g the re su lts o f th e ir e ffo rts to le g is la to rs , to the cour t s , to th e sa d la and u ltia a te ly to th e p u b lic a t la r g e . Emmas 1 . C a x sM lli, D ., L . H o fb a rr, J . I b s e i c , and B.W. orga n . 1981. A Cass C r a h n l S tu d y o f t h e R e la tio n s h ip B etw een Brpns>n e t o 2,4--0 i d S p o n ta n so u s A b o rtio n s I n B u n in s. SRI In te rn a tio n a l R ep o rt. to tlo n a l F o re st P ro d u cts A s s o d s tio n , tfesh ln g to n , D.C. 116 p . 2 . O aaaaittae a n t h e E f f e c t s o f H e rb ic id e s i n S o u th V ietnam - 197 4 . I h e Ef f e c ts o f H erb icid es in South V ietnan. M r t At Siasaary and C onclusions. N atio n al Aesdesy o f S c ie n c e s , W ashington, D.C. 398 pp. 3 . C u t ti n g , R .T ., T .a . Ib u o c , J J f . B e ll o , M.W. B eneneon, and C .H . B re n s. 1970. C ongenital M alform ations, B y d ad d lfo n s Holes and S t i ll b i r th s in th e R epublic o f Vietnam , 1 9 6 0 -1 9 6 9 . Dac m e n t Ho. 9 0 3 .2 3 3 . CJ.S. Gover nm e n t P r i n t i n g O f f i c e , W ashington, D .C . 29 PP- 4 . B a td e ll, L. 1980. R e la tio n o f S o f t- tis s u e Sarcom a, M alignant lyaphcma and Colon Cancer to Phenoxy A cids, O ilorophenols and O ther A gents. Scandinavian Jo u rn al o f Hark and E--nv ir-onm--en ta l H e a lth , 7 :1 1 9 -1 3 0 . y 5 . Bfcnchar, P . 1982. H ealth Hazard E v alu atio n R eport HSEA-80-039-117J. N ational I n s titu te fo r O ccupational S afety and H ealth, C in cin n a ti, Ohio. 12 p . >4 < cr> C3 Cd H \ i l% l 6 . Jo h n so n , EU,. 1979. D ecisio n and Bnsrgency O rder Suspending R e g is tra tio n s f a r th e F o re st, Rights-of-W ey, and P astu re Uses o f 2 ,4 .5 -T . F ederal R e g is te r, 44(52): 15B74 -1 5 9 2 0 . 7 . MesaI s o n , M .S ., A.H. t k a t l a g , and J .D . C o n sta b le . 1071. Background M a te ria l R elev an t t o P r e s e n t a t i o n s a t t h e 1970 Annual H a stin g o f t h e AAAS Concer n i ng t h e H e rb ic id e A sia a m e n t Ccmnisalon f o r th e American A s so c ia tio n f o r th e Advancement o f S c ien c e. Am erican A sso ciatio n fo r th e Advancement o f S cien ce, W ashington, D.C. 47 p p . 8 . N e ls c n , C - 7 . , J . F . B a ls n , H.G. Greca*., and D.W. Gayl o r . 197 9 . R e tr o s p e c tiv e S tu d y o f t h e R e la tio n s h ip Between A g r i c u l t u r a l Dee o f 2 , 4 ,5 -T a n d C l e f t P a l a t e O ccurrence i n A rkansas, te ra to lo g y , 19:377-384. rfC t &* i *r. ]t BW XL82G7 IJ cI. * =159 9 . R iih ijaak i, V. 1982. f e r t i l i t y o f 2 ,4-O ichlorophnoxyaoetie Acid and 2 ,4 ,5 -T tic ttio ro p henoxyacetic Acid H erbicide A p p licato rs in F in lan d . T hird In te rn a tio n a l S yoposiw on C h lo rin a te d D ioxins and R elated Compounds, S alzb u rg , A u s tria , O ctober 12>14, A b s tra c t No. 54. 1 0 . t o s a , B.A . and S .P .R . Jtoee. 1972. C handra! S p ra y in g a s Rep o r te d by R efugees F ra n South V ietnex- Science, 177 710-712. 1 1 . S a d th , A .H ., D .O . F i s h s r , N. P e a rc e , C.A. T begue. 1982. Do A g r i c u lt u r a l r e -- C a u se S o f t T is s u s Sarcoma? I n i t i a l F in d in g o f a C a s e -C o n tro l S tudy i n New T eel end. C ow unlty H ealth S tu d ies, 6(23)J114-119. 1 2 . t o w a i d , J . C . , K.N. B odnar, P.JU D . van Peenon, R J3. O lacn and R.R. Cook. 1982. S urvey o f R ep roductive E v en ts o f Wives o f ttp lo y e a s t o C h lo rin a te d D io x in s. Am erican Journal o f fpldanioloqy, 115(5)<695-713. 1 3 . Tung, T .T ., T .X . Anh, B.Q. Tuyen, D.X. T ta , and N.X. ftjg e n . 1971. Q i n i c s l E f f e c ts o f M assive and Continuous O tillx a tio n o f D efo lian ts on C iv ilia n s . Vietnam ese S tu d ie s , 29:53-81. 14. W o lfe , W.H. 1980. "A gent O range i n P e r s p e c t i v e " . P r e s e n t a t i o n t o th e In la n d A g ric u ltu ra l Chemical A sso c ia tio n . Spokane W ashington, Deoaaber 10. A v a ila b le from E p id o a io lo g y D iv is io n , OAT S chool o f A erospace M e d icin e, Broc k s AFB, T exas 78235. 6 pp. 15. Young, A .L ., J .A . C alcag n i, C .2 . Thaik e n , and J.W . T tam baly. 1978. The T bxicology, B iv ir o n a a n ta l F a ta , ro d Human R isk o f H e rb ic id e Grange and I t s A s s o c ia te d D io x in . T e c h n ic a l R e p o rt OEHL-IR-78-92. OSAF O c c u p a tio n a l and E n v iro t .m ental H e a lth L a b o ra to ry , B rooks AFB, T ex as 78235 . 247 p p . 4 1 1 0 9 I 2 MO# Abstract. T C O D (2,3,7.8-tetrachlorodibenzo-p-dioxin), a toxic balogenatcd aromatic hy drocarbon, acts in the body as a cumulative poison. The chronic immunoioxic effects of 1T C D D were studied in C57BI/6 male mice. Total doses of 100 n t f c t r freater produced cellular depletion in thymus, spleen, and lymph nodes, and the animals appeared sick. No. cellulardepletion was seen following 0.4 ug/kg. and only thymus was affected by 4 and 40 Ug'kg. The antibody response to SRB C and T N P Srucrlla abortus was impaired following 40 Mg'kg TCDD. the delayed hypersensitivity response to oxazalone was impaired by 4( Mg/kg and the generation of alloantigen-soeciflc cytotoxic T cells (CTLI was sensitive to as littleas 0.004 uc/kg TCOD. In vitro analvsis of the mechanism of suppression using limning dilution tecr.maues showed that T C O D did not deplete the precursors of C T L but generated ceils ctinapie of suppressing C T L generation in vitro. BOH2I80III' TABLE I. Effect of TCDD Treatment on Delayed Hyfesensitivity Reactions reaimcnt given N Increment in footpad swelling 24 hr after oxazaione challenge" (x 10"' mA/. mean s SEM) Increment in ear thickness s 24 hr after oxazaione challenge* * (x 10"* tnAf. mean 2 SEM) Immune recipient Control recipient 1 1 Control (corn oil) 0.4 ig T C D D 4 ftg T C D D 40 M g T C D D 23.7 = 5.2 (10) 24.3 = 3.1 (10) 34.3 = 4.1 (10) 14.7 - 4.9 (10) 10.9 2 1.9 (ft) 16.8 2 4.1 (8 ) 6 .8 2 1.4 (8 )r 7.4 2 0.7 (8 r 0.17 2 0.87 -0.83 r 1J5 " -0.83 2 1.33 0 2 1.29 * The mice were sensitized by intravenous infection of 1 - 2 x 10* SRB C and tested 5 days later by injection '2 x 10* SRBC intothe left hind footpad. A similarvolume of saline was injected into the righthind footpad to mtrol for the nonspecific effects of the fluid injection. Parentheses enclose the number of measurements. * The mice were sensitized to oxazalonc in ethanol and ear swelling in sensitized and nonsensitized animals s measured 24 hr after application of oxazaione in olive oil. rSignificantly lower than control by student's / and Mann-W hitney U tests. P < 0.05. T A B LE II. Efff.ct o r T C D D o n Tin: P i :q u i ;n o y o r CTLp a n d Gi n i h a i i o n o f C.TL i n h V in to Mais IL C G ultuxes Treatment of mice CTLp frequency* Number of cells per culture* O I0 ') Cytntotic activity per culture (<Vur x 10') Estimated C T Lp * " per culture Yield C T L /C T L p 1 Control PLN 0.4 Mg PLN 4 pg PLN 40 pg PLN 400 pg PLN \ Control spleen \ 0.4 pg spleen \ 4 pg spleen 40 pg spleen 400 Mg spleen 2 Control PLN M Mg PLN 4 PLN 0.4 jig PLN Mixture Control PLN + 0.4 pg PLN 1/292 1/211 1/235 1/130 NC 1/1660 1/1561 1/1469 1/1607 1/206 1/1463 1/1006 1/1296 1/1229 1 1 1 1 NC 2 2 2 2 2 3 3 3 3 1.3 + 1.5 4633 2 394 2305 z 317 2617 233 4590 2 197 NC 4156 2 351 1408 2 97 2347 2 170 1745 2 104 2007 2 100 326 2 18 184 2 20 116 2 17 130 2 43 131 2 21 3425 4739 3922 6667 NC 1205 1282 1361 1245 9709 2051 2982 - 2313 . ; 2441 2246 1.33 0.53 0.67 0.69 3.45 1.10 1.72 1.40 0.21 0.159 0.062 0.050 0.053 / / / 0.058 Frequency of precursors of C T L was determined using limiting dilution micrncttlltircs as described under Materials and Methods. NC = insufficient ells In perform culture. * The test lymph node or spleen cells from the treated mice were cultured with irradiated F, stimulator cells in M L C lube cultures and the resulting cyitoxic activity was measured after 5 days incubation. The number of initial CTLp per culture was calculated by multiplying cell number by the CTLp cqucncy. Fig. 1. Effect of TCDD on cellular content of >use lymphoid organs. Data arc given as mean = 1 M for spleen (A). thym ui(B). and peripheral lymph des (C) studies in four to six independent experims. 4 sig/kg represents a dose of approximately 4 rts per billion. In three experiments done I - 6 weeks er t st dose of TCDD, the proportion o f thy !-b e...ig cells was determined in PLN by mplement-dependenl lvi ttwo experiments) or by munofluorescent staining (one experiment! as dcibcd under Materials and Methods. The PLS front ntrol mice contained 70.4-74'.? thy l.2-positi\e Is whereas PLN from mice given 0.4 pgkg T C D D .s 4 3 -4 6 S 7 . from mice given 4 pg>kc T C D D - 6 0 Ct. and from mice given 40 pg/kg T C D D . -5571. The doited line in C shows the estimated mbcr of thy 1.2-positivt cells in lymph nodes. _ o evs after im m unization Fig . 2. Eil'ecr ot t CDD treatment on theanttoody response. Control mice t o or mice treated tin a total of 0.4 ( _ j. 4 (0 ). or 40 ( -.t Mg xg were given an intraperitoneal immunization with SRJBC (A) or TNP-BA <Bi. I nc spicens .ere removed at the times indicated and the number of PFC was measured as descnoec unoer tenais ano Metnoos. The toul JgM JgG -r igA PFC number = SE.M is shown, for groups containing tnrce to four pooled mouse spleens. W: iiiaiZMa DAYS AFTER IMMUNIZATION Fig . ffect of TCDD treatment on the generation CTL/' vivt following ailoantigen challenge. Control ce (1 or mice given 0.-: (A ). 4 ( O k or 4 0 ( 0 kg :rc immunized with irradiated allogeneic P-815 mor cells as described under Materials and Methods. TL activity was measured in spleen cell suspensions m i groups of three to four mice at the times ind*> `7- hiUMBifl 0 * THYMOCYTES a o o 0 R CULTUA6 : FiC. 4. Thymocytes from TCDD-treated mice suppress C T L generation in m ro. The effect of different numbers o f thymocytes from control mice t # l or from .. mice given 0.004 ( v i. 0.04 kg (C ). or 0.4 <) MS Pr k? on the generation of CTL from 2 x iff1normai PLN is -shown. 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SZEWCZUK, a n d GEORGE SWEENEY I I Departments o f Medicine and Pathology, Host Resistance Program, McMaster University. 1200 Main Street West, Hamilton, Ontario L&N 3Z5. Canada Abstract. TCDD (2J,7,8-Utr*chk>rodibenzo-f>-clioxin). a toxic halogcnalcd aromatic hy drocarbon, acts in the body as a cumulative poison. The chronic immunoioxic effects o f TCDD were studied in C57B1/6 male mice. Total doses of 100 pglkg or greater produced cellular depletion in thymus, spleen, and lymph nodes, and the animals appeared sick. No cellular depletion was seen following 0.4 pg/kg, and only thymus was affected by 4 and 40 pg/kg. The antibody response to SRBC and TNP-Brucella abortus was impaired following 40 pg/kg TCDD, the delayed hypersensitivity response to oxazalone was impaired by 4 pg/kg and the generation of aOoantigen-specific cytotoxic T cells (CTL) was sensitive to as little as 0.004 pg/kg TCDD. In vitro analysis of the mechanism of suppression using limiting dilution techniques showed that TCDD did not deplete the precursors of CTL but generated cells capable of suppressing CTL generation in vitro. ___ Halogenated aromatic hydrocarbons lesions developing after TCDD exposure (8, (HAHs)1 such as polychlorinated biphenyls 10, 11), a consistent observation in all (PCBs)1, polychlorinated naphthalenes, animal species which have been studied is polybrominated biphenyls (PBBs), poly atrophy o f the thymus. TCDD-treated ro chlorinated dibenzofurans, Pentachlorphe dents show impairment o f delayed hyper nols (PCP), and polychlorinated dibenzo-p- sensitivity responses, reduced ability to dioxins, pose a serious man-made environ mount a graft-versus-host reaction and re mental threat to health (1--9). They are fat duced ability to reject allografts, and show soluble, poorly metabolized, and tend to an increased susceptibility to certain infec accumulate in the environment and in the tious agents (8 ,1 0 ,1 2 -1 5 ). Thus, one of the body where they act as persistent poi potentially important aspects o f toxicity o f sons (1). Polychlorinated dibenzo-p-dioxins TCDD and related HAHs is the effect on such as TCDD (2,3,7,8-tetrachlorodibenzo- the immune system. In order to better un (p)-dioxin) formed industrially by pyrolysis of derstand the mechanism of TCDD im- chlorphenols, a particularly toxic subgroup of munotoxicity, we have analyzed the effects the halogenated aromatic hydrocarbons, pro o f different doses o f TCDD on several types vide a good model system for studying HAH o f immune function o f C57B1/6J male mice. toxicity. Although species vary in the types of Particular attention has been directed to the chronic effects o f TCDD since chronicity typifies human exposure. We find that the 1 Presented in pari at the 23rd Annual Meeting of the generation o f cytotoxic T lymphocytes Canadian Federation of Biological Societies, June 11, (CTL) following alloantigen challenge is 1980, and published in abstract form Vol 23, ab particularly susceptible to impairment by stract 293. low doses o f TCDD, and this impairment is 1 Abbreviations used: HAHs, halogenated aromatic caused by a disturbance in the cells which hydrocarbons: PBBs, polybrominated biphenyls: TCDD. 2.3.7.8-tetrachlorodibcnzo-p-dioxin; SRBC. sheep red blood cells; TNP-BA, irinitrophenylated regulate the CTL response. Measurement and characterization o f this disturbance Brucellas abortus organisms: PLN, peripheral lymph may provide a sensitive test for presence of nodes (axillary, brachial, inguinal): CTL, cytotoxic T biologically significant amounts o f TCDD in lymphocyte: CTLp, precursor of CTL: PFC, plaque- the body. forming cell: oxazalone. 4-ethoxymethylcne.2-phenyl. Materials and Methods. Mice. C57BI/6 0037-9727/81/100290- IOS01.00/0 9(1Copyright (T > by be Society for Experim ent*] | Ail n|M < reserved. 18273 IMMUNOSUPPRESSION BY 2J,7>TETRACHLORODIBENZO*-D!OXIN 291 - male mice age 6 - 8 weeks were purchased o f " Cr release was calculated and percent-' from the Jackson Laboratory, Bar Har- age lysis by anti-thy 1.2 plus complement - bour, Maine, and housed six per cage, calculated by subtraction of the comple i TekJad chow (6% fat) and water were pro- ment control. Since TCDD treatment might vided ad lib. DBA/2J mice were similarly have altered the sensitivity of the cells to f- obtained and were used for maintenance of complement, a second method was used. |r F-815-X2 (H ^ ) mastocytoma cells by se- PLN were incubated in a 1/4 dilution of h rial ascities transfer. C3H/HeJ x DBA/2J anti-thy 1.2 serum for 45 min at 4s, washed, - % F, and C57BI/6J x DBA/2J F, hybrids were and then incubated in a 40-p.g/ml solution of \ produced in our own colony. RNC nude fluorescein-labeled protein A (Pharmacia). L mice were purchased from the Ontario Vet- After washing, the proportion o f cells ' erinary College, Guelph, Ontario. showing membrane immunofluorescent .- TCDD treatments. TCDD Lot No. 851- staining was determined by counting (*' 144-2 purity >99% was obtained from Dow 200--400 ceils under indirect ultraviolet il Chemical, Sarnia, Ontario, Canada. Two lumination (Leitz). k.- hundred and fifty micrograms was dis- A n tib o d y response. The antibody re 51' solved in p-dioxane and diluted in com oil. sponse to two different antigens was mea . Mice were injected ip weekly with 100 pi o f sured. Mice were injected intraperitoneally *- com oil containing specified amounts o f with either 5 x 107 washed sheep red blood k,' TCDD and studied 1 week after the fourth cells (SRBC) or, 0.1 ml of an 8% suspen f=> dose. sion of TNP-conjugated Brucella abortus C ell preparations. The TCDD-treated (TNP-BA). Spleens were removed from and control mice were killed by cervical SRBC- or TNP-BA-challenged mice 3 - 7 & " 'lo ca tio n . The spleen, thym us, and days later and tested for antibody-forming Spheral lymph nodes (axillary, brachial, cells against SRBC or TNP-SRBC, respec i- and inguinal) were aseptically removed, tively, using a plaque assay as modified for < and a single-cell suspension was obtained slides by Dresser and Greaves (16). Freshly -- by pressing minced tissue through a 60- frozen guinea pig serum absorbed with 50% f m esh stainless-steel screen into cold packed SRBC was thawed and added at a Z- medium. Culture medium consisted o f final dilution of 1/30 as a source of comple -MEM supplemented with 10% v/v fetal ment to detect " direct" plaque-forming bovine serum (Grand Island Biological Co., cells. To detect IgG and IgA PFC, direct - Grand Island, N.Y.), 100 units penicillin G IgM PFC were first blocked by adding a and 100 pg slreptomycin/ml and 20 mM 1/200 dilution of goat anti-mouse p during H ep es buffer. 2-M ercaptoethanol was the initial incubation o f target and spleen added at a concentration of 5 x 10- i M for cells. Indirect IgG PFC were then devel cell culture experiments. oped by adding rabbit anti-mouse IgG at a Anti-Thy 1 2 treatment. AKR anli-C3H predetermined optimal 1/200 dilution, and serum was purchased from Cedarlane IgA PFC were developed using a 1/100 di Laboratories, Hornby, Ontario (Lot 5101). lution of rabbit anti-mouse IgA. The IgG- The proportion of thy 1.2-bearing cells jn developing serum was prepared by im peripheral lymph node (PLN) cell suspen munizing rabbits with purified mouse IgG sions was determined in two ways. PLN (Miles, Lot 51), and showed only a single suspension initially prepared in Dulbecco's line against mouse serum and IgG on phosphate-buffered saline with 0.2% Frac Ochterlony plates. The IgA-developing tion V bovine serum albumin was labeled serum was prepared in rabbits using pure by incubation with Na241C r04 (New En mouse myeloma MOPC 315 (IgA A.2) and gland Nuclear) and washed. Labeled cells was depleted o f anti-light-chain activity at 107/ml were incubated with antiserum at by passage through a murine IgG--Seph- a 1/20 dilution at 4 for 45 min and resus arose-immunoadsorbent column. Pure mouse pended in a 1/12 dilution o f Low-Tox rabbit IgA or IgG added to the PFC assay inhib com plem ent (Cedarlane Laboratories). ited only IgA or IgG PFC, respectively, After 90 min of incubation at 37, the degree when the developing sera were used. -27' 'I 6909 I Z M00 nL IMMUNOSUPPRESSION BY 2J.7.-TETRACHLORODIBENZO*-DIOXIN Delayed hypersensitivity response. In the bation period at 37 in a 4-hr "Cr-release first method used, TCDD-treated and con assay using 2 x 10 P-815 target cells. trol mice were sensitized by injecting 1--2 In some experiments, thymocytes from x 10s SRBC iv and 5 days later the mice treated or control mice were added to the were tested by injecting 2 x 10* SRBC in 20 cultures. Second, the frequency o f precur /xl into one of the hind footpads (17). Saline sors o f CTL (CTLp) was measured using (20 p\) was injected into the other footpad the limiting dilution microculture method of and the degree of swelling was measured Teh et ai. (20). Briefly, small numbers of after 24 hr. The net amount o f swelling ob C57BI/6J spleen or PLN cells were cul served following SRBC challenge was cor tured in V-bottom microwells (Linbro 96- rected for swelling produced by medium MVC-TC) in 0.2 ml culture medium with 2 alone. In a second method, TCDD treated x 10* irradiated Ft spleen and 2 x 10* RNC or control mice ,were sensitized by paint nude spleen cells. Here, the nude spleen ing both ears with 3% oxazalone (4-eth- cells provide " help" which allows the de oxymethyiene-2-phenyl, BDH Chemicals, velopment of CTL from single precursor Poole, England) dissolved in 95% ethanol. cells (20). After 6 days incubation at 37, Nine to sixteen days later, sensitized and 2000 "Cr-labeled P-815 target cells were unsensitized control animals were chal added to each well and the amount of lysis lenged with 1% oxazalone in olive oil (18). was determined after 5 hr. Those wells Ear thickness was measured prior to chal showing release exceeding the 95% confi lenge and at 24 hr using a constant-pressure dence interval o f spontaneous release from micrometer. negative control wells containing F, stim G eneration and a ssay o f cytotoxic T ulators and nude spleen alone were scored cells. CTL were generated against the H-2d positive. Under limiting dilution condi alloantigen in vivo and in vitro. The in vivo tions, the number o f lymphoid cells yield response to TCDD-treated mice was de ing 37% negative wells defines the cell termined following injection o f 2 x 107 number containing, on the average, 1 CTLp P-815 tumor cells (H-2d) that had been according to the Poisson distribution: P(0) treated with 5000 rad " Co to prevent prolif = e -", where P(0) represents the fraction of eration. Spleens were removed from the re negative wells and u the mean frequency of cipients 3 -6 days later and the CTL activ CTLp per well.\The value of u was calcu ity was assayed in a 6-hr " Cr-release assay lated using a computer program derived against 4lCr-labeled P-815 targets as de from Porter et al. (21). TTie value of u al scribed in detail elsewhere (19). The titra lowed an estimate o f the number o f CTLp tion curve (percentage specific J,Cr release present in the tube MLC cultures and thus, vs concentration of sensitized spleen cells) a determination of the yield of CTL per was analyzed by computer for the parame CTLp (N a t x 103 per 1 CTLp). ter N a t which is proportional to the number R esults. E ffe c t o f d ifferen t d oses o f of CTL lytic units in the cell suspension. TCDD on cellularity o f lymphoid organs. 693 N at x 103 units is equi":Jent to 1 lytic Figure 1 shows the effects of different doses unit (the number of cells required to pro o f TCDD on the cellularity of different or duce 50% lysis in the absence of interfering gans of the mouse immune system. The re bystanders (19)). ported LDjo in mice for TCDD is approxi CTL were generated in vitro in two dif mately 100 /tgOs'g (8). although the time of ferent ways. First, mixed lymphocyte cul death is quite variable and often delayed. ture (MLC) was performed in 17 x 100-mm When a total dose exceeding 40 /ig/kg was polystyrene tubes (Falcon Plastics, No. administered, cellularity o f the spleen, 2057) in 3 ml culture medium. Briefly, 1 -3 thymus, and peripheral lymph nodes was x 10 spleen or PLN cells from the markedly reduced and the animals ap C57B1/6J mice were mixed with 10s C3D2F, peared ill. In contrast, doses o f 0.4--40 pg or B6D2F, spleen cells that had been inac did not reduce cell numbers in the spleen tivated with 1500 rad. CTL content o f the and peripheral lymph nodes and the animals culture was determined after a 5-days incu appeared healthy. Nevertheless, cellular 1o n e; FiO. tnou>e SEM fc r nodes m enis. pars p after t l.:-b e is' compW irnmuc . scribe contre cells i was * 43-6- 40-5! numb fx* L . depli fxg'kl in e d i post TCE IMMUNOSUPPRESSION BY 2J.7.S-TETRACHLORODIBENZO^OIOXIN 293 DO H 2 1 60693 experiments showed that the percentage thy 1.2-positive cells decreased following TCDD treatment at all doses studied (see - Fig. 1). The dotted line in Fig. 1C shows that the estimated number o f thy 1.2- positive cells in mouse lymph nodes also decreased after TCDD treatment. A de crease in the absolute number of thy 1.2- positive cells was also seen in spleen in one experiment where this organ was studied. Effect o f TCDD on immune responses. Figure 2A shows a representative experi ment illustrating the effect o f TCDD on the humoral immune response to SRBC. The number o f background PFC in the spleens of unimmunized mice was consistently increased by TCDD treatment and the greatest increase was observed, at 4 /ig/kg dose (Fig. 2, Day 0). Following immuniza tion with SRBC, PFC number increased in control (com-oQ-injected) mice reaching a peak on Day 5. TCDD at a dose o f 40 /ig/kg o f TCDD produced a marked suppression of the PFC response. This suppression was reproducible and all three isotypes (IgG, IgM, IgA) were affected (data not shown). The result shown in Fig. 2A could have re 3. I. Effect of TCDD on cellularconlenl of sulted from an effect o f TCDD on the T- mouse lymphoid organs. Data are given as mean I dependent component o f the anti-SRBC re SEM for spleen (A), thymus (B). and peripheral lymph nodes (C) studies in four to six independent experi ments. 4 tig/kg represents a dose of approximately 4 parts per billion. In three experiments done I- 6 weeks after the last dose of TCDD, the proportion of thy sponse (22). Therefore, the response to a relatively T-independent antigen (23), TNP-fl. abortus was tested. Similar to the result obtained using SRBC as antigens 1.2-bearing cells was determ ined in PLN by (Fig. 2A), Fig. 2B shows that the 0.4 and 4 complement-dependent lysis (two experiments) o r by /ig/kg doses o f TCDD had little effect on the immunofluorescent staining (one experiment) as de anti-TNP-BA response and the 40 /ig/kg scribed under Materials and Methods. The PLN from dose suppressed the response. We con control mice contained 70.4--74% thy 1.2-positive cells whereas PLN from mice given 0.4 fig/kg TCDD was 43-46% . from mice given 4 (ig/kg TCDD 43 --60%. and from mice given 40 /ig/kg TCDD. 40-53%. The dotted line in C shows the estimated number of thy 1.2-positive cells in lymph nodes. cluded that the dramatic suppression o f the antibody response to SRBC by 40 /ig/kg TCDD could not be explained by elimina tion o f T helper cells. The effect of various doses of TCDD on the delayed hypersensitivity response is shown in Table I. Footpad swelling elicited depletion was already apparent at the 4 by SRBC was only slightly reduced by the /g/kg dose when the thymus was exam 40 /ig/kg dose o f TCDD but this reduction ined. Since these data suggested that the was not statistically significant. The ear thymus was particularly sensitive to the swelling response to oxazalone appeared to effects of TCDD compared to other lym be more sensitive. It can be seen that the 4 phoid organs, we tested whether TCDD and 40 /ig/kg doses impaired the ear swell affected the peripheral T-cell population, by ing response whereas little effect occurred determining the proportion of thy 1.2- at the 0.4 /ig/kg dose. positive cells in PLN suspensions from The effect of TCDD on the generation of TCDD-treated and control animals. Three CTL following alloantigen challenge in vivo fc' 294 IMMUNOSUPPRESSION BY 2J,7.(-TETRACHLORODIBENZQ-?-Q!OX!N DAYS AFTER IMMUNIZATION Fig. 2. Effect of TCDD treatment on the antibody response. Control mice ( ) or mice treated with a total of 0.4 (A), 4 (O), or 40 (O) ig/kg were given an intraperiioneal immunization with SRfiC (A) or TNP-BA (B). The spleens were removed at the times indicated and the number of PFC was measured as described under Materials and Methods. The total IgM + IgG + IgA PFC number = SEM is shown, for groups1containing three to four pooled mouse spleens. is shown in Fig. 3. Even the 0.4 figfkg dose o f TCDD impaired the ability to generate a maximal response on Days 6 and 7. We concluded that the CTL response was par ticularly sensitive to functional impairment by TCDD. We therefore decided to direct our subsequent investigation s toward elucidation o f the mechanism o f impairment of CTL generation by TCDD. In vitro a n a ly sis o f e ffe c t o f T C D D on TABLE I. Effect of TCDD T reatment on Delayed Hypersensitivity Reactions Treatment given Increment in footpad swelling 24 hr after oxazalone challenge" (x l0 `l mM, mean SEM) Increment in ear thickness 24 hr after oxazalone challenge* (x 10"1 mAt. mean ~ SEM) Immune recipient Control recipient Control (corn oil) . 0.4 fig TCI)!!1 4 Mg TCDD , 40 Mg TCDD . 23.7 * 5.2 (10) 24.5 5.1 (10) 34.5 4.1 (10) 14.7 4.9(10) 10.9 = 1.9(8) 16.8 4.1 (8) 6.8 1.4 <8V 7.4 0.7 (8)r 0.17 0.87 -0.83 1.35 -0.83 1.35 0 1.29 * The mice weVe sensitized by intravenous injection of 1- 2 x 10s SRBC and tested 5 days later by injection of 2 x 10* SRBCjnto the left hind footpad. A similar volume of saline was injected into the right hind footpad to control for the nonspecific effects of the fluid injection. Parentheses enclose the number of measurements. * The mice were sensitized to oxazalone in ethanol and ear swelling in sensitized and nonscnsitized animals was measured 24 hr after application of oxazalone in olive oil. ' Significantly lower than control by student's t and Mann-Whitney U tests. P < 0.05. ,. q ^ .y -k.O 7 IMMUNOSUPPRESSION BY 2J,7.S-TETRACHLORODIBENZO*-DIOXIN 295 D 0 H 2 160695 it . m w MVS AFTER IMMUNIZATION - Fic. 3. Effect of TCDD (realmem on the generation of CT1_in vivo following ailoantigen challenge. Control mice ( ) or mice given 0.4 (A), 4 (O), or 40 ( ) Mg/kg were immunized with irradiated allogeneic P-813 u tumor cells as described under Materials and Methods. CTL activity was measured in spleen cell suspensions from groups of three to four mice at the times indi cated. CTL generation. The suppressive effect of TCDD treatment on the generation o f CTL was analyzed in vitro using the same methods that have been employed to characterize the mechanism of immunosuppression in pregnant mice (24). Experiment 1 in Table II shows that TCDD treatment impaired the ability of spleen and PLN cells from TCDD-treated mice to generate CTL against the H-2* ailoantigen in vitro in tube cultures. The frequency o f CTLp, how ever, was normal or slightly increased in TCDD-treated animals as determined by limiting dilution microcultures. This obser vation indicated that TCDD did not have a direct toxic effect on CTLp. In agreement with this conclusion was the marked in crease in CTLp frequency seen in the spleens o f animals given a 400 /g/kg dose of TCDD that produced gross cellular deple tion in this organ. It can also be appreciated from the experiment that the yield of cytotoxic activity per unit precursor was usually subnormal when lymphoid cells from TCDD-treated animals were studied. Experiment 2 shows that CTL generation was affected by doses of TCDD as low as 0.004 fig/kg. Furthermore, mixing cells from TCDD-treated mice with those o f normal mice appeared to suppress the CTL response o f the untreated cells. Demonstration o f suppressor cells in the thymus o f TCDD-treated mice. The above observations suggest that TCDD may en hance suppressor cell activity. We exam ined this possibility by adding thymocytes from TCDD-treated or control mice to MLC cultures containing normal PLN cells and quantitated the number of CTL gener ated in vitro. The effect o f different num- . bers of thymocytes on the generation of CTL from normal PLN is shown in Fig. 4. Dramatic suppression of CTL generation was observed when 4 --8 x 10* thymocytes from mice given 0.4 /g/kg and 0.04 p.g/kg TCDD were tested. Less suppression was seen when thymocytes from mice given 0.004 /g/kg were used. Direct addition of TCDD up to doses o f 0.025 /g to the cul tures produced no suppression (data not shown). If the entire dose of TCDD given to a mouse was concentrated in the thymus (containing on the average 8 x 107) cells then 4 x 10* thymocytes from a mouse given 0.4 /g/kg would add 0.00050 /g TCDD to the culture. These data suggest that the suppression of CTL generation in TCDD-treated mice represented suppressor cell activity and was not a direct toxic ef fect of TCDD. Discussion. The data in this paper show that the type of immunotoxicity produced by chronic exposure to TCDD depends on the dose. In agreement with the results of Faith er at. (25), we found reduced delayed hypersensitivity reactions as assessed by ear swelling at a 4 /g/kg dose of TCDD that had little effect on the antibody response. The most interesting finding, however, was the exquisite sensitivity o f the CTL re sponse to TCDD. CTL generation was im paired in vivo at the 0.4 /g/kg TCDD dose which produced no thymic atrophy or de tectable impairment of antibody or delayed hypersensitivity responses. Impairment-of CTL generation could be demonstrated in vitro with TCDD doses as low as 0.004 /g/kg when washed spleen or lymph node cells from TCDD-treated animals were stimulated in tube cultures with ailoantigen. Some insight into the cellular basis for IMMUNOSUPPRESSION BY 2J.7.S-TETRACHLORODfBENZO?-DIOXIN TABLE II. Effect of TCDD on the Frfoucncv of CTLp and Generation of CTL in in Vitro Mass MLC Cultures Treatment of mice .1 Control PLN 0.4 /ig PLN 4 Mg PLN 40 MS PLN 400 Mg PLN Control spleen 0.4 MS spleen 4 ms spleen 40 ms spleen . 400 ms spleen 2 Control PLN 0.004 MS PLN 0.04 ms PLN 0.4 MS PLN Mixture Control PLN + 0.4 ms PLN CTLp frequency* 1/292 1/2II 1/255 1/150 NC 1/1660 1/1561 1/1469 1/1607 1/206 1/1461 1/1006 1/1296 1/1229 Number of cells per culture1 (x|0*) 1 1 l 1 NC 2 2 2 2 2 3 3 1 3 1.3 + 1.3 Cytotoxic activity per culture (No/ x 10*) 4635 394 2503 317 2617 * 233 4590 191. NC 4156 351 1408 97 2347 170 1745 X 104 2007 X 100 326 x 18 184 l 20 116 17 130 x 43 131 x 21 Estimated CTLp per culture 3425 4739 3922 6667 NC 1205 1282 1361 1245 9709 2051 2982 2315 . 2441 ; 2246 Yield CTL/CTLp 1.35 0.53 0.67 0.69 3.45 1.10 1.72 1.40 0.21 0.159 0.062 0.050 0.053 0.058 * Frequency of precursors of CTL wns determined usin limiting dilution microcultures as described under Materials nnd Methods. NC insufficient cells to perform culture. * The test lymph node or spleen cells from the treated mice were cultured with irradiated F, stimulator cells in MLC lube cultures and the resulting cy totoxic activity wns measured niter 5 days incubation. The number of initial CTLp per culture was calculated by multiplying cell number by the CTLp frequency. ZO R 3 C Roq.. ----boor\ t/sav*s s ->G g - T l i j I i ^ o J a p S . C . 2 - S . - . 3 7 =r - g 3r> o-fa3- s-fi 6909IZMOG I IMMUNOSUPPRESSION BY 2.3.7.-TETRACHLORODrBENZO-f*-DIOX!N 297 DON 2 1 60697 _ jS ' NUM BER O f T H Y M O C fT tl , 1 J j;? ' AOOCO W l CULTURE ' 5* " Fig. 4. Thymocyte* from TCDD-treated mice- sup- prts CTL generation in vitro. The effect of different numbers of thymocytes from control mice ( ) o r from t micc given 0.004 (V), 0.04 leg (O), or 0.4 (A) per kg on the generation of CI L from 2 x 1 0 * normal PLN is shown. Five to six thymus donors were used per ?- group. & this impairment was obtained by measuring ! j;' the frequency of CTLp in limiting dilution i i microcultures. It was found that the fre- ?' quency of CTLp was not reduced by treat5- ment with TCDD. Thus, the impaired CTL , *. generation in vivo and in tube MLC cultures : represented a defect in the development of CTLp into CTL. This observation suggested that TCDD treatment affects :rr those cell types which regulate the generaX*- tion o f CTL in tube MLC cultures. For -T- example, TCDD could either reduce the ' amount of " help" available from T helper ~ cells and accessory cells, or increase supr pressor T-cell activity that can be detected under in vitro conditions allowing sufficient c e ll-cell interaction (24). The reduction - in thy 1.2-positive cells in the PLN of ' TCDD-treated mice (Fig. 1) is consistent with a reduction in T helper subpopula tions. On the other hand, several observa tions have suggested that suppressor cells may be generated. Figure 3 shows that the 5- generation o f CTL in vivo to allogeneic P-815 tumor cells that lack la antigens needed to activate T helper cells (26, 27) ' was normal in TCDD-treated animals at V early times but seemed to " shut off" as the response continued. Secondly, mixing lymph node cells- from TCDD-treated animals with those from normal mice pro duced suppression (experiment 2, Table II). Thirdly, thymocytes obtained from mice treated with low doses o f TCDD (0.004-0.4 Hg per kg) suppressed the generation of CTL/Vi vitro. This suppression could not be explained by the amount o f TCDD that would be present in the thymocytes. Addi tional experiments have shown that the presence of thymic suppressor activity par allels the impairment o f CTL generation in PLN and that both alterations ultimately disappear as the dose o f TCDD is progres sively reduced to 0.00004 (ig/kg. ((31), manuscript in preparation). Taken together, these data indicate that TCDD. may impair the generation of CTL through'the genera tion o f suppressor T cells. The mechanism by which TCDD alters the thymocyte population to produce sup pression i^ unclear. It is unlikely that the effects o f low dose TCDD on thymus can be attributed to malnutrition or to an increase in cortisone levels in the body. Malnutrition tends to deplete cells in the murine spleen and thymus and to increase the cellular and hu moral immune response (28). Our mice showed normal antibody responses and DTH but impaired CTL generation at TCDD doses that caused neither lymphoid organ atrophy (Fig. 1) nor significant weight loss (data not shown). Serum cortisol levels in TCDDtreated animals have been found to be nor mal by Vos et al. (10). TCDD is known to interact with a cytoplasmic binding protein or " receptor" and this " receptor" is pres ent in the thymus as well as liver (29, 30). Indeed, in the rat, the highest concentration o f " receptor" is found in thymus (30). Thus, it is possible that TCDD may act di rectly on thymus to alter T-cell differentia tion or perhaps the life span o f certain Tcell subpopulations thereby promoting the generation of suppressor T cells. Alter nately, the alteration in thymus could be mediated indirectly through effects of TCDD on hepatic enzyme activity (5, 31). ` It can be seen from Table I that there was a slight (but not statistically significantHncrease in oxazaione-induced ear swelling 298 IMMUNOSUPPRESSION BY 2J,7.t-TETRACHLORODIBENZO-j>-DIOXIN after 0.4 /ig/kg TCOD and a similar effect on footpad swelling after 4 ftg/kg TCDD, The stimulatory effect o f 0.4 and 4 fig/kg TCDD on the background level o f PFC has already been noted. We also found that these doses o f TCDD increased the number of spleen colony-forming ceils in bone mar row by 25--39% (data not shown) and thus it is possible that the 0.4 and 4 jsg/kg doses of TCDD have a small nonspecific stimu latory effect on a variety o f different cell types in addition to suppressor cells that influence the immune system. Our data showing that low doses of TCDD in mice impairs the generation of CTL may have some relevance to the ef fects o f exposure to other HAHs. We have recently found that PCBs also impair CTL generation in mice ((31), manuscript prepa ration). Bekesi et al. (32) have reported that Michigan residents exposed to PBBs in their diet show a reduced number of T cells in blood and impaired mitogen and MLC responses in vitro. The effects o f TCDD on the murine immune system may therefore have some generality. The biologic significance of the immunotoxicity detected in vitro following exposure to low doses of TCDD and HAHs is not yet known. Our data show that a suf ficiently high dose of TCDD produces im pairment of the antibody response to SRBC in mice. Cattle heavily exposed to PBBs develop mastitis and furunculosis due to bacterial infections (33), and TCDD-treated mice are reported to be more susceptible to Salmonella infection (14). However, we and others (25) find that lower doses of TCDD impair primarily the cellular arm of the immune response, and as shown in this paper, the generation o f CTL appears to be particularly sensitive. One would predict from these observations that low doses o f TCDD and related HAHs should increase susceptibility to only certain types of infec tious agents. For example, CTL appear to play an important role in the defense against certain types of virus infection (34, 35). Susceptibility to duck hepatitis virus (15) and susceptibility to herpesvirus infec tion in mice (36) is increased by PCB expo sure. Experiments are currently in progress in our laboratory to determine if the in creased thymic suppressor cell activity produced by very low TCDD doses in mice confers an increased susceptibility to lethal viral infections. We thank Ann Robson, Angela Mulvibfl]. Dalgeet Banwat, Fred Krestynsk, Dr. Mark McDermott, and Dr. David Basfurd for expert technical assistance and Dr. W. E. Rawls for helpful comments during prepa ration of the manuscript. We thank Roberta Axler for typing the manuscript and Rita Campbell for preparing the figures. Crant support from the Ontario Ministries of Environment and Labour is acknowledged. David A. Clark and Myron R. Szcwczuk hold scholarships from the"lMedical Research Council of Canada. 1. Kimbrough, R. D., Ann. N.Y. Acad. S ci.320,415 (1979). 2. Urabe, H., Koda, H., and Asahi, M., Ann. N.Y. Acad. Sci. 320, 273 (1979). 3. Warehaw, R., Fischbein, A., Thomioa, !.. Miller. A., and SelikofT, J. L , Ann. N.Y. Acad. Sci. 320, 277 (1979). 4. Valciukas, J. A., Lilis, R., Anderson. H. A.. Wolfe, M. S., and Petrocci, M.. Ann. N.Y. Acad. Sci. 320, 337 (1979). 3. Strik, J. J. T. W. A., Ann. N.Y. Acad. Sci. 320, 308 (1979). 6. Dickson, D., Nature (London) 283, 418 (1980). 7. Hailing. H.. Ann. New York Acad. Sci. 320. 426 (1979). 8. Greig, J.B., Ann. Occup. Hyg. 22. 4M (1979). 9. Kociba, R. J., Keyes, D. G., Beyer. J. E., Car rent, R. ., Wade, C. E., Dittenber, D. A., KaJnins, R. D., Ftauson. L. E., Park, C. N., Bar nard, S. D., Hummel, R. A., and Hutnision. C. G., Toxicol. Appl. Pharmacol. 46, 279 (1978i. 10. Vos, J.G.. Moore, J.A., and Zinkl, J. C., Envi ron. Health Perspect. S, 149(1973). 11. Thomas, P. T ., and Hinsdill, R. D .. Toxicol. Appl. Pharmacol. 44, 41 (1978). 12. Vos, ]. G., and Moore, J.A., Int. Arch. Allergy Appl. Immunol. 44, 777 (1974). 13. Moore. J. A., and Faith, R. .. Environ. Health Perspect. 18, 123 0 976). 14. Thigpen, J. E., Faith, R. E., McConnell. E. E.. and Moore, J. A., Infect. Immunity 12. 13)9 (1973). 15. Friend, M,, and Trainer, D. O., Science 170. 1314 (1970). 16. Dresser. D. W., and Greaves. M. R., in "Hand book of Experimental Immunology" (D. M. Weir, ed.), p. 271. Blackwell Scientific. Oxford (1973). 17. Lagrange, P. H .. Mackaness, G. B.. and Miller. T. E., i . Exp. Med. 159, 528 (1974). 69091 2)(00 IMMUNOSUPPRESSION BY 2J.7.S-TETRACHLORODIBENZO-?-DIOXIN 299 Ashenon. G. L., and Plak, W.. Immunology IS, . 29. Poland. A., Greenlee, W. F ,, and Rende. A. S., #fct- 405 (1968). Ann. N.Y. Acad. Sci. 320, 214 (1979). tt]! Clark, O. A., Phillips. R. A., and Miller, R. G., 30. Caristedt-Duke. J. M. B., Cancer Res. 39, 3172 g s . Cell. Immunol. 34, 25 (1977). (1979). ^20. Teh, H. S., Harley, E.. Phillips, R. A., and Miller, 31. Clark, D. A.. Gauldie, 5., Sweeney, G. D., and i .- L R. G.. J. Immunol. 118. 1049(1977). Szewczuk, M. R., Fed. Proc. 40, 1096 (1981). $ 21. Porter, E. W., Hewitt, H. B., and Blake, E. R., 32. Bekesi, J. G., Anderson, H. A., Roboz. J. P., Brit. J. Cancer 27, 55 U973). Roboz, J., Ftschbein, A., SelikofT, I. J., and Hol 2 2 . Gershon. R. K.. and Rondo. K., Immunology 21, land. J. F., Ann. N.Y. Acad. Sci. 320.717(1979). : 903(1971). 33. Jackson, T. F.. and Halbert. F. L .. J. Amer. Vet: 5^23. Mono, J. J., Sher, Mosier, D. E., Baese, M.. Med Assoc. 165. 437 (1974). g : and Paul, W. E., Eur. J. Immunol. 8, 459 (1980). 34. Zinkemagel, R. M., and Althage, A., J. Exp. Clark, D. A., McDermott, M. R.. and Szewczuk, Med. 145, 644 (1977). M. R.. Cell. Immunol. 52. 106 (1980). 35. Howes, E. L.. Taylor, W., Mitchison. N. A., and Faith, R. E., Luster. M. I., and Moore, J. A., Simpson, E., Nature (London) 277, 67 (1979). Cell, lmaiunol. 40, 275 (1978). 36. Im anishi, J., N om ura. H ., M atsubaru, M., Frelinger, J. A.. Niederhuber. J. E., David, C. S., Masakazu. K., Won, S.-J., Mizutani, T ., and and Schrefiler. D. C-. J. Exp. Med. 140. 1273 Kisltida. T., Infect. Immunity 29, 275 (1980). (1974). Roehm, N. W,, Alter, B. J.. and Bach, F. H ,, J. Immunol. 126, 353(1981). 28. Bongiomi-Malave. I., and Pocino, M., Clin. Im9;'.z munol. Immunopathol. 16, 19(1980). Received April 16, 1981. P.S.E.B.M. 1981, Vol. 168. I X % OC 9 | - i 1 1SO9 IZHOfl Chlorinated Dibenzodioxins and Dibenzofurans* il Janes Edward Hair aid Join S. Wasson* 186 The problems and universal concern about chlorinated dibenzodioxin and dibenzofuran compounds were brought to the forefront by the scientific community during the National Institute of Environmental Health Sciences' (NIEH S) Conference on this subject which was held at Research Triangle Park, North Carolina, April 2 and 3, 1973. This idea for this literature collection emanated from that scientific gathering and resulted in this an notated bibliography of 242 references. These references are categorized by year and ar ranged alphabetically by author. The number of references per year are: 56/1973; 67/ 1972; 66/1971; 24/1970; 29/1969-1934. Sources searched are summarized in Table 1. Due to the time available to complete this collection, some errors and omissions were inevitable; we apologize for these and hope those using this literature survey will supply us with past, present, and future topical * Work supported by Toxicology Information Pro gram, National Library of Uedicina; National Insti-torts of Environmental Health Science; and the National Cancer Institute under contract with the Union Carbide Corporation. _t_BiomedIcal Studiea and Toxicology Information Response Center. ^Environmental Mutagen Information Center, P.Ot Box Y, Bldg. 9224, Environmental Information System Office, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830. September 1973 reprints or citation information. We plan to maintain and up-date this file continually. Most of the nomenclature or terms searched are listed in Table 2. Each particular author, journal, secondary abstracting serv ice, and news copy unfortunately utilizes separate and distinct terminology when re porting on the chlorinated dibenzodioxins and dibenzofurans. As can be seen from Table 2, it is vitally important, therefore, to become thoroughly familiar with the sources being utilized before mounting a massive effort to collate all that is written or reported about a particular compound, series, or class of compounds, or subject. The magnitude of the search effort for this report is selfevident when noting all the necessary terms used. The original papers were annotated when ever possible; some were gleaned from ab stract journals. We did not alter author's remarks or conclusions; the facts are pre sented in these annotations as they appeared in the literature. As many salient points as possible, due to space limitations, were taken from each paper or report. Many papers and reports were referred to, after the fact, by more recent authors as having dealt intimate ly with the chlorinated dibenzodioxins and dibenzofurans; these were not included in this bibliography unless the dioxins or furans were mentioned specifically *. most of the 283 18283 RISOSIZMOQ articles were consulted and read however to determine if these compounds were pres ent, For example, numerous articles reporting on the adverse effects of 2,4,5-T were screen * ed but not included because direct mention of TCDD and other dioxin derivatives was * Tabi* L Source* and tisi perioda searched. Source Multidisciplinary Information Resource* Bibliography of Agriculture Biological Abetracte Biological and Agricultural Index Bioroctarek Index Chemical Abetraete Chemical-Biological Aativitiee Food Chemical Neue Health Aspects of Peetieidee Abetract Bulletin Health Effects of Environmental Pollutante Index htedicue Peeticide Chemical Neue Science Citation Index Teratology Lookout Toxicology Bibliography Specialized Information Center* and Libraries Environmental Mutagen Information Center (EMIC) Environmental Information System Office (EISO) Oak Ridge National Laboratory (ORNL) Toxicology Information Response Center (TIRC) On-Line Computer Data Bases MEDLine TOXLine Journals Amino Archive of Environmental Health Bulletin of Environmental Contamination and Toxicology Clinical Toxicology Environment Environmental Health Perspective* Federation Proceedings Food and Coemetio Toxicology Journal of Agriculture and Food Chemietry Journal of the Association of Official Analytical Chemists Journal of Chromatography Mutation Research Residue Reviews Science Teratology Toxicology and Applied Pharmacology Period VoL 21 (1957) to VoL 37(1) (1973) VoL 31 (1967) to VoL 55(8) (1973) VoL 19 (1964) to VoL 24 (1970) VoL 1 (1966) to VoL 9 (4 ) (1973) VoL 1 (1907) to VoL 78(20) (1973) 1965 to 1971 VoL 13 (42) 1972 to VoL 15(9) (1973) VoL 1 (1986) to VoL 6(4) (1973) VoL 1 (1972) to VoL 2 (3 ) (1973) VoL 60 (1966) to VoL 14(5) (1973) VoL 1 (1-24) (1973) VoL 1 (1961) to VoL 6 (1965); 1966 to 1972 VoL 3 (1972) VoL 1 (1968) to VoL 6 (1) (1973) VoL 1 (1972) to VoL 2 (1973) VoL 7 (1963) to VoL 26(2) (1973) VoL 1 (1966) to Voi. 7 (1973) VoL 1(1) (1971) to VoL 8 (1 ) (1973) VoL 12 (1970) to VoL 15(3) (1973) No. 1 (1972) to No. 5 (1973) Voi. 16(1) (1957) to VoL 32(4) (1973) Vol. 1 (1969) to VoL 10(6) (1972) VoL 1 (1953) to VoL 21(2) (1973) Voi. 40 i 1957) to VoL 56 (1973) Vol. 1 (1958) to VoL 73 (1973) Vol. 1 (1964) to Voi. 10(8) (1972) VoL 1 (1962) to VoL 41 (1972) VoL 157 (1967) to VoL 179 (1972) Vol. 1 (1968) to Voi. 6(2) (1972) Vol. 1 (1959) to Voi. 24(3) (1973) 284 Environmental Health Perspectives 18284 DON2I 606 U Anonymous. New look may be taken a t trichlorophenol compounds. Peat. Chem. .kiroi.v..o. 1 /f>1 \ . Ov --IxAw Anv.i.l 4u Ct/ f A1fwl<iTw9yk. Work showing "surprisingly high" levels of di oxin (ppt) in fish caught in Vietnam has placed doubt on earlier government consideration about dioxin residues. It had been previously thought that the dioxin content of 2,4,5-T was so low that there was little opportunity of residues appearing. In ad dition to 2,4,5-T and silver, any compound using trichlorophenol intermediates "may be suspect". Anonymous. Correction. Pest. Chem. N ew s 1(22): 2 (May 2,1973). The National Academy of Science's Advisory Committee on 2,4,5-T recommended that the regis tration of 2,4,5-T be restored with the following exceptions: (a) a permissible residue of not more than 0.1 ppm 2,4,5-T on edible parts of food products and In water for human consumption and (b) a limit of 0.5 ppm of contamination with TCDD, ex cept that In all formulations to be used around the home and recreational areas, TCDD contamination should be limited to 0.1 ppm. Anonymous. Recall program, another look at trichlorophenols urged on EPA. Peat Chem. N ew s 1(23): 3-6 (May 9, 1973). The General Accounting Office urged the En vironmental Protection Agency (EPA) to imple ment full-scale recall procedures for suspended pesticides and raised questions on the dioxin content of trichlorophenol herbicides. GAO stated that "be cause silvex, ronnel, erbon, and hexachlorophene can contain the tame level of dioxin as 2,4,5-T and because a safe level of dioxin has not been de termined, we believe EPA should establish a stand ard for dioxin content and prohibit the use of all pesticides containing dioxin in excess of the es tablished standard." Baughman, R. W., and Meselson, M. S. An analytical method for detecting TCDD (d io x in ): levela of TCDD in samples from Vietnam. Environ. H ealth Per- sped. (No. 5 ): 27 (1973). An analytic procedure involving extensive clean up and mass spectroscopy detects approximately one picogram of TCDD. The method separates TCDD from DDE, PCB's, and other chlorinated hydrocar bon residues. Bowes, G. W., Simoneit, B. R., Burlin game, A. L., DE Lappe, B. W., Peakall, D. B., and Risebrough, R. W. The search for chlorinated dibenzofurans and chlorinated dibenzodioxins in wildlife populations showing elevated levels of 286 embryonic death. Environ. Health Per f e c t . (No. 5 ): 191 (1973). Embryonic deaths have been recorded in the lab oratory among birds treated with PCB. These deaths have been attributed to chlorinated dibenzofuran contaminants. Also, birth defects in wild populations of birds and sea lions are believed caused by chlorinated dibenzodioxins. High-resolu tion mass spectrometry was used to examine pre pared from aborted sea lions and dead embryos of the herring gull and osprey. Crosby, D. G., Moilanen, K. W,, and Wong, A. S. Environmental generation and de gradation of dibenzodioxins and diben zofurans. Environ. Health Perspect. (No. 5 ): 259 (1973). Both the chlorinated dibenzodioxins and dibenzo furans are unstable to light in the presence of or ganic substrates. Even if generated under environ mental conditions, light provides a mechanism for rapid destruction. Crummett, W. B., and Stehl, R. H. Determi nation of chlorinated dibenzodioxins and dibenzofurans in various materials. E n viron. Health Perspect. (No. 5 ): 15 (1973). Chlorinated dibenzo-p-dloxins and chlorinated dibenzofurana can be determined in chlorinated phen ols,- chlorinated phenoxy herbicides, ronnel, fat, and conbustion products by such analytical techniques as gas chromatography, liquid chromatography, thin-layer chromatography, and gas chromatogra phy-mass spectrometry. Dougherty, W. H .; Coulston, F . ; Golberg, L. Non-teratogenicity of 2,4,5-trichIorophenoxyacetic acid in monkeys (Macaca m ulatto). Twelfth Annual Meeting, So ciety of Toxicology, New York, NY (March 18-22, 1973), Abstract 9, p. 7. Technical grade 2,4,5-T which contained less than 0.05 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin was ad ministered orally to forty pregnant Rhesus monkeys daily from day 22 through day 33 of gestation. Dose levela used in the experiment were 0.05, 1.0, and 10.0 mg/kg. Hematology, clinical. chemistry, and urinalysis data were recorded for ail females before and at various times following treatment until par turition; no toxicity was observed. Examination of live born infants revealed no terata. E nvironmental Protection Agency, Pub lications and Information Section. Toxicology and pharmacology of 2,4,5-T Environmental Health Perspectives I O O <r~ OD 0 0 N 2 160615 Tabi* t Taras Marched. Nomenclature Chemical A bttra eU registry number Chlorinated Chlorinated dibenzofuran(s) lorodibenzobenzo jbenzonzofuran sn `alogenated dibenzofuran(s) 'olychlorinated dibenzoiurans Dibenzo-p-dioxin (diphenylene 262-12-4 tt34f,31oxide) (phendloxLn) 2.3-Dichloro- 29446-16-9 2,7-Dlchloro- 33867-26-0 !12>7.Trichloro- 33867-28-2 ,2,3,4-Tetrachloro- 30746-68-8 ^3,6,8-Tetrachloro^3,8,7-Tetraehloro- 33423-92-6 2z3,7,8-Tetrachloro 1746-01-6 entachloro- 36088-22-9 exachloro- 34466-46-8 3,7,8,9-Hexachloro 19408-74-3 eptachloro- 1.2,3,4,6,7,8,9-OctaehIoroJBenzo- 3268-87-9 Biphenyl- t-. Chlorinated Chlorinated dibenzodioxin(s) Chlorinated dibenzo-p- J L __ dlozin(s) Dibcnxo- Dibcnzo Chlorodibenzo dioxin(s) 3D-g Chlorodibenzo-p-dioxin (s ) Dibenzodioxin(a) q> K__Dibenzo-p-dioxinz Dibenzo-p-dioxins, tetra- chloro- Dibenzo-p-dloxin, other chloro- Dioxin Halogensted dibenzo dioxin(s) ~5alogenated dibenzo-p- __ (Jiaxin(s) OCDD "Polychlorinated dibenzodioxin Polychlorodibenzodioxin Di ofuran (biphenylene oxide) (3-Chloro- 2,4-Dichloro1^,4-Trichloro- trachlororj/(2^<4-Tetrachloro&Psluachloro- BenzoBipheyl- 132-64-9 25074-67-3 24478-74-8 24478-73-7 23076-57-6 24478-72-6 32076-58-7 September 1973 1973 Anonymous. Herbicides better for birds than bacteria. Food Comer. Toxicol. 11(1): 149-150 (1973). Teratogenic effects of the 2,4,5-T contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), are well known and the effect at this compound on liver enzymes resembles that of certain carcinogens. Di oxin has also been associated with mutagenic prop erties through its possible intercalation with DNA; the observed effects resemble thoM at acridine. Anonymous. TCDD residue disappears. D ow n To E a rth 28(4): 18 (Spring 1973). TCDD was not detected (<1 ppm) in 3-ft soil core samples in a sandy area where a total at 947 lb at 2,4,5-T per acre was applied over a 3-yr period. No TCDD residues were found (<0.06 ppm) in bald eagle tissue gathered from 16 states. Anonymous. 2,4,5-T comes to public atten tion again. Peat. Chem. News 1(19): 6-8 April 11, 1973). EPA was asked to suspend the remaining uses of 2,4,5-T until the extent, if any, of TCDD-contaminated food chains is determined. TCDD was labeled a cumulative poison and. one of the most potent agents of birth defects In animals ever dis covered. Anonymous. Health hazard of dioxins still uncertain. Chem. Eng. N ew s 51(16): 12 (April 16, 1973). A selected summary report of the National In stitute of Environmental Health Sciences meeting on chlorinated dibenzodioxins and dibenzoiurans held at Research Triangle Park in North Carolina on April 2-3, 1973. The conference as a whole, however, seemed to raise as many questions as it answered. From the data discussed there is no doubt that these contaminants are highly toxic and teratogenic, but there is still some doubt as to how much of an actual hazard they represent to human health. 285 18286 inn (includes dioxins). Bibliography Num ber 73-04 (February 1973). A bibliographic listin g of 62 r u ir tn c s a pertain in g to 2,4,5-T and dioxins. E nvironmental Protection Agency, Pub lications and Information Section. Chemistry and residues of 2,4,5-T (in cludes dioxins). Bibliography Number 73-05 (February 1973). A bibliographic coilaction of 58 refercr.ce on 2,4,5-T containing a limited namber of citations on dioxins. E nvironmental Protection Agency and Institute of Rural E nvironmental Health. Environmental chemicals: hu man and animal health. Fort Collins, CO (July 23-27, 1973). The Institute of Rural Environmental Health, Colorado State University, and the Office of Pes ticide Programs, U. S. Environmental Protection Agency, conducted a one-week course on environ mental problems, contaminants, toxicants, and chem icals (Including dioxins); and human and animal health problems; and poisoning. Firestone, D. Etiology of chick edema dis ease. Environ. H ealth Perspeet. (No. 5) : 59 (1973). Early work indicated that chick edema factors (CEF) were chlorinated aromatic compounds; later, the compounds were shown to belong to a family of chlorodibenso-p-dioxins. Further investigation showed that (a) chlorophenols were precursors of the chlorodioxins and (b> chlorodioxins and related compounds are commonly present as minor com ponents in commercial chlorophenols. Characteristic chick edema disease symptoms include excessive fluid in the heart sac and abdominal cavity fol lowed by high mortality starting in the third week. Fowler, B., Lucier, G., Brown, H., and Mc Daniel, 0 . Ultrastructural changes in rat liver cells following a single injec tion of TCDD. Environ. Health Perspeet. (No. 5) : 141 (1973). Ultrastr ucture changes in rat liver microsomes and mitochondria were examined at various inter vals from 1 to 30 days following a single TCDD in jection of 0, 5, or 25 ug/kg. No histologic difference was noted between groups. Observed changes in cluded: proliferation of smooth endoplasmic reticu lum (SER), mild increase in rough endoplasmic reticulum (RER), moderate swelling of mitochon dria; at 7 days, large aggregates of SER, massive September 1973 amounts of RER, and small numbers of moderately swollen mitochondria were seen from the 25 mg/kg dosed rats. Greig, J. B. Biochemical toxicity of TCDD in rat liver. Environ. Health Perspeet. (No. 5 ): 211 (1973). The penistent toxic effect of 2,3,7,8-tetrachlorodlbenzodioxin in rats is evidenced by death as long as 15 weeks after a single oral dose. Alterations, how ever, in liver constitution (microsomes and cyto chrome P-450) and drug metabolism (zoxazolamine and hexobarbital) occurred within 24 hr after dosing. Gupta, B., Vos, J., Moore, J., Zinex, j ,, and Bullock, B. C. Pathologic effects of TCDD in laboratory animals. Environ. H ealth Perspeet. (No. 5 ): 125 (1973). Gross snd microscopic examinations were per formed on rats, guinea pigs, and mice treated with TCDD. A spectrum of dose ranges and schedules were used. Lymphoid organs (thymus, spleen, and lymph nodes) were affected consistently. Thymus atrophy (dose related decrease in weight) was found to be a sensitive index of TCDD exposure. The most severe hepatic effects were seen in rats that received a lethal dose of TCDD. The magnitude of the degenerative and necrotic liver changes were diminished in guinea pigs and mice. Harris, M., Moore, J., and Vos, J. General biological effects of TCDD in laboratory animals. Environ. Health Perspeet. (No. 5) : 101 (1973). Albino rats were grouped and treated with single oral doses of 0, 5, 25, 50, or 100 M g / k g TCDD in an acetone-corn oil mixture. Animals that eventually died continued to lose weight until death while sur vivors exhibited a depressed weight gain. Ruffled hair coat, hunched posture, inactivity, and jaundice were the overt signs seen in the high dose group. Daily oral administration of 10 M g / k g caused death in 15/16 rats with a mean time of 21.8 days. Death resulted in 9/10 female guinea pigs after receiving an oral dose of 3 M g / k g ; the mean survival time was 18.1 days. A single oral dose of 1, 10, or 50 M g / k g to adult mice hsd no effect on appearance or body weight. Hutzinger, 0 ., Safe, S., Wentzell, B. R., and ZlTKO, V. Photochemical degradation of di- and octachlorodibenzofurans. E n viron. Health Perspeet. (No. 5) : 267 (1973). Irradiation of 2,8-dichlorodibenzofuran (low chlo rine content) and octachlorodibenzofuran (high chlorine content) in hexane and methanol caused 287 D0H2160615 DO H 2 I 606 11 decomposition to compounds which are formed by reductive dechlorination aa well as polar substances. H wang, S. W. Effect of TCDD on biliary excretion of indocyanine green. Environ. Health Perrpect (No. 5): 227 (1973). Bile flow and biliary excretion of indocyanine green (ICG) in male rats 1, 7, and IS days after receiving a single oral dose of S or 25 pg/kg TCDD. Bile flow rate increased at day 1 through day IS. During a 20-min. collection period, both the concen tration and total ICG excreted in bile decreased. ICG disappearance rate decreased with time. These effects were dose related. In contrast, the 5 Mg/kg dosed rats accumulated more ICG in liver. J en sen , S., and Renberg, L. Various chlori nated dimers present in several technical chlorophenols used as fungicides. E n viron. H ealth Perrpect. (No. 5 ): 37 (1973). The presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in 2,4,5-trichlorophenoxy acid esters originates from 2,4,5-triehlorophenol during the manufacturing process. Dimerization occurs when the phenol is produced by the action of alkali mi tetrachlorobenxene. Ail products originating from alkali-treated chlorinated benzenes logically may contain chlori nated dibenzo-p-dioxins. Dimers in pentachlorophenal and in 2,4,3-tri- and 2,3,4,6-tetrachlorophenols from direct chlorination of phenol were presented. J ohnson, R. L., Gehring, P. J., and Kociba, R.J. Chlorinated dibenzodioxins and pen- tachlorophenol. E nviron. H ealth Per rpect. (No. 5 ): 171 (1973). Pentachlorophenol enjoys widespread use as a wood preservative. Commercial grades have been found to contain up to 2500 ppm chlorinated dibenzop-dioxins. The predominant dioxin is octachlorodibenzo-p-dioxin, one of the least toxic members. Eval uating pentachlorophenol toxicity in animals re vealed that some untoward effects (chloracne, chick edema disease, and histopathologic alterations) were caused by chlorinated dibenzo-p-dioxin content. Purified pentachlorophenol did not produce these effects. A new procedure was capable of producing pentachlorophenol containing lowered concentra tions of chlorinated dibenzo-p-dioxin and devoid of dioxin-like toxic effects. Kearney, P. C., Woolson, E. A. Isen see, A. R., and HELLING, C. S. Tetrachloro- dibenzodioxin in the environment: sources, fate, and decontamination. E n viron. Health Perrpect. (No. 5 ): 273 (1973). TCDD does not leach in soils, does not reside in the economic portion of plants growing in contam inated soil, degrades to about 50% after 1 yr in soils, and does not result from microbial or chemical condensation of 2,4,5-trichlofophenoi in soil. Kende, A. S., and Wade, J. J., Synthesis of new steric and electronic analogs of 2, 3,7,8-tetrachlorodibenzo-p-dioxin. E n v i ron. Health Perrpect. (No. 5): 49 (1973). Structural-activity relationships for a series of TCDD analogs were accomplished emphasizing chemical studies in an attempt to distinguish steric from electronic requirements for toxicity. Catechol condensation was used to explore the scope and lim itations of polyhalobenzene electrophiles. King, M. E., and SHEFNER, A. M., Carcino genesis bioassay of chlorinated dibenzo dioxins and related chemicals. Environ. Health P enpect. (No. 5 ): 163 (1973). Chlorinated dibenzodioxins were dissolved in ace tone and applied to the backs of mice three times a week to assess the activity of dioxins as complete carcinogens and/or promoting agents. Octachlorodioxin caused skin tumor formation in only one female mouse. No othar dioxin produced papillomas. Langer, H. G. Formation of dibenzodioxins and other condensation products from chlorinated phenols and derivatives. E n- . viron. Health Perrpect. (No. 5 ): 3 (1973). Chlorodloxins are formed in a two-step condensa tion reaction from ortho-substituted halophenoxy radicals or anions. Reaction of chlorine with penta chlorophenol at elevated temperature proceeds by radicals; anionic condensation products result from strongly exothermic reactions of alkali metal salts of chlorinated phenols above 300* C. Reaction pro duct distribution depends on the total number of halogen substituents, the crystal lattice arrangement of the molecule, steric effects, and an electronic ef fect. Dioxin formation was the major condensation product only for sodium pentachlorophenate. L ucier, G. W., McDaniel, 0 . S., F owler, B. A., F aeder, E., Hook, G., and Sona- wane, B. R. Studies on TCDD-induced changes in rat liver microsomal and mitochondrial enzymes. Environ. Health Perrpect. (No. 5 ): 199 (1973). A single oral dose of 5 or 25 g/kg was adminis tered to male rats and time-course measurements were made on some hepatic microsomal and mito chondrial enzymes. Cytochrome P-450 and b-5 contents were increased, hydroxylation of aniline 288 Environmental Health Perspectives DOH21BOB 18 / r7 w u induced, microsomal protein content* wen in- concentration was found to be more important in created, aminopyrine demethylstion rate were de causing death than duration of exposure. The coho creased, and moat strikingly UDP glucuronyitrans- salmon fingerlings threshold response level for ail ferase waa increased about 3-fold. exposure periods was between 0.066 snd 0.56 ppt Martin, R. L., Porter, M. L., P omerantz, I. H. Studies on the formation potential and presence of chlorinated dibenzofu- dioxin. Mosquito larvae, oligochaete worms, and pulminator snails were maintained in water initially dosed with 0.2 ppb dioxin. These aquatic organisms were less sensitive than fish. rans in chlorinated biphenyls. NIEHS Moore, J .; Gupta, B.; Vos, J.; Zinkl, J. conference, April 2-3, 1973. Postnatal effects of maternal exposure to Chlorinated dibanaofurana may arise by photo i chemical alteration of chlorinated biphenyla under appropriate conditions; this did not occur with TCDD. Environ. Health Perapect. (No. 5 ): 81 (1973). pentschlorobiphenyi or 2,2'-dlchlorobiphenyl. Maternal exposure of C57S1/6 mice to TCDD Matsumura, F., and Benezet, H. J. Studies on the bioaccumulation and microbial caused dose-related variations in fetal kidney matu ration and development. Thymuses were reduced in size; cystic kidneys developed. Mean body weights degradation of 2,3,7,8-tetrachlorodiben- snd thymus snd spleen weights (sbsoluts snd rela zo-p-dioxin. E nviron. H ealth Perapect. tive) were reduced in litters whoss mothers received (No. 5 ): 253 (1973). Moat organisms capable of degrading other chlo rinated hydrocarbons showed no ability to metabolize 10 Mg/kg TCDD; the 3 Mg/kg dose group exhibited no weight deviations. Kidney effects ware seen st both dose levels. TCDD; a few exhibited a limited degree of TCDD- N eubert, D., Zen s, P., and Rothenwallner, metabolixing activity. TCDD leached from sand A. Survey of the teratogenic effects of to organic soil much leas than did DDT. With pes ticide-coated sand in aquaria containing various or ganisms, TCDD had the lowest biologic accumulation and affinity. 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian species. Environ. Health Perapect. (No. 5 ): 67 (1973). Meselson, S. Vietnam dioxin contamination. Center for Short-Lived Phenomena, The frequency of deft palate induction waa used as s criterion of the teratogenic effects of TCDD. Dose-response relationships snd potentiating effects Event 51-73, No. 1611, Smithsonian In of TCDD with other agents were presented. stitute, Cambridge, Mass. (April 19, 1973). Varions fish snd shellfish collected in 1970 from the Dong Nai and Saigon Rivers and along the Can Guo Coast contained dioxin; catfish had the highest concentration. Meselson, S. Vietnam dioxin contamination. Center for Short-Lived Phenomena, Event 51-73, No. 1627, Smithsonian In stitute, Cambridge, Mass. (8 May 1973). Samples of fish and crustaceans caught in Viet nam in September 1970 were analyzed for dioxin content by using mass spectrometry. Dioxin con centrations ranged from 18 to 814 ppt; Dong Nai river carp averaged 640 ppt dioxin. Miller, R. A., Norris, L. a ., and Hawkes, C. L. Acute and chronic toxicity of 2,3,7,3-tetrachlorodibenzo-p-dioxin (dioxin) in aquatic organisms. Environ. Health Perapect. (No. 5 ): 177 (1973). Guppies and coho salmon fingerlings were exposed to dioxin concentrations ranging from 0.066 ppt to more than 0.2 ppb for 24, 48, and 96 h n . The initial NIEHS Conference on Chlorinated Dibenzo- dioxins and Dibenzofurans. National In stitute of Environmental Health Scien ces, Research Triangle Park, N.C. (April 2-3, 1973). The two-Jay conference reviewed critically and summarized the world literature and research ac tivities on the chlorinated derivatives of dibenzodioxin and dibenzofuran. Approximately 36 papers were presented on all aspects ranging from chemical nature to ultimate biologic effects. Mora than 110 scientists were in attendance. The proceedings are given in full in Environmental Htaltk Perspectives, Experimental No. 5, (1973). (this issue). NORBACK, D. H., and E ngblom, J. F. Chlori nated dibenzo-p-dioxin distribution with in rat tissues and subfractions of the liver. Fed. Proc. 32(3): 236 (1973) Ab stract 138. Radioactivity from orally intubated "Cl-labeled octachiorodibenzo-p-dioxin in rata was confined to the liver, adipose tissue, and skin after 7 weeks on s control diet; concentrations were about 20Vc that in tissues of rats after 21 days. September 1973 289 182C9 D O H 2 I 60S I 9 Norhack, D. H., Engblom, J. F., and Allen, J. R. Chlorinated dibenzo-p-dioxiir dis tribution within rat tissues and subfrac tions of the liver. Environ. Health Per f e c t . (No. 5 ): 233 (1973). Male rata received daily lor 21 days 100 ug "Cllabeied octachlorodibenzo-p-dioxin by gastric intuba tion. Feces contained 95% of the total dose and urine 4%. Significant levels were found in the kid neys, heart, and serum. The liver contained the highest concentration per unit weight; adipose tis sue had 1/3 that at the liver. Microsome* (rough and smooth fractions) had 96% of the Uver radio activity. Urine radioactivity resided in the lipid fraction. Novicic, S. Dioxin. E nvironm ent 15(4) : 23-- 24 (May 1973). A news item reports the detection of dioxin in fish and shellfish used for food in Vietnam. Plimmer, J. R., Ruth, J. M., and Woolson, E. A. Mass spectrometric identification of the hepta- and octa-chlorinated dibenzo-p-dioxins and dibenzofurans in technical pentaehlorophenol. J. Agr. Food Chem. 21(1); 90-93 (1973). The presence of contaminant dioxins and dibenzofurana in some samples of technical pentachlorophenoi were confirmed by using mass spectrometry. Three samples collected in 1970 contained hexachlorodibenzo-p-dioxin (0.5 to 37 ppmw) and heptaehlorodibenzo-p-dioxin (90 to 135 ppmw). It was reemphasized that high-resolution spectra do not provide chlorine orientation information. Plimmer, J. R. Technical pentachlorophe- nols--origin and analysis of base-insol uble contaminants. Environ. H ealth Per f e c t . (No. 5 ): 41 (1973). Fats used as feed additives from hides preserved with technical pentaehlorophenol have been impli cated as a source of chick edema factor. Polychlori nated dibeniodioxlns and dibenzofurans have been identified in the neutral fractions of pentachlorophenol by gas chromatography, mass spectrometry, and a combination of these two. Pohland, A. E ,, Yang, G. C., and Brown, N. Analytical and confirmative techniques for dibenzo-p-dioxins based upon their cation radicals. Environ. Health Per- epeet. (No. 5): 9 (1973). Chlorinated dibenzo-p-dloxins form cation radi cals when dissolved in strong acids such as trifluoromethane sulfonic add, in the presence of ultraviolet light, or an oxidizing agent like potassium nitrate. These cation radicals are quantified by using elec tion spin resonance and visible spectroscopic tech niques. A general bathochromic shift was observed with increasing chlorine content. These shifts were dependent upon the position of the chlorine atoms. P oland, A. P., and Glover, E. 2,3,7,8-Tetra- chlorodibenzo-p-dioxin: A potent inducer of {-aminolevulinic acid synthetase. Science 179(4072) : 476-677 (Feb. 2, 1973). As little as 4.56 X 10-* mole (1.5 ng) of TCDD per egg induced hepatic-aminolvulinic add synthe tase (ALAS) activity in the chick embryo. Enzyme induction was dose-related and prolonged in time: 70% of the maximum Induced activity was present 5 days after a single 160 ng dose. TCDD was linked to an outbreak of porphyria cutanea tarda where 2,4,5-T was synthesized and manufactured. At least three of the 2,3,7, and 8 positions on the ring must be occupied to induce ALAS. Poland, A. P,, and Glover, E. Studies on the mechanism of action of the halogen- ated dibenzo-p-dioxins. E nviron. Health Perepect. (No. 5 ): 245 (1973). Aminolevulinic add synthetase (ALAS) was stimulated by TCDD in the chick embryo liver; 4.56 x 10'" mole/egg (1.5 ng) caused doubling of ALAS activity and 1.55 x 10-* mole/egg (0.5 jig) caused a 35-fold stimulation of enzyme activity. A single dose of TCDD stimulates hepatic aryl hydro carbon hydroxylase (AHH) and cytochrome P-450 for 35 days and more in the rat. AHH activity was induced in chick embryo liver. It is suggested that the chemically inert parent compound is not the toxic moiety, but that a highly reactive intermed iate causes cell damage. Schwetz, B. A., Norris, J. M., Spahschu, G. L., Rowe, V. BL, Gehring, P. J., E merson, J. L .; Gerbig, C. G. Chlorodi- benzo-p-dioxin toxicology. Environ. Health Perepect. (No. 5): 87 (1973). 2,7-Dichlorodibenzo-(DCDD), 2,3,7.8-tetraehlorodibenzo-(TCDD), hexachlorodibenzo-(HCDD). and octachlorodibenzo-p-dioxin-(OCDD) were evaluated toxicology. TCDD and HCDD were acnegenic. embryo toxic (TCDD markedly so), teratogenic, and positive for chick edema factor (CEF). DCDD and OCDD were negative for acnegenidty, teratogen icity, and CEF; OCDD was embryotoxie, while DCDD was not. The lethal dose range for DCDD, TCDD, HCDD, and OCDD was g/kg, >*g/kg, mg/kg, and g/kg. respectively. Suapley, D. Herbicides: AAAS study finds dioxin in Vietnamese fish. Science 180 (4083): 285-286 (April 20, 1973). 290 Environmental Health Perspectives x 8290 Fish and shellfish from anas of South Vietnam that wen heavily sprayed during the U. S. defolia tion campaign contained significant quantities of dioxin. This is s news report on the data R. Baugh man and M. Meselson presented to the NIEHS Con ference on chlorinated dibenzodioxins and dibenzodifurans. SHAPLEY, D. Herbicides: Agent Orange stockpile may go to the South Americans. Science 180(4081): 43-46 (April 6, 1973). The U. S. Air Force has a surplus stockpile of 2,338.900 gal of Agent Orange (60% 2,4,5-T and 50% 2,4-D); some of these mixtures contain as much as 28 times the maximum acceptable safety limit of dioxin. Presently, dioxin concentrations per missible for new herbicides are 0.1 ppm and 0.5 ppm for stocks already manufactured. Vos, J. G., Moore, J. A., and Zinkl, J. Effect of TCDD on the immune system of labo ratory animals. Environ. H ealth Per spect. (No. 5 ): 149 (1973). TCDD at sublethal dose levels caused atrophy of the thymus, suppressed the cell-mediated immunity in both guinea pigs and mice, but did not affect the humoral immunity in guinea pigs. Waldbott, G. L. E ffects o f E nvironm ental Pollutants. C. V. Mosby Co., St. Louis, Mo., 1973. Dioxin compounds are listed with other environ mental pollutants as examples of airborne pollu tants. These substances are contaminants of the popular weed killer, 2,4J-T. Dioxins caused death and gastrointestinal hemorrhage in rat fetuses when mothers were treated with doses of 0.125 to 8 Mg. The mutagenic effects are due to its intercalation with DNA. Dibenzofurans were implicated as being responsible for some of the toxic effects attributed to PCBs. Webber, T. J. N., and Box, D. G. The exam ination of tetrachlorvinphos and its formulations for the presence of tetrach- lorodibenzo-p-dioxins by a gas-liquid chromatographic method. A n a ly st 98: 181-189 (1973). A gas-Uquid chromatographic analytical method was developed for use in detecting tetrachlorodibenzo-p-dioxin impurities in samples of the insecti cide tetrachlorvinphos and its formulations. Tetra chlorvinphos is the Z- or trans-styrene isomer of 2chloro-l-(2,4,5-trichlorophenyl)vinyl dimethyl phos phate. Sequential use of silica gel and aluminum oxide column chromatography followed by concen trated sulfuric acid treatment of the resultant September 1973 eluate made it possible to detect TCDD levels down to a limit of 0.025 ppm with electron-capture detec tion. Tetrachlorodibenzo-p-dioxins were not detected in any of the 21 tetrachlorvinphos samples or any of its formulations selected for analysis either be fore or after accelerated storage at 55*C for 2 weeks. Weissberg, J., and ZlNKL, J. Effects of TCDD upon hemostasis and hematologic func tion in the rat. E nviron. Health Perspect. (No. 5 ): 119 (1973). Daily oral doses of 10 Mg/kg TCDD to female rats for 10 and 14 days caused nonspecific altera tions of hematopoietic function, thrombocytopenia, thrombocytoasthenia, and derangements in blood co agulation. Platelet aggregation, bleeding time, and platelet factor III activity were normal, but clot retraction was abnormal. Wilson, J. G. Teratological potential of 2,4,5-T. Down to Earth 28(4) : 14-17 (Spring 1973). A chronology of the hazards of 2,4,5-T is pre sented. The report issued by the President's Science Advisory Committee appointed to study the 2,4,5-T question served as the basis for this review. The dioxin contaminant of 2,4,5-T, 2,3,7,8-tetrachlorodibenzo-p-dioxin, is discussed briefly as a toxicogen. Woods, J. S. Studies of the effects of 2,3,7,8t.etrachlorodibenzo-p-dioxin on mamma lian hepatic 5-aminolevulinic acid synthe tase. Environ. Health Perspect. (No. 5) : 221 (1973). No differences in hepatic 4-aminolevulinic acid syn thetase activity were seen in control rats and those receiving orally 5, 25, or 100 Mg/kg TCDD for up to 30 days. Mice and guinea pigs were also nonreactive. Thus, TCDD is not porphyrogenic in mam mals, even at several times the LD,, dose levels. Zinkl, J., Moore, J. A., Vos, J. G., and Gupta, B. N. Hematologic and clinical chemical effects of 2,3,7,8-tetrachlorodi- benzodioxin in laboratory animals. E n viron. H ealth Perspect. (No. 5 ): 111 (1973). TCDD-induced changes observed in female rata after 10 days treatment with 10 Mg/kg and 17 days at 1 Mg/kg were increases in serum glutamic oxalo acetic transaminase. After 13 days treatment with 10 Mg/kg serum glutamic pyruvate transaminase was increased. Platelet depression was observed after 10 days at all dose levels. Zitko, V., Wildish. D. J . ; Hutzinger, O., and Choi, P. M. K. Acute and chronic oral 291 DOH2I608Z0 I I Z909IZMOG I toxicity of chlorinated dibenzofurans to salmonid fishes. Environ. Health Persped. (No. 5 ): 187 (1373). Dry fish food was fad to juvenile Atlantic salmon contaminated with a mixtora ot 2.7 itg/g di-, 5,7 tri-, 2.8 tetra-, and 9.1 octachlorodibenzofuran. Mad* ian mortality was 12 ~ 30 days. Only octachlorodlbenxofuran was found in tissues of dead fish (0.03 ng/g in muacla and 0.2 jig/g in the gut). Fish sur viving 110 days feeding contained corresponding values of 0.01 and 0.02 ng/g. 1972 Abelson, P. H. Pollution by organic chemi cals. Our Chemical E nvironm ent, J. C. Giddings and M. D. Monroe, Eds., Canfield Press, San Francisco, 1972, Chap. 28, pp. 183-184. The moat toxic chlorine-containing compound known is 2,3,7,8-tetrachlorodibenzodioxine (C.JLOrCl.), often called dioxin. The acute oral LD. dose in male guinea pigs is about 10"* g/kg. In spite of its toxicity, the behavior of dioxin in the food chain has not been worked out. Anonymous. TCDD residues disappear. A gr. Res. 21(4): 6 (1972). The 2,4,5-T contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can be formed during syn thesis of some chlorinated phenols if high tempera tures are used. Anonymous. Research heightens concern over PCB's. Chem. Eng. N ew s 50(10): 27-28 (1972). Certain polychlorinated biphenyls may contain traces of tetrachlorodibenzofuran. A contaminant found in PCB's, not conclusively identified, has the same mass spectrum as tstrachloro-p-dibenzofuran. Anonymous. Dioxin with a b ang! Food Cosmet. Toxicol. 10(1): 110-111 (1972). During a manufacturing plant explosion, dioxin was formed by the interaction of sodium 2,4,5-trichlorophenate molecules under the influence of the exothermic decomposition of sodium 2-hydroxyethoxide. Anonymous. . . . But 2,4,5-T is in the dock again. Food Cosmet. Toxicol. 10(5): 722 (1972). Negative teratogenic results have been reported for rats and rabbits treated with 2,4,5-T samples with very minute levels of dioxin impurity. Terato genic effects have been detected, however, in three mouse strains treated with 2,4,5-T containing as little as 0.06 or 0.5 ppm dioxin. 292 Anonymous. 2,4,5-T and dioxins accused of teratogenicity. Food Chem. N ew s 13 (43): 24-27 (Jan. 17, 1972). Responding to challenges on 2,4,5-T cancellation, EPA concentrated its replies on lack of proof that 2,4,5-T and contaminants are not teratogens. The dose-response curves for 2,4,5-T and dioxin (TCDD) have not been determined, and the possibility of no effect levels is only a matter of conjecture. Anonymous. FDA annual report shows in crease in enforcement. Food Chem. News 13(47): 20 (Feb. 14, 1972). In the herbicide section of its 1971 annual report, FDA said the toxicity of 2,4,5-T is chiefly due to dioxins. Anonymous. Senate settlement expected on" pesticides bill. Food Chem. N ew s 14 (27) : 53-54 (Sept. 25,1972). A wide range of teratogenic dioxins can be pro duced both in the manufacture of 2,4,5-T and during pyrolysis (incomplete combustion). Boer, F, P., Neuman, M. A., and Aniline, O. 2,8-Dichlorodibenzo-p-dioxin. A d a Crystallogr. B 2 8 (9 ): 2878-2880 (1972). Crystals of 23-dichlorodibenzo-p-diaxin ire or thorhombic; molecules are slightly nonplanar with an unusual packing arrangement. Boer, F. P., and North, P. P. Crystal and molecular structure of 2,7-dichlorodibenzo-p-dioxin. Acta Crystallogr. B28(5) ; 1613-1616(1972). Three-dimensional single-crystal x-ray diffraction data revealed the crystal and molecular structure of 2,7-dichlorodibenxo-p-dioxin. The C-Cl bond dis tance is 1.742 A, the C-0 distances are 1.380 and 1.382, and the 6 C-C distances range between L370 and 1.397. The C-O-C angle in the heterocyclic ring is 118.3*. Boer, F. P., Van Remoortere, F. P., and MUELDER, W. W. Preparation and struc ture of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,7-dichlorodibenzo-p-dioxin. J. Amer. Chem. Soc. 94(3) : 1006-1007 (1972). The preparation, isolation, and isometric struc tures of 2,3,7,8-tetrachloro and 2.7-dichlorodibeniop-dioxin are described. Boer, F. P., Van Remoortere, F. P., North, P. P., and Neuman, M. A. Crystal and molecular structure of 2,3,7,8-tetrach- Environmental Health Perspectives V R /VwR<0 X9*' 2 2 9 0 9 1 ZHOfl lorodibenxo-p-dioxin. A cta Crystallogr. transaminase. Effects in homeostasis indicate the B 2 8 (4 ): 1023-1029 (1972). liver is one of the main targets for dioxin intoxica 2,3,7,8-Tetrachlorodibenzo-p-dioxin was studied by tion. using three-dimensional single-crystal x-ray diffrac Buu-Hoi, N. P., Chanh, P-H., Sesque, G., tion. The four unique C-Cl distances range from Azum-Gelade, M.C.; Saint-Ruf, G. Or CJ 1.726 to 1.730 A, the 4 C--0 distances from 1,37? to 1.379, and the 12 C-C bonds are all between 1.374 and 1.388. The C-O-C angles are 115.6' and 115.8*. gans as targets of "dioxin" (2,3,7,8-tefcrachlorodibenzo-p-dioxin). N aturw iss. Brenner, K. 3., Muller, K., and Sattel, P. Detection and determination of 2,3,7,8- 59(4): 174-175 (1972). Organ damage was found along with weight loss and hematologic effects following IP injection of 1 CT- I 1I tetrachloro dibenzo-p-dioxin in chlorosubetituted phenoxyalkane acids. J. Chrnmatogr. 64: 39-48 (1972). Dioxin separation from the herbicide phenoxy- and 10 mg/kg dioxin to rats: Ten days after treat ment, damage was observed to the liver, thymus, heart. Less damage was observed in the lungs and blood cells. aikane acids was accomplished by extractive distil* Buu-Hoi, N. P., Saint-Rot, G., and Man- lation of the potassium salts with n-hexane in the gane, M. Fragmentation of dibenzo-p- j Bleidner apparatus. Quantitative determination of the hexane extracts for dioxin was done by gas chromatography. Sensitivity was about 0.1 ppm. dioxin and its derivatives under electron impact. J. Heterocycl. Ckem. 9 (3 ): 691- 693 (1972). Brooks, 6. T. Pesticides in Britain. In: En Mass spectra were reported and discussed on five vironmental Toxicology of Pesticides, F. chloro derivatives of dibenzo-p-dioxin: 2,7-dichloro-, Matsumura, G. M. Boush, and T. Misato, 1,6-dichloro-, 1,3,8-trichloro-, 2,3,7,8-tetrachloro-, and Eds., Academic Press, New York, 1972, 1,3,4,6,7J ,9-octachlorodibenzo-p-dioxin. pp. 61-114. CHEN, J. T. Infrared studies of chlorinated The reported teratogenic effects of 2,4,5-T in mammals and the isolation of the highly toxic con taminant tetrachlorodibenxo-p-dioxin are cautionary, but 2,4,5-T has been used in Europe for nearly 15 years without evidence of il\ effects. More studies are needed in comparative detoxication between mammals, birds, fishes, and insects. dibenzo-p-dioxins and structurally re lated compounds. Division of Pesticide Chemistry, 164th National Meeting of the American Chemical Society, New York, Aug. 27-Sept. 1, 1972, Abstract No. 12. Brownrigg, J. T., Eastwood, D,, and Hornig, A. W. Identification of polychlori nated biphenyls in the presence of DDTtype compounds. Office of Research and Monitoring, U.S. Environmental Protec tion Agency, Washington, DC (Oct. 1972), EPA-R2-72-004. Low temperature (77*K) luminescence tech niques could in principle be applied to a wide variety of compounds including the highly toxic chlorinated dibenzofurana and dibenzo-p-dioxins. Buu-Hol N. P., CHANH, P-H., SESQUE, G., A zum-Gelade, M. C., and Saint-Rot, G. Enzymatic functions as targets of the toxicity of "dioxin" (2,3,7,8-tetrachlorodibenzo-p-dioxin). N aturw iss. 5 9 (4 ): 173-174 (1972). Following a single I.P dose of 10 mg/lcg dioxin to rats, deep pertubations in several enzymatic sys tems were observed, i.e., a decrease in cholinesterase or an increase in serum glutamic oxaloacetic acid Data are reported on the reference infrared spectra of 24 chlorinated dibenzo-p-dioxins and the observed characteristic frequencies are tabulated, Courtney, K. D. The teratogenic evaluation of the herbicide 2,4,5-T and dioxin. Item 4.0785, Page 1-632. Part 4. Pesticides, Environmental Protection Research Catalog. Research Information Division, U.S. EPA, Washington, D.C., Jan. 1972. Phenoxyacetic acid herbicides and dioxins are be ing evaluated for teratogenic and perinatal toxic effects. Crosby, D. G., Moilanen, K. W., Nakagawa, M., and Wong, A. S. Photonucleophilic reactions of pesticides. Environm ental Toxicology of Pesticides, F. Matsumura.i G. M. Bousch, and T. Misato, Eds., Aca demic Press, New York, 1972, pp. 423- 433. Photonucleophilic displacement of chloride by chiorophenoxide ion in o-chiorophenols introduced September 1973 293 ;; r ? q s DO H 21 606 23 the possibility of the photochemical feneration of chlorinated dlbenzo-p-dioxins. Sunlight wavelength* exposure to sodium pentaehlorophenate yielded l,23,4,6,?33-octachiorodibenzo-p-dioxin; no 2,3,73TCDD reiuited from irradiated 2,43,-T or 2,4,5-triehlorophenoL Cunningham, H. M., and Williams, D. T. Effect of tetrachlorodibenzo-p-dioxin on growth rate and the synthesis of lipids and proteins in rats. Bull. E nviron. Con tain. Toxicol. 7 (1 ): 45-61 (1972). Protein concentration in the rat liver waa alightly reduced 3 daya after dioxin treatment. Thia decreaae waa accompanied by a significant increase in "C-ieucin* incorporation into liver proteins. The lowest single dose of dioxin that caused an increase in rat liver weight waa 0.1 ag/kg Curley, a ., Burse, V. W., and J ennings, R. Metabolite or contaminant of Aroclor 1254 found in rat urine. Division of Pesticide Chemistry, 163rd National Meeting of the American Chemical So ciety, Boston, April 9-14, 1972, Abstract No. 5. Mass spectra of urine collected from rata on die tary levels of 100 or 500 ppm Aroclor 1254 for Intervals up to eight months indicated the presence of a molecular ion at m/e 304 and the characteristic isotopic cluster of 4 chlorine atoms. Others have shown the presence of tetra and pentadibenxofurans (mass numbers 304 and 338) as contaminants in the foreign products. Environmental Protection Agency, Pub lications and Information Section. Chemistry and residues of 2,4,5-T (in cludes dioxins). Bibliography Number 72-59 (1972). A reference list of 48 references is given. E nvironmental Protection Agency, Pub lications and Information Section. Toxicology and pharmacology of 2,4,5-T (includes dioxins). Bibliography Num ber 72-58 (1972). A list of 75 references is given. E pstein, S. S. Environmental pathology. A review. Am er. J. Pathol. 66(2) : 352-373 (1972). Toxicity testing must not be confined to the test agent per *e, but should be extended to its chemical and metabolic derivatives, its pyrolytic and degrada tion products and its contaminants and reaction products, especially when various derivatives or de gradation products are of toxicologic or environ mental consequence. Dioxin pyrolytic products in phenoxy herbicides are illustrative. F irestone, D., Ress, J., Brown, N. L., Bar ron, R. P., and Damico, j . N. Determi nation of polychlorodibenzo-p-dioxins and related compounds in commercial chlorophenols. J. Assoc. Offic. Anal. Chem. 55(1) : 85-92 (1972). Twenty-one commercial chlorophenols were dis solved separately in aqueous alkali, extracted with petroleum ether, fractionated on an alumina col umn, and examined by electron capture gas chrom atography and combined gas chromatography-mass spectrometry. The 2,3,7,8-tetrachlorodioxin was found in 3 of 6 samples of 2,4,5-trichlorophenol but not in any of the 11 samples of tetra- and pentachlorophenoi. Hexachlorodioxin, present in all 8* pentachlorophenols tested, ranged from 0.17 to 39 ppm. Hexa-, hepta-, and octachlorodioxins and chlorofurans were present in most of the tetra- and pentachlorophenols. F ishbein, L. Human directed aspects of PCBs. In : Polychlorinated Biphenyls and the Environm ent, Report No. IT F- PCB-72-1, (COM-72-10419), Inter departmental Task Force on PCBs, Washington, D.C., May, 1972, pp. 122- 151. Work reported previously in the literature was summarized. Emphasis was placed on the tetra- and pentachlorodibenzofuran impurities in commercial PCB products; the tri- and tetraehlorodibenzofuran single oral liver necrotic dose (0.5 to 1.0 mg/kg) in rabbits; and the 2,3,7,8-tetrachlorodibenzo-p-dloxin contaminant in 2,4,5-T and 2,43-trichlorophenol caused lethal liver necrosis and chloracne in rabbits at a dose range of 0.06 to 0.1 mg/kff. Fishbein, L. Chromatographic and biological aspects of polychlorinated biphenyls. J. Chromatog. 68(1) : 345-426 (1972). Chromatographic methods of analysis for chlori nated dibenxofurans were reviewed as part of a more extensive review of polychlorinated biphenyls. F ishbein, L., and Flamm, W. G. Potential environmental chemical hazards. Part II. Feed additives and pesticides. Sci. Total Environ. 1: 31-64 (1972). The action of alkali on 1,2,4,5-tetrachlorobenzone, a by-product from lindane synthesis, produces 2,4,5trichlorophenol which, when interacted with sodium monochloracetate, yields 2,4,5-T. The ehloracnegen, 2,3,7,8-tetrachlorodibenzo-p-dioxin, is an impurity produced in the manufacture of 2,4,5-T: 1,2.4,5-tetra- 294 Environmental Health Perspectives 282S- k Z 90 9 1 Z MOS chlorobenzene under the influence of high pressure and temperato n , methanol, and odium hydroxide, ia converted to sodium trichlorophenate which re acts with another molecule of sodium trichlorophen ate which reacts with another molecule of sodium trichlorophenate with high temperatures to form TCDD; or two moleculea of trichlorophenol combine to form TCDD. Goldmann, P. J . Severe acute chlorine acne caused by trichlorophenol decomposition products: A contribution to the perna problem. Arbeitam ed. SozuUmed. A rbeit- ahyg. 7(1) : 12-18 (1972) (Ger.). Occupational and can* histories are r.ported de scribing 2,3,6,7-tatrachiorodibenzodioxin as the causative agent in 42 cases of serious skin changes, 14 cases of internal organ damage, and 7 cases of nervous system disturbances. Greig, j . B. Effect of 2,3,7,8-tetrachlorodi- benzo-l,4-dioxin on drug1metabolism in the ra t Biochem. Pharmacol. 21(23): 3196-3198 (1972). Rata given a single oral 200 sgAg dose of dioxin exhibited a decreased duration of soxasoiamine (100 mgAg IP) induced paralysia by 54%. After 200 g/kg oral dioxin, sleeping time induced by bexabarbital, 150 mgAg (male rats) or 75 mgAg (fe male rata), was prolonged--more than double at 3 days. These results indicate that dioxin haa simul taneous stimulatory and inhibitory effects on dif ferent pathways of oxidative drug metabolism in the rat liver. Hammond, A. L. Chemical pollution: poly chlorinated biphenyls. Science 175 (4018) : 155-156 (Jan. 14, 1972). Some commercial PCB mixturea, especially those manufactured in Europe or Japan, may contain trace amounts of dibenzofurans or other toxic im purities. These may be the cause of some toxic effects of the PCBs. Hussain, S., E hbenberg, L., Lofroth, G., and GEJVALL, T. Mutagenic effects of TCDD on bacterial systems. Am bio 1 (1): 32-33 (1972). Results from three distinct bacterial assay sys tems showed TCDD to be mutagenic: (1) reversion to streptomycin independency in E. eoli SD--4, (2) reversion to histidine prototrophy in Salmonella typhimurium strains, and (3) prophage induction in E. eoli K-39. The results indicated that an acridine like behavior of DNA intercalation may have caused these genetic effects. Huston, B. L. Identification of three neutral contaminants in production grade 2,4-D. September 1973 J. Agr. Food Chem. 20(3) : 724-727 (1972). Three chemical impurities of 2,4-D are profiled. These contaminants interfere with the gas-liquid chromatographic analysis of 2,4-D for 2,3,73-tetrachlorodibenzo-p-dioxin. J ackson, W. T. Regulation of mitosis. III. Cytological effects of 2,4,5-trichlorophen- oxyacetic add and of dioxin contami nants in 2,4,5-T formulations. J. Cell Set. 10: 15-25 (1972). Inhibition of mitosis and development of cytolog ical abnormalities observed in dividing endosperm cells of the African blood Uly were believed caused by 2,3,7,3-tetraehlorodibenzo-p-dioxin, a contaminant of 2,4,5-T, rather than the herbicide itself. In con trast to 2,4,5-T. which haa no ffect, dramatic in hibition of mitosia was observed in cells subjected to 0.2 to 1.0 sg/I dioxin, 0.2 >g/l dioxin plus lO^.V 2,4,5-T, or lO^Af 2,4,5-T containing dioxin as a contaminant. These preparations also induced for mation of dicentric bridges and chromatin fusion with formation of multinuclei or a single large nu cleus. J ensen, S. The PCB story. A m bio 1(4) :123- 131 (1972). The work of Vos et. ah is reported. Tetra- and pentachlorodlbensofuran impurities in commercial PCBs may have been formed from a phenolic con taminant in PCB in similar manner as 2,3,7,8-tetrachlorodibenzo-p-dioxin is formed from 2,4,5-T. J ensen, S., and Renberg, L. Contaminants in pentachlorophenol : chlorinated diox ins and predioxins (chlorinated hydroxy- diphenylethers). Ambio 1(2) : 62-65 (1972). Octaehlorodioxin and 2-hydroxynonachlorodipheny! ether (labeled predoxin) were identified in technical pentachlorophenate using ion exchange, diazome thane treatment, gas chromatography-mass spec trometry, and thinlayer chromatography. Predioxin have not been discovered or detected previously be cause: (1) clean-up procedures remove the sub stance, e.g., alumina column chromatography or concentrated sulfuric acid treatment of extract, (2) predoxin spontaneously forms dioxin in a gas chromatograph. Kearney, P. C., Woolson, E. A., and El lington, C. P. Persistence and metabo lism of chlorodioxins in soils. Environ. Set. Technol. 6(12) : 1017-1019 (1972). Persistence of 1,10, or 100 ppm 2,3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined by elec tron-capture gas chromatography in soils after 20, 295 5X Q O Q ..i. D O N 2 I 606 25 40, 80, 100, and S60 dir*. After 1 yr, 50-88% TCDD n i recovered. Neither 2,7-dichlorodibenxo-pdioxin (DCDD) nor TCDD i u detected in soil re ceiving 10, 100, or 1000 ppm 2,4-D or 2,4,5-T after 70 day*. TCDD ia degraded slowly in soils. TCDD and DCDD are not bioayntheaized by microbial condenaation reactions. Khera, K. S., and McKinley, W. P. Pre- and postnatal studies on 2,4,5-trichloro- phenoxyacetic acid, 2,4-dichlorophenoxy- acetic acid and their derivatives in rats. ToxicoL AppL Pharmacol. 22: 14-28 (1972). 2,4,5-T (containing leaa than O.fi mgAg 2,3,73tetrachlorodibenzo-p-dioxin) indnced fetopathy and skeletal anomaliea in progeny from females treated with a single daily oral doae of 100 to 150 mgAg on geatation days 0 to 15. Nomber of conceptions and numbers of viable and dead fetuses per Utter gave no indication that m utero treatment of offspring with up to 100 mgAg 2,4,5-T had impaired fertility. Kimbrough, R. D. Toxicity of of chlorinated hydrocarbons and related compounds. A review including chlorinated dibenzodi- oxins and chlorinated dibenzofnrans. Arch. Environ. Health 25(2) : 125-131 (1972). Trace amounts of chlorinated dibenzofnrans and dibenzodloxins were identified as contaminants in many chlorinated technical compounds, e.g., 2,43trichlorophenol, 2,4,5-trichorophenoxyacetie acid (2,4,5-T), and European chlorinated biphenyla (Phenoclor DPS and Clophen A0O). Toxic fat con taining chick edema factor also contained chlorinated dibenzodioxina. Kins, N., and BaRandy, J. Short method for the detection of chick edema factor in fats, oils, and fatty acids by electron capture gas chromatography. J. Am er. Oil Cham. Soc. 49(2): 115-117 (1972). A modification of the official electron eapture-gaa chromatographic AOAC assay for chick edema fac tor, e.g., hexa-, hepta-, and octachlorodibenzo-p-dioxins, was presented to shorten the aaaay time and eliminate problems in the alumina column frac tionation. In place of the alumina fractionation foUowing 2,2,4-trimethylpentane extraction, a sec ond sulfuric add treatment and a caustic wash were done before the final sulfuric acid wash. Kraybill, H. F. Chemical and physical prop erties of PCBs. In: Polychlorinated Bi phenyls and the Environment, Report No. ITF-PCB-72-1, (COM-72-10419), Interdepartmental Task Force on PCBs, Washington, D.C. (May 1972). pp. 2240. Tetrachiorodibenzofuran (mass number 304) and pentachlorodibenzofuran (mass number 338) were identified from a fractionated PCB sample. Maier-Bode, H. Contribution to 2,4,5-T ques tion. Anz. Schaedlingakd. Pflanzenschutz 45(1) : 2-6 (1972) (Ger.). The teratogenic effect of 2,4,5-T In an earlier U.S. experiment ia attributable to the content (30 ppm) of 23,73-tatrachlorodlbenzo-p-dioxin in the test sample. Martin, H. (E d.). Pesticide Manual. Basic Information on the Chemicals used as Active Components of Pesticides, 3rd. ed. British Crop Protection Council, Worcester, England (1972). A contaminant of 2,4,5-T (2,3,7,8-tetrachlorodl- benzo-p-dloxin) caused serious acne in man and pro duced fetal death in hamsters at 9.1 MgAg. Modern methods of synthesis now limit the dioxin concen tration in 2,4,5-T to leaa than 0.5 ppm. Matsumura, F. Biological effects of toxic pesticidal contaminants and terminal residues. In: Environmental Toxicology of Pesticides, F. Matsumura, G. M. Boush, and T. Misato, Eds., Academic Press, New York, 1972, pp. 525-548. Chlorinated dibenzo-p-dloxina have been classified as terminal residues-chemicala which accumulate in biologic material in the environment as a result of pesticide introduction. The presence of terminal residues in the environment ia due to stable pesti cides, conversion products, and chemical impurities (dioxins) that remain in the environment longer than the principal pesticides. The importance of such terminal residues in relation to the final mag nitude of environmental hazardousnesa needa fur ther definition. Neubert, D., and Dillmann, I. Embryotoxic effects in mice treated with 2,4,5-tri- chlorophenoxyacetic acid and 2,3,7,8-tet- rachlorodibenzo-p-dioxin. Naunyn- Sehmiedeberg's A rch. Pharmacol. 272: 243-264 (1972). Oral doaes of 1 Mg/kg 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDBD) in NMRI mice on days 6 to 15 of geatation produced cleft palate; embryo mortal ity was significant for doaea of 4.5 Mg/kg or more. Cleft palate without pronounced embryo lethality was produced in mice with high single doaes (2050 MgAg) TCDBD given between days 7 and 13 of pregnancy. A maximum teratogenic effect was 296 Environmental Health Perspectives .. IS256 DOM2 I 60616 observed on day IX of gestation, bat another peak in deft palate frequency occurred when TCDBD * u given on day 8 of pregnancy. Neuman, M. A., North, P. P.f and Boer, F. P. Crystal and molecular structure of octachlorodibenzo-p-dioxin. A cta Cry- atailogr. B28(8) : 2313-2317 (1972). Three-dimensional tingle-crymtal x-ray diffraction waa need to elucidate the crystal and molecular structure of octachlorodibenzo-p-dioxin. The four unique C-Cl distances ranged from 1.714 to 1.718A. the two C-O distances were 1.373 and 1.374, and the six C-C bonds ranged between 1.882 and 1.398. The C-O-C angle was 115-8". Norback, D. H., and Allen, J. R. Chlorinated aromatic hydrocarbon induced modifica tions of the hepatic endoplasmic retic ulum : concentric membrane arrays. En viron. Health Perapect 1: 137-143 (1972). Separate groups of rats were fed diets containing 1% highly chlorinated triphenyls (PCTs, Arbcior 5460), 0.02% polychlorinated biphenyls (PCBs, Arochlor 1254), and 0.002% chlorinated diphenyl-p-dioxin. The dioxin and PCT groups ate readily and attained 80% of control weight in 3 weeks. Liver hypertrophy varied from moderate enlargement (di oxin group) to an increase In relative liver weight of 8/100 body weight for the PCB and PCT groups. Pobland, A. E., and Yang, G. C. Prepara tion and characterization of chlorinated dibenzo-p-dioxina. J. A g r . Food Chem. 20(6) : 1093-1099 (1972). A series of 13 chlorinated dibanzo-p-dloxins (nine of these were new and previously unreported) was prepared containing from 1 to 8 chlorine atoms to provide pure standards of the various chlorinated derivatives for use in methods development and tox icology studies. Synthesis, utility, yield, parity, and physical and chemical properties were presented and discussed. Stability, color reactions, and in frared, ultraviolet, nuclear magnetic resonance, and phosphorescence spectra were also reported. Phos phorescence and triplet state lifetime wavelengths were dependent on the number of chlorine atoms and their positions on the dibenxo-p-dioxin nucleua Rappe, C., and Nilsson, C .-A. An artifact in the gas chromatographic determina tion of impurities in pentachlorophenol. J. Chromatogr. 67: 247-253 (1972). The main impurity of commercial samples of pen tachlorophenol was 3,4,5,S-tetrachloro-2-(2,3,4,5,pentachlorophenoxy)phenol; this compound under went ring closure during gas chromatography to 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin. September 1973 Saint-R uf, G. Form ation of "dioxin" in the pyrolysis of sodium -(2,4,5-trichloropnenoxy) -propionate. Naturwiss. 59 (12) : 648 (1972). A residue of 2,3,7,8-tetrachlorodibenzo-p-dioxin re sulted from the pyrolysis (500*C for 5 hr) of sodium--<2,4,5-trichlorophenoxy)-propionate, a com pound used in the synthesis of Sylvex. The dioxin impurity yield was much less than that observed during 2,4,5-T synthesis. Task F orce. Polychlorinated biphenyls and the environment. Report No. ITF-PC B- 72-1, (COM -72-10419), Interdepart mental Task Force on PCBs, Washing ton, DC (May, 1972). This report emanated from a six-month review on the polychlorinated biphenyls by five Federal agencies. The main emphasis was PCBs; however, numerous statements and discussions centered on chlorinated dibenzodloxina and dibenzofursns. For example, fractionated samples of some PCBs of foreign manufacture contained as contaminants tetra- and pentachlorodibenzofurans. The task force recommended: (1) toxicological evaluation of a se lect number of representative, purified PCB isomers as well as purified trace contaminants such as the chlorinated dibenzofursns; (2) definitive mammalian elaboration of the kinetics, absorption, distribution, metabolism, and excretion of the technical PCBs as well as a number of key isomers and the chlorinated dibenzofursns; and (3) elaboration of the subcelluJar and intracellular actions of the technical PCBs as well as a number of representative isomers and chlorinated dibenzofursns. Vasiliu, G-, and Baciu, I. Acetylation of some 2,7-dihalodibenzo-p-dioxins. Rev. Chim. (Bucharest) 23(9) : 523-525 (1972) (Rom.). Reaction of 2,7-diehlorodibenzo-p-dioxin with chloroacetic acid in the presence of aluminum chlo ride and carbon disulfide gave the corresponding 3monoacetyl derivative. Confirmation was by infrared spectra. Vos, J. G. Toxicology o f PCB's for mammals and for birds. E nviron. H ealth Persp e d . 1: 105-117 (1972). Polychlorinated dibenzofursns or other toxic im purities in crude PCB preparations caused difficul ty in interpreting toxicity studies. Vos, J. G. Toxicology of polychlorinated bi phenyls (PCB's) and impurities. Tijdaehr. Diergeneesk. 97(22): 1378- 1385 (1972) (N eth). 297 182S7 D0N2160627 The mot toxic imparities in polychlorinated bi phenyls were found to be chlorinated benzofurans. Vos, J, G., and Notenboom-Ram, E. Com parative toxicity study of 2,4,5,2?A ',5'.hexachlorobiphenyl and a polychlorinat ed biphenyl mixture in rabbits. Toxicol. A p p l. Pharmacol. 23: 563-578 (1972). The major acnegenie action of crude PCB mix tures originates from chlorinated dibenzofurans. The probable contribution of chlorinated dibenzofuran and pure PCB to the toxicity of technical PCB preparations in rabbits is discussed: chloracne (--furan + + , PCB + ). edema formation (+ + , --), liver damage ( + + , 4-), and hepatic porphyria ( - , + + ). Wabmbbunn, K. Considerations regarding the ban or limitation on the use of some pesticides imposed July 23, 1971. Gesunde Pflanz. 24(1) : 6-8 (1972) (Ger). The dioxin content of 2,4,5-T produced in Ger many is approximately 1 ppm, a level at which mal formation hazards are practically eliminated. Williams, C. S. The current status of phenoxy herbicides. D ow n to E a rth 27 (4) : 20-24 (Spring 1972). A chronology of events from April 13, 1966 to December 6, 1971 pertaining to phenoxy herbicides is presented, with particular attention devoted to TCDD. Williams, D. T., and Blanchfield, B. J. Screening method for the detection of chlorodibenzo-p-dioxins in the presence of chlorobiphenyls, chloronaphth&lenes, and chlorodibenzofurans. J. A ssoc. Offic. Anal. Chem. 55(1) : 93-95 (1972). The chlorodibenzo-p-dioxins were chlorinated to octachlorodibenzo-p-dioxin and identified by electron capture-gas-liquid chromatography. 2,3,7,8-Tetrachlorodibenzo-p-dioxin was determined at the 1 ppm level in corn oil. This method should be considered as a screen for dioxins, since it does not distinguish among the large number of isomers. Williams, D. T., Cunningham, H. M., and Blanchfielj), B. J. Distribution and ex cretion studies of octachlorodibenzo-pdioxin in the rat. Bull. E nviron. Contam. Toxicol. 7(1). 57-62 (1972). Absorption, distribution, and excretion of octachlorodibenzo-p-dioxin in the rat were examined. The AOAC method was used to analyze organs and tissues for dioxin content. The only gross pathology observed was congestion of the liver. Octachlorodi- benzo-p-dioxin was absorbed by the rat mainly in the liver with small amounts in the adipose tissue and bile. Williams, D. T., and Blanchfield, B. J. Improved screening method for chlorodibenzo-p-dioxins. J. Assoc. Offic. Anal. Chem. 55(6): 1358-1359 (1972). Conversion of 2,3,7,8-tetrachlorodibenzo-p-dioxin to octachlorodibenzo-p-dioxin allowed detection by electron capture-gas-liquid chromatography of as low as 0.06 ppm in corn oil. The author's previous method was modified to include different chlorination conditions and a later alumina column cleanup. Wilson, J. G. Teratolo^ical potential of 2,4,5-T. Proceedings Twenty-Fifth An nual Meeting Southern Weed Science Society, Dallas, TX, Jan. TS-20, 1972. A chronology of the hazards of 2,1,5-T was pre sented. The report issued by the President's Science Advisory Committee appointed to study the 2,4,5-T question served as the basis for this review. The dioxin contaminant of 2,4,5-T, 2,3,7,8-tetrachIorodibenzo-p-dioxin, was discussed briefly as a toxicogen. Woolson, E. A., Thomas, R. F., and E nsob, P. D. J. Survey of polychlorodibenzo-pdioxin content in selected pesticides. J. A gr. Food. Chem. 20(2) : 351-354 (1972). Electron capture-gas chromatography was used to examine 129 samples of 17 different pesticides de rived from chlorophenols for polychlorinated dibenzo-p-dioxins. Clean-up entailed concentrated sul furic acid extraction of impurities from hexane and mild nitration of the ehlorophenol extracts. 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) was detected in the samples analyzed: 76% contained less than 0.1 g/g, 7% between 0.1 to 1.0 >*g/g, and 9% had greater than 10 >g/g. The 20 tri-, tetra-, and pentachlorophenols examined contained no TCDD. Phenoxy herbicides from current production had less than 0.5 *g/g TCDD. Yang, G. C., and Pohland, A. E. Electron spin resonance studies of cation radicals in trifluoromethane sulfonic acid. J. Phys. Chem. 76(10) : 1504-1505 (1972). Electron spin resonance spectra were obtained for chlorinated dibenzo-p-dioxins in trifluoromethane sulfonic acid (MSA); no oxidizing agents were used to produce the spectra in sulfuric acid. An oxidizing agent or ultraviolet light was needed for octachloro dibenzo-p-dioxin to yield a specturn in MSA; no spectrum was shown in sulfuric acid. Zitko, V. Absence of chlorinated dibenzodi- 298 Environmental Health Perspectives 3 o3 D0M2160628 t1II cr o O' I I oxins and dibenzofurans from aquatic animals. Bull. Environ. Contam. Toxicol. 7 (2 /3 ): 105-110 (1972). Chlorinated dlbenzodioxin and dibenzofuran resi dues were not detected in any at the aquatic samples analyzed. Zitko, V., Hutzingeb, 0 ., and Choi, P. M. K. Contamination of the Bay of FundyGulf of Maine area with polychlorinated biphenyls, polychlorinated terphenyls, chlorinated dibenzodioxins, and dibenzo furans. E nviron . H ealth P erspect. 1: 4 7 50 (1972). No residues ot chlorinated dibenzodioxins and di benzofurans were found in the samples analyzed: muscle and liver of white shark, eggs of doublecrested cormorants and herring gulls, commercial herring oil, and ground fish herring meal. Detection limits ranged from 0.01 itg/g octachlorodibenzofuran wet tissue to 0.04 Mg/TCDD/g wet tissue. 1971 ANONYMOUS. A close look at TCDD. A gr. Res. 20(4) : 3-10 (1971). The toxicological properties of tetrachlorodlbenzo-p-dioxin were investigated following the discovery that the mutagenic and teratogenic potential of 2,4,5-T may be due to thia contaminant. Anonymous. Herbicides: More research on 2,4,5-T. Chem. E ng. N ew s 49(20) : 11 (1971). The President's Science Advisory Committee's re port on 2,4,5-T and ita dioxin impurity is reviewed. The panel made a number of recommendations, in cluding one asking for a mechanism that would temporarily restrict the use of certain registered pesticides on the basis of information that impli cates the chemical as a possible health hazard, pend ing the collection of more conclusive information. Anonymous. Herbicides: Secret 2,4,5-T re p o rt Chem. E ng. N e w s 49(29) : 15 (1971). An advisory committee's report submitted to the Environmental Protection Agency concerning 2,4, 5-T and its dioxin contaminant was criticized by a dissenting advisory committee member and the Com mittee for Environmental Information. Specifically stated was that a level at which TCDD is not tera togenic haa not been established and that the report did not consider the consequences of the fate of TCDD in food chains and animal tissue. Anonymous. A taste of honey (flavoured September 1973 with 2,4,5,-T ). Food C o m e t. Toxicol. 9: 152 (1971). This is a commentary on the teratogenic evalua tion of 2,4,5-T by K. D. Courtney, et al.; a note added in proof indicate the 2,4,5-T contained 30 ppm dioxin. Thus, conclusions from the paper label ing 2,4,5-T teratogenic must be considered tentative. Anonymous. The PCB story unfolds. Food C o m et. Toxicol. 9(4): 568-571 (1971). Lesions resembling those produced by chick edema factor were traced to a PCB contaminant, chlori nated dibenzofurans which is present in some com mercial PCB*. Anonymous. Working with 2,4,5-T. "Food C o m e t. Toxicol. 9: 908-909 (1971). Chloracne, characterized by inclusioircysts, come dones, and pustules, was found in 13 workers, and was correlated in severity with the presence of scarring, hyperpigmentation, hirsutism, and com plaints of eye irritation. A. P. Poland et aL. com mented that chloracne was not correlated with oc cupations within plants manufacturing 2,4,5-T, du ration of employment, or coproporphyrin excretion. Anonymous. Tetrachlorodibenzodioxin-- in timations of carcinogenicity? Food Cos- m et. Toxicol. 9: 909 (1971). Although such effects as described by Buu-Hoi, et al. of the carcinomimetic activity of TCDD exist, the relationship is by no means exclusive. For example, stimulation of hydrcxylating enzymes can be induced by many compounds, including BHT, which haa been shown not to be carcinogenic in long term feeding studies. Anonymous. PSAC hiccoughs over 2,4,5-T. N ature 231(5300): 210-211 (1971). The President's Science Advisory Committee (PSAC) report on 2,4,5-T is questioned: justifica tion by the PSAC concerning permissable levels of dioxin in 2,4,5-T was not based on scientific review; also, the need for establishing a dose-response curve for the teratogenicity of 2,4,5-T should have been cited. Anonymous. 2,4,5-T report attacked. N a ture 232(5308): 218 (1971). The response of the Committee for Environmental Information (CEI) to the scientific advisory com mittee's recommendation that 2,4,5-T be restored was one of strong opposition. With respect to dioxins, the CEI indicated that dioxins may accumulate in the soil and that present analytical techniques were not sensitive enough to detect them. Furthermore, lack of concrete proof that the use of 2,1,5-T was correlated with the occurrence of birth defects in Vietnam did not mean that no correlation existed. 299 82^9 6 909 IZMOQ Baughman, R. W., and Meselson, M. An improved analysis for 2.3.7.8-tetrachlorodibeuzo-p-dioxin (TCDBD). Division Pesticide Chemistry, 162nd National Meeting of the American Chemical So ciety, Washington, D.C., Sept. 12-17, 1971, Abstract No. 89. Current analytical method* for the detection of potentially hazardous levels of 2,3,7.8-tetrachlorodibenzo-p-dioxin are inadquats. A combined gas chromatogTaphy-maas spectroscopy method was utilized with modifications in extraction concentration and detection. Bevenue, A., and K awano, Y. Pesticides, pesticide residues, tolerances, and the law (U .S.A .). Residue Rev. 35: 103-149 (1971). Politics and science surround the 2.4,5-T and hence dioxin controversy. Contents include: Federal In secticide, Fungicide, and Rodenticide Act (FIFRA), as amended through 1969; the DDT episode; the 2,4,5-T episode; Federal Food, Drug, and Cosmetic Act (FDCA); and food imports and international pesticide control. Mention is made of the USDA's investigation of possible excessive contamination of 17 polychlorophenolic pesticides by tetrachlorodibenzo-p-dioxin. Boer, F. P., Neuman, M. A., van Remoobtebe, F. P., North, P. P., and Rin n , H. W. X-ray diffraction studies of chlori nated dibenzo-p-dioxins. Division Pesti cide Chemistry, 162nd National Meeting of the American Chemical Society, Wash ington, D.C., Sept. 12-17, 1971 ; Abstract No. 82. The crystal structures of four chlorinated dibenzo-p-dioxin compounds (2,7-dichloro; 2.8-dichloro; 2,3,7,8-tetrachloro; and octachloro) were determined by x-ray diffraction. The structures were elucidated in order to provide absolute standards for isomeric structure assignment. Buu-Ho, N. P., Hien , D. P,, Saint-Ruf, G., and SERVOEN-SiDOlNE, J. Canceromimetic properties of tetrachloro-2,3,7,8 dibenzo-p-dioxin ("dioxin" ). C. R. Acad. Set. (Paris) D272(10): 1447-1450 (1971) (F r). Tetrachloro-2,3,7,8-dibenzo-p-dioxin in rats caused induction of zoxazolamine hydroxylase, marked re duction in phnobarbital sedative effect, and a de crease in hepatic arginase. Dioxin therefore, has a pronounced inhibitory effect on the enzymatic sys- terns as do the carcinogens benzo [a]-pyrene and pdimethylaminoazobenzene. Buu-Hot, N. F,, Saint-Ruf, G., Bigot. P., and Mangane, M. Preparation, pronerties and identification of "dioxin" in pyrolysates of defoliants containing 2,- 4.5- trichlorophenoxyacetic acid, their esters, and contaminated plants. C. R. Acad. Sci. (Paris) D 273(7): 708-711 (1971) (Fr). Synthesis and physico-chemical properties of 2, 3,7,8-tetrachloTodibenzo-p-dioxin and its less toxic isomer 1.3,6.8-tetrachlorodibenzo-p-dioxin are de scribed. Caution was urged about the burning of wooded areas treated with 2,4,5-T or its butyl ester because of possible dioxin formation. Campbell, A. D., and F irestone, D. Chick edema factor-- toxic dioxins. In : Interna tional Symposium on Identification and Measurement of Environmental Pollu tants, June 14-17, 1971, Ottawa, On tario, Canada, B. Wesley, Ed., National Research Council, Canada, 1971, pp. 195-198. The formation, occurrence, toxicity, tissue distri bution in the chick, and analytical methods for the detection of polychlorodibenzo-p-dioxins are de scribed. Dioxins were implicated in chick edema disease. Dioxins belong to one of the most toxic classes of chlorinated compounds known, being al most 1000 times as toxic as most pesticides. Collins, T. F. X., Williams, C. H., and Gray, G. C. Teratogenic studies with 2,- 4.5- T and 2,4-D in the hamster. Bull. Environ. Contain. Toxicol. 6(6): 559- 567 (1971). 2,4,5-T commercial samples given on days 6 to 10 of organogenesis were feticidal and teratogenic in the golden Syrian hamster. The incidence of the observed effects increased with an increasing con tent of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dioxin impurity caused edema and hemorrhages in new born animals. Committee for E nvironmental Informa tion. Critique of the report of the ad visory committee on 2,4,5-T. E nviron m ent 13(7): 24, 29 (1971). Criticism by the Committee for Environmental Information levied against the Advisory Commit tee's report on 2,4,5-T centered on the following areas: no effect levels; environmental accumulation, transmission and degradation; human hazards from 300 Environmental Health Perspectives 18300- D O M2 ! 6 06 30 dioxin*; 2,4,5-T teratogenicity ; and the benefits vs. risks of 2,4,5-T usage. bit. Food Cosmet. Toxicol. 9 (3 ): 395404 (1971). Courtney, C. D.t and Moore, J. A. Terat Treatment of rabbits and rats with 2,4.5-T con ology studies with 2,4,5-trichlorophen taining 0.5 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin oxyacetic acid and 2,3,7,8-tetrachlorodi- did not cause any teratogenic or embryotoxic effects. O benzo-p-dioxin. Toxicol. Appl. Phar Rats received 1, 3, 6, 12, or 24 mg 2,4,5-T/kg on c macol. 20(3) : 396-403 (1971). days 6 to 15 of pregnancy. Rabbits received 0, 10, 20, or 40 mg 2,4,5-T/kg on days 6 to 18 of pregnancy. 2.4.5-T and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were teratogenic in three strains ot mice; F irestone, D. Determination of polychloro- combinations ot these compounds did not potentiate dibenzo-p-dioxins (dioxins) in chloro- observed remits. Both produced cleft palates and Iddney malformations. Studies with rats gave nega tive results with 2,4,5-T; TCDD Induced kidney anomalies. Crosby, D. G., Wong, A. S., Plimmer, J. R., and Woolson, E. A. Photodecomposition of chlorinated dibenzo-p-dioxins. Science 173(3998): 748-749 (1971). phenols and lipids-. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Wash ington, D.C., Sept. 12-17, 1971; Ab stract No. 80. High resolution mass spectrometry was reported to offer a sensitive method for detecting specific dioxins after adequate sample cleanup, e.g., chroma 2.4.5-T and 2,3,7,8-tatrachlorodibenso-p-dioxin tography and gas chromatography. homoiogs photodecompose rapidly in an alcohol solu tion exposed to artificial light and natural sunlight. The decomposition rate was correlated with the de F irestone, D. Fuck, D. F., Ress, J., and H igginbotham, G. R. Distribution of gree of chlorination. Negligible photodecomposition chick edema factors in chick tissues. J. was observed in aqueous suspensions and on wet or dry soil. Assoc. Offlc. Anal. Chem. 54(6): 12931298 (1971). Davring, L., and Sunner, M. Cytogenetic Chickens fed toxic animal fat containing 9.9 ppm effects of 2,4,5-trichlorophenoxyacetic chlorinated dibenzo-p-dioxins excreted over 90% the acid on oogenesis and early embryogenesis in Drosophila melanogaster. Hrdi t 68(1) : 115-122 (1971). ingested hexa-, hepta-, and octachlorodioxins. De creased chlorination resulted in greater body reten tion. Residues were found in many tissues but was most prominent in the liver. 2.4.5- T with dioxin contaminants less than 0.1 ppm was evaluated in a wild-type Drosophila population, Hansen, W. H., Quaife, M. L., Habermann, Canton-S 109. Adult flies were exposed to 250 ppm R. T., and F itzhugh, O. G. Chronic 2,4,5-T in their food within or later than 24 hr of toxicity of 2,4-dichlorophenoxyacetic closion. Results indicated that this 2,4,5-T formula tion effected early oogenesis and caused chromosome disturbances which could result in sterility. Elvxdge, D. A. The gas-chromatographic de acid in rats and dogs. Toxicol. Appl. Pharmacol. 20: 122-129 (1971). 2,4-D preparations, having undetectable levels of 2,7-dichlorodibenzo-p-dioxin or 2,3,7,8-tetrachlorodi- termination of 2,3,7,8-tetrachloro-p-di- benzo-p-dioxin, were tested for chronic toxicity in oxin in 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-T ethyl-hexyl ester, for mulations of 2,4,5-T esters and 2,4,5-tri- rats and dogs. No significant effect on growth, sur vival, organ weight, or hematologic values were noted. chlorophenol. A n a lyst 96(1147) : 721- Hays, H., and Risebrough, R. W. The early 727 (1971). warning of the terms. N atural History A gas chromatographic method is described for 80(9): 39-46 (Nov. 1971). the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin Possible health implications to humans and wild impurities in herbicides. Dioxin recoveries ranged life from environmental pollution with chlorinated from 89 to 98% ; limit of detection was 0.05 ppm. dibenzofurans are reviewed. E merson, J. L., T hompson, D. J., Strebing, R. J., Gerbig, C. G., and Robinson, V. G. Teratogenic studies of 2,4,5-trichloro phenoxyacetic acid in the rat and rab HELLING, C. S. Pesticide mobility in soils. II. Applications of soil thin-layer chro matography. Soil Sci. Soc. Am er. Proc. 35(5): 737-743 (1971). September 1973 301 1 O: 'JsL DO M2 1 6 0 6 3 1 Relative mobility by thin-layer chromatography and diffusion in moist and air-dry soils were used to test pesticide movements. The two chlorinated dlbenzo-p-dlozins were immobile. Holden, C. Critics weigh EPA herbicide re port, find it wanting. Science 173(3994): 312 (1971). The EPA's Advisory Committee on 2,4,5-T recom mended that the ban on 2,4,5-T be lifted with cer tain qualifications, i.e., no more than 0.1 ppm 2,4,5-T in drinking water and formulations containing no more than 0.1 ppm dioxin. The Committee for En vironmental Information voiced five major objec tions. Isensee, A. R., and JONES, G. E. Absorption and translocation of root and foliage applied 2,4-dichlorophenol, 2,7-dichloro- dibenzo-p-dioxin, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. A gr. Food Chem. 19(6): 1210-1214 (1971). Measured uptake of "C-labeied 2,7-dichloro- and 2,3.7,8-tetrachlorodibenzo-p-dioxin from nutrient so lution, soil, and foliage by oats and soybeans indi cated that accumulation of TCDD in plants via the soil is unlikely. J ohnson, J. E. The public health implica tions of widespread use of the phenoxy herbicides and picloram. Bioscience 21 (17) : 899-905 (Sept. 1, 1971). A review presenting the myriad historical aspects of the dioxin-2,4,5-T( and other herbicides) picture. The potential adverse health effects from phenoxy herbicides were documented. One conclusion reported was that impurities--particularly the chlorodibenzoP-dioxins--can be an important factor, but these can be controlled by proper manufacturing techniques. J ohnson, J. E. Safety in the development of herbicides. Proc. Ann. Calif. Weed Conf. 23: 43-67 (1971). Experiments using 2,4,5-T alone and in combina tion with dioxin allowed the conclusion that com mercially produced 2,4,5-T containing less than 1 ppm dioxin does not present a hazard to health. J ohnson, J. E. Safety in the development of herbicides. Down to E arth 27(1): 1-7 (1971). After the discovery of highly toxic dioxin resi dues in the herbicides 2,4,5-T, precautionary control measures were instituted to insure greater quality control in pesticide production. Pregnant rats were treated orally with 2,4,5-T at doses of up to 100 mg/kg/day. No symptoms of teratology were pro duced in the offspring. Dioxin was toxic to embryos at 0 125 Mg/kg/day, whereas 0.03 Mg/kg/day was be low the "no-effect" level. Some aspects of production and costs are discussed, Kearney, P. C., and Woolson, E. A. Per sistence and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in soils. Division Pesticide Chemistry, 161st Na tional Meeting of the American Chemical Society, Los Angeles, March 28-April 2, 1971; Abstract No- 21. TCDD applied 1, 10, and 100 ppm to two soils held at 30*C remained relatively unchanged (80-85% of original samples were recovered) after 20, 40, 80, and 160 days. Incubation of 10, 100, and 1000 ppm, 2,4-dichlorophenol. and 2,4,5-trichtorophenol in soils for 70 days produced no detectable di- or trichlorodioxins. Kearney, P. C., Isensee, a .. Helling, C. S. Woolson, E. A., and Plimmer, J. R. Environmental significance of chloro- dioxins. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Washington, D.C., Sept. 12-17, 1971; Abstract No. 90. Studies on TCDD under controlled environmental conditions revealed that TCDD was persistent and immobile in soil, not readily absorbed by plants, sub ject to photodecomposition, and slowly degraded to polar metabolites. Other studies revealed that the environmental contamination is small and not detect able in biological samples. Khera, K. S., and Ruddick, J. A. Perinatal effects of dibenzodioxins in Wistar rats. Division Pesticide Chemistry, 162nd Na tional Meeting of the American Chemi cal Society, Washington, D.C. Sept. 12- 17, 1971; Abstract No. 87. Oral administration of 16 to 0.125 Mg/kg 2,3,7,8tetrachlorodibenzo-p-dioxin to pregnant rats on days 6-15 of gestation caused a reduction in viable litter size and a high incidence of resorptions. No effect was observed at the 0.125 level. Histological exami nation of fetuses revealed brain and intestinal hemorrhages and edema of subcutaneous tissue. Khera, K. S., Huston, B. L., and McKinney, W. P. Pre- and postnatal studies on 2, 4.5-T, 2,4-D, and derivatives in Wistar rats. Toxicol. Appl. Pharmacol. 19:369- 370 (1971). Results from teratogenic studies with rats treated with 2,4,5-T, 2,4-D, and several derivatives were 302 Environmental Health Perspectives it 2909 lZMQQ either negative or inconclusive. The 2,3,7,8-tetrachlorodibensbo-p-dioxin levels in these preparations were not known in all eases, but most were suspected to be iess than 0.5 ppm. Danger, H. G., Brady, T. P., Dalton, L. A. Shannon, T. W., and Briggs, P. R. Thermal chemistry of chlorinated phe nols. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Washington, D.C., Sept 12-17, 1971; Abstract No. 83. At the decomposition temperatures of chlorinated phenols and derivatives, the following: conditions are ideal for dioxin formation: (a) high concentration of the phenol, (b) high degree of chlorination of the phenol, and (c) presence of base. Loefroth, G. Environmental poisons, man and the physician. L aekartidningen 68 (18): 2113-2133 (1971) (Swed.). Possible sdverse health effects from environmental poisons demands caution. Tetrachlorobenzo-p-dioxin was mentioned for its adverse effects of fetal develop ment. MlLNES, M. H. Formation o f 2,3,7,8-tetra- chlorodibenzodioxin by thermal decom position of sodium 2,4,5-trichlorophen- nate. N ature (London) 232(5310): 395-396 (1971). Hydrolysis of 1,2,4,5-tetrachlorobenzene in methyl alcohol with sodium hydroxide at high pressures or in ethylene glycol at 1 atm formed sodium monochlorophenate. This allowed exothermic decomposi tion of 2,4,5-trichlorophenate sodium to 2,3,7,8-tetrachlorodibenxodioxin. Muelder, W. W-., and Shadoff, L. The prep aration of uniformly labeled MC-2,7-di- chlorodibenzo-p-dioxin and 2,3,7,8-tetra- chlorodibenzo-p-dioxin. Division Pesti cide Chemistry, 162nd National Meeting of the American Chemical Society, Washington, D.C., Sept. 12-17, 1971; Abstract No. 78. 2,7-Dichlorodizenzo-p-dioxin was prepared from isotopic potassium 2,4-dlchlorophenate uniformly labeled with "C. Chlorination of the dioxin in a chloroform solution containing FeCL and Ii gave a mixture of tri-, tetra-, and pentachlorosubstitution products. Purification by digestion in boiling chloro form, fractional sublimation, and recrystallization from anisole yielded a product containing 92% 2,3,7,8-tetrachlorodibenzo-p-dioxin. Piper, W. N., and Rose, J. Q. The excretion and tissue distribution of 2,3,7,8-tetra- chlorodibenzo-p-dioxin in the rat. Divi sion Pesticide Chemistry, 162nd National Meeting of the Amercian Chemical So ciety, Washington, D.C., Sept. 12-17, 1971; Abstract No. 88. TCDD was administered to male rats in a single oral dose of 50 *gAg. The primary excretion route was via the feces and the highest tissue concentra tion was in the liver. Pummer, J. R., Crosby, D. G., Wong, a . S., and KlinGEBTEL, U.' I. Photochemistry of dibenzo-p-dioxins. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Wash ington, D.C., Sept. 12-17, 1971; Ab stract No. 85. Ring closure and photochemical reaction of o-chlorophenols yield dioxin. Dibenzo-p-dioxins were decomposed in solution by light or sunlight. Absorp tion of dioxin on soil reduced its rate of photode composition. PLIMMER, J. R., and Kllngebiel, U. I. Ribo flavin photosensitized oxidation of 2,4- dichlorophenol : assessment of possible chlorinated dioxin formation. Science 174(4007): 407-408 (1971). Chlorophenols in aqueous solution were exposed to the action of light in wavelengths greater than 280 nm. Products were characterized by gas chromatog raphy and mass spectrometry. Possible chlorinated dibenzo-p-dioxins from ring closure of a 2-phenoxyphenol derivative were not detected in the photolysis products. Under environmental conditions, dioxins are unlikely products of lower chlorinated phenols or phenoxy-aikanoic acids. P ohland, A. E., Yang, G. C., and Hansen, E. A. The preparation and characteriza tion of chlorinated dibenzo-p-dioxins. Di vision Pesticide Chemistry, 162nd Na tional Meeting of the American Chemi cal Society, Washington, D.C., Sept. 12-17, 1971; Abstract No. 77. Twelve chlorinated dibenzo-p-dioxin standard com pounds containing from one to eight chlorine atoms were prepared as standards to develop snalytical techniques and to use in toxicity studies. The wave length associated with the observed phosphorescence and the triplet state lifetime were dependent upon the number of chlorine atoms and their positions on the dibenzo-p-dioxin nucleus. Poland, A. P., Sm ith. D., Metter, G., and POSSICK, P. A health survey of workers September 1973 303 183C 00H 2160633 in a 2,4-D and 2,4,5-T plant. Arch. E n viron. Health 22:316-327 (1971). A study &mu rsTii of ssploys health in &plant producing 2,4,5-T and 2,4-D. Chloracne was found in 13/73 male workers and was believed caused by chlorinated dioxins. The plant has begun several programs to reduce dioxin levels in 2,4,5-trichlorophenol; the contaminant concentration has dropped from 10 to 25 ppm to 1 ppm. Poster, M. L., and Burke, J. A. Separation of three chlorodibenzo-p-dioxins from some polychlorinated biphenyls by chro matography on an aluminum oxide colum. Chlorodioxina (2,3-di-, 2,3,7-tri-, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) were separated from poly chlorinated biphenyls by column chromatography on aluminum oxide. Recoveries of both dioxins and the polychlorinated biphenyls were approximately 100%. Risebrough, R. W. Determination of poly chlorinated biphenyls in environmental samples. In International Symposium on Identification and Measurement of En vironmental Pollutants, Ottawa, Ontario, Canada, June 14-17, 1971, B. Westley, Ed., National Research Council, Canada, 1971, pp. 147-153. Even though it ia not known whether dibenzofurmns can be formed from environmental residues bf polychloronated biphenyls (PCB), they should be considered as a part of the PCB problem. Dibenzofurans have been found in commercial PCB mixtures sampled from France, Germany, and the United States in concentrations ranging from 5 to 20 ppm. The European samples had higher levels than the U.S. sample. Roll, R. Teratogenic effect of 2,4,5-T [2,4, 5-trichlorophenoxyacetic acid] in mice. Food C o m et. Toxicol. 9 (5 ): 671-676 (1971) (Ger). 2,4,5-T having a 2,3,7,S-tetrachlorodibenzo-p-dioxin content of less than 0.1 ppm caused embryotoxic effects and a significant increase in the incidence of cleft palate in mice. Concentrations used in the study ranged from 35 to 130 mg/kg 2,4,5-T/dioxin which were given orally on days 5-15 of pregnancy. The teratogenic "no-effect" level of 2,4,5-T/dioxin was found to be 20 mg/kg. Rowe, V. K., Norris, J. M., Sparschu, G. L., Schwetz, B. A., and Gehring, P. J. Toxicology of chlorinated dibenzo-p-dio- xins. Division Pesticide Chemistry, 162nd National Meeting of the Ameri can Chemical Division, Washington, D.C., Sept. 12-17,1971, Abstract No. 86. The toxicology of several chlorinated dibenzo-pdioxins were investigated in rats, guinea pigs, and rabbits. The compounds studied were 2,7,-dichlorodibenzo-p-dioxin (DCDPD); 2,3,7,3-tetrachlorodibenzo-p-dioxin (TCDPD); hexachlorodibenzo-pdioxin (HCDPD), and octachlorodibenzo-p-dioxin (OCDPD1. TCDPD waa found to be the most toxic of the compounds investigated. DCDPD and OCDPD were the least toxic. For instance, little if any fetotoxic effects were observed with these two com pounds ; doses of 2 to 4 g/kg were not lethal to rats. On the other hand, TCDPD and HCDPD induced chloracne, fetotoxicity, and teratogenic effects. Schwetz, B. A., Sparschu, G. L., and Gehr ing, P. J. The effect of 2,4-dichlorophe- noxyacetic acid (2,4-D) and esters of 2,4,D on rat embryonal, foetal and neo natal growth and development. Food C om et. Toxicol. 9:801-817 (1971). The teratogenicity of 2,4-D was investigated in the rat. Formation of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in the manufacture of 2,4-D is not theoretically possible. The 2,4-D sample used was analyzed for the TCDD contaminant with a method having a 0.2 ppm sensitivity and none was found. Doses of 12.5, 25, 50, 75, and 87.5 mg/kg 2,4-D were administered orally on days 6-16 of gestation. High dose levels caused signs of embryotoxieity and feto toxicity. Somers, E., and Smith, D. M. Source and occurrence of environmental contami nants. Food C om et. Toxicol. 9(2) : 185- 193 (1971). The sources, occurrences, and some of the toxi cological effects of polychlorinated biphenyls and chlorodioxina were discussed. Chlorophenols exposed to high temperatures may form chlorinated deriva tives of dibenzo-p-dioxin. Inadequate methodoigy prevents analysis for these contaminants in foods. Sparschu, G. L., Dunn, F. L., Lisowe, R. W., and Rowe, V. K. Study on the effects of high levels of 2,4,5-trichlorophenoxyace tic acid on foetal development in the rat. Food C o m e t Toxicol. 9(4) : 527-530 (1971). Commercial-grade 2,4,5-T containing 0.6 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin did not induce a teratogenic response when administered orally to rats on days 6 to 15 of gestation at a dosage level of 50 mg/kg/ day. 304 Environmental Health Perspectives * 8 9 0 9 1 ZM0Q Spabschu, G. L., Dunn, F. L., and Rowe, V. K. Study of the teratogenicity of 2.3.7.8- tetrachlorodibenzo-p-dioxin in the rat. Teratology 4: 247 (Abstract) (1971). 2,3,7,8-Tetrachlorodlbenzo-p-dioxin wai adminis tered orally to pregnant rats during days 8 to 15 of gestation in doses of 0, 0.03, 0.125, 0.5, 2.0, and 8.0 gAg/day (sic]. No effect was observed on the fetus or mother at the 0.03 dose level. However, at the 0.125 level and above, fetal mortality, early and late resorptions, and fetal intestinal hemorrhags occur red. The effects became more pronounced with the increase dose. Maternal toxicity was noted be ginning at the 0.5 level which also increased with the dose. Spabschu, G. L., Du n n , F. L., and Rowe, V. K. Study of the teratogenicity of 2.3.7.8- tetrachlorodibenzo-p-dioxin in the rat. Food C o m et. Toxicol. 9 (3 ): 405- 412 (1971). Investigations of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rata revealed that this compound is capable of inducing a high degree of adverse effects in the fatus and mother. Doses of 0, 0.03, 0.125, 0.5, 2.0, and 8.0 pg kg/day were given orally to pregnant rata during days 8 to 15 of gesta tion. Effects such as fetal mortality, intestinal hemorrhage, and early and late resorptions began appearing at the 0.125 level and increased with dose. It is suggested that the teratogenic effects of 2,4,5-T observed in other studies may have been due to this contaminant (30 ppm). STEHL, R. H., Papenfuss, R. R., Bbedweg, R. A., and Roberts, R. W. The stability of pentachlorophenol and chlorinated dioxins to sunlight, heat, and combus tion. Division Pesticide Chemistry, 162nd National Meeting of the Ameri can Chemical Society, Washington, D.C., Sept. 12-17, 1971; Abstract No. 92. Degradation of pentachlorophenol in the environ ment could theoretically lead to the formation of chlorinated dibenxo-p-dioxina. Several simulated en vironmental conditions were checked for possible dioxin formation. Pentachlorophenol-treated wood and paper indicated no increase in dioxins when burned; likewise photolysis of sodium pentachloro phenol yielded only a trace of the compound. 2,7-Dland 2,3,7,8-tetrachlorodibenxo-p-dioxin were rapidly decomposed under artificial sunlight, whereas octachlorodibenzo-p-dioxin was not. Stehl, R. H., Wilke, E., Papenfuss, R. R., and Matalon, R. Determination of non phenolic impurities in chlorinated phe nols and related compounds. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Divi sion, Washington, D.C., Sept. 12-17, Abstract No. 81. Analytical techniques for the detection of neutral impurities (chlorinated dibenzo-p-dioxins) in chlori nated phenols, were ion-exchange chromatography for removal of the matrix, liquid-liquid hromatography for separation of the various neutral com ponents, followed by gas chromatographic examina tion of the separated components using electron capture detection. Storhebb, R. W., Watts, R. R., Gardner, A. M., and Osgood, T. Steam distillation technique for the analysis of 2,3,7,8- tetrachlorodibenzo-p-dioxin in technical 2.4.5- T. J. Aatoc. Offic. Anal. Chew.. 54(1): 218-219 (1971). 2,3,7,8-Tetrachlorodibenzo-p-dioxin was separated from 2,4,5-T by steam distillation and analyzed by microcoulometric gas-liquid chromatography. Thompson, D. J., E merson, J. L., and Spab schu, G. L. Study of the effects of 2,4,5- trichlorophenoxyacetic acid (2,4,5-T) on rat and rabbit fetal development. T e r o tology 4:243 (Abstract) (1971). 2,4,5-T was investigated for its effects on fetal development in the rat and rabbit. Doses ranging from 1 to 50 mg/kg were administered during days 8 to 15 of gestation. No clinical or gross pathology was observed. Detailed examination of the visceral and skeleton revealed no teratogenic effects. How ever, rats receiving 100 mg/kg during day 6 to 10 of gestation did produce maternal toxicity and death, early fetal resorptions, fetal toxicity, but no mal formations. Cukboer, W. F., Graham, C.. Gratkowski, H., and Norris, L. A., U.S. Forestry Service. Report on background informa tion for the phenoxy herbicides 2,4-D-- 2.4.5- T--2,4,5-TP. U.S. Forestry Service Report, pp. 1-165 (1971). A through review is given on the phenoxy herbi cides, 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), and 2,4,5-TP [2(2,4,5-trichlorophenoxy)propionic acid], and their potential contaminant, e.g., dioxin. Vinopal, J. H., Yamamoto, I., and Casida, J. E. Preparation of tritium-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structure-activity investigations of September 1973 305 TCDD and other related dibenzo-pdioxina. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Washington, D.C., Sept. 12-17, 1971, Abstract No. 79. Preparation of tritium-labeled 2,3,7,8-tetraehlorodlbenzo-p-dioxin was investigated. One approach uaed waa the vitiation of 2,4-dichlorophenol or 2,4,5trichlorophenol, lithiummetalation and subsequent aqueous hydrolysis of a chlorinated dibenzo-p-dioxin, and the catalytic reduction of a chlorinated dibenzop-dioxin. WESTING, A. H. Herbicides as agents of chemical warfare: Their impact in re lation to the. Geneva Protocol of 1925. Environ. Affair 1(3): 573-586 (1971). The use of herbicide* as chemical warfare agents Is reviewed with respect to the Genera Protocol of 1925. The total impact of these agents on the en vironment and man is not usually known or con sidered before widespread use. For example, 47 mil lion lb of 2,4,5-T was used in Indochina over an 8yr period before it waa discovered that about 1000 lb dioxin was also being applied as an unsuspected contaminant. Williams, D. T., and Blanchfield, B. J. Thin layer chromatographic separation of two chlorodibenzo-p-dioxins from some polychlorinated biphenyls and organochlorine pesticides. J. Assoc. Offlc. Anal. Chem. 54(8): 1429-1431 (1971). Thin-layer chromatography waa used for the iso lation of 2,3,7,S-tetraehlorodibenzo-p-dioxin and octachloTodibenzo-p-dioxin from polychlorinated biphe nyls and organochlorine pesticides. After isolation, these dioxins were analyzed by the electron capturegas chromatography. Wilson, J. G.t Boutwell, R. K., Davis, D. E., Dost, F. N., Hates, W. J., Kalter, H., Loomis, T. A., Schulert, A., Sterl ing, T. D., and Bowen, D. L. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency. (Submitted May 7, 1971). The 2,4,5-T Scientific Advisory Committee's report to the U.S. Environmental protection Agency was stimulated after experimental result* and environ mental indicators pointed to a potential health threat to humans. This nine-member committee was selected from a list of names supplied by the National Aca 306 demy of Science. The Committee recommended by an 8 to 1 vote to restore registration of 2,4,5-T with certain conditions; those applicable to TCDD were: (i) a limit of 0.5 ppm TCDD on existing 2,4,5-T in ventories and 0.1 ppm on future 2,4,5-T production, and (2) conduct specific research on TCDD for po tential soil accumulation and food drain magnifica tion. Williams, C. S. The status of 2,4-D, 2,4,5-T, Silvex and MCPA herbicides. D own to E arth 26(4): 12-15 (1971). 2,3,7,8-Tetrachlorodibenzo-p-dloxin (TCDD) is the causative agent in experiments reporting terotogenic effects from 2,4,5-T. TCDD was found in 2,4,5-T formulations but not in 2,4-D samples. The pre cursor for 2,4,5-T is 2,4,5-trichlorophenol which is prepared from 1,2,4,5-tetrachlorobenzene; the high temperature and alkaline conditions necessary to produce this compound also favor dioxin formation. Since 2,4-D is formed by direct chlorination of 2,4dichlorophenol and not by alkaline hydrolysis of 1,4-trichlorobenzene, no dioxin impurities are theo retically possible. Woolson, E. A., Reichel, W. L., and Young, A. L. Dioxin residues in lakeland sand and eagle samples. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Wash ington, D.C., Sept. 12-17,1971. Abstract No. 91. Between 1962 and 1969 a lakeland region in Flori da waa treated with 912 lb 2,4,5-T per acre. To assess the ecological importance of chlorinated dioxins in the environment, samples of soil to a depth of 3 ft, and tissues from eagles were analyzed for dioxins by electron capture-gas chromatography. No dioxins were detected at a minimum detection limit of 50 ppb for the eagle tissue samples and 5 ppb for the soil samples. Small residues of about 20 ppb 2,4,5-T were found in soil samples. Yang, G. C., and P ohland, A. E. Cation radicals from chlorinated dibenzo-p-dioxins. Division Pesticide Chemistry, 162nd National Meeting of the American Chemical Society, Washington, D.C., Sept. 12-17, 1971. Abstract No. 84. Characteristic blue coloration and electron spin resonance (ESR) spectra of cation radicals were ob tained following dissolution of chlorinated dibenzop-dioxins in trifiuoromethanesulfonic acid. The monoand dichlorodibenzo-p-dioxins showed well resolved ESR by hyperflne lines; the tetrachloro analogs ex hibited single broad ESR lines. Environmental Health Perspectives DON 216 0 6 3 5 D0M2I 60638 1970 Abelson, P. H. Pollution by organic chemi cals. Science 170(3957): 496 (30 Oct. 1970). When manufacture of 2,4,5-T U controlled care fully, dioxin contamination is less than 1 ppm. Dioxin was identified in 1962 as the culprit of damage and death in 1957 of uncounted numbera of chicks. Dioxin produces neurological disturbances and is terato genic. The acute LD for male guinea pigs is 0.000001 g/kg. Anonymous. HEW, USDA hold Arm; 2,4,5-T ruling postponed. Chem. Ena. N ew s 48(7):11-12 (1970). Because early studies indicated that most of the adverse toxicological effects of 2,4,5-T were due to high dioxin contamination (27 ppm) of test samples, the USDA did not feel obligated to cancel registra tion of currently manufactured 2,4,5-T containing about 1 ppm dioxin. Preliminary tests have shownthat 2,4,5-T containing 1 ppm dioxin is not terato genic. However, problems may still exist, since ex cessive heat exposure of the tri- or pentachlorophenols, used as Intermediates in 2,4,5-T production, liberates dioxins. Anonymous. Government steps up pressure on pesticides; recent joint action restrict ing use of 2,4,5-T herbicide portends changes in pesticide regulations and testing. Chem. Eng. News 48(18): 60- 61 (1970). Evidence from Congressional hearings catalysed the government's eventual suspension and cancella tion of consumer and food crop usage of 2,4,5-T and curtailment of its use in Vietnam. HEW and the USDA issued a joint statement declaring that both 2.4.5- T and its dioxin contaminant may cause birth defects. The pesticide industry reacted adversely to these actions and statements. Anonymous. Pesticide policies scored at Ag meeting. Chem. Eng. N ew s 48(42): 7 (1971). The politics and pressures brought about by the 2.4.5- T/dioxin controversy between government and industry were reviewed. The Chairman of the Sec retary's Pesticide Advisory Committee (SPAC) casti gated both the federal government and the pesticides industry for failure to avoid the panicky crises which brought about an "era of chemical McCarthy!sm." Anonymous. The strange case of the gov ernment vs. 2,4,5-T. II. F arm Chem. 133(3): 26 (1970). A review of the government involvement in the 2.4.5- T controversy which began after preliminary research reports indicated 2,4,5-T was teratogenic. These government studies were instituted in an at tempt to determine whether 2,4,5-T or its dioxin contaminant was the causative agent of these effects. Anonymous. The 2,4,5-T identification p ar ade. Food Coemet. Toxicol. 8: 596-597 (1970). Inconsistent teat data on the teratogenicity of 2.4.5- T were thought resultant of the level of its chief contaminant, 2,3,7,8-tetrachlorodibenxo-p-dioxin. Levels of 27 ppm have been found in some 2,4,5-T samples. Anonymous. Defoliants, deformities: What risk? Med. World N ew s 11(9): 15-17 (1970). Fertilised chicken eggs were injected with 2.5 pg (1 5 x l0 '"g) (50 ppt) of 2,3,6,7-tetrachlorodibenzop-dioxin. The dioxin caused leg deformities, cleft palates, and beak defecta in 11 of 15 survivors from 25 eggs. Unhatched birds showed tissue edema, necrotic livers, and deformities similar to hatched birds. Anonymous. Another herbicide on the black list. Nature 226(5243): 309-311 (1970). A commentary was presented to clarify the con troversy of whether 2,4,5-T or 2,3,7,8-tetrachlorodibenzo-p-dioxin was the causative teratogenic and mutagenic agent. Hearings with government and industrial representatives were held before the Sub committee on Energy, Natural Resources and the Environment of the Senate Committee on Com merce. Anonymous. The tangled tale of 2,4,5-T. PANS 16(3): 421-422 (1970). Use of 2,4,5-T has been restricted where food stuffs for human consumption might be contami nated. The possibility exists that dioxin (2,3,7,8tetrachlorodibenzo-fMlioxin) may be the contaminant in 2,4,5-T formulations or that there may be a syner gistic effect between 2,4,5-T and dioxin. Hypothetically, if dioxin contaminated the 2,4,5-T reported in food in the U.S. to the extent of 1 ppm. it would take 100 years for a normal person to accumulate a dose 600 times less than that which produced toxic affects during the pregnancy of a rat. Courtney, K. D., Gaylor, D. W., Hogan, M. D,, F alk, H. L., Bates, R. R., Mit chell, I. Teratogenic evaluation of 2,4,5-T. Science 168(3933): 864-866 (May 15, 1970). 2,4,5-T samples were evaluated in rata and mice for teratogenicity. Results indicated that 2,4,5-T s.c. or orally administered was teratogenic and fetotoxic September 1973 307 1 f 9 0 9 i^Hnn to the moose end orally to ths r a t Cleft pelate and cystic kidneys were among the effects observed- A note added in proof indicated that the 2,4,5-T sam ples contained 30 ppm 2,3,7,8-tetrachlorodibenzo-pdioxin. Dabsow, G., and SCHNELL, H. Chlorodibenzo- p-dioxins. Ger. Patent 1,930,259, Appl. 13 June 1969, Granted 17 Dec. 1970, 10 PP (1970). Chlorodibenzo-p-dioxins were prepared from tetri or hexachiorobenzene. The compounds were useful for flameprooflng polymers, e.g. polyesters. Day, B. E. 2,4,5-T and government decisions. Proc. W est. Soc. Weed Sei. 23: 4-6 (1970). Unpublished material which influenced the 2,4,5-T ban was held as questionable and suspect since pre parations tested contained 27 ppm dioxin. It was stated that 2,4,5-T containing 1 ppm dioxin is not teratogenic; therefore, the alarm about the adverse effects of 2,4,5-T was unjustified. E merson, J. L., T hompson, D. J., Gerbig, C. G., Robinson, V. B. Teratogenic study of 2,4,5-trichlorophenoxyacetic acid in the rat. Toxicol. A ppl. Pharmacol. 17: 317 (1970). The teratogenicity of 2,4,5-T containing 1 ppm of 2,3,7,8-tetrachlorodibenzo-p-dioxin was evaluated in rats. No teratogenic effects from 1,3,6,12, or 24 mg/ kg/day 2,4,5-T or other pathological signs were observed. These results do not substantiate the ad verse effects observed with 2,4,5-T containing 27 ppm dioxin. E pstein, S. S. A family likeness. E nviron m ent 12(6): 16-25 (July/August, 1970). The human and environmental implications of the widespread chemical exposure problem have been accentuated by the increased use of herbicides dur ing the past ten years. For example, although re cently dioxin impurities in 2,4,5-T have been re duced, dioxin is still present; even minute concen trations are potential haxarda. The analysis, forma tion, and teratogenic and other toxicological consequendea of dioxins were reviewed. E pstein, S. S. Testimony on teratogenic effects of 2,4,5-T formulations. U.S. Senate Hearing before the Subcommit tee of Energy, Natural Resources and the Environment of the Committee on Commerce (April 15, 1970). Polychlorophenol contaminants and their dioxin pyrolytic products in phenoxy herbicides are of toxi cologic and environmental consequence. Some dioxins are highly toxic and teratogenic at the microgram per kilogram level; most dioxins are, however, toxicologiealiy Galston, A. W. Herbicide usage. Science 168 (3939) : 1607 (1970). The phenoxyacetic add herbiddes should not be used until all questions regarding the teratogenidty of 2.4,5-T have been resolved. A tetrachlorodibenzodioxin impurity found in some commercial prepara tions of 2,4,5-T was the teratogenic agent in 2,4,5-T samples tested for teratogenidty by the Bionetics Research Laboratories. The possibility that phenoxy acetic adds may be degraded in plants and soil or by fire or bright sunlight into dioxin-like teratogens should be studied. Extensive research should be ac complished on all aspects of the biomedical and en vironmental consequences of dioxins. Hearings Before the Subcommittee on Energy, Natural Resources and the En vironment of the Committee on Com merce, U. S. Senate, Serial 91-60, Gov ernmental Printing Office, Washington, D.C. (1970). An impurity in 2,4,5-trichlorophenol was found to be 2,3,7A-tetrachlorodibenzo-p-dioxin, a compound with a high mammalian toxicity and teratogenic effect. Horwitz, W. (E d .). Official Methods of ' Analysis, 11th Ed. Association of Official Analytical Chemists, Washington, D.C., 1970, p. 468 (28.109). The official first action gas chromatographic meth od for chick edema factor is presented in Section 28 on oils and fats. Fat, oil, fatty add, or lipid is treat ed with H.SO,, and extracted with petroleum ether. The extract is purified on an Al.O. column, further treated with H,SO,, and examined by electron capture-GLC. Peaks with retention times relative to aldrin (S.) between 8 and 45 indicate presence of chick edema factors (hexa-, hepta-, and octachlorodibenzo-p-dioxins). Kearney, P. C. Paper presented before a joint (United Kingdom, Canada, and United States) meeting on Pesticides, Washington, D.C., (November 5, 1970). The Agricultural Research Service, USDA, began a program to assess the significance of chlorinated dioxin impurities in currently registered pesticides. Theoretically, any pesticide with a chlorinated phenoxy nucleus' or which is derived from s chlo rinated phenol precursor could contain chlorinated dioxin contaminants. 308 Environmental Health Perspectives 88909 IZH00 Ress, J., Higginbotham, G. R... and Fo e stone, D. Methodology for chlorinated aromatics in fats, oils, and fatty scids J. Aaaoe. Offie. A nal. Chem. 53(3): 628634 (1970). Following review of the current itatus of chemi cal and biological methods for chlorophenols and chlorinated dibenzo-p-dloxins In fata, oila, and fatty acid, the modified electron capture-gas liquid chro matography method for the detection of chick edema factora was recommended for adoption as official first action to replace all existing gas-liquid chroma tography methods. Robson, J. M. Testing drugs for teratogeni city and their effects on fertility: The present position. B rit. M ed Bud. 26(3): 212-216 (1970). The problems of interpreting test results involving teratogenic compounds were outlined using data gathered on the teratogenicity of 2,4,5-T and its dioxin contaminant. Spabschu, G. L., Du n n , F. L., and Rowe, V. K. Teratogenic study of 2,3,7,8-tetrachlorodibenzo-p-diozin in the rat. T o xi col. AppL Pharmacol. 17(1): 317-318 (1970). 24,73-Tetrachlorodibenzo-p-dioxin was studied to determine whether this 2,4,5-T impurity could ac count for fetal abnormalities in test animals. Bats received 0, 0.03, 0.125, 0.5, 2.0, and 8.0 jig/kg body weight /day on days 8 to 15 of gestation. At the 0.5 level, the number of fetuses was reduced, and the number of resorptions and fetal deaths was increased. Vos, J. G., and KOEMAN, J. H. Comparative toxicologic study with polychlorinated biphenyls in chickens with special ref erence to porphyria, edema formation, liver necrosis, and tissue residues. ToxicoL A ppl. Pharmacol. 17: 656-668 (1970). Briefly stated were the concerns and evidence pertaining to dioxins, particularly to the chick edema factor and whether PCBs and/or the dioxins were the culprit(a). Voa, J. G., Koeman, J. II., Van deb Maas, H. L., Ten' Noeveb de Bbauw, M. C., and DE Vos, R. H. Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphtha lene in two commercial polychlorinated biphenyls. Food Cosmet. Toxicol. 8 (6 ): 625-633 (1970). Three commercial polychlorinated biphenyl com pounds (Phenodor OPS, Clophen, and Arodor 1260) were analyzed for impurities. In two of these (Phenodor DPS and Clophen A60) pentachlorodibenzofuran and tetrachlorodibenzofuran were among the polar compounds found. 1969 Cantbell, J. S., Webb. N . C., and Mabis, A. J. Identification and crystal structure of a hydropericardium-producing factor: 1 ,2 ,3 ,7 ,8 ,9-hexachloro-dibenzo-p-dioxin Chem. Abatr. Acta. Cryatrallogr. B 2 5 ( l ) : 150-156 (1969); 70: 51305u ( 0000). Chemical identity of hexachlorodibenxo-p-dioxin, one of the toxic substances capable of pTodudng the chick edema disease, was established by x-ray crys tallography of a sample of edema-inducing lipid material RlED, W., and Eng, J. T. S. Reactions with diazocarbonyl compounds. A nn. Chem. 727: 219-221 (1969) (G er.); Chem. Abatr. 72: 12458e (0000). o-Quinoce diaxidea added benzene with nitrogen elimination to yield chloro substituted dibenzofurans: 1,2,3,4-tetrachloro-, 1,2,3,-trichloro-, and 1,3-dichloro-. 1968 Higginbotham, G. R., Huang, A., F ibestone, D., Vebbett, J., Ress, J., and Campbell, A. D. Chemical and toxico logical evaluations of isolated and syn thetic chloro derivatives of dibenzo-pdioxin. N ature 220(5168): 702-703 (1968); Chem. Abatr. 70:18509c (0000). Commercial chlorophenols Including 2,4-dichlorophenoi, 2,4,5,-trichlorophenol, and 2,3,4,8-tetrachlorophenol, were pyrolyzed to chlorinated dibenzo-pdioxina, These were examined by electron capturegas liquid chromatography and tested bibliogically by the chick embryo assay. 2,3,7,8-Tetrachlorodibenzop-dioxin was prepared by direct chlorination of dibenzc-p-dioxin. These dioxins proved to be chick edema factors (hydropericardium factors) and toxic. - 1967 Anonymous. Search for chick edema factor. Chem. Eng. N ew s 45(5): 10 (Jan. 30, 1967). September 1973 309 r r q091ZMGG Chicks afflicted with edema, or hydropericardinm, (offer an accumulation of fluid in the heart sac and gross ladney and liver damage; as little as 5 Mg can hill a chick. X-Ray crysiailograpaers have deter mined the molecular structure of one ot the toxic compounds known as the chick edema factor. Singlecrystal structural analysis showed that the structure is 1,2,3,7,8,9-hexachlorodibenso-p-dioxin. Two crys tals, each about 0.2x 0.1x 0.1 mm and weighing about Sag were recrystalised from a benzene - hexane solution. The Anal data showed that the 1,2,3,7,8,9hcxachlorodibenzo-p-dioxin molecules are nearly planar, and are packed in the 044 crystallographic planes, with an interplanar separation of about 3.3 A. No unusual bond lengths or angles were apparent. 1965 Cox, J. M., Wright, B. A., and Wright, W. W. Thermal degradation of poiy(pheny- lene oxides). J. Appl. Polym er Sci. 9 (2 ): 515-622 (1965). The thermal degradation in vacuum ot various phenylene oxide polymers and copolymers was studied by a weight-loss method. Thermal stability decreased with increasing substitution in the aro matic nuclei. This may be due to the presence of 2.3.7.8- tetrachlorodlbenxo-p-dioxin, formed as an al ternate reaction product. Chloro derivatives were more stable than the corresponding bromo deriva tives, except for the tetra-substituted phenylene oxides. Oetahalodibenzo-p-dioxins were prepared by heat ing pentahalophenols in the presence of catalytic amounts of halogen or halocyclohexadiene. For oxample, 40 g pentachlorophenol and 2 g 2,3,4,4,5,6hexachloro-2,5-cydohexadienone in 120 ml 1,2,4trichlorobenzene was refluxed at 213*C to give 30 g of octachlorodibenzo-p-dioxin. 1961 Bauer, H., Schulz, K. H., and Speigelberg, U. Occupational intoxications in manu facturing chlorophenol compounds. A rch. Gewerb&path. Gewerbehyg. 18: 538-565 (1961). Thirty-one workers in an industry manufacturing 2,4,5-trichlorophenol by sodium hydroxide saponifica tion of 1,2,4,5-tetrachlorobenzene exhibited acne and symptoms of psychopathoiogic disturbances. Solu tions of 0.01 to 0.05% of 2,3,6,7-tetrachlorodibenzo-pdioxin painted on rabbit ears produced acne; oral administration of 0.05-0.1 mg/kg caused serious liver damage. Kulka, M. Octahalodibenzo-p-dioxins. Can. J. Chem. 39: 1973-1976 (1961). Nearly 100% conversion from pentahalophenols to l,2,3,4,S,7,8,9-octahalo-dibenzo-p-dioxins was accom plished by using reaction initiators such as halogens or halogen-generating compounds. Kulka, M., Perhalodibenzo-p-dioxins. Can. Patent 702,144, Appl. May 3, 1961; Granted Jan. 19, 1965, 13 pp. (1965) Octahalodibenzo-p-dioxina, useful as chemical in termediates, biocides, and flame retardants, were prepared by using a pentahalophenol in the presence of a catalytic amount of halogen or a halogenated cydohexadienone at 200-400*C. An 83% yield was attained for octachlorodibenzo-p-dioxin. 1962 Jones, E. L., and Krizek, H. A technique for testing; acnegenic potency in rabbits, ap plied to the potent acnegen 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Invest. Derma tol. 39: 511-518 (1962). Hyperkeratinization of the inner surface of the rabbit ear was induced by topical application of 2.3.7.8- tetrachlorodibenzo-p-dioxin in acetone. The weight of keratin served as a criterion of acnegenic activity. Kulka, M., Octahalodibenzo-p-dioxins. Belgr. Patent 616,197, Appl. May 2, 1961; Granted July 31, 1962, 9 pp. (1962) 1960 Denivelle, L., Fort, R., and Van Hai, P. Octachloro- and octabroraodibenzo-pdioxins and decachloro- and decabromodiphenyl ether. Bull. Soc. Chim. France 1960. 1538-1543 (1960). Pentachlorophenol was pyrolyzed at 300'C ex tracted with carbon tetrachloride and aqueous al kali, recrystalized from phenyl nitrite, and yielded "perchlorophenylene oxide" (melting point of 326'C). Steric considerations dictated ring fusion in the Im position leaving octachlorodibenzo-p-dioxin the only acceptable structure for perchlorophenylene oxide. Sodium 2,4-dichlorophenate gave dichlorodibenzo-pdioxin. Kaupp, J., and Klug, H. Perchlorodiphenylene dioxide. Ger. Patent 1,092,480, Appl. June 27, 1956, Granted Nov. 10, 1960 (1960). Octachlorodibenzo-p-dioxin was prepared by heat ing pentachlorophenol and 2,3,4,4,5,S-hexachloro-2,5cyclohexadien-l-one; the yield was 84%. 310 Environmental Health Perspectives 8310 DO N 2 1 6 0 6 4 6 1959 Tomita, M., Ueda, S., and Narisada, M. nil.TM --j : v vtttt i~ /i& jc u x ir7 /-u iu ju u u c x i v a u v e a . a a v 11. Synthesis of polyhalodibenzo-p-dioxin. Y akugaka Zasshi 79: 186-192 (1969). Pentachlorophenol t u hutted at 300'C for 12 hr. ReerystalUzation of the product from benzene, yielded octachlorodibenzo-p-dioxin needle. 1958 Sandermann, W., Casten, R., Krasting, W., and Pieper, J. Studies on wood-protec tion chemistry. IX. Investigations con cerning oily blue stain protection agents. H olzfonch. HoUverwert. 10(4): 57-66 (1968). Compound formed by heat treatment of pentachlorophenol inch a octachlorodiphenylene oxide were inactive for preventing blue tain. Tetrachlorodiphenylene oxide was eapecially toxic to man and beast 1957 Gilman, H., and Dietrich, J. J. Halogen de rivatives of dibenzo-p-dioxin. J. A m er. Chem. Soe. 79: 1439-1441 (1957). Dibenzo-p-dioxin was synthesized and reacted with chlorine gas to form 2-chlorodibenzo-p-dioxin; a similar run coupled with ultraviolet light gave 2,7dichlorodibenzo-p-dioxin. Kimmig, J., and Schulz, K. H. Chlorinateda- romatic cyclic ethers as a cause of the so-called chloracne. N aturw ise. 44: 337- 338 (1957) (Ger.). Trichlordibenzo furan, tstrcchlorodibenzofuran, and 2,3,6,7-tetrachlorodihenzodioxin were especially effective acne-inducing agents on rabbit ears. 2,3,6,7Tetrachlorodibenzodioxin proved most toxic; the characteristic changes were achieved with concentra tions of 0.01 to 0.002%. Higher concentrations caused liver necrosis and death. Single oval administration of 0.06 to 0.1 mg/kg body weight caused death in 1-2 weeks. Autopsy revealed necrosis and fatty de generation of the liver. Pure 2,4,5-trichlorophenol and pentachlorophenol did not cause chloracne. Kimmig, J., and Schulz, K. H. Occupational acne (so-called chloiacne) due to chlori nated aromatic cyclic ethers. Dermato- logia 115 (4 ): 540-546 (1957) (Ger.). The agent causing occupational chloracne in 31 employees of chlorophenol producing factories was found to be 2,3,6,7-tetrachlorodibenzodioxin. The toxic action in animals was due to the alkaline hydro lysis product of 1,2,4,5-tetrachlorobenzene. Tetraehlorodibenzo-p-dioxin and tri- and tetrachlorodibenzofuran were active skin irritants. 2,3,6,7-Tetraehiorodibenzodioxin formation from sodium trichiorophenoiacetata was established as was isolation from the by-products. Sandermann, H. S., Casten, R., and Stock mann, H. Pyrolysis of pentachlorophe nol. Chem. Ber. 90: 690-692 (1957). Octachlorodiphenylene dioxide was formed after pentachlorophenol was heated at 300*C for 24 hr, distilled at 320*C, and crystallized from benzene or extracted with benzene and fractional crystalliza tion. 2,3,73-Tetruchlorodlphenylene dioxide wa; ac tive against insects and wood-destroying fungi; octa chlorodiphenylene dioxide was inactive. Schulz, EL H. Clinical and experimental stu dies on the etiology of chloracne. Arch. Klin. E xp. Derm. 206: 589-596 (1957). The acnegenic properties occurring as a refractive dermatologic condition in factory workers involved in the production of chlorinated aromatic compounds were ascribed to TCDD, an unwanted side product in the sythesis of 2,4,5-T. 1955 Oita, K., J ohnson, R. G., and Gilman, H. The chlorination of dibenzofuran and some of its derivatives. J. Org. Chem. 20: 657-S67 (1955). Step-by-step reaction procedures were presented far the chemical synthesis of chlorinated dibenzofurans, i.e., 2-chloro-, 23-dichloro-, 3-chloro-, and a chiorodibenzofuran. 1954 Cerniani, A., Passerini, R., and Righi, G. Ultraviolet spectra of some dibenzofurans. Bod. Sci. Foe. Chim. Ind. 12: 75-79 (1954). 2-Chloro- and 3-chloro-dibenzofurans were charac terized by ultraviolet spectrometry. These derivatives had little effect on spectra of the parent compound. 1953 J ulia, M., and Baillarge, M. Growth factors in plants. III. (l-Carboxymethyl-2-naphthoxy) acetic acid and (2-carboxymethyl4-chlorophenoxy) acetic acid. Bull. Soc. Chim. France. 640--643 (1953). In the preparation of 2,4-dichlorophenol. a portion of the mixture was insoluble in benzene-wacer; the substance was extracted with boiling ligroine and recrystallized to yield 2,7-dichiorodibenzo-p-dioxin. September 1973 311 D 0 H 2 I 606 41 1952 Shibata, I. S., Natobi, S., and SUMI, Y. An tibacterial effects of lichen substances and their related compounds. J. P h a m . Soc. Japan 72: 133S-1336 (1952). The chlorinated dlbenxofurans synthesized and tested for antibacterial activity included: 2-chloro-, 3-chloro-, 2,8-dicbloro-, and 3,8-dichloro-. The chlo rine atom, when in the 3-poaition ot the dibenzofuran ring, increased its antibacterial effect. 1951 T omita, M., and Watanabe, W. Antibac terial activity of some organic com pounds in vitro. II. Antibacterial activi ty of dibenzo-p-dioxin, phenoxathiin, and 1,4-benzodioxan derivatives on Mycobac terium tuberculosis, Staphylococcus au reus, and Escherichia colt. J. Pharm . Soc. Japan 71: 1204-1206 (1951). 2,7-d:chlorodibenzo-p-dioxin was tested for antibac terial activity and found to be inactive. 1941 Higasi, K. The molecular structures of diphenylene dioxide and phenoxathliin as revealed by dipole-moment data. Set. Papers Inst. Phys. Chem. Research 38: 331-34C (1941). 2,6-DichIorodiphenylene dioxide exhibited a dipole moment of 0.62 Debyes. This indicated a folded molecule and no optical isomers. Higasi, K., and Uyeo, S. Polarity and mole cular structure of diphenylene dioxide. J. Chem. Soc. Japan 62: 396-399 (1941). 2,6-Dichlorodiphenylene dioxide was found to have a small but finite dipole movement. This molecule and the parent probably fold along the line joining the two oxygen atoms. UEO, S. 2,6-Dichlorodiphenylene dioxide. Bull. Chem. Soc. Japan 16: 177-179 (1941). > -V Synthesis of 2,6-dichlorodiphenylene dioxide, a light yellow compound with a melting point of 207*C and a dipole moment of 0.62 was described. The di pole movement of 2,6-dichlorodiphenylene dioxide was somewhat less than the dipole moment of di phenylene dioxide which was 0.64. 1939 British Thompson-Houston Co. Ltd. Electric insulation. Brit. Pat. 506,560 (May 31, 1939). Direct chlorination of diphenylene oxide in the presence of a catalyst produced solid, waxlike pro ducts of high dielectric constants; i.e., mixture of. chlorinated diphenylene oxide and pentachlorobiphenyl. Clark, F. M. Chlorinated diphenylene oxide composition for treating paper in capa citors, U.S. Patent 2,198,473 (April 23, 1939). Chlorinated diphenylene oxide containing 1 to 4 atoms of chlorine per molecule imparts a constant electrical capacity when used to impregnate paper in capacitors. 1936 Bell. F. Pyrolysis of chlorophenols. J. Chem. Soc. 1936: 1244. o-Chlorophenol was pyrolyzed at 180-340'C: di phenylene oxide and 3,6-dichlorodiphenyl oxide were isolated from the distillate. 1934 Gilm a n , H., Bkown, G. E., Bywater, W. G,, and Kirkpatrick, W. H. Dibenzofuran. III. Nuclear substitutions. J. Am er. Chem. Soc. 56: 2473-2477 (1934). 2-Chloro- and 2,8-dichlorodibenzofuran were syn thesized. Dibenzofuran and chlorine in carbon tetra chloride gave 38% of the 2.8-dichloro derivative which had a melting point of 185*C. Dibenzofuran in alcohol at 60'C gave the 2-chloro derivative which had a melting point of I02.5*C. 312 -- -- - Environmental Health Perspectives 18312 ' ' \J \ A ppearing m o n th ly - V o lu m e 46 No 3 - Septem ber 15m 1983 ^ C o m p le tin g V olum e 46 \' k- (ab(o8! ~ Clpo) -;\1 Lun1 ISSN 0009-2797 CBINA 46 131 283-383 188 j l 7***Lr4-*Pc Chemico - Biological In te ra c tio n / :i moLECULAR mECHflni/m/ bv uuhich EHOGEflOU/ CHEiillCflL/ EFFECT CHARGE/ in BIOLOGCflL /V/TEfTV EDITORS P. Moldeus (Stockholm), J. DePierrt (Stockholm) R.M. Philpot (Research Triangle Park), J.R, Bend (Research Triangle Park) ELSEVIER K - b b b t f l - C ic c ) 8fS09IZMOO Chem.-Biot Interaction*. 46 (1983) 335-346 Elsevier Scientific Publishers Ireland Ltd. 335 COMPOUNDS IN URBAN AIR COMPETE WITH 2,3,7,8-TETRACHLORODEBENZO-p-DIOXIN FOR BINDING TO THE RECEPTOR PROTEIN RUNE TOFTGRD*, GRAN LFROTH*. JAN CARLSTEDT-DUKE*. RABINDEX KURL* and JAN-KE GUSTAFSSON* 'D epartment o f M edical N utrition, Karohnska Irutitutet, Huddinge U niversity Haepital P69, S-141 38 H uddinge and *Nordic School o f Public H eath, S ~ tl3 48 Gothenburg and Department of Radiobiology, Wallenberg Laboratory, U nivenity o f Stockholm, S-106 91 Stockholm (Sweden) (Received November 29th, 1962) (Revision received April 7th, 1963) (Accepted April 8th, 1963) SUMMARY Acetone extracts o f filter-collected urban airborne particulate matter contain compounds which can competitively inhibit 2,3,7,8-{l,6-*H}tetrachlorodibenzo-p-dioxin (TCDD) binding to the rat liver TCDD-recaptor pro tein. The concentration of conventional polycyclic aromatic hydrocarbons (PAHs) or chlorinated dioxins and dibenzofurana cannot account for more than 1-30% of the observed competition for PHJTCDD binding to the receptor protein. The difference in potency between samples collected in urban areas during different periods of the year and a background sample is 25-400-fold. Collecting samples in the presence of increased concentrations of nitrogen dioxide, nitrous acid, nitric acid or ozone did not increase the amount of compounds with receptor affinity. However, with nitrogen dioxide and espe cially with nitric acid, a substantial increase of the mutagenic effects in the Ames Salmonella assay in the absence of mammalian activation as well as a degradation of several PAHs were noted. Affinity for the TCDD-receptor protein, mutagenicity in the absence of mammalian metabolic activation in the Ames Salmonella assay and PAH-content are characteristics of urban particulate matter showing the presence of compounds, that represent poten tial health risks. The compounds with affinity for the receptor may constitute a group of substances different from both conventional PAHs and directacting mutagens. Abbreviations: AHH, aryl hydrocarbon hydroxylaaa; BfaIP. benzofalpyronc; DMSO, dimethylsulfoxide; PAHs, polycyclic aromatic hydrocarbons; TCDD, 2J,7.S-totrnehlorodibenap-diaxia; TCDBF, 2^.7,8-tetrachlorodibenioforaa. 0009-2797/83/J03.00 1963 Elsevier Scientific Publishers Inland Ltd. Printed and Published in Ireland 1 DOW2I60579 336 Key word: Air pollution--2,3,7,8-Tetrachlorodibenzo-p-dioxin-- binding--Mutagenicity--Polycyclic aromatic hydrocarbons f INTRODUCTION Urban airborne particulate matter contains a complex mixture of compounds. The mutagenicity of extracts of such particulate matter in the Ames Salmonella assay in the absence of rat liver metabolic activation [1--4] indicates the presence of compounds different from conventional polycyclic aromatic hydrocarbons. Presently much interest is focused on nitroaromatica. Several compounds belonging to this class, e.g. nitropyrenes and dinitropyrenes, have been shown to be extremely powerful 'direct-acting' mutagens [5,6] and are believed to represent a major fraction of the 'direct-acting* mutagens in particulate emissions from diesel engines [7-11]. Furthermore, experiments with simulated atmospheres indicate that nitroaromatics can be formed when certain PAHs are exposed to nitrogen dioxide [12]. With respect to chemical carcinogens and mutagens, e.g. PAHs, requiring metabolic activation, the level and substrate specificity of the specific r cytochrome P-450 isozymes that are present in the target tissue are of great importance. The induction of izozymes capable of activating PAHs, generally known as aryl hydrocarbon hydroxylase (AHH), is thought to be mediated by the interaction of the inducer with a highly specific intracellular receptor protein [13,14]. This receptor, often called the TCDD-receptor or Ah-receptor, is present in several tissues including lung [15]. It is also known that certain PAHs as well as cigarette smoke induce AHH in the lung [16]. The present study was undertaken to investigate the possible presence in urban particulate matter of compounds with an affinity for the TCDDreceptor protein and to monitor the effect of adding nitrogen dioxide, nitrous arid, nitric acid and ozone during environmental sampling of particulate matter on mutagenicity and levels of compounds having an affinity for the receptor. . METHODS 1 i Particulate matter was collected on glass fiber filters by high volume sampling at roof top levels for 24 h in central Stockholm and a suburban site. Collection, Soxhlet extraction with acetone, extract preparation in dimethylsulfoxide (DMSO) and mutagenicity testing of the extracts in the Salmonella assay have been described earlier [3,4]. The Salmonella strains TA98 and TA100 were obtained from Dr. B.N. Ames, Berkeley, CA and the nitroreduc tase deficient strains TA98NR, TA98/1, 8 DNP and TA100NR were obtained from Dr. H.S. Rosenkranz, Cleveland, OH. For the addition of nitrogen dioxide, nitric acid, nitrous arid and ozone, one high volume sampler equipped with a dosing system was run in parallel with S09IZMOQ 337 a conventional sampler. Details of the dosing system, concentration measurements and filter extraction are given elsewhere [17,18]. These sam ples were collected in central Gothenburg. A background sampler was col lected at Rorvik located on the west coast of Sweden. Affinity of compounds present in particulate extracts for the rat liver TCDDreceptor Liver cytosol was prepared as described [19] and diluted to 3.5 Ajgo-jio/ml (2-2.5 mg protein/ml). Each experiment consisted of a series of ten in cubations of 1 ml of cytosol with 1.5 nM [*H]TCDD. [1,6-5H]TCDD (spec. act. 36.6Ci/mmol) was a kind gift from Dr. A. Poland (McArdle Laboratory for Cancer Research, WI). Two incubations were carried out in the presence of 20 p 1 of DMSO (total binding). Two incubations contained 150 nM radioinert 2,3,7,8-tetrachlorodibenzofuran (TCDBF) (non-specific binding). The remain ing six incubations contained varying amounts of the extract of the parti culate fraction of an air sample. A quantity of the extract (2.5-20 p 1), dissolved in DMSO, was added to the incubation mixture and the volume of DMSO was then adjusted to 20 tl/incubation. After incubation at 0C for 60 min, the incubations were treated with dextran-coated charcoal and the amount of PH]TCDD bound to the receptor in each of the ten incubations was measured by isoelectric focusing carried out in polyacrylamide gel as des cribed previously [19]. The non-specific binding of PHJTCDD was obtained from the two in cubations in the presence of 150 nM radioinert TCDBF and was substracted from the total binding in each of the other incubations (specific binding). The specific binding of [*H]TCDD in the presence of extracts of the air samples was expressed' as a percentage of the specific binding in the control in cubation (I*H]TCDD + DMSO). The relative binding affinity for the in dividual air samples was calculated from log-logit plots of the competition for TCDD-binding where logit b =*In (b /l-b ). The log-logit plots were calculated using linear regression analysis. Each extract was analysed 2-4 times at a suitable dilution. An unused glass fiber filter was extracted in the same manner as the other samples and tested for competition for receptor binding. Benzo(a]pyrene (B[a]P) and TCDBF were dissolved in dioxane and the competition for binding to the receptor measured. Chemical analysis Some samples (790410, 790705 and 800204) have been analyzed for TCDD, TCDBF and PAHs by the Swedish Water and Air Pollution Research In stitute, Gothenburg [20]. Other samples reported in Table II were analyzed for PAHs by three laboratories (Swedish Water and Air Pollution Research Institute, Gothen burg, Central Institute for Industrial Research, Oslo and Department of Analytical Chemistry, University of Stockholm). The results are presented as the average concentration reported by the different laboratories. D0N2160581 -r I* | r I :i || h i . irfc ii{t .b-I i I ;| 338 RESULTS AND DISCUSSION The results shown in Fig. 1 and Tsble I describe the mutagenic effects in Salmonella strains TA98 and TA100 in the absence of mammalian metabolic activation and the relative binding affinities expressed as the concentration of air sample extract that competes for 50% of the specific TCDD-binding to TABLE I MUTAGENICITY AND COMPETITION FOR RECEPTOR BINDING BY EXTRACTS OF URBAN PARTICULATE MATTER nd, not determined Sample Receptor affinity ECwIm1air/mi cytoaol) Mutagmic response* (revertanta/m*) TA98 TA100 790410 790410 Inner city Suburban 0.05 =0.01 0.04 =0.02 19 11 14 8 790705 790705 Inner city Suburban 0.14=0.07 0.30 = 0.15 3 ad 1 ad 800204 800206 800206 800207 Inner city Innw city Inner city Inner city 0.02 = 0.01 ad 0.04 =0.01 0.05 = 0.03 59 79 92 102 30 20 97 800106-11 800310-14 800414-18 800414-18 800414-18 800609-13 Inner city Inner city Inner city Inner city* Suburban* Inner city 0.05 = 0.03 0.06 = 0.04 0.08 = 0.01 0.14= 0.09 0.19 = 0.10 0.20= 0.12 80 74 48 50 30 52 31 53 19 32 12 17 810408-14 Background* 7.8= 2.4 <0.5 <0.5 TCDBF BlnlP 2.7= 1.9nM 18.2=9.9 nM 'Samples were collected at roof top levels for 24 h Starting about 6a.m. in central Stockholm and at a suburban site 22km NNW Stockholm. The EC* is the concentration of competitor that fwwpftiis for 50%of the specific binding of l^HflCDD. The ECts fin nM) for TCDBF and BfalP are given for corapariaon. The values represent the meaner S.D., n " 3-4. `The mutagenic effect was determined with 1plate/does level at 3 or more doae levels in at least 3 independent testa and the reeponae wee calculated from the linear or ilmnot linear part of the lines response curve. The mutagenic reeponae is given in the absence of mammalian metabolic activation. Three or four 24-h samplee were pooled. `Sample collected with a lower air flow (34 o'/h) than normal (68m*/h). *Sampla coilactad with as alsctrostatic prscipitatioo ampler at Rfirvik on tha wast coast of 7*'5*s'> DOHZI 60581 concentration o f competitor Fig. 1A: Competition for tho binding of PHJTCDD to the TCDD-receptor protein in rat liver cytoeol by extract of filter-collected urban particulate matter. For description of the different eamplea eee Table I. Typical valuaa for total and noo-epecifie binding in the aaaay are 110b-13)0 and 200-300rpm. leapirtively. Deeignatlana are: IC, inner city; 3, euburban. B: log-logit plota of the data from (A). The relative affinity ia arpraaeni aa the omcentratioo of extract that compete for 60%of the pacificTCDD-binding (BCm, logit b " 0). The valuea givm in the upper right corner are the raeaho of the 9 individual experiment ahown in thia figure. 183 Q T*^ f>W O O * w w etC M ' DOH2I60583 340 i the TCDD-receptor protein. These results demonstrate the presence of both i "direct-acting* mutagens and compounds competing with TCDD for receptor binding in extracts of particulate air samples collected in central Stockholm and at a suburban site. Control experiments with unused filters carried through the entire extraction procedure showed that the measured effects were due to components extracted from the particulates. In Fig. 2 the com petitive nature of the inhibition of TCDD-binding to the receptor protein is shown by the use of Lineweaver-Burke plots. The absence of significant differences in the slope of the log-logit plots for the different air samples indicate that they compete for the same binding site. There seems to be a gross correlation between the binding affinity and the pollution level as measured by mutagenic effects. Samples collected during the winter have lower ECao*values than samples collected during the summer and late spring as illustrated by a maximal 15-fold variation. The very marked difference (25-400-fold) between a back ground sample collected on the west coast of Sweden and the samples collected in greater Stockholm indicate that local emissions may be of major importance with regard to receptor binding components. Samples collected with the electrostatic precipitation sampler used for collection of the back ground sample have in separate experiments been shown to give mutagenic effects and binding affinities comparable to samples collected with the con ventional high-volume Bampler (unpublished observations). Several compounds are known to' bind to the TCDD-receptor protein including many PAHs [13,21,22] present in urban particulate matter. Some of the particulate samples have been analyzed for PAHs (Table II). Assuming that all analyzed PAHs present at concentrations equal to or above 0.1 ng/m1 have the same binding affinity for the receptor protein as B(a]P, between 1% Fig. 2. Linsweaver-Burite plots of dots obtained when incubating different concentrations of PHTTCDO in the absence and presence of a constant concentration at particulate air sample extract. The samples used in these experiments were collected in central Gothenburg. F and B^> represent the concentration at free and specifically bound PHJTCDD, respectively. s4(WW-,v' >- TABLE U CONTENTS OF PAHb IN EXTRACTS OF FILTER COLLECTED URBAN PARTICULATE MATTER** PAH content (ng/m1) Sample 790410 790706 auburban inner city 800204 inner city 800108-11 800310-14 inner city inner city 800414*18 800414*18 800414-18 800609-13 inner city inner city4 suburban inner city Pbenanthrone 1-Methylphenanthrene 2-Methylpbenanthrene 3-Methylphenanthrene 4-/9-Methylphenanthrenee Fluorene Dibenxothiophene Pyrene 1,2-Dihydropyrene 1-Methylpyrene 4-Methylpyrene Bena(a lanthracene Chryaene j Trphenylene ) Fluoranthene Bcnanfa(fluorene Benxefblfluorene Batuolelpyreae Bento(a(pyrene Beiuo(b/h)fluoranthene BenxoO(fluoranthene Beuolg.h.ilfluoranthene I Benio(ctphenanthrene 1 Indeno(l,2,3-cd(pyrene Beno(l.h.ilperylene Coronane Total* 0.6 - 0.6 - 0.5 0.6 0.6 0.2 - - 2.8 6.6 2.2 4.8 0.6 -- - -- - -- - 1.7 0.7 0.8 0.4 0.2 1.2 3.2 3.8 0.4 0.2 -- - 7.3 2.1 0.1 4.6 2 8 0.1 9.8 3.2 L I 1.4 1.4 0.6 2.3 1.6 1.7 1.0 6.4 4.4 --- 0.6 1.2 3.8 2.1 - - 0.8 0.3 68.6 28.6 2.2 0.2 0.2 0.2 0.2 0.2 2.6 0.4 0.1 0.1 0.9 2.0 3.1 0.4 0.2 1.1 0,6 2.8 1.3 1.1 1.6 0.9 23.6 0.8 0.7 0.2 0.1 0.1 0.1 - - 0.3 1.0 1.2 -- - - 0.3 0.3 0.8 1.6 0.9 1.6 0.2 - - 0.6 0.6 0.4 0.3 1.3 1.6 0.4 - 0.6 0.6 0.9 0.8 0.3 0.3 9.6 9.2 0.4 0.1 0.1 0.1 * 0.1 0.4 - 0.1 0.6 0.6 0.3 0.2 0.8 0.3 0.3 0.6 0.2 5.2 0.1 0.2 0.3 0.4 0.2 0.3 0.2 0.8 0.2 ni 0.3 0.5 - 3.9 'All PAHe preeent at a concentration equal to or above 0.1 ng/m' are included, not determined in the actual eample. Total PAH content calculated by adding the amounta of each analyzed compound preaent at a concentration above 0.1 ng/m1. `Sample collected with a lower air flow (34 m'/h) then normal (60 m'/h). 341 U S 0 9 I ZMOQ !'iV li{; i : ! j 4 342 and 30% of the observed binding can be accounted for. The above assumption is a conservative one based on the fact that when 16 of the PAHs in Table n were tested for receptor affinity only two showed a higher binding affinity than B[a]P (our unpublished observations). This indicates that other types of compounds might be more active. TCDD and TCDBF have the highest binding affinities for the receptor protein among investigated compounds (23). Two samples (790410 inner city, 790705 suburban) have been analyzed for TCDD and TCDBF and the concentrations were below the detection levels of 2 pg/m*. This is equivalent to a maximal concentration of 2pM in an incubation where the specific PHJTCDD binding is reduced by 50%. Pure TCDBF has an ECu-value of 2.7 nM (see Table I). Consequently these compounds cannot account for the binding. At present we cannot, however, exclude the presence of other halogenated hydrocarbons with receptor affinity. The experimental series with increased concentrations of reactive gases present during sampling was performed with the aim to (1) investigate the possible role of chemical transformation for the appearance of both mutagenic components and receptor binding compounds and (2) investigate the importance of artifact formation due to the sampling methods. From previous studies it is known that certain nitro>PAHs have a high affinity for the TCDD-receptor protein [24]. However, the results from the present study (Table HI) failed; to demonstrate ah increased competition with TCDD for receptor binding;in extracts of samples collected*with increased concen trations of nitrogen dioxide, nitrous acid, nitric acid or ozone. Chemical analysis, details of which will be published elsewhere [17,18], showed that an extensive degradation of PAHs and formation of mono-nitro-PAHs took place in the experiment with nitric acid and to a sm aller'extent with nitrogen dioxide whereas nitrous acid and ozone were without effects. Thus, it seems that the nitro-aromatics are not the major receptor binding species in extracts of particulate air samples and that the artifact formation during sampling is negligible in this respect. The mutagenic response of the samples in the Salmonella assay in the absence of exogenous metabolic activation were in the range found earlier, Tables I and m [3,4]. The presence of mammalian activation in the form of liver S9-mix from Aroclor 1254-induced male rata mostly, but not always, decreased the response (data not shown). The experiments with nitrogen oxides show that nitrogen dioxide and nitric acid, but not nitrous acid, when present in the air can increase the mutagenic response in the absence of mammalian activation. This increase is probably due to formation of a variety of nitro compounds. The types of mutagenic compounds formed in the samples exposed to elevated concen trations of nitrogen dioxide and nitric acid are probably also similar to those present in the ambient samples as judged by the relative response in nitro-reductase proficient and deficient strains (Table HI). The increase of the mutagenic activity at about 1 ppm of added nitrogen dioxide is on the average fourfold with a range between 2- and 7-fold. DOH2I6058S table m EFFECT OF NITROGEN DIOXIDE, NITROUS ACID. NITRIC ACID AND OZONE PRESENT DURING SAMPLING OF URBAN PARTI CULATE MATTER ON MUTAGENIC RESPONSE AND RECEPTOR AFFINITY* Sample Mutagenic reapoaae (revartanta/mV TA96 TA98NR TA98/1.8 DNP, TANK) TANK) NR Receptor affinity' ECm(m'/ml cytoaol) 800225 Ambient 82 37 28 76 36 nd 960 ppb NO, 87 48 32 142 62 nd 800228 Ambient 14 8 3.2 14 6 0.17 x 0.04 60 ppb NO, 37 24 6.5 41 20 0.09x0.04 800228 Ambient 11 6 5 20 6 0.07x0.02 MOppb NO, 76 30 20 110 41 0.10x0.03 800328 Ambient 4.3 nd nd 2.9 nd nd 200ppb O, 4.6 nd nd 3.3 nd nd 800327 Ambient 28 u 4.6 21 6 0.08x0.02 200 ppb O, 31 12 4.4 26 7 0.06x0.04 800331 Ambient 10 nd nd 8.4 nd nd 200ppb O, 16 nd nd 14 nd nd 810108-22 Ambient' 10 nd nd 10 nd 60* 100 ppb HNO, 9 nd nd 10 nd 53* 810126-0208 Ambient' 8 nd nd 7 nd 70* 1000ppb NO, 26 nd nd 35 nd 68* 810224-0312 Ambient' 11 6.4 4.7 11 6.6 64* 120 ppb HNO, 40 29 20 70 32 54* 'Sample* were collected et roof top levele in centre! Gothenburg during 24 h period* with two imullaneoualy operating high volume ampler. One of the ampler* we* equipped with a doaing eyetem enhancing the concentrationa of nitrogen dioxide, nitroua acid, nitric acid or oione. Th* particluate extract* were aaaayed for mutagenicity on at leaat three different oocaaiona by the Salmonella plate incorporation method with bacterial culture* fully grown overnight. The teat were performed in the abeence of mammalian metabolic activation. The reapmae we r - i calculated from the linear or almoet linear part of the doee-reaponae curve. coOC The value* repreaent the meanaS.D., n - 3--4. 'Eight 24-h ample* were pooled. ro The data repreaent the remaining percentage apecifically bound PHJTCDD compared to the apecific binding obtained in incubation without added particulate extract. Particulate extract* equivalent to 0.075, 0.060 and 0.065 m1air were added in the experiment* with HNOj, NO] and u HNOj, reapeclively. The reaulla are the mean of 2 aeparate experiment*. U S09IZMOS 2 8 9 0 9 1Z M0 0 \ i - Assuming that the formation of mutagenic components during sampling has a first-order dependence on the concentration of nitrogen dioxide, it can be calculated that only exceptionally high ambient concentrations of nitrogen dioxide, i.e. >200 ppb over 24 h, would cause a pronounced increase of. tire mutagenic response. Nitric acid seems to be more effective than nitrogen dioxide in increasing the mutagenic response. It is, however, possible that the effects observed from nitrogen dioxide are in fact caused either by nitric acid and nitrogen dioxide in combination or by nitric acid alone as the latter compound also would have been present in the nitrogen dioxide preparation. Ambient concentrations of nitric acid are generally much lower than the concentration used in the experiment suggesting that only exceptionally high ambient concentrations would cause a pronounced increase of the mutagenic response. With the exception of one sample, addition of ozone during sampling did not change the mutagenic response (Table III). The exceptional sample, 800331, was, however, collected during a period with a high nitric oxide concentration and an episode with an influx of a long-range transported air mass normally containing an acid sulphate aerosol [17]. This may have increased the ozone oxidation of nitrogen oxide to higher oxides and enhanced the nitration reactions. The affinity of a compound for the TCDD-receptor is well correlated to the magnitude of aryl hydrocarbon hydroxylase induction caused by that com pound [23]. This is especially true for halogenated aromatic hydrocarbons. Strains of mice with high AHH inducibility are more susceptible to pul monary cancere caused by 3-methylcholanthrene than strains with low in ducibility indicating a link between AHH inducibility and incidence of pulmonary tumors [25]. The inducibility of AHH in cultured explants of human bronchus [26,27] indicates the presence of the receptor protein also in this tissue. Animal experiments suggest that also other responses such as thymic atrophy and incidence of cleft palate correlate to receptor affinity [28]. Although TCDD has been shown to be a potent carcinogen in chronic feeding studies [29] it is apparent that TCDD shows no or very little mutagenic activity in in vitro test systems such as the Ames Salmonella test [36-32] and a very low covalent binding to DNA in vivo [33,34]. In two recent studies, however, it has been shown that TCDD is a potent tumor promotor in the rat liver and mouse skin two-stage models of carcinogenesis [35,36]. If this effect of TCDD is mediated via binding to the receptor is not yet known. Affinity to the TCDD-receptor protein, mutagenicity in the absence of mammalian metabolic activation in the Ames Salmonella assay and PAHcontent are different characteristics of urban particulate matter showing the presence of compounds, which represent potential health risks. The com pounds with affinity for the receptor may constitute a group of substances different from both conventional PAHs and the direct acting mutagens. ACKNOWLEDGEMENTS This study was supported by grants from the National Swedish Environ- 1833 i gesoauwo 345 ment Protection Board; Project: Air Pollution in Urban Areas, and the Swedish Tobacco Company. J.C.-D. is a recipient of a research fellowship from the Swedish Cancer Society. We thank Birgitta Fernstrom, Keratin Svensson and Ann Ehnborg for skillful technical assistance. REFERENCES 1 J.N. Pitts Jr., D. Groejean, T.M. Mischka. V. Simmon usd D. Pool, Mutagenic activity of airborne particulate organic pollutant. Toxicol. Lett., 1<1977) 66. 2 R. Talcott and E. Wei. Airborne mutagena bioaaaayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58 (1977) 449. 3 G. Ldfroth, Comparison at the mutagenic activity in carbon particulate matter and in dieael and gaaoliae exhaust, in: M.D. Waters, S3. Sendhu. J. Lewtaa Huiaingh, L. Claxtoa and S. Nesnow (Eds.). Short-tarm Bioaaaays in the Analysts of Complex Environmental Mixtures II. Plenum Publ. Corp., New York, 1961, p. 319. 4 G. Ldfroth. SalmoneUa/micmeome mutagenicity asseye of exhaust from diesel and gasoline powered motor vehicles. Environ. 1st., 5 (1961) 255. 5 H.S. Roaankranz, E.C. McCoy. DJI. Senders, M. Butler, D.K. Kiriaxidee and R. Marmelstein. Nitropyranm: Isolation, identification and reduction of mutagenic imparities in carbon black and toner. Science, 209 (1960) 1039. 6 J. Campbell, C.G. Crumplin, J.V. Garner, R.C. Garner, C.N. Martin and A. Rutter, Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagms and stimulators of DNA repair synthesis in cultured human cells. Carcinogensais, 2 (1961) 559. 7 T:C. Pedersen and J . S . Siak, Tho role of nitroaromatic eompoends la tha direct-acting mutagenidty ef (basal partiels extracts. J. Appl. Toxicol. 1 (1961) 54. 8 O. Schuatxle, T i . Riley, T J. Prater, T_M. Harvey end D.F. Hunt, Analyaia of nitrated poly cyclic aromatic hydrocarbons in disaal particulates. Anal. Chenu, 54 (1982) 265. 9 S.M. Rappeport, Zl>. Jin and X_B. Xu, High-performance liquid chromatography with reductive electrochemical detection of mutagenic nitm-eubatituted polynuclear aromatic hydrocarbons in diasal exhausts. J. Chromatogr. 240 (1962) 145. 10 J.N. Pitu Jr., D M . Lokaoagard. W. Hargor, T3. Piahsr. V. Maija, J J . Schuler. GJd. ScorxiaU end Y-A. Kattanatain. Ifutagmu in dieael exhaust particulate. Identification and direct activities of 8-nitrobenxo(a)pyTene, 9-nitroanthracana. 1-nitropyrane and 5H-phenan- thxo-(4.5-bcdlpyrmn-5-ooa. Mutt. Ran, 103 (1962) 241. 11 L Salomon. AM. Dnriaia, TJ. Prater. T. Riley and O. Schuatxle. Contribution of 1- nitropyrono to direct acting Amas aaaay mutagenicities of dieael particulate extracts. Mutt. Res.. 104 (1982) 17. 12 J.N. Pitta J r, K-A. VaoCauwenbergbe, D. Groejean, JJ*. Schmid, D.R. FUx, WX. Baiser Jr., G.B. Knudeon and P M Hynda, Atmospheric reactions of polycyclic aromatic hydrocarbons: Facile firmattira of mntaganir nitra derivatives, Scianca. 202 (1978) 515. 13 -P. N aad, E. dovm- and A3. Kande, Stareoepedfie high affinity binding of 2,3,7,8- tatrachlorodlbaam p rtinria by hapatic cytosol: Evidenca that tha binding spades is tha receptor far indartlmt of aryl hydrocarbon hydroxylase, J. Biol. Cham.. 251 (1978) 4936. 14 D.W. Nsbert and NkL Jm na, The Ah-locus: gmadc regulation of the metabolism of carrinogenasia, drags and other anvinnmantal chemicals by cytochrosaa P-46C-oaadiatad rnnnrvrrygenaaaa. CBC Crit Rev. Biochanu, 8 (1979) 401. 15 J.M3. Caristsdt-Duka, Tianua distribution of tha receptor for 2.3,7,B-t*trachlorodibaaxo-p- dtmdn in tha rat, Cancw Ban, 39 (1979) 3172. 16 P li. Penestte K. Aiaxandrov, R. Axared and C.H. Frayes'net. The effect of some mixed' function indnema on aryl hydrocarbon hydroxyLeas and epoxide hydraee in nuclei and mieroaomae from rat liver and lung. Tha affect of dgaratie smoke. Sur. J. Cancer, 15 (1979) 915. 17 E. Brorstrtau P. Greonfelt and A Lindskog, The effect of nitrogen dioxide end oaone on the dacompoeitioa of pertirto eosociitoH polycyclic erotnetic hydrocarbons during sampling from the atmosphere. Atm. Environ., (1383) in precs. DOM 2 l 6 0 5 8 9 346 ,,18 E. Brorstrdm, P. Grennfelt, A. Lindakog. A. Sjodin and T. Nielsen. Transformation of polycyclic aromatic hydrocarbons during aampling in ambient air by azpoaura to different nitrogen compounda and oaone. in: Seventh International Sympoeium on Polynuclear Aromatic Hydrocarbons. Batteile-Columbua Laborstoriea, Columbus. OIL 1962, in preaa. 19 J.M.B. Carlatadt-Duke, G. Elfatpm, M. Snocbowaki, B. Hqgberg and J.-A. Guatafaeon, Detection of 2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liven by isoelectric focusing in polyacrylamide gels. Toxicol. Lett., 2 (1978) 368. 20 A. Bjorseth, G. Lundhe and A. Lindakog, Long-range transport-of polycyclic arom^iic hydrocarbons. Atm. Environ.. 13 (1979) 48. 21 A.B. Okey. G.P. Bondy, M.E. Mason. G.F. Kahi. HJ. Eiaen, T.M. Gueathner and D.W. Nebert, Regulatory gene product of the Ah-locus. J. Biol. Chem., 254 (1979) 11636. 22 M.F. Argus, SC. Myers and J.C. Arcoe, Apparent absence of requirement of hydrocarbon metabolism for induction and repression of mixad-function -tririsers Cbam.-Biol. Internet.. 29 (1980) 247. 23 AJ*. Poland and E. Glover. 2^,7,8-tetrachlorodibenxo-p-diaxin: Studies on the mechanism of action, in: H. Witachi (Ed.). The Scientific Basis of Toxicity Assessment. Elsevur/NorthHolland Biomedical Press, Amsterdam. 1960, pp. 223-239. 24 T. Greibmkk. G. Ldfroth. L. Nilsson, R. Toftgird, J. Carlstadt-Duke and J.-A. Guatafeeon, Nitroarenaa: Mutagenicity in the Ames Salmonalla/microaoma assay and affinity to the TCDD-receptor protein, in: D.E. Rickart (Ed.), Toxicity of Nitroaromatic Compounds. Hemisphere Pub). Corp., Washington D.CM1963, in prase 25 R.E. Kouri, L.R Billups, T.H. Rude, C-E. Whitmire, B. Seas and CJ. Henry, Correlation of indudbility of aryl hydrocarbon hydroxylase with susceptibility to 3-mathyicbolanthreosinducad lung cancers. Cancer Lett., 9 (1960) 277. 26 C.C. Harris, H. Autrup, G. Stoner, S.K. Yang, J.C. Louts, K.V. Golbcan, JJC. Selkirk. RJ. Conner, L_A_ Baiett, R.T. Janas, EJ4. McDowell and B J. Trump, Metabolism of benzofalpyrene and 7.12-dimethylbanx<a)anthrarana in cultured human bronchus and pan creatic duct. Cancer Ran., 37 (1977) 3349. 27 M.W. Kabng, M.W. Smith and B J. Trump, Aryl hydrocarbon hydroxylase in human bronchial epithelium and blood monocyte, J. Natl. Cancar Inst., 66 (1961) 227. 28 A. Poland and E. Glovar. 2^3,7,8-leCracblorodibeiixo-p-dioxin: segregation of toxicity with tbs Ah-locus, MoL Pharmacol., 17 (1966) 86. 29 RJ. Kodba, P.G. Keyes, JJL Boyer. R-M. Cerraoo, CX. Wade. D.A. Ditteoberg. R_P Kalnia. LX. Fraason, C.N. Park. SJ3. Barnard. R.A. Hummel and C.G. Humiston, Results of a two-year chronic toxicity and oncogenicity study of 2J,7,8-ttrachlorodib#nio-p-djoxin in rats, Toxicol. Appi. Pharmacol., 46 (1978) 279. 30 S. Hussain, L. Ehrenberg, G. Ldfroth and T. Getvall, Mutaganic effects of TCDD on bacterial syatama. Amble, 1 (1972) 32. 31 J-P-A. Sailar, Survey on the mutagenicity of variooa pesticides, Experentia, 29 (1973) 622. 32 L.E. Gdg- and R-A. Neel, Mutagmidty tasting of 2-3.7.8-tetrachlomdibenxo-p-dioxin in histidine auxotropha of Salmonella typhimurium, Toxicol. Appl. Pharmacol., 89 (1961) 128. 33 J.Q. Rose, J.C. Ramsey, T.H. Wentaler, R-A. Hummel and PJ . Gehring. The feta of 2.3.7,8. tatrachiortidihenuvpdioxin following single end repeated oral doses to the rat, Toxicol. Appl. Pharmacol.. 36 (1976) 209. 34 A. Poland and E. Glover, An eatimara of the maximum in vivo oovalent binding of 2A7X4atncblorodibanxo-pdioxin to rat livw protein, ribosomal RNA and DNA, Cancer Raa^ 39 (1979) 3341. 35 H.C. Pitot, T. Goldsworthy. H.A. Campbell and A. Poland. Quantitative evaluation of tba promotion by 2J.7,8-tatmchlorodibanao-p-dioxin of hopatocardnoganaaia tram diathylnitmaamina. Cancer Res.. 40 (1960) 3616. 36 A. Poland, D. Palen iui E. Glover, tumour promotion by TCDD in akin of HRS/J hairless mica. Nature, 300 (1962) 271. -11 DQM2I605SI K ' S I ~ /cc Chemico - Biological In te ra c tio n / moLECUwp m ECHfinim r bv ujhich EHOGEAOU/ CHEilICAL/ EFFECT CHARGE/ H BIOLOGICAL /V/TEfTl/ EDITORS P. Moldeus (Stockholm). J. DePierre IStockholm) R.M. Philpot (Research Triangle Park). J.R. Bend (Research Triangle Park) ELSEVIER C h e m . - B i o l . I n t e r a c t i o n s , 45 (1983) 393--399 Elsevier Scientific Publishers Ireland Ltd. K- c ? ( ? V ! - ( i c o ) [M W ] 393 Short Communication THE BINDING OF 2,3,7,S-TETRACHLORODIBENZODIOXIN TO PLASMA LIPOPROTEINS MAY DELAY TOXICITY IN EXPERIMENTAL HYPERLIPIDEMIA MARINA MARINOV1CH, CESABE R. SIRTORI, CORRADO L. GALLI and RODOLFO PAOLETTI In stitu te o f P h a rm a co lo g y a n d P h a rm a c o g n o sy , via A ndrea D el S a rto 21. U niversity o f Milano, 2 0 1 2 9 Milan (Italy) (Received November 17th, 1982) (Revision received February 2nd, 1983) (Accepted February 6th, 1983) Key words: 2, 3 ,7 , 8--Tetrachlorodibenzodioxin binding -- Lipoproteins -- Experimental hyperlipidemia -- LDS0 Introduction The association between reduced blood lipid levels and increased inci dence of tumors, particularly o f the gastrointestinal tract, has been recently described by many authors (1--3 ], The apparent increase of risk, particularly in hypocholesterolemic subjects, has been linked to: reduced plasma trans port of carotene [4 ], loss o f cell membrane cholesterol, with consequent increased cell vulnerability (5 ) and to a deficiency of immunoregulatory lipoproteins [6 ]. Lipophilic xenobiotic compounds, including most of the known carcinogens, are carried by plasma lipoproteins [7]; such is the case of chloroethylnitrosoureas [8], methylcholanthrene [9 ], benzopyrene [lO j, dimethylaminoazobenzene [11], etc. The toxicity of compounds of this type may be, therefore, modified, in conditions of hyper- or hypolipidemia. 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) intoxication has raised con siderable interest in recent years, particularly in view of numerous industrial accidents [1 2 ], with contamination o f many thousands of individuals. The acute administration o f non-lethal doses of TCDD has been associated, in studies by us and by other groups [13,14], with hyperlipidemia and increased high density lipoprotein cholesterol levels in rodent models. Since Abbreviations: HDL, high density lipoproteins; LDL, low density lipoproteins; VDHL, very high density lipoproteins; VLDL, very low density lipoproteins; TCDD, 2,3,7,8tetrachlorodibenzodioxin. 0009-2797/83/503.00 Q1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland JSS09IZHOQ w - -1 0SSO9IZMOO 394 the mode of TCDD transport in plasma has not been completely elucidated, we examined the binding pattern o f this compound in normal and hyper lipidmie humans and rodents, also attempting to verify changes in toxicity in this latter experimental model. Materials and Methods TCDD was acquired from C. Erba (Milan, Italy) and dissolved in com oil/acetone (9 :1 , v/v) prior to administration. [ 14C]TCDD was from Eco.Control, Inc., Cambridge, MA, U.S.A. For the in vitro experiments, plasma was obtained from human subjects, either normal or with hyperlipoproteinemias types IIA and IV (1 5 ]. Rat plasma was from normal Sprague--Dawley animals (CD-I Albino, Charles River, Calco, Italy) or from rats of the same strain treated for one week with a cholesterol-cholic acid diet [16] (Altromin Rieper, Vandoies, Bol zano, Italy). Samples from human or rat plasmas (3.0 ml) were incubated with 0.5 jjCi [ UC]TCDD (spec, act., 47.5 pCi/mmol) for 90 min at 37C. The TCDDplasma solution was then eluted on a Bio Gel A-15 m agarose column (2.5 X 100 cm) with 0.01 Tris--HCl buffer (pH 7.4), containing 0.15 M NaCl, 0. 01% EDTA-Na2 and 0.02% NaN3. The collected fractions were monitored for absorbance at 280 nm and for radioactivity in a liquid scintillator (Pack ard Tri-Carb 3255). For the comparative analysis o f multiple plasma samples, and to allow a complete radioactivity recovery, plasmas from hyperlipidmie patients were also separated by sequential ultracentrifugation [17]. Lipoprotein mass, calculated by the chemical compositional data, and radioactivity were determined for each separated lipoprotein. To evaluate any changes in TCDD toxicity in the presence of hyper lipidemia, CD-I Albino rats (C. River), body wt. 200--250 g, were injected 1. p. with 30, 45, 67.5, 101, 152 pg/kg of TCDD. At least 8 animals were used for each dose level. Rats were kept oh either the standard or the hyper lipidmie [16] regimen for 1 week prior to the administration o f TCDD and kept on the same diet thereafter. Mortality data were collected for 34 days. The method o f probits was employed for the calculation o f theLD50 at different time intervals during the toxicity study. Results and Discussion Incubation of [ 14C]TCDD with plasma and with the lipoprotein (d = 1.225) fraction from normo- and hyperlipidmie individuals, resulted in a negligible binding to albumin, most of the radioactivity being associated with very low and low density lipoproteins (VLDL and LDL) (Fig. 1). To evaluate the different binding patterns in normo- and hyperlipidemia, representative sera from 3 normolipidemic individuals, 3 subject- with type IlA hyperlipidemia and 3 with type IV hyperlipidemia, i.e. characterized, respectively, by increased LDL and VLDL in plasma [15], were analyzed for the binding of labelled TCDD. As shown in Table I, both in normals 18328 i fi SO9 IZ HOa 395 m Fig. 1. Elution profile of plasma lipoproteins ( d > 1.225 g/ml), isolated from a normolipidemic subject by ultracentrifugation and incubated with (" CJTCDD. The separation was carried out on a Bio Gel A-15 m agarose column (2.5 X 100 cm). A 3.0 ml sample was eluted with 0.01 M Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl, 0.01% EDTA Na, and 0.02% NaN,. (------) A ,,, ; (-------) radioactivity (cpm/fraction), associated with VLDL, LDL and HDL. Similar findings were obtained with w h o i f plasma, radioactivity associated with albumin being less than 10% of the total. TAByS 1 PERCENTAGE DISTRIBUttM B f ['*C]TCDD AMONG HUMAN PLASMA LIPO- PROTEINS SEPARATED BlY U W RaAiCENTRIFUGATION VaJues represents the percentages of radioactivity associated to the different lipoprotein fractions. Values in brackets indicate the molar ratio TCDD/lipoprotein. Duplicate samples from 3 normolipidemic subjects, 3 type 1IA and 4 type IV patients (X t S.E.M.). P < 0.01 vs. LDL N; **P < 0.05 vs. VLDL N; **/> < 0.01 vs. VLDL N. VLDL (d < 1.006) LDL (d 1.006-1.063) HDL ( d 1.065--1.21) Normolipidemic subjects Type p a tie n t s Type IV patients 17.0 s 1.7 (5.4 2 1.3) 5.2 : 1.9** (2.4 : 0.3) 41.5 t 2.4***_ (4.2 0.5) 55.1 r 2.6 (0.6 t 0.03) 69.1 s 0.6* (0.3 - 0.05) 36.8 2 2.7* .(.3 2 0.04) 15.1 s 2.3 (0.02 t 0.003) 14.4 x 2.3 (0.01 0.001) 10.4 x 1.5 (0.02 x 0.01) -s \ 183539 D0W2160598 396 and type IIA subjects, LDL are the major binding fraction. In contrast, VLDL are the quantitatively most important carrier in type IV patients. Relatively minimal amounts are bound to high density lipoproteins (HDL) and to very high density lipoproteins (VHDL). The binding of TCDD was also calculated per mole of lipoprotein. Molar lipoprotein concentrations are derived from chemical compositional data and published molecular weights [18]. From these data (Table 1), it appears that the maximal binding capacity is exerted by VLDL, followed by LDL and HDL, thus confirming recent findings for benzopyrene in normolipoproteinemic male donors [10]. A similar evaluation o f the lipoprotein binding o f TCDD in normal and cholesterol-cholic acid fed rats, clearly showed that also in this species the toxic chemical is preferentially bound to plasma lipoproteins, a higher percentage of binding being observed in hyperlipidemic animals (Table II). Based on these data, the acute toxicity o f TCDD was evaluated in Sprague--Dawley rats, both normolipidemic and after dietary induction of hyperlipidemia (plasma cholesterol levels: 89 8.1 mg/dl for controls and 1586 z 249 mg/dl for hypercholesterolemics; plasma triglycerides: 118 11.1 mg/dl for controls and 478 * 121 mg/dl for diet treated rats at the end of the experiment). The lipoprotein distribution in this experi mental model o f hyperlipidemia is characterized by a marked increase, particularly of VLDL cholesterol and triglycerides [19]. The LD,o for rats, calculated after 3 weeks from the administration of TCDD, was significantly higher in hyperlipidemic animals as compared to controls on a normal diet (Fig. 2a). At a later interval, however, the LDS0 tended to approach that observed in normal animals (Fig. 2b). The possible relationship between changes in LD:o and metabolic turnover o f lipopro teins in hyperlipidemic.rodents [2 0 ], resulting in the release of the bound toxic chemical, needs further investigation. The reported findings indicate that most o f circularing TCDD is bound to plasma lipoproteins; LDL carry the highest percentage o f the compound; however, with elevations o f VLDL, as in type IV plasmas, also this latter lipoprotein may carry a significant amount of the chemical. In normal rodents, which have markedly lower plasma lipids, compared to humans, TCDD is distributed almost equally among lipoproteins and a relatively TABLE II PERCENTAGE DISTRIBUTION OF (14C]TCDD AMONG RAT LIPOPROTEINS ISOLATED BY SEQUENTIAL ULTRACENTRIFUGATION Three duplicate samples from each gtoup of animals (x t S.E.M.) *P < 0.01 vs. controls. VLDL LDL HDL VHDL Normolipidemic animals Hyperlipideuiic animals 20.S : 2.2 39.9 . 1.3* 17.8 t 1.4 45 4 i 1.6* 19.4 t 1.8 5.6 t 0.09* 41.9 : 5.4 S.9 i 0.3* D0H2160559 397 AB Fig. 2. LD,,-values for normo (-- --- ) and hyperlipidmie (-- --- ) rats calculated at two different time intervals. A: 23 days; B: 34 days. The LD,,-values for normo- and hyper lipidmie animals, i.e. 76.9 (58.6--100.9 c.i.) and 151.2 (105--217.2) pg/kg, respectively, at 23 days, differ significantly (P < 0.01); at 34 days LDl(-values were 73.7 (58.5--92.8) and 90.9 (63.1--128.8) ug/kg, respectively (P , njs.). high percentage is also carried by VHDL. In this animal species, almost equal amounts are carried by VLDL and LDL after the experimental diet. The possibility that binding to plasma lipoproteins may modify the long term toxic effects of lipophilic chemicals, is of compelling interest; if a chemical, in fact, is slowly released from the lipoprotein fraction to which it is bound, plasma and tissue concentrations might not reach sufficient levels to induce damage. Species differences in TCDD toxicity are well established: rats, which have significantly lower plasma lipid levels, com pared to hamsters show a higher sensitivity to the toxic effects o f TCDD [211- The experimental induction of hyperlipidemia in rats, in our experi ments, delayed the development o f overt toxicity. The protocol of the in vivo studies did not allow examination o f the binding of TCDD to lipo proteins; on the other hand, it is likely that the release of the bound chem i cal may occur after an interval of time, related to the metabolic turnover of lipoproteins. In hyperlipidmie rodents, turnover o f VLDL and LDL is significantly delayed [20] compared to normolipidemic animals. If plasma lipoprotein binding may modify toxicity, humans, which have the highest baseline lipids among known animal species, should, for this reason, be less sensitive to the development o f parenchymal lesions. The general human toxicity from TCDD is still the object of debate; the only available long t^rm report after an acute intoxication does not show in creases in tumor and, indeed, cardiovascular accidents are reduced in con taminated individuals [22]. The deposition of lipophilic chemicals in tissues 398 DOM2 I 6 0560 does not, apparently, follow the lipoprotein receptor pathway, being similar in receptor-positive and negative cells [23]. If lipoprotein binding and consequent recirculation of chemicals is an important factor in the induction of toxicity, then delayed -and possibly reduced toxicity may be at hand in hyperlipidemic individuals. The con verse, i.e. that hypolipidemic animals or individuals may be at higher risk of tissue damage remains, o f course, to be established. Experiments were carried out in the Special Research Laboratory o f the Regione Lombardia in Seveso. The generous cooperation and support of the Regione Lombardia is gratefully acknowledged. 1 J.D. Kark, A.H. Smith and C.G. Him , The relationship of serum cholesterol to the incidence of cancer in Evans Country, Georgia, J. Chronic Dis., 33 (1980) 311. 2 R.R. Williams, P.D. Sortie, M. Feinleib, P. McNamara, W.B. Kannel and TJl. Dawber, Cancer indicence by levels of cholesterol, J. Am. Med Assoc., 245 (1981) 245. 3 B. Peterson, E. Trell and N.H. Sternby, Low cholesterol levels as risk factor for noncoronary death in middle-aged man, J. Am. Med. Assoc., 245 (1981) 2056. 4 R.B. Shekelle, M. Lepper, S. Liu, C. Maliza, W.J. Raynor Jr., A.H. Rossof, P. Oglesby, A.M. Shryock and J. Stamler, Dietary vitamin-A and risk of cancer in the Western Electric Study, Lancet, ii (1981) 1185. 5 R.A. Demel and B. Dekruyff, The function of sterols in membranes, Biochim. Biophys. Acta, 457 (1976) 109. 6 F. Chisari, L.K. Curtiss and F.C. Jensen, Physiologic concentrations of normal human plasma lipoprotein inhibit the immortalization of peripheral lymphocytes-B by the Epstein-Barr virs, J. Clin. Invest., 68 (1981) 329. 7 O.G. NOsen and S. Jacobsen, The influence of salts and differences in protein iso lation procedure on the binding of quinidine to human serum albumin, Biochem. Pharmacol., 25 (1976) 1261. 8 R.J. Weinkam, A. Finn, V.A. Levin and J.P. Kane, Lipophilic drugs and lipopro teins -- Partitioning effects on chloroethylnitrosourea reaction rates in serum, J. Pharmacol. Exp. Ther., 214 (1980) 318. 9 E.P. Benditt, The origin of atherosclerosis, Sci. Am., 236 (1977) 74. 10 H.P. Shu and A.V. Nichols, Uptake of lipophilic carcinogens by plasma lipoproteins -- Structure activity studies, Biochim. Biophys. Acta, 665 (1981) 376. 11 T.C. Chen, W.A. Bradley, A.M. Gotto Jr. and J.D. Morrissett, Binding of the chemical carcinogen, p-dimethylaminoazobenzene, by human plasma low density lipoproteins, FEBS Lett., 104 (1979) 236. 12 B. Holmstedt, Prolegomena to Seveso -- Ecclesiastes 1, IS, Arch. Toxicol., 44 (198C) 211. 13 A. Poli, G. Franceechini, L. Puglisi and C.R. Sirtori, Increased total and high density lipoprotein cholesterol with apoprotein changes resembling streptozocin diabetes in tetrachlorodibenzodioxin (TCDD) treated rats, Biochem. Pharmacol., 29 (1980) 835. 14 P.W. Albro, J.T. Corbett, M. Harris and L.D. Lawson, Effects of 2,3,7,8-TCDD on lipid profiles in tissue of Fischer rat, Chem.-Biol. Interact., 23 (1978) 315. 15 J.L. Beaumont, L.A. Carlson, G.R. Cooper, Z. Fejfar, D.S. Frederickson and T. T. Straaser, Classification of hyperlipidaemias and hyperlipoproteinaemias. Bull. Wld. Hlth. Org., 43 (1970) 891. 16 N. Nath, R. Weiner, A.E. Harper and C.A. Elvehjem, Diet and choiesterolemia. Part 1 (development of a diet for the study of the nutritional factors affecting choles- terolemia in rats), J. Nutr., 67 (1959) 289. 17 R.J. Havel, H.A. Eder and J.H. Bragdon, The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 83il JL n. TW 399 18 R.W. Shen, A.M. Scanu and F.J. Kezdy, Structure of human serum lipoproteins inferred from compositional analysis, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 837. 19 N.L. Lasser, P.S. Roheim, D. Edelstein and H_A. Eder, Serum lipoproteins of normal and cholesterol-fed rats, J. Lipid Res., 14 (1973) 1. 20 A. Van Tol, Hypertriglyceridemia in the diabetic rat -- Defective removal of serum low density lipoproteins, Atherosclerosis, 26 (1977) 117. 21 J.R.'Olson, A. Myron, M A. Holscher and R.A. Neal, Toxicity of 2,3.7,8-tetrachIorodibenzo-p-dioxin in the Golden Syrian Hamster, Toxicol. Appl. Pharmacol., 55 (1980)67. 22 J.A. Zack and RJt. Suskind, The mortality experience of workers exposed to tetrachlorodibenzodioxin in trichlorophenol process accident, J. Occup. Med., 22 (1980) 11. 23 J.F. Remsen and R.B. Shierman, Effect of low-density lipoprotein of the incorpora tion of benzo(a)pyrene by cultured cells, Cancer Res., 41 (1981) 3179. O rvj "" C3 Crt 09 18333 0 0 H 2 I6055* i y k ~ iffkfe s i - ( C C Cheroico - Biological In te ra c tio n / moLECump mECHnnimr bv uuhich EHOGEfiOULrCHEfilCfL/EFFECT CHfflGE/IHBIOLOGICAL /V/TEfT/ EDITORS P. Moldeus (Stockholm), J. DePierre (Stockholm) R.M. Philpot (Research Triangle Park), J.R. Bend (Research Triangle Park) ELSEVIER eerrw mgm .83: K- b b b % i - ... \ ( i c `c bow2 i 6053# C h e m . - B i o l . I n t e r a c t i o n s , 45 (1983) 393--399 Elsevier Scientific Publishers Ireland Lid. 393 Short Communication THE BINDING OF 2,3,7,8-TETRACHLORODIBENZODIOXIN TO PLASMA LIPOPROTEINS MAY DELAY TOXICITY IN EXPERIMENTAL HYPERLIPIDEMIA MARINA MARINOVICH. CESARE R. S1RTORI, CORRADO L. GALLI and RODOLFO PAOLETTI In stitu te o f P h a rm a co lo g y a n d P h a rm a c o g n o s y , via A n d re a D el S a rto 21, U niversity o f Milano, 2 0 1 2 9 Milan (Italy) (Received November 17th, 1982) (Revision received February 2nd, 1983) (Accepted February 6th, 1983) Key words: 2, 3, 7, 8--Tetrachlorodibenzodioxin binding -- Lipoproteins -- Experimental hyperlipidemia -- LDi0 In troduction The association between reduced blood lipid levels and increased inci dence of tumors, particularly o f the gastrointestinal tract, has been recently described by many authors [1--3 ], The apparent increase of risk, particularly in hypocholestrolmie subjects, has been linked to: reduced plasma trans port of carotene [4], loss of cell membrane cholesterol, with consequent increased cell vulnerability [5 ] and to a deficiency of immunoregulatory lipoproteins [6 ]. Lipophilic xenobiotic compounds, including most of the known carcinogens, are carried by plasma lipoproteins (7]; such is the case of chloroethylnitrosoureas (8 ], methylcholanthrene [9 ], benzopyrene [10], dimethylaminoazobenzene [11], etc. The toxicity of compounds of this type may be, therefore, modified, in conditions of hyper- or hypolipidemia. 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) intoxication has raised con siderable interest in recent years, particularly in view of numerous industrial accidents [1 2 ], with contamination o f many thousands of individuals. The acute administration of non-lethal doses of TCDD has been associated, in studies by us and by other groups [13,14], with hyperlipidemia and increased high density lipoprotein cholesterol levels in rodent models. Since Abbreviations: HDL, high density lipoproteins; LDL, low density lipoproteins; VDHL, very high density lipoproteins; VLDL, very low density lipoproteins; TCDD, 2,3,7,8tetrachlorodibenzodioxin. 0009-2797/83/503.00 01983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland y 18335 DON2I60558 394 the mode of TCDD transport in plasma has not been completely elucidated, we examined the binding pattern of this compound in normal and hyper lipidmie humans and rodents, also attempting to verify changes in toxicity in this latter experimental model. Materials and Methods TCDD was acquired from C. Erba (Milan, Italy) and dissolved in com oil/acetone (9 :1 , v/v) prior to administration. [ 14C]TCDD was from Eco.Control, Inc., Cambridge, MA, U.S.A. For the in vitro experiments, plasma was obtained from human subjects, either normal or with hyperlipoproteinemias types IIA and IV [15]. Rat plasma was from normal Sprague--Dawley animals (CD-I Albino, Charles River, Calco, Italy) or from rats o f the same strain treated for one week with a cholesterol-cholic acid diet [16] (Altromin Rieper, Vandoies, Bol zano, Italy). Samples from human or rat plasmas (3.0 ml) were incubated with 0.5 pCi [ 14C]TCDD (spec, act., 47.5 pCi/mmol) for 90 min at 37C. The TCDDplasma solution was then eluted on a Bio Gel A-15 m agarose column (2.5 X 100 cm) with 0.01 Tris--HCl buffer (pH 7.4), containing 0.15 M NaCl, 0. 01% EDTA-Na2 and 0.02% NaN3. The collected fractions were monitored for absorbance at 280 nm and for radioactivity in a liquid scintillator (Pack ard Tri-Carb 3255). For the comparative analysis of multiple plasma samples, and to allow a complete radioactivity recovery, plasmas from hyperlipidmie patients were also separated by sequential ultracentrifugation [17]. Lipoprotein mass, calculated by the chemical compositional data, and radioactivity were determined for each separated lipoprotein. To evaluate any changes in TCDD toxicity in the presence o f hyper lipidemia, CD-I Albino rats (C. River), body wt. 200--250 g, were injected 1. p. with 30, 45, 67.5, 101, 152 pg/kg of TCDD. At least 8 animals were used for each dose level. Rats were kept oh either the standard or the hyper lipidmie [16] regimen for 1 week prior to the administration o f TCDD and kept on the same diet thereafter. Mortality data were collected for 34 days. The method of probits was employed for the calculation of the LDS0 at different time intervals during the toxicity study. R e su lts a n d D iscu ssio n Incubation of [ 14C]TCDD with plasma and with the lipoprotein (d 1.225) fraction from normo- and hyperlipidmie individuals, resulted in a negligible binding to albumin, most o f the radioactivity being associated with very low and low density lipoproteins (VLDL and LDL) (Fig. 1). To evaluate the different binding patterns in normo- and hyperlipidemia, representative sera from 3 normolipidemic individuals, 3 subjects- with type IlA hyperlipidemia and 3 with type IV hyperlipidemia, i.e. characterized, respectively, by increased LDL and VLDL in plasma [15], were analyzed for the binding of labelled TCDD. As shown in Table I, both in normals i!.8 O O' Fig. 1. Elution profile of plasma lipoproteins (d > 1.225 g/mi), isolated from a normolipidemic subject by ultracentrifugation and incubated with (" CJTCDD. The separation was carried out on a Bio Gel A-15 m agarose column (2.5 x 100 cm). A 3.0 ml sample was eluted with 0.01 M Tris--HC1 buffer (pH 7.4) containing 0.15 M NaCI, 0.01% EDTA Na, and 0.02% NaN,. (------) A ,,,; (-------) radioactivity (cpm/fraction), associated with VLDL. LDL and HDL. Similar findings were obtained with wholiplasma, radioactivity associated with albumin being less than 10% of the total. TABIE 1 PERCENTAGE DISTRIBU ['*C)TCDD AMONG HUMAN PLASMA LIPO- PROTEINS SEPARATED BYY O ffR aACENTRIFUGATION Values represents the percentages of radioactivity associated to the different lipoprotein fractions. Values in brackets indicate the molar ratio TCDD/lipoprotein. Duplicate samples from 3 normolipidemic subjects, 3 type IIA and 4 type IV patients (if i S.E.M.). P < 0.01 vs. LDL N; **P < 0.05 vs. VLDL N; ***P < 0.01 vs. VLDL N. VLDL (d < 1.006) LDL (d 1.006-1.063) HDL (d 1.065--1.21) Normolipidemic subjects Type^yatiente Type IV patients 17.0 i 1.7 (5.4 : 1.3) 5.2 i 1.9** (2.4 : 0.3) 41.5 t 2.4 *. (4.2 : 0.5) 55.1 : 2.6 (0.6 t 0.03) 69.1 i 0.6* (0.3 : 0.05) 36.8 : 2.7* (fl.3 : 0.04) 15.1 t 2.3 (0.02 l 0.003) 14.4 t 2.3 (0.01 t 0.001) 10.4 t 1.5 (0.02 * 0.01) BOMZI 6 055 f ! 00 W2160598 396 and type IIA subjects, LDL are the major binding fraction. In contrast, VLDL are the quantitatively most important carrier in type IV patients. Relatively minimal amounts are bound to high density lipoproteins (HDL) and to very high density lipoproteins (VHDL). The binding of TCDD was also calculated per mole of lipoprotein. Molar lipoprotein concentrations, are derived from chemical compositional data and published molecular weights [18], From these data (Table I), it appears that the maximal binding capacity is exerted by VLDL, followed by LDL and HDL, thus confirming recent findings for benzopyrene in normolipoproteinemic male donors [10]. A similar evaluation o f the lipoprotein binding o f TCDD in normal and cholesterol-cholic acid fed rats, clearly showed that also in this species the toxic chemical is preferentially bound to plasma lipoproteins, a higher percentage of binding being observed in hyperlipidemic animals (Table II). Based on these data, the acute toxicity o f TCDD was evaluated in Sprague--Dawley rats, both normolipidemic and after dietary induction of hyperlipidemia (plasma cholesterol levels: 89 8.1 mg/dl for controls and 1586 249 mg/dl for hypercholesterolemics; plasma triglycerides: 118 11.1 mg/dl for controls and 478 s 121 mg/dl for diet treated rats at the end o f the experiment). The lipoprotein distribution in this experi mental model o f hyperlipidemia is characterized by a marked increase, particularly of VLDL cholesterol and triglycerides [19]. The LD;o for rats, calculated after 3 weeks from the administration of TCDD, was significantly higher in hyperlipidemic animals as compared to controls on a normal diet (Fig. 2a). At a later interval, however, the LDS0 tended to approach that observed in normal animals (Fig. 2b). The possible relationship between changes in LDS0 and metabolic turnover o f lipopro teins in hyperlipidemic.rodents [2 0 ], resulting in the release of the bound toxic chemical, needs further investigation. The reported findings indicate that most o f circularing TCDD is bound to plasma lipoproteins; LDL carry the highest percentage o f the compound; however, with elevations of VLDL, as in type IV plasmas, also this latter lipoprotein may carry a significant amount of the chemical. In normal rodents, which have markedly lower plasma lipids, compared to humans, TCDD is distributed almost equally among lipoproteins and a relatively TABLE II PERCENTAGE DISTRIBUTION OF [ " C]TCDD a m o n g r a t l ip o p r o t e in s ISOLATED BY SEQUENTIAL U LTRACENTRIFUGATION Three duplicate sam ples from each gTOup of animals (5 - S.E.M .) *P < 0.01 vs. controls. VLDL LDL HDL VHDL Normolipidemic animals Hyperlipidemic animals 20.5 : 2.2 39.y . 1.3* 17.8 : 1.4 45 4 I 1.6* 19.4 s 1.8 5.6 I 0.09* 41.9 : 5.4 S.9 I 0.3* P D O H 2 16 05 5* 397 AB Fig. 2. LD,,,-values for norm o (----- ) and hyperlipidm ie (--- -- ) rats calculated a t tw o different time intervals. A: 23 days; B: 34 days. The LD,,-valus for norm o- and h y p er lipidmie animals, i.e. 76.9 (58.6--100.9 c .i.)a n d 151.2 (105--217.2) jig/kg, respectively, at 23 days, differ significantly (P < 0.01); at 34 days LD l(-values were 73.7 (58.5--92.8) and 90.9 (63.1--128.8) ug/kg, respectively (P, n s .). high percentage is also carried by VHDL. In this animal species, almost equal amounts are carried by VLDL and LDL after the experimental diet. The possibility that binding to plasma lipoproteins may modify the long term toxic effects c f lipophilic chemicals, is of compelling interest; if a chemical, in fact, is slowly released from the lipoprotein fraction to which it is bound, plasma and tissue concentrations might not reach sufficient levels to induce damage. Species differences in TCDD toxicity are well established: rats, which have significantly lower plasma lipid levels, com pared to hamsters show a higher sensitivity to the toxic effects of TCDD [21]. The experimental induction o f hyperlipidemia in rats, in our experi ments, delayed the development o f overt toxicity. The protocol of the in vivo studios did not allow examination o f the binding o f TCDD to lipo proteins; on the other hand, it is likely that the release of the bound chemi cal may occur after an interval of time, related to the metabolic turnover of lipoproteins. In hyperlipidmie rodents, turnover of VLDL and LDL is significantly delayed [20] compared to normolipidemic animals. If plasma lipoprotein binding may modify toxicity, humans, which have the highest baseline lipids among known animal species, should, for this reason, be less sensitive to the development of parenchymal lesions. The general human toxicity from TCDD is still the object of debate; the only available long t^rm report after an acute intoxication does not show in creases in tumor and, indeed, cardiovascular accidents are reduced in con taminated individuals [2 2 ]. The deposition of lipophilic chemicals in tissues 8r* o *3 O' 00M2160580 398 does not, apparently, follow the lipoprotein receptor pathway, being similar in receptor-positive and negative cells [23]. If lipoprotein binding and consequent recirculation of chemicals is an important factor in the induction o f toxicity, then delayed -and possibly reduced toxicity may be at hand in hyperlipidemic individuals. The con verse, i.e. that hypolipidemic animals or individuals may be at higher risk of tissue damage remains, of course, to be established. Experiments were carried out in the Special Research Laboratory o f the Regione Lombardia in Seveso. The generous cooperation and support of the Regione Lombardia is gratefully acknov/ledged. 1 J.D. K irk, A.H. Smith and C.G. Hames, T he relationship of serum cholesterol to the incidence of cancer in Evans C ountry, Georgia, J. Chronic Dis., 33 (1980) 311. 2 R .R . Williams, P.D. Sorlie, M. Feinleib, P. McNamara, W.B. Kannel and T J t. Dawber, Cancer indicence by levels o f cholesterol, J. Am. Med Assoc., 245 (1 9 8 1 ) 245. 3 B. Peterson, E. Trell and N.H. S tern b y , Low cholesterol levels as risk facto r for noncoronary death in middle-aged man, J. Am. Med. Assoc., 245 (1961) 2056. 4 R.B. Shekelle, M. Lepper, S. Liu, C. M&liza, W.J. R aynor Jr., A.H. Rossof, P. Oglesby, A.M. Shryock and J. Stam ler, D ietary vitamin-A and risk of cancer in the Western Electric S tu d y , Lancet, ii (1 9 8 1 ) 1185. 5 R.A. Demel and B. D ekruyff, T he function o f sterols in m em branes, Biochim. Biophys. Acta, 457 (1976) 109. 6 F. Chisari, L.K. Curtiss and F.C. Jensen, Physiologic concentrations of normal human plasma lipoprotein inhibit the im m ortalization o f peripheral lymphocytes-B by the Epstein-BarT virs, J. Clin. Invest., 68 (1981) 329. 7 O.G. NQsen and S. Jacobsen, T he influence o f salts and differences in protein iso lation procedure on the binding of quinidine to hum an serum album in, Biochem. Pharmacol., 25 (1976) 1261. 8 R.J. Weinkam, A. Finn, V.A. Levin and J.P. Kane, Lipophilic drugs and lipopro teins -- Partitioning effects on chloroethylnitrosourea reaction rates in serum , J. Pharmacol. Exp. Ther., 214 (1980) 318. 9 E J \ Benditt, The origin of atherosclerosis, Sci. Am., 236 (1977) 74. 10 H.P. Shu and A.V. Nichols, U ptake of lipophilic carcinogens by plasma lipoproteins - Structure activity studies, Biochim. Biophys. Acta, 665 (1981) 376. 11 T.C. C hen, W.A. Bradley, A.M. G o tto Jr. and J.D . M orrissett, Binding o f the chemical carcinogen, p-dimethylam inoazobenzene, by hum an plasma low density lipoproteins, FEBS Lett., 104 (1979) 236. 12 3. H olm stedt, Prolegomena to Seveso -- Ecclesiastes 1 ,1 3 , A rch. Toxicol., 44 (I98C ) 211. 13 A. Poll, G. Franceschini, L. Puglisi and C.R. Sir tori, increased total and high density lipoprotein cholesterol with apoprotein changes resembling streptozocin diabetes in tetrachlorodibenzodioxin (TCDD) treated rats, Biochem. Pharmacol., 29 (1980) 835. 14 P.W. A lbro, J.T. C orbett, M. Harris and L.D. Lawson, E ffects o f 2,3,7,8-TCDD on lipid profiles in tissue of Fischer rat, Chem.-Biol. Interact., 23 (1978) 315. 15 J.L . Beaum ont, L.A. Carlson, G .R . Cooper, Z. Fejfar, D.S. Frederickson and T. T. Straaser, Classification o f hyperlipidaem ias and hyperlipoproteinaem ias, Bull. Wld. H lth. Org., 43 (1970) 891. 16 N. N ath, R. Weiner, A.E. Harper and C.A. Elvehjem, Diet and cholesterolem ia, Part 1 (development of a diet for the study of the nutritional factors affecting choiesterolemia in rats), J. N utr., 67 (1959) 289. 17 R .J. Havel, H.A. Eder and J.H . Bragdon, The distribution and chem ical com position of ultracentrifugally separated lipoproteins in hum an serum , J . Clin. Invest., 34 (1955)1345. I9S09IZMOQ 399 IS R.W. Shen, A.M. Scanu and F.J. Kezdy, S tru ctu re o f hum an serum lipoproteins inferred from compositional analysis, Proc. N atl. Acad. Sci. U.S.A., 74 (1977) 837. 19 N.L. Lasser, P.S. Roheim , D. Edelstein and H_A. Eder, Serum lipoproteins of normal and cholesterol-fed rats, J. Lipid Res., 14 (1973) 1. 20 A. Van Tol, Hypertriglyceridemia in the diabetic rat -- Defective removal of serum low density lipoproteins, Atherosclerosis, 26 (1 9 7 7 ) 117. 21 J.R .'O lson, A. Myron, M.A. Holscher and R.A . Neal, T oxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Golden SyTian H a m sttr, T oxicol. Appl. Pharmacol., 55 (1980) 67. 22 J.A. Zack and R J l. Suskind, The mortality experience of workers exposed to tetrachlorodibenzodioxin in trichlorophenol process accident, J. O ccup. Med., 22 (1980) 11. 23 J.F . Remsen and R.B. Shierm an, Effect of low -density lipoprotein of the incorpora tion of benzo(a)pyrene by cultured cells. Cancer Res., 41 (1 981) 3179. 18341 ,,Jfeu .-* - Iolden s r a p p o r t R I V: <$> V e~ o .\^ . C c>w --Ck N IA mMI p e i ... - . Staatsin4.r**il,*Jnrt)H' 0U.*ri. 1. b io x in e n ie t v o lle d ig 5'?>'=' t i` O l i K Ujo ii Netherlandish Drawings >**l| n itateli* <>jnKia, IkbMf I* C*M l|M < MI .nm.it 10101 1... ilv im tm tn iM III.Im m u n i kankerverw ekkend Ik jai>\;uiUDare 'lagelijU c apuani? v..n 2 .3 .7 .y.-T*l)D ( Diamine) Jwi le mcirt (AD I) -fauiSI .lXM iMiioyr.im ( IO \> per k ilofiain lwha.ui.*;* u h i. Dio ine nuel. l o eli hci KijkintinuH oor c V!k'`c/<iiui!ieid iKIVj ard en b c eh ou d ii> een n.ct vollt-iliy k.->ikcreiekKcnde taf, m iar al ccn ioyciia.ii.nde koiikciIreur* JeicnJc >ii>l & 5ov- N4^r ' Ddt iU.it w ccn (ktu4iic(4fivn Li1 hcl IMV op bui voli de boclokharc lowiicilktfrtftvct* hccfl cnu.Al luJdt in de T*ecdc Kmcr oj uugu%<u lutti vrugen wjiei jcvtcld m ir de |(/oihllicilmh;u<k vuoi de oniwotkcndc vande Vi>l|jificcipvldcf, In he. nlnuunl op Jcec Wajcn dui d op 2V>epicnbcr iVaTwccd c^cvcri. i% de coiHrtkivie vun hei ooJcuuck tccJ> CCItfCtfCVCll. 16th centuries in the Ri*jksmusv.V"m"ft';u-l?u`n*i"ut*etiae*Z`1H*u', Voor u iu ifi, lu io tlm io rw i, v e m u u l i j i y u tu tl, ebbri n v* ' binnen* cu buitctliiu! ccn unici? bruii cau tuiurnunc cu duvuntctiurtc Tw rt JrUu hu UU u ! Ari(r/ttr. /*#*#* / /''.iW * I t riZriiJilUJi wrtdtti tu i I Oak t i ijji k.'filuiUil KONINKUJX HUIS laichte d atin g Ilei un vcudicncn luppoli voluti de zieklcvtT /uiin ctet'Wluppclijke xn)crt>ou*if vuit de bcoordelitti wtt de kjitkei *<r*ci- itiucmciivciliiiy Ilei Ccmiual Bureau uur Je keude e nau*cne ci'envwtuppeii tui\ Ilare Maicvicil de Kouinfin lai op Stalilick en hcl NcJcil.iiiJv 2.J,7>*TCD) (I)ioAtficl op /n i/frcun vuoi de meni Ilei <*ppoi! u <mii de TciJ i Kmtucr (jeeuiuieii. * In in h lappati Iplac i itiaarl a t. le Ulcitv.ijk hcl mcuw)>oubcumple ande Caapcfaiievt tllacinciivcilinn 'Heikel cr. Uiiivlrekci)' W .A. ullicicel in ckiruk lullen. liivliluul vuur l`i .icvciii it c CicioiulliciiJviarg 7 NO iciJcfl val dal hei vtriciii.,a.-iC cni_.e ui hcl iculc ktvariaal |y i oor iiMimeii K.d en our v ia im c n I !.< all J< CclIHUlllChiMiUil `AJviC. bedrueg. iutukc CalCiilogciiileil vali vliiinivc'hc luii.-n1van Iti no.cwflcr IH?, ucrd en mi li w giucp our JcvkunJigcn Haags Ditrencenlruni, Iircil|ihjking mel hcl icidt koaiiaal an IHIU*een de iufr> en Ja.'mg le gClaaJpliv.iJ Deli aJ hoc giucp ktiiuiHli ili nini ili. f Hmmelot, pilli, di. 1. Il va Gemici!, pini, tir. I. II. Kucinaii. Ji M Kro< en pto(. tir. H, Il ..hcl licci! eh vcrcnigJ II. M. de Konin^m la) op dinvdag ?} maar| a. de utlwick apcnin errich ten van hi'l nieuttc purl van de . Siichiinp H . . | i Dieienccniruin. en van .i% oor mannen cn O.i'X oor iouv.cn. 0c mctdingtpcrccaugc in hcl vicide ktoanval |yl v.icn v,i per IMI mannen tn 7H.S per lau raui.cn legen rep. 4H.2 oor ntannen n.ci le ia l.cl lappali gtvlcMt conciline n 71 6 voor vrouwcn in hcl icrdc dal fCHI) MCI al* V0l!tji| kailkerCI- koallaal (HkU. OC CjO r.ar<-a/j,v%*ifonmenArtnaie/Ln>\ J r Iv ig tim rr'^ o lJ t 'najrg io ir U ^ .n H .tJ rr. liadmenortr.t u r g t t h j j r n u'loi-ri i t n u t r ii u.iU.lou wckkenJc vial, luaai al ccn ugcilaamde kankcibcvnidciciidc lai hiocl Muidcn bcvvhoutvd Hit bclckcnt Jet voi lliu.iin- ccn voor de mcn ciligc doi> kan a ai deil uilgcvlitj. . Nlatlurodain De genudile1Je ciluunJoui m ' kalendcldagcn in hcl icl Je .attuai Il M. de Kamr.pin tal op ucnvdaf 7 aprii a , de ollieielc heropcmiip veiK'hlen an 'MaJurodam', lei eelc.ci.heiJ ari Ilei 111-jatif bcUan . alide mmialuulvlaj 19*1 vuoi'ntannen l?.9eii IJ.I Oor vroonvcn. In ergel.jkiug mei nei vierJe kaaiiaal Ivvo hciccriit Jii aal nunnen cn uar ruuacn ecu Juhng au icp. 0.6 cn ll.) Jagen. >i>- (nhoud "u ri. 1 i1H .i.Jm 2an 2.3 (lsitucnreuupntjsitbd1Cceozemwlnmneianmr'i:nteggeinnVveorlIvhgoo&gAiBnlgTWOfl 1>< l:ulwic>c Commi', c n . u u M N e J e il u iiJ * kcccimc cn \!CC->~' -- IlaagscliL' Sipiash Kacket Club Z K l I'm! flau der NcdciUmdcn lai op ri)Jag ly maait a v. Je uilbrcidmg au hcl compie an de liaagwhc Scoatll kackrl. l'-V ----- - Zeepost Mel Je algende .liepen kan ecp.>i orJcn ecionJen De Jala,waaiapJ coiicvpunJcniic uilcrlijk lei |a<i iliaci in bciorgd. laan aclaer Je iiaiiicu r...ii. ..mi i j . u n 1! DOH2I6Q5S4 FROM: S ta a tsco u ra n i. 9 Feb 82 II REPORT OF R .I .V . STATES: DIOX1NE NOT A COMPLETE CARCINOGEN The d a ll y a c c e p ta b le In tak e o f 2 , 3 , 7 , 8-TCDD ( d lo x in e ) fo r humans (AOI) _g I s 0 .004 nanogram (10 ) per kilogram body w e ig h t. D loxine sh o u ld , ac cording to the N ation al I n s titu te fo r P u b lic H ealth (RIV ), not be con sidered to be a com plete carcinogen, but Is a so -c a lle d cancer promotor. T h is i s th e e s s e n c e o f a r e p o r t is s u e d by RIV based on th e e v a lu a tio n o f a v a i l a b l e t o x i c i t y d ata a t a r e q u e st o f th e p a rlia m en t (2nd Chamber) o f 26 August 1981, In view o f the h e a lth r is k s o f the p o p u la tio n liv in g around the V olgerm eerpolder. This recen t report is the b a sis of the s c ie n t if ic ev alu ation of the c a r c in o g e n ic and m u tagenic p r o p e r t ie s o f 2 , 3 , 7 , 8-TCDD and th e im p li c a tio n fo r humans. I t has been se n t to the p arliam en t. In aggreement w ith the document of the H ealtli C ouncil "Advice in Case of C a rcin o g en ic C h em icals" d ated 10 November 1978, an 'ad h oc' group o f s p e c i a l i s t s was c o n s u lt e d . Titi s group o f s p e c i a l i s t s , c o n s i s t i n g o f P ro f. Dr. P. Emotel o t . P r o f . Dr. J . H. van G cudcren, P r o f. D r. j . H. Kooman, Dr. R. K roes, and P r o f . Dr. F. II. Sob*I s , i s in agreem ent w ith the con c lu s io n s t a t e d in th e r e p o r t th ac TCDD sh ou ld n o t be c o n sid e r e d a s a 'com plete' carcinogen, but as a so -c a lle d cancer promotor. This Im plies th a t s a f e ex p o su re l e v e l s o f d io x ln e fo r humans can be e s t a b l i s h e d . S ta n tsco u ra n t: D aily paper which p ublished the p roceed in gs o f prime m in is t e r and c a b in e t , and p a r l i intone, new laws and r e g u l a t i o n s , ( c f . Federal R egister.) 1 Q -i -> 195 Htoai7.Qi British Journal o f Industria! Medicine l<W3:4A: 11< K-66681-(100) ' 1983 .1 u i Notes and miscellanea Tetrachlorodibenzodioxin I would like 1 lake issue with some nl the conclusions nM ay,sp ap cr."T cu ach lo ro d ih en /o d to x in . a survey of subjects ten y ean after exposure'' (May l^ s j) Since Wl people were originally exposed sullicicntly I*develop chloracne hut only 46 remameU employ cil III years la te r. of w hom 4 1were stu d ied . u is prolut'lv misleading lo conclutlc that " ih eie Ii.is Iv e il no le.ii!i lim n neoplasm in the relevant popul Hum " Siikc the "relevant population'' constitutes active (surviving) w orkers and ahour half were lost to lollow -up. lock ot deaths should nor he surprising, even ignoring III years as on inadequate latent period for nu*st tumours to become clinically apparent. The scattcrgtims which show greater gammagiuumyliransfcrase in the exposed groups are not accompanied by any calculations of statistical signifi cance. but judging from the scatter, the difference may be significant. Further, the author assumes that this must be an artifact and that changes induced by dioxin must be transient once exposure was ceased. No evidence in support of this conclusion is given, merely an analogy with phcnobarhitol enzy me induc tion. His conclusion may or may not be correct: the evidence is not convincing. The differences in fetal outcomes also have no calculations of statistical significance, although they may well be borderline. Again. Ihcy are dismissed without adequate evidence. In short, the study is not designed to evaluate carcinogenesis, the numbers arc probably in* adequate to evaluate fetal outcomes, and there is a difference in enzyme induction between exposed and unexposed groups, which may be hut is not neces sarily related to exposure. (iXAO. /ItM School o f IlyKtene and I'uhhc Health. Johns H opkins University:. Halttmorr. Marvluiul 21205. USA h r M m tt`p l:r\. In answer to /Oeni's tirst paragraph f most point out that the text of my article includes th e phrase " th e w hereabouts of all living cases (W out id W ) being know n" and mcanscxactly what it says. Though I was prcclmlcd from mcorporating in my study those employees who hod fell the com pany. I was given every assistance in locating their current w here abouts. Ih e death which did occur was due to cardiac failure in a retiree. I am at a loss to u nderstand, therefore, how it can be misleading to sav th c ic has been no ileath ftom neoplasm. The comment about the scallcrgrams itoes not dispute, indeed, appears to support, my own tiudings it would have been easy lo say. "there are significant variations m some o f the hiochem u >1 icmiIis. and I attribute then* lo dioxin exposure a llv that evposurv was ten sea ts ago" 114 seats now i Sueh an assumption would pr.ibahly mil have been disputed and tor years to come reference would have heen made lo it hy o th er authors. A case in point is the fact that a passing reference to cholesterol concentrations in my 1V7.V article is quoted widely and regularly and out of context. I believe, however, that the biochemical variations are unlikely to he due to d io n n exposure. I have therefore considered it necessary to offer an alternate and more acceptable proposition, not a statem ent of fact. I di not see how. with such very small numbers and in the absence of national statistics, it would be possible to make significant calculations on fetal outcomes I accept /Jem 's last paragraph. The study was undertaken (or the henctit of the employees as a purely internal matter without thought of publica tion . The results were considered to he so interesting, however, that wider distribution was urged from the highest levels. I make no claims to he either an epidemiologist or a statistician hut have endeavoured to acquaint the medical community with my findings, being careful to avoid deductions which the small sample was incapable of sustaining. The continued reassuring passage of time reinforces the tenor of my article and increasingly defies the ovcrrcaction with which some greeted the original measurem ents. References * (i I'h U u j.'n c tmm t h e iw c tJc A U I pmiSKIma itiruathk.*.- iliisn/iijhuin Hr i I m i S I * J Jts-vl Ills I I I :! II \ I I I B9N2I 80311 >.*< *** i-*J 1 v-if i I Ifcl ** I / * - 4 J I. ; m .><# Pr u. ; . . i i. $ * tw \ to H .. \ . 1 * |m M h \ HI S f * sjr M 4 1 * - " 4 H r f t I.** k ! 4 ft I?- H >* !. ! # |lV' %H 4*04. II . 4- * h* | I > H I I J M !* 4 \ b* -` * ! # * . 1 M* ft 4 \t I A\,4* * ft J*#j W4'> I4 .` :i ('arcinoyciucity Ivsling of herbicide 2,4,5-lrichloropheno\yelluinol containing dioxin and of pure dioxin in Swiss mice Sl H i m e in ir o n m e n la i .otitam n:.ition in.- h a/a id in the uve id i lr m i tu c N of 2 .4 .<-ttih lo i.vh eiio | i' I I T i as w eedkiller-1 n r drluluHlN ami in a .v n ie n l\ tturmc ihi' m am d.ulure id I <1 ' h n in S r ir v i. Ila k in I mT*" * ..1 .'.* I r i u i h lu im lik -n /ii'filu n in *1 (1 )1 ) " I ilH>\in> in an niculaW c lnie leiati'cem ." anil e a k li m u ia p e n n ''h v p rin lu .l n u ll in alayN p ic 'cn l as a eontaminant VNhen 1(1* i l r n u m c v im hidiny 2.4.? -irichlorophcnox\aeti: i> id 1 2 .4.' I 1. t i e levied lor I'aivm**p 'l i in l i in it V I U .h ('.M l A.'ifd-- ami l O ' H I ** A K K *F i m uc icxpeiimenlv la-.line I n m o n iim mint' imreaved lum nuf im iJ e m c ' 1. while in another xPuI n Ihete .in a virmlieant nwreave in Ihi- ineuieme lum otrv in O I K m in' Healed u h 2 .4 .? -1"- D unne a chioni.' to v u it. Ntudy u h 1< 1)1) ia potent indueci n/m ieiiNiiinaleit/\ineN, '.in Sprague D a le \ ratssome lum outxereid'N er\ed'*. In another 2->rNiud It'D D m g e x le d al o .l * f per kg hodv eight per day cau>ed an incrraxcd im'HieiKc id hcpaloccikilar and V|U.immix yartinomax in ... . , . .in lr t Nft. K.1 leiWeUtbe____ ____ _ ___ tunvHjfv Snullrt Ju\ of Imumi Ui mT^ifir. t lunvmf' muiriHe* \fc.* re;mrl brir Ifiat ' h..lh ' l ^ In lil.iropfH'nntu II anl . I<I'l .iik in /n r.iC II |'. jih|<iti! rnluiHC Inn IvnmuiN iti nub' amr mi a ! <l<|-!i.|tiit manner in a brnc lerm Noniplai4i idten a i- elated III M-vrtr vkin lev*,min imimr.l In I t | ) | ) Ite ia u M - l< I 'l in j l n c n l in v o ln M e in a l r i il a . N in p e m ir d . Nall Vi l u l l 'd I.U lllllll' i4 llk i in K 'tlll!,i'llll|.n r !* i.' iil j ' ili v i |i v i l iii Nuii!`<Nr m l a m ! s l d r i i i i i i i i 'u !* paN 4I IlliillU t'ir!.ll'liV VlfHIUN i!a<M'M'l 4 u n il'i'M " .H IIIif | ( J*| :,,! di**Ni*i. of p u le i>" \ n i aii*i ie!i.i lv. alone e ;e ad m im -an e d I a l ' * ! I' i*l*'ii;' i. J* 1*1 :!?.i.nt-.i 11 % nl . :;lafi.in.i"lN le i! m i l I'l.l. n u ti'in l S a n - II K.'p Mine -2* * rle used it: i'Ii *u| in nl l*in n u l l ' 'un l'lo t e m a l e v eN irpl l.*f i*T'i.i| 'n ad n im iv lrted p u le iIi*nii<an.I ..i:i* o e l m! alm ie. l a .! i h i.h iniiM 'leil nl 4 ' n ialcM dlx A:::n..dv e ie k e p t in p la\l:. ..i.r v al 2? : 2 I Staiid.iid iliet ami a le : e re provided u J lib' M T l- .o:itanim ated illi d;o<im p u le d.oxm or velm le al- e re adiuiiiivleied In raN in. luN ; i - u e a r r l f*n 1 w Ammalv e re lo |lo e d up(*`i tlie lev !id IIk' ii l n e \ Anii-psies e re peifo rm ed a lle r N ponuneouN tieaSSor h e n il.e m . * e rr m o n l'u n d . and.illo:vaiiN erceN ani.!U `d iiiN :o io e:iai.. I .1 *'1 m iiio v io p s . veitHiiiN e r e vta:nr<t i:h haerr.atiivvlii. r v : A m vloid dep>*NitN * c i e nieidiiied :!!. < K ii'. le d ..'id- : poiariNed liy h l'' am i In rie itr o n m iv l'm o p v I'a ih -Jo .'iia l limiinitN e r e aNNeNNed and a r a lw e d .Tativiuaj'.: '1 he e tV .M e num h ei anim atv j v th e auinl-er >*i vurvnotN al the in:::- ilie lirNl tu n m u r .in oinerN ed S ii'r.th iam e a v e ai. ul.iled l<\ :n-.- 1 ' tcM. A illtier e rn e in lu in o u : iie tiu c n ii l*eteen eiout'v as cotiNideied Nicfiith.aiil only !ieii / ' a s levs in.i`> I Hie raivim*eeneNiN viud'. 4 1 t j r r . e d .-ul in In ree piiavev I - ? . 4 K anil *1-12* a n d nntpjriNnnN e i e m ade ith the fCN|H-ilive m n lro l y m u p v I h e I I ). id an ai'u te. p e ro ia l d *ve of K 'I'I* 1ic ii.iru n e ii 1 p p in tin 1(in in l..)2 < im c k e ' I lie lafeevi doNe I* I'l-. th al m d u ie d th n o lu e a h le IINNUC le 'io n s in the >*:ear). 1 ! i^e m n'e durinp r> m onihN id ad.-n:niNtralMHi aN 7o me pel ky 1 cal a \ th erefo re tak en a> th e toleraicd dine I [wept 1 H fi im- per k |i 4* 11 3 7no 3 4 7 11 4 7 11 6 Ii ? 7- * H ! n 1; laU e 1 t umbldior Udtk idi *.am. ji Iirdimroi Oi**tiit < r< 1< 1*n ; 1 ppf* Ibi* * 1 r r 1 ( .. .11.4 MIi* :|.* n " f 11* n*n- <> | p p m * -- < fS Itlli ! ' 1 ..* \ rhulr* m t re* kt . 4i* <0 4| 4 II <11 Jt. |M l* V* M f M t M 1 M 1 M 1 M I- M 1 M M \t M M | l*Mif > 4 mill th 1 V % M Sr*. 4 l* *#* >4 #* N* * 4> 44 44 U N.. W luflMur t**4finc Kf b*>d M *4 1. v* "v Mi *7 "I - >4 *4 14 -* 4* U. w .r c Oi* 4.'%* 4 n. \ +l 4. e V ; 2k 4 4, ;* \ ; ; .*4 s * .*4 11 4 * .. 44 4 ;. * * 4it*m fiIinI.elluk*vr n "'up\ | I .-.a > . -| i- , i r- 1 . :p-< iv ; 1 ... 4. ^ 4a 1# 44 4] 4 1 41 4.' 4 1 4 ** A 1. ;s ; ; f:. - ;. 4 . .; IA ! : ;; ,4 ; ;; - '1 *1 l *'.( a- \ n.. ; '4 ,f, i* - - ; 4 ;; ; 4 .. * X. .r- n,- , 4 .... 'i 4- : - . . *>1: * N. *i '*> 4lv 4 r4 Nii *4. 18345 I DOW 2 I e 0 3 SI w .*'a i ArnHiitS) \ I>Mt 2 1l,.*irwr *<4 xMh4mlrt**MtiM^rJ n 4nf S i1.-ni*i I K jIW II .. ------------ ffc>. in \ch * lr *4pc# kf* " V f-f l| V * i* M. 1!* " I.IH 11j : 7 !* * |..4I m * 4 MkC 4 44 44 A U a S4 A 1 4 p 1* ! 54 11 41 1 he cumulatitc data in 'I j Wc I ho- that unit I h r in* I.k-m c I if lit Cl lUlllnUtt Ilf I lit llu ll'IM irn itliJ IllIl llllclvd lH'tt'4'll t'll<U|<S Sul'M'qui-nt iiim p jiiM n n therefore iclaic l<> llic males lh c incidence i'f lite r tumours ingroups I . 2 and In as l i.c tli.il in the respective . " n i i " h groups 1 jik ! I 2 i H it 'IH j I H th*>t ul I t 1*1- and dioxin gixcn In gtoups * and > ' 1 In and l . !<' of maximum tolerated dosci caused less frequent tumours. I here t i t no significant differences from groups ? anil s 1 here t i e no significant ihttcfcnccs in lhc frequency ul lo c i tumours h t t t t n groups I and 2 igtscn d xiu n at 0 112 and <>i n i ' per kg. 1 groups 4 .mil I h t lalSCI t o groups K ic n c d tiju a l amounts of K .'l'l hut group 4 t c .r n i'd ltm limcs.ni.' i t tlioxin than did |!li>up 5 Thus, dunlin in this dose taupe ilui not inllutntc tumour frequency. (roups 1 1 and 2 received equal amnunls of diosm id in i* Vf pet k c hut there as no sigmkcant difference in liter tumour iniid c n ic heteen croup I I and its control, group 12 Thus diosm has no l o t i tumour enhancing ctfcci at this dose The iiHicuscd incidence ol liter tumours in group 2 as caused by 1 ( T l . and not by diosm In gioup I " , inch received ItMi times n io it vliosin than did group I I <> ? ug per kgi. Iite i tumour incidence as licc that in the control, group 12 11)0 dioxin has a Ittet tumour enhancing v tle tt at a higher dose. Diosm tnitiutnmalion in gioup 4 as less than n t jh-i k g - the thieshold dose ol ' I ( ' D I ) in nut espcrimcnt --and its hepatocarcinogenic cried could n>t he ohsetted. Sp>>ntancous and induced lite r tumours ere not histologic al] Jitlercni. The latio of l>cnien hepatomas to hepatocellular carcinomas in the control groups as not atlected by treatm .:U. onlt the absolute number >( lite r tumours increased S o cirr hosis as seen concurrently irh tumours. Average life span decreased considerahls in group *1iTat 'c 11 hich received the largest dose of 1 ("D O . T ( 'D I ) caused severe chronic, ulcerous skin lesions iprohahly similar to thloracre in humansi followed ht generalised lethal amyloidosis iTahlc 2i. h u h tan he regarded as a ptiHess secondary to chronic skin lesion This group as not evaluated in the carcinogenic hioassat for early death l 'n lil more is known about the people h o have been exp*>vcd to them, the carctnogenicily of 2.4.5 -T and structurally related chemicals in humans cannot he et aluated*1* Hut it is clear that not only dioxin contamination, hut also exposure to `lC P L during Us production or use should he controlled and reduced Amyloidosis should he considered a p**ssihle later complication of the most setere fc.in.sof human chioraenc induced ht I I 1)1) A ptclimmary report of this studs a t ptetented at the International Conference on l-.cologicai I'cl'pcclncs on ( arcinogent and Cancer Control in l remona in | v ? l " This ork a t initialed ht the laic I'roft-ttor H iscliner We thank Mrs Hose (iaa l Sraho lor technical assisiancc i ' M m l ` ? \4 **** I ' 1 M* H 4/ I*.; *. iTJ 2 4 | M . I** .* 14 * IV* *4 H . 4 \ *--H*i4 .* * m H ..*: . * # . 445 < 4. 4k. * I I*!. i \ j* 4 n I 'm M * . fW *# i f I t w I , Sk M . a v * ** .. I I | 4 l i - M 4 Ik f . . %.i* 4 4 i ; - l M i V I U m < H i M e t h , , | I | . .*..* t l U | t ^ i I 4 ^ * , I *; t IV * ; ! V i * I I I f * , a ' h a t * ** * I*' * II t+f I0 -- / *M*Ma # 4 |. : |*| 11*4 lv* * 1: M t< ( ! ' - I I* U . ( i 1 ^ ` H I | 4 r j I . # M . . # M . | * * A . 1I S 4 4 k A A 14 *.M r I I 1 . I J* I k -.* ! ] / . IA k.m*. /* 1 A U p # I I . A 4 IV' S | A A . - . | k 4 w -M !*# V ` * 44 ; **. 4. # -*-.a 4 ;# l-.** C l |4* | .. M ^ in SH (. a \ A*. V* ) I*l " 4 . e x . * , 4 . 4 . A r # 1 I .* f s a - M M. I l l a | , # . 4* 4- f . M a k ^ t -#A 1| a a m * U h. - ( a * . . * *.*4 . . .A - , # * . * r . . . - I . , , , . . . U a ` 1 1M>. iUalf. . UA. . I* l!i , . . 'I t* ' * 2 * .#.* 4 / 4 -Am . J t* . : I x . 'j M o t IV M . t \ m'.M 2 II \ \ mmtV ; i ii# . a Nm. . m |AV 4 4 * . 2AV. *4 ** n . s ' * : . ' | / l f . It ' A t i t | N. ! . 1 , 1 V A 4 f U , l A* Hxe. |.v* I n V A / 'S a '- t a 14. 4 l V4 K Tom M r .St 'SAN S 'lS Il-A I-K l t I I J ah Jt t >1I 111 M I Rr\tunh InMituU- ..r ( r>u..p.iih...`..ct. Ruth fn.agi ?y. / / - / / . V l l u J u f f jr. /fu 'ic j m , M* - ,, I ..irv I I I 1 * 197 K-66681-G00) 1983 ; 0OH2l6O5it B ritish Jou rn al-ef Industrial Medicine 0 0 M2 160513 9^1 British Journal o f Industrial Medicine 1983;40:318-324 Medical and morbidity surveillance findings among employees potentially exposed to TCDD G G B O N D ,' M G O TT ,' F E B R E N N E R .1 AND R R C O O K ' From US Area Medical' and Michigan Division Medical,* Dow Chemical USA, Midland, Michigan 48640 USA a b s t r a c t Available medical and morbidity surveillance findings from 1976 to 1978 for two employee cohorts potentially exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were compared with those of matched unexposed employees. The medical surveillance findings were derived from a screening programme offered to all active employees and included an analysis of various,medical history questions and blood chemistry results. Group medical insurance claims served as the source of morbidity surveillance data and the period prevalence of selected diseases was analysed..Few significant differences between the exposed and unexposed were detected. Among. the-cohort of employees potentially exposed during the manufacture of 2,4,5trichlorophenoxyacetic acid (2,4,5,-T), a significantly greater frequency of x-ray proved ulcer was reported and significantly more members of this group had diseases of the digestive system diagnosed. Such findings Were absent in the more highly TCDD-exposed cohort engaged in 2,4,5-trichlorophenol production, making it unlikely that dioxin was a cause. The trace impurity 2,3,7,8-tetrachlorodibenzo-pdioxin (T C D D ) is formed as a byproduct during the manufacture of 2,4,5-trichlorophenol (TCP) under alkaline conditions at' raised tem peratures and high pressures. Since TCP is a precursor for the produc tion of the herbicide 2,4,5-trichlorophenoxy acetic a d d (2,4,5-T), low level contamination of the herbicide by T C D D is expected. Thus the potential for occupational exposure-'to TCD D may arise among employees.making either TCP or 2,4,5-T. Recent evidence suggests that TCD D may also be formed during the routine combustion of car bonaceous fuels.1 Chlorinated dioxins have been found in trace amounts in samples gathered from incinerator stacks as well as from chemical ta r bur ners and a fossil fuelled pow er plant, vehicle silencers, fireplaces, chimneys, dgarette smoke, and portions of charcoal broiled steaks. Mortality studies and case summaries of occupa tional cohorts presumably exposed to high levels o f TCDD during the production of TCP, or exposed to lower levels during the synthesis of 2,4,5-T, have been published*- * (J A Zack, unpublished observa tions). Reports have appeared of soft tissue sarcoma among employees with exposure severe enough to Received 11 A u fu u 1982 Accepted 30 September 1982 cause chloracne*- '; however, no consistent unique mortality patterns have been noted among those with lower level exposure* (J A Zack). A survey of reproductive outcom es among wives of employees exposed to dioxins was also recently completed and indicated no biologically im portant assoaations.1* Available evidence suggests that occupational cohorts exposed to low er levels of T C D D in the manufacture of 2,4,5-T experience no important long term health risks. Investigations, inducting the evaluation of health examination findings and morbidity data for TCD D -exposed individuals with long latency, have only recently been sta rte d ." Q inical follow up 10 years later of 41 o u t o f 79 individuals who developed chloracne in a 1968 TCP production aeddent, showed that other than minor persistent chloracne, there was no evidence of adverse health effects." While relatively little data are available from sound epidemiological follow up of other exposed cohorts, reported systemic health effects after exposure to large amounts of TCDD indude impaired liver function, nephropathy, gastrointesti nal irritation, myopathy, and neuropathy, inducting depression and irritation of the central nervous sys tem. These symptoms have not been progressive and have been reported to disappear with time. Vietnam w ar veterans, presumably exposed to lower levels of 318 18348 .... ... i.. * Z S 0 9 l l NOS Medical and morbidity surveillance findings among employees potentially exposed to TCDD 319 TCOD and for shorter periods than industrial been considered separately in the analysis to allow cohorts, have alleged a number of medical for differences in their potential for TCDD expos complaints--for instance, weight loss, liver damage, ure, and for differences in their exposures to other recurrent rashes, deformed offspring, stillbirths, agents. cancer, sterility, personality changes, and 'other" Data of interest were derived from two separate illnesses--which they attribute to their exposure to sources: health examination findings from the ' Agent Orange." To date, there have been no well routinely administered medical surveillance pro , controlled epidemiological studies to substantiate gramme and morbidity surveillance as reflected in the allegations that these complaints are attributable diagnoses from external medical service providers to exposure to the trace amounts of TCDD in Agent reported for payment of fee to the group insurance Orange. department. Medical follow up of inhabitants of Seveso The medical surveillance examination has been exposed to TCDD as a result of an explosion of a offered to all employees since 1967. The time trichlorophenol reactor has continued to document required for the programme to cycle through the a lack of severe lasting health effects. While the entire manufacturing complex is about two years. findings are still regarded as preliminary, no organs Only cohort members who had participated in a or body functions, except the skin, have been found medical surveillance examinadon between 1976 and to be impaired; no excessive derangement of gesta 1978 were eligible for consideradon for this pordon tion, fetal lethality and loss, gross malformations, of the study. growth retardation at term or cytogenetic abnor Controls for the medical surveillance analyses malities have yet occurred." were selected from among other white men em The present study reports the cross sectional ployed at this location who had also participated in medical and morbidity surveillance findings from such an examination between 1976 and 1978. Em 1976 to 1978 among employees at Dow Chemical, ployees considered at high risk because of a history Michigan, who were considered potentially exposed of a job assignment as a pipe coverer, in the produc to TCDD and for whom mortality surveys have been tion or packaging of arsenical pesticides, considered reported.2' The objective of the present investiga as potentially exposed to high levels of vinyl tion was to determine whether patterns of selected chloride, or potentially exposed to dioxins were health examination findings from the routinely excluded from consideration as potential controls. administered medical surveillance examination, and Four controls were then matched from those eligible indices of morbidity as reflected in medical insur to each exposed on year of birth five years, ance claims for employees potentially exposed to whether hourly or salaried, smoking habit (never TCDD, differed from those of employees not smoker, ex-smoker, current smoker) and, when exposed. possible, month and year of the most recent medical Methods and materials surveillance examination taken. When more than four controls were eligible for matching, the four The study population was assembled from two pre with the date of hire nearest that of the exposed viously identified cohorts: (1) a group of employees were selected. who had been engaged in the manufacture of Based on a review of relevant reports on animal 2.4.5- T for at least one month between 1950 andand human exposure to TCDD, seiected medical 1971 and (2) a cohort of employees involved in a history questions and blood chemistry results were 1964 chloracne incident in the area of TCP produc considered for analyses. The matching was retained tion. throughout. For the dichotomous questionnaire The methods for the selection of these cohorts responses (yes or no), the analysis was that which have been described previously.2* Briefly, the 204 has been proposed for a fixed R:1 matching ratio white men comprising the 2,4,5-T cohort were with R = 4.14 Exact confidence limits were calcu : identified from annual department census lists and lated with a = 0-10 using a packaged set of pro work history records as having worked one month or grammes for a programmable calculator.12 Blood longer between 1950 and 1971 on one of four job chemistry values for each exposed subject were con assignments (reactor operator, salt wheel operator, trasted with the mean for the four matched controls, add wheel operator, or dryer operator) in the and a paired (-test was used to test the hypothesis 2.4.5- T process. The TCP cohort of 61 white male that the mean difference between the exposed and employees was identified from monthly department matched controls was zero. Linear regression was census lists and lists of maintenance personnel who done to test the correlation between log, (days of were known to have worked in the process area dur employment in 2,4,5-T production) and the differ ing a 1964 chloracne incident. The two cohorts have ence in blood chemistry values. i 8349 | 320 Bond, On, Brenner, and Cook G roup medical insurance claims filed by em Tabic 1 Vital and employment status o f exposed cohorts ployees for medical expenses incurred between 1 as o f 3 1 December 1978. (Percentage o f original cohorts in January 1976. and 31 D ecem ber 1978 served as the parentheses) IZfi09 IZMOQ source of morbidity data. M embers of the original study cohorts who were eligible for benefits on 31 Status TCP cohon 2,4.5' T cohon wX D ecem ber 1978 from the Medical C are Programme Original administered by the group insurance department Employed Rented 61 204 40 116 II 21 were included for study. This time restriction Deceased 4 11 ensured that morbid events reported were indepen Left other than through itttremenc 6 Deceared 0 56 2 dent of m ortality as reported in earlier studies of Known alive 62 these cohorts.1* Controls for morbidity comparisons were selected from among all white male employees Unknown 0 Pattidpated in tnctfcal surveillance exam 27(44) 52 87(43) eligible for Medical C are Program m e benefits as of Eligible foe medical care 31 D ecem ber 1978. Em ployees with a history of programme and morbidiry surveillance 48(79) - 135(66) certain job assignments were excluded from con sideration as controls, as has been described above. From among the pool of eligible controls, four were additional follow up with company records of the matched to each exposed on the basis of year of 2,4,5-T cohort identified two deaths not previously birth - five years, and w hether hourly o r salaried. reported--one from cerebrovascular disease and the When m ore than four controls were eligible, the other from cardiovascular disease. Some 44 % of the four with the date o f hire nearest that of the exposed original 61 members of the T C P cohort and 43% of were selected. the 204 members of the 2,4,5-T cohort participated Diagnoses and expense dates were abstracted in medical surveillance exam ination between 1976 from Medical Care Programme claims by a regis and 1978. Of those who were actively employed and tered nurse, who was unaware of the exposure status thus eligible to participate, 68% of the TCP cohort of the subjects. Only diagnoses for.ilaims for hospi and 72% of the 2,4,5-T cohort did so. These partici tal inpatient care, hospital outpatient care, surgery, pation rates are significantly lower than the partici ! diagnostic expenses, o r emergency first aid were pation rate of 80% among all o th e r white men em considered. This excluded diagnoses from physician ployed at this location (p m 0-04 and p 0-02, office visits, unless diagnostic expenses were incur respectively). O f the 49 individuals in the original red or surgery was done, and inform ation from drug TCP cohort who had evidence o f acne like lesions in prescriptions. No validation of diagnoses with any 1964, 22 participated in a medical surveillance external source was attempted.: examination. Nearly all mem bers of both cohorts Coding of diagnoses to the ' international (94% of TCP and 97% of 2,4,5-T), who were active classification o f diseases-- ninth revision-- clinical or retired, were eligible for medical care programme modification (IC D -9-C M ) was done by trained cod benefits and thus could be surveyed for morbidity in ers w ithout know ledge of the exposure status of sub this study. jects. Diagnostic categories of interest were derived Table 2 presents a comparison of demographic based on the relevant animal and human toxicology data for the subsets of each original cohort eligible data. for the medical surveillance examination and for An individual could contribute, at most, only one morbidity surveillance and their matched controls. observation to each diagnostic category. Period pre The exposed cohorts and their matched controls valence o f all cancer, various site specific malignan com pared well with respect to all variables consi cies, diseases o f porphyrin metabolism , digestive dered. In the analysis, potential confounding by system diseases, hepatic disorders, renal disorders, cigarette smoking, age, year of hire, and and diseases of skin and subcutaneous tissue was socioeconomic status was controlled by matching compared, using an analysis that maintained the and stratification on these variables or correlates. m a tc h in g .14 M EDICAL SURVEILLANCE FINDINGS Results Table 3 presents a comparison of the frequency of posidve screens with selected questions from the DEM OGRAPHIC CHARACTERISTICS medical surveillance examination for the two Table 1 presents the vital and employment status exposed cohorts and their m atched controls. The distributions for the two cohorts, together with frequency of positive screens was low for nearly all eligibility for the medical surveillance and morbidity the questions, and the exposed and controls surveillance portions of the study. Two years of responded comparably. The only statistically 4 . 8 - 3 . 0 - ........ ................... P0W2160526 M edical an d m orb id ity surveillance findings am ong em p lo yees p o ten tia lly exposed to T C D D Tabic 2 Comparison o f exposed cohorts and their matched controls on selected demographic variables Demographic variable M ed ial surveillance: No eligible for study Mean ace - SD Year h ir e Salaried Paid by (be hour Current smoker Ex-smoker Non-smoker Morbidity surveillance: No eligible for study Mean age s SD Y evofhire Salaried P u d by the hour TCP cohort 27 45-* 2 7-6 1953-6 2 7-9 5 22 6 10 ii 48 49-2 2 9-5 1951-4 = 8-0 5 43 C ontrols 108 45-4 = 7-8 1953-3 2 9 2 20 88 24 40 44 192 492 2 96 1951-7 2 10-5 20 172 2 .4 ,5 -T cohort 87 42-8 - 8-2 1957-1 = 8-5 14 73 25 37 25 135 45-3 2 10-2 1956-7 2 7-9 17 118 Controls 348 42-8 = 8-3 1956-6 2 10-0 56 292 100 148 100 540 45-3 2 10-2 1955-0 * 10-6 68 472 321 Table 3 Frequency o fpositive screens to selected medical surveillance examination questions among TCP and 2,4,5-T cohorts and matched controls, most recent exam 1976--8 Q uestion Since your last Dow physical exam or health inventory, have you had any of the following? Skin trouble Yellow jaundice Problems with urination Albumin or sugar in urine Blood in urine Frequent indigestion or heartburn Intestinal trouble L o u oi wdght Coughing up of blood Change in bowel habits Blood in stools - Tany stoob Do you have or have you had any of the following? High blood pressure Liver trouble (hepatitis) Kidney or bladder trouble Cancer or malignant growth x-ray proved uteer TCP cohort N e In 27) 2 0 o .; 0 0 0 0 0 0 0 0 0 1 1 2 0 2 Nc In - 108) 2,4 ,5 -T cohort jVf (n - 87) Nc (n - 348) 5 6 14 00 0 0l 5 00 3 l0 2 5 4 19 0 1* 2 43 6 00 1 00 0 10 2 10 1 7 6 24 4 2 12 7 5 19 00 3 7 9* 18 *RmL ' M l : Xm h * ITS. 9 0 * a - 1-06-4-20. Ng - Number of cipaecd responding " Yea." Nc - Number o( controls responding "Yes." significant difference was the greater frequency of regarded as clinically significant. Given the number I positive screens for x-ray proved ulcer among mem of comparisons done and the level of accepting a bers of the 2,4,5-T cohort ( R ^ " 2-11, 90% Cl = type 1 error, some statistically significant differences 1-06-4-20), based on nine of those exposed having were to be expected. reported such a history. As asked, the question of When the mean difference between blood chemis x-ray proved ulcer does not discriminate between try values for the exposed and their matched con incidence or prevalence, so the temporal relation trols was regressed against log,, (days of employ between exposure and disease is not known. ment in 2,4,5-T), there was a lack of significant Table 4 presents a comparison of results for nine association for all laboratory tests except for a selected blood chemistry tests performed as a part of significant positive slope for blood urea nitrogen the medical surveillance examination. Mean total (p 0-022); however, the independent variable bilirubin was slightly lower in both exposed cohorts explained only 6% of the variability in the depen and mean serum aspartate transaminase was lower dent variable, suggesting that duration of employ in the 2,4,5-T cohort than in their respective con ment in 2,4,5-T was not an important determinant. trols; and while these differences were statistically Analyses of medical surveillance findings among significant at a =* 0-10, two tailed, they were not only those members of the TCP cohort who had 1i U Q K JLQ DON 2 1 6 0 5 1 7 322 B o n d , O n , B r e n n e r, a n d C o o k Table 4 Comparison o f exposed cottons widt matched controls for nine selected laboratory tests, most recent medical surveillance examinations 1976-8 T a t procedure Trichlorophenoi cohort M sSD Total bilirubin (mg/100 mi) T o u i protein (f/100 ml) P roton albumin (g/lUO ml) Blood urea nitrogen Lactic dehydrogenate (mi-u/ml) Serum aspartate inum m inatc (Wmi) Seram alanine transaminase (u/mJ) Alkaline phosphatase Albumin-globulin ratio 051 x 017 (n - 26) 7-17 * 0 4 5 (n - 26) 4*16 0 4 3 (n - 26) 1008 x >79 (a - 26) 114-7 2 30*4 (n - 26) 27-69 - 8-84 (a - 26) 32-26 2 13-34 (d - 23) 7 0 8 8 x 21-52 (a - 26) 1-38 x 0 2 3 (n - 23) (n Number o( matched quintuplets) Trichlorophenoi cohort lest cootrob d 2 90%a -008 r 007 004 2 016 0002 2 012 0 3 7 2 1-34 4-08 2 6-75 5-32 2 6-39 1-21 2 > 20 - 0 3 6 2 6-18 -0008 008 2 .4 .5 -T co h o n M s SD 0 5 0 - P-21 (a -84) 7-10 x 0 3 3 (a - 84) 4-11 x 0 3 3 (a - 84) 16-07 x >64 (a -84) 108-3 x 2 > 9 (a - 82) 29*99 2 7-72 (n - 85) - 33-24 x 14-66 (a - 79) 7 1 0 5 x 24-13 (a - 84) 1*40 2 0 3 2 (n - 78) 2 ,4 ,5 -T cohon le u control d 2 90% a -0 0 6 2 004 -O O l 007 -005 x 006 061 x 065 -->67 2 4-49 -2-53 x 1-83 0 5 9 2 2-95 0 21 2 4-54 -0003 2 006 Table 5 Comparison o f number o fprevalent cases o fselected disease conditions by exposure group: diagnoses from MedicoI Care Programme, 1976-8 Discaic category /Vo o f prvoient cases TCP cohort N e (ft - 4 8 )' N c (n - 192) Malignant neoplasms (140-209) Mn oi fiver (155) Mn of trachea, broochus. and hing (162) Mo of connective and other soft osane (171) Ma ot kin (172-173) Malignane lymphoma (200-202) Diseases of porphyrin metabolism (277-1) Diseases of the digestive system excluding fiver disease (530-535. 553-558. 564-1) Disorders of the liver (570-573) GkHnenilooephrids (580-583) Renal failure (584-586) Infecboos of kidney (590) Diseases of skin and subcutaneous dames (680-686. 692. 695) 1 0 1* 0 0 r 0 3 0 0 0 0 0 4 0 0 1 1 0 0 10 5 0 0 0 2 Refer to a angle individual. t RML " M l *U H " 2-957, 90% a - 1-50-4-18. Nf: - Number a< pcevmietK canea amoog expoacd cohort. Nc - Number a l prevaleat cases among controls. 2,4,5-T cohort N g <n - H i ) 4 0 1 0 0 0 0 17 2 0 0 0 2 N c (n - 540) 9 1 1 0 2 0 0 27 5 0 1 0 11 exhibited acne like lesions in 1964 produced similar results to the analysis of the total eligible cohort. MORBIDITY SURVEILLANCE FINDINGS Of the 913 members of the exposed cohorts and the matched control groups eligible for Medical Cars Programme benefits, 656 (71-7%) filed at least one claim for medical services during the study period 1976--8. A total of 6539 separate diagnostic entries were abstracted and coded and constituted the total morbidity data base from which the analyses of con ditions of interest were carried out. The diagnostic entries were distributed fairly evenly across the study years with 2126 entries in 1976,2245 in 1977, and 2168 in 1978. Most of the diagnoses were on claims for physician's services (58-4%) and daims for hospital services (35*3%), with few diagnoses provided solely from laboratories (4-7%) or other sources (1-5%). A bout one quarter of all diagnoses provided were preceded to some disease categorisation scheme by the service provider. Table 5 presents a comparison of the number of prevalent cases of disease conditions of interest for each o i the exposed cohorts and their matched con- DOW?I 6 0 5 2 8 Medical and m orbidity surveillance findings am ong em ployees potentially exposed to TC D D 323 Table 6 Detailed breakdown o f diseases o f digestive system among 2,4,5-T cohort and matched controls. Diagnoses from Medical Care Programme, 1976-8 irritating, and were believed to have resulted primarily from finishing operations where the end product was dried and fed through a ham m er mill. Disease category bio o f prrvaUni eases biE NC O ne could postulate th at ingested dust could lead to a greatet frequency of diseases of the digestive sys tem. The 2,4,5-T exposures, however, were incurred Diseases of oesophagus (530) Gastric ulcer (531) Duodenal ulcer (532) Pepc ulcer, rite unspecified (533) GastrojejunaJ ulcer (534 ) Gastrins and duodeuris (535) Regional entends (555) Other non-iofeebous gasrroentcrios and cott (558) Irritable colon (564-1) 2 2 3 2 0 3 0 6 4 1 3 3 7 0 4 1 10 5 by subjects in this study a minimum of five years before the indication of disease prevalence, thus making latency an im portant issue. A lim itation of cross sectional studies is the uncer tainty surrounding the tem poral sequence in the exposure disease relationship. A s was the case in this study, it is usually not possible to discriminate between disease incidence and prevalence, so that Note: An individual may have more than one condition. Ng Number of prevalent cases among 2.4,5-T cohort. N c " Number of prevalent cases among matched controls. one is uncertain w hether or not the exposure p re ceded the disease and could possibly be a cause. Data on health outcome from medical and mor bidity surveillance of the two cohorts in the present trol groups. The period prevalence of m ost'condi study were limited to the active or recently retired tions was low and was comparable between the w orkforce. M em bers of the two cohorts who left the exposed and their controls. An exception was the company for reasons other than retirem ent could significantly greater prevalence of diseases of the not be studied. Inferences from the present study digestive system excluding liver disease among the concerning the health status of form er employees 2.4.5- T cohort (RML = 2-51,90% C l = 1 -5 0 -4 -1 8 ) must be draw n carefully owing to possible retire based on 17 prevalent cases among the exposed. m ent of form er employees on the basis of health. Table 6 gives a detailed breakdown of the distribu The study was further limited to medical and tion of prevalent cases among the 2,4,5-T cohort morbidity surveillance for a three year period and their controls by disease within this category. (1 9 7 6 -8 ). Owing to the large am ount of time The difference in prevalence cannot be explained on required to locate and code archived medical claims, the basis of a preponderance of any one specific dis a larger sample was deem ed unfeasible. ease entity; instead, the 2,4,5-T cohort had rela With the completion of this study, these em tively m ore cases of each condition. ployees have now been surveyed for morbidity, m or tality, and reproductive outcomes. To date, the Discussion available data do not indicate serious long term health risks associated with exposures to TCD D at Results from this cross sectional examination of the levels encountered by these two industrial medical and morbidity surveillance findings among cohorts. C onsideration is being given to continued two cohorts who were potentially exposed to TCDD medical and morbidity surveillance of these cohorts, at some point in the past and their matched unex and we recom m end that oth er cohorts with similar posed controls showed few differences. Those em exposures also be surveyed. ployees who were engaged in 2,4,5-T production at some point, between 1950 and 1971, reported the We gratefully acknowledge the contributions of Mr prevalence of x-ray proved ulcer significantly more Bernard Lasich, Mr Stewart G udm undsen, Mrs often than their matched controls, and recorded Janice C anm ill, Mrs Jean Townsend, Mrs Elsie significantly more diagnoses of diseases of the diges McLaren, D r Robert Kolesar, and Mrs Angie tive system. The lack of a similar finding among the Grzegorczyk of Dow Chemical, U SA, and of Mr more highly TCD D -exposed in the TCP cohort Larry Silverstein, formerly of Dow Chemical, USA, makes it unlikely that the dioxin is associated with and now with the Dow Com ing C orporation. this finding. The departmental unit under which the 2.4.5- T process was first organised was also respons ible for manufacturing various other products. Thus many of the individuals in the 2,4,5-T cohort were References potentially exposed to numerous other substances during their employment with this unit. The 2,4,5-T dust concentrations found in the 1Bumb RR. Crummetl WB. Cur.a SS. u at. Trace chemistries o( fire: 1 source of chlorinated dioxins. Science 1930:210:385^-9. Cook RR. Townsend J C O il MG. Silverstein LG. Mcriality plant were often high enough to be noticeably experience of employees exposed lo 2.3,7,8-ietrachloro- cL 83- t*gyB4Qg"iiy,';' rra^y~ 324 dibcazo-p-dioxiti (TCDD). JOM 198003:330-2. *Zack JA. SuxkinJ RR. The monaliry experience of workers exposed to tetradborodibm&Ddioxins in a cnebiorophenoJ process a c o d n t. JOAf 1980:22:11-4. *Theu s AM, Frc&Ocl-Bcymc R. Mortality study of persons exposed to dioxin in s irichlorophenol-process scddenl that occurred in the BASF AG on November 17, 1933. Am erican Journal o f Industrial M edian* 1982;3:179-89. * Honchar PA, Halpenn WE. 2.4.3-T. Trichiorophenol and soft-dsnie sarcomas. Lancet 198l;i:268-9. *Cook RR. Dioxin, chlorscne, and soft-tissue sarcoma. Lancet 1981;1:618-9. *Cook RR. Soft-tm oe sarcomas: chics and caution. In: Tucker R, Young A L, Gray AP. eds. Environm enm i science research end theory. Vo4 26. Human and environm enm i ricks o f chlorinated dioxins and related com pounds. New York: Plenum Publishing Corporation, 1983. 1 International Agency for Research on Cancer. Evaluation o f the cardnogenie risk o f chem icals to hum ans. Som e fum igants, the herbicides 2 ,4 -0 end 2.4,J-T , chlorinated dibentodioxins and m iscellaneous industrial chem icals. Lyon: (ARC, 1977. (Monograph 13.) Bond, Ott, Brenner, and Cook * Ott MG, Holder BB, O bon RD. A mortality analysts of em ployees engaged in the manufacture of 2.4.5-mchloro phenoxyaeebe add. JOM 198002:47-50. __ " Townsend JC, Bodner KM, Van Peenen PFD, O boo RD, Cook O RR. Survey of reproductive events cf v iv o of employees ^ exposed to chlorinated dioxins. Am J Epidem iol 1982:115:693-713. * 11 C ounbl on Socnoiic Alfairs Advisory Paoei on Toxic Sub PO stances. The health effects o f "Agent Orange'* end potycH or. - nosed dioxin contam inants. Chicago: American Medical Association, 1981. 03 " May G. Tctrachlcrodbcnsodioxin: survey of subjects ten years after exposure. Br J Ind Med 1982:39:128-35. 11 Huff JE, Moore JA, Saracri R, Tocisb L_ Long-tens hazards of ispolychlorinated dibeszsdioxiss and polychlorinated dibeo- zofunas. Environ H eath P c n p e c i 1980;36:221-40 14 Miettinen OS. Estimabon of reiaave rule from individually matched series. B io m e o ic s 1970:24:73-86. 11 Rothman K. Boies J. Epidem iologic analysis widt a programme able calculator. Washington: US Government Printing Office, 1979:19-24. (N1H Pubfccaoon No 79-1649.) I Q.K. D0N2I 60S IF lit. i D^olc book is | o c ^ < - d ir\ + h c J o / Library Jfco3 Building 198 ^-ijuiK ioo) n ^ i ] Strtdies on the effect of A 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on vitamin A: a new aspect concerning the _mechanism of toxidty by Tuuia Thunberg ier u^- STOCKHOLM 1983 DON21605II STUDIES ON THE EFfECT OF 2,3,7,8-TETRACHLOROOIBENZO--OIOXIN ON VITAMIN A: A NEU ASPECT CONCERNING THE MECHANISM OF TOXICITY by Tuula Thunberg, Dr.Med.Sci., Department of Toxicology, Karolinska Institute, S-10A 01 Stockholm, Sweden ABSTRACT Chlorinated dibenzo-p-dioxins have been claimed to be ubiquitous in the environment. The aost toxic of totally 75 possible isoaers is 2,3,7,8tetraehlorodibenzo-p-dioxin (TCDO). TCDO is formed during the industrial preparation of 2,4,5-tetrachlorophenol (TCP), which is used as a starting Material for the Manufacture of the herbicide 2,A,5-T and the gernicide hexachlorophene. The presence of TCDD has also been demonstrated in fly ash from Municipal incinerators. This thesis summarizes studies on the effect of TCDD on vitaain A. TCDD causes a dose-related reduction of hepatic vitamin A in the rat. The potency of TCDD in reducing the vitaain A level in the liver is aore than A x 10^ tines that of phnobarbital (PB), and 3 x 104 times that of 3-methylcholanthrene (3-MC), two well-known enzyme inducers. However, no correlation between the UOPGT or AHH-induction and the reduction of hepatic vitamin A can be found. Dietary vitamin A can partly coapensate for the TCDO-induced reduction of vitaain A in the liver, suggesting chat TCDD does not cause a reduction in the hepatic storage capacity of vitaain A. The level of vitaain A in serum is not decreased by TCOO-treatment, indicating that the synthesis of the specific transport protein for vitaain A in the liver is not impaired by TCDO. Studies in guinea pig, rabbit, hamster, Sprague-Oawley and Gunn rats reveal a corre lation between the TCDO-induced vitamin A reduction and its toxicity in these animals. Further evidence for TCDO-induced vitamin A deficiency was obtained by the finding that TCDO-trcataent produces a similar effect on renal vitaain A level as does a vitaain A-deficienc diet. The discovery of the TCDD-induced vitamin A deficiency may explain some of the signs in TCDD-intoxication, and may also prove to be important in evaluating the carcinogenic properties of TCDD. Key words: TC00, Vitaain A, Dioxin, Toxicity, Liver, Kidney, Serum, Enzyme induction ISBN 91-7222-588-2 0 0 H2 I 605 IB 0 STUDIES OH THE EFFECT OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ON VITAMIN A: A NEW ASPECT CONCERNING THE MECHANISM OF TOXICITY I Akadeai sk avhandling ! so fdr avlggande av doktorsexaaen i aedicinsk vetenskap vid Karolinska Institutet offentligen frsvaras i Rtts*edicinska Institutionen* frelasningssal, Doktorsringen 16, Karolinska Institutet tisdagen den 24 aaj 1983 kl 9 av Tuula Thunberg nat.kand. STOCKHOLM 1983 Ov v & 2 0 9 \ ZHQ PRESENT STUDY AIN II COMMENTS ON METHOOOLOGY 1. Animals, treatment and diet 2. Assessment of vitamin A status 3. Preparation of microsomes 4. Enzyme assays 29 29 29 III RESULTS AND DISCUSSION 1. Dose and time-related effect of TCDD on vitamin A 2. Effect of dietary vitamin A 3. Comparison of TCDD with other xenobiotics 4. Studies in different species 5. The role of enzyme induction 6. Comparison with dietary vitamin A deficiency 33 IV CONCLUSIONS REFERENCES 42 43 APPENDIX SEPARATE PUBLICATIONS 183 K CONTENTS PREFACE 5 LIST OF SEPARATE PUBLICATIONS 7 ABBREVIATIONS 8 INTRODUCTION 9 I TOXICOLOGY OF TCDO 1. Wasting syndrome 2. Immunosuppression and lymphoid involution 3. Epithelial lesions 4. Hepatotoxicity 5. Reproductive abnormalities and teratogenicity 10 II CARCINOGENICITY OF TCDD 17 III VITAMIN A DEFICIENCY 1. Growth 2. Immune response 3. Epithelial cells 4. Hepatic lesions 5. Reproduction 19 IV VITAMIN A AND CANCER 25 V MECHANISMS OF TCDD-TOXICITY AND VITAMIN A FUNCTION 24 -a. - * ' v V ' f. DQH2I6052I f -r 199 D0 H2 I 60 5 1 British Journal of Industrial Medicine t K - 6 6 6 8 1 --f l 0 0 ) :" l 983 9IS09U100 B r itish J o u r n a l o f I n d u s tr ia l M e d ic in e 1983;40:116 Notes and miscellanea Tetrachlorodibenzodioxin I would like to take issue with some of the conclusions in May's paper, "T etrachlorodibenzodioxin: a survey of subjects ten years after exposure" (M ay 1982). Since 90 people were originally exposed sufficiently to develop chioracne but only 46 rem ained employed 10 years later, of whom 41 w ere studied, it is probably misleading to conclude that "there has been no death from neoplasm in (he relevant population." Since the "relevant population" constitutes active (surviving) w orkers and about half were lost to follow -up, lack of deaths should not be surprising, even ignoring 10 years as an inadequate latent period for most tumours to become clinically apparent. The scattergrams which show greater garamaglutamyltransferase in the exposed groups are not accompanied by any calculations o f statistical signifi cance, but judging from the scatter, the difference may be significant. Further, the author assumes that this must be an artifact and that changes induced by digxin must be transient once exposure was ceased. No evidence in support of this conclusion is given, merely an analogy with phcnobarbitol enzyme induc tion. His conclusion may or may not be correct: the evidence is not convincing. The differences in fetal outcomes also have no calculations of statistical significance, although they may well be borderline. Again, they are dismissrd without adequate evidence. In short, the study is not designed to evaluate carcinogenesis, the numbers are probably in adequate to evaluate fetal outcom es, and (here is a difference in enzyme induction between exposed and unexposed groups, which may be but is not neces sarily related to exposure. GACc. ZIEM S ch o o l o f H ygien e a n d P u blic H ealth, Johns H o p k in s U niversity, B altim ore, M arylan d 21205, USA Dr May replies: In answer to Ziem 's first paragraph I must point out that the text of my article includes the phrase "the whereabouts of all living cases (89 out of 90) being knqwii and means exactly what it says. Though I was precluded from incorporating in my study those employees who had left the company. 1 was given every assistance in locating their current w here abouts. The death which did occur was due to cardiac failure in a retiree. I am at a loss to understand, therefore, how it can be misleading to say there has been no death from neoplasm. The comment about the scattergrams does not dispute, indeed, appears to support, my own find ings. It would have been easy to say, "th e re are significant variations in some of the biochem ical results, and I attribute these to dioxin exposure albeit that exposure was ten years ago" (14 years now). Such an assumption would probably not have been disputed and for years to come reference would have been made to it by other authors. A case in point is the fact that a passing reference to cholesterol concentrations in my 1973' article is quoted widely and regularly and out of context. I believe, however, that the biochemical variations are unlikely to be due to dioxin exposure. I have therefore considered it necessary to offer an alternate and more acceptable proposition, not a statem ent o f fact. I do not see how, with such very small numbers and in the absence of national statistics, it would be possible to make significant calculations on fetal outcomes. I accept 23em's last paragraph. The study was undertaken for the benefit of the employees as a purely internal matter without thought of publica tion. The results were considered to be so interesting, however, that wider distribution was urged from the highest levels. I make no claims to be either an epidemiologist or a statistician but have endeavoured to acquaint the medical community with my findings, being careful to avoid deductions which the small sample was incapable of sustaining. The continued re-assuring passage of time reinforces the tenor of my article and increasingly defies the overreaction with which some greeted the original measurem ents. References 1May G- ChJoracne from (he accidental production of tccrachloro dibenzodioxm. B rJ In d M td 1973J # : 276-43. ) ! 116 ,836 00 5 0 9 1 ZM01 fir j \ >. . v 'mp*! T ' ' W *** 5!? ,, 1 . ........................ , K--66681 --(100)Nl [l984j 9 nirv.v/ -a- ..T V ** OhiciaLcugan o fthe fntrXUonl ..x ^ Societyfor Environmental Tqxiaology*. , and Cancer ,: W ' * v . ^ V t * \ . : W . 0506 D J WROBLEWSKI DOW CHEM CO TOXI I NFO BLDG MIDLAND J PO292 1803 MI 48640 Volume 5, Number 4/5 JULY, 1984 ISSN 0731-9898 ar 1836* onuctetc tense. i microoJvems. *tddiois vaciv rene to y$u bv >4 2-SO $ 709 IMMUNOTOXICOLOGIC EFFECTS OF 2,3,7, - TETRACHLORODIBENZOp-DIOXIN (TCDD) IN RABBITS R. P. Shamu Toxicology Program, Utah State University, Logan, U T 84322 R .). Koriba and P. J. Gehring Toxicology Research Laboratory, D ow Chemical USA, M id land, M l 48640 M a le white N e w Zealand rabbits were exposed orally to 0, 0 . 0 1 , 0 . 1, I and 10 fig/kg/wk o f 2,3,7,8-tetrachlorodibenzo-pdioxin (TC D D ) for a period o f 8 weeks. After 4 and 6 weeks of first T C D D administration, the rabbits were inoculated with a mixture o f tetanus toxoid and Freund's adjuvant. TC D D expo sure reduced the serum antitoxin titers, skin sensitivity to tuber culin, and the number o f antibody producing cells in popliteal lymph nodes. At the end o f the treatment period serum IgC levels were increased at the lowest dose of T C D D treatment w hile a marked depression was noticed at the highest dose level. An increase in the thymidine uptake by splenic lympho cytes in culture was noted at all levels of T C D D treatment whereas the response o f these cells to phytomitogens was decreased at high levels o f T C D D exposure. A ll different im munologic effects were not altered at the lowest T C D D treat ment but both humoral and cell-mediated immune responses were depressed at the highest level o f T C D D exposure. INTRODUCTION The depression of immunologic responses by 2,3,7,8-tetrachlorodibenzop-dioxin (T C D D ) has been described in a number of mammalian species. O ne of the sensitive measures of T C D D toxicity is a dose-related atrophy of thymus (Buo - H o i ef a/., 1972; Vos et a!., 1973, 1978), although it is questionable whether this effect is related to the mortality induced by this chemical. Consid erable species and age-related variation has been reported regarding the imm unosuppressive effects of T C D D . For example, T C D D depressed the delayed type tuberculin skin reactivity in young guinea pigs but not in rats (Vos et a!., 1973). Vos and M oore (1974) showed the suppression of cellular im m unity in rats and mice after maternal and post-natal exposures of offspring to T C D D . Thigpen et al. (1975) reported that T C D D at sublethal doses in- Please send requests for reprints to: Or. R. P.Sharma, Department of Animal, Dairy and Veterinary Sciences. Utah State University, UMC 56, Logan. UT 84322 '' ; lEfTO 5-4/5:321-328 Copyright (; 1984 by Chem-Orbital 00*12 (60501 r.- ...- 18363 Ui SHAJtMAET Al_ creased mortality in mice when challenged by Salmonella infection and also shortened the incubation time of the infecting organisms. O n the other hand, no effect on the mortality of pseudorabies virus (PRV) infected mice was demonstrated. Vos et a/. (1978) recently demonstrated that the increased sensitivity of TC DD-treated mice to Salmonella was because of endotoxin hypersensitivity, rather than the altered phagocytic action o f macrophages. The thymic atrophy by T C D D , which resembles the neonatal thymectomy, was not prevented by the simultaneous thymosin treatment. O u r recent studies with adult mice indicated that a continued treatment with T C D D may depress the immune response but the effects were less pronounced after an 8-week exposure, as compared to the early periods (Sharma and Gehring, 1979). This suggested a possible adaptation to the T C D D effects. Low doses of TC D D also produced an enhancement of splenic lymphocyte responsiveness in culture. The present study deals with the dose-related immunologic effects of TC DD in mature rabbits when the animals were repeatedly treated with this toxic chemical for a period of 8 weeks. MATERIALS AND METHODS M a le white N ew Zealand rabbits (Langshaw Rabbitory, Carson City, Ml), weighing approximately 3 kg each, were housed individually and provided free access to feed and water. After equilibration to the surroundings for one week, the animals were randomly divided into 5 groups of 5 animals each and were administered 0 .0 ,0 .0 1 , 0 .10 , and 10 /ig /k g o f T C D D in com oil by oral gavage, once a week for 8 weeks. The animals were kept in environmentcontrolled rooms with a 12-hour light/dark cycle throughout the experiment T C D D solutions for dosing these animals were prepared by dissolving 5.0 mg of the chemical (> 9 9 % purity) initially into 10 ml acetone (glass distilled) and mixing this solution with 190 ml of com oil (Nutritional Biochemicals, Cleveland, O H ). This stock solution containing 25 /ig TC D D /m l was diluted with corn oil containing 5% acetone to provide appropriate dosing solutions. The control group was dosed with 5% acetone in com oil. Concentrations of all dosing solutions were adjusted to provide 0 .4 ml/kg. The animals were weighed once a week. After 4 and 6 weeks o f the first dose the animals were injected with a 1:1 mixture o f tetanus toxoid (Jensen-Saisbury Laboratories, Kansas City, M O ) and Freund's complete adjuvant (G1BCO, Grand Islands, NY), given 0.3 m l/ animal in their foot pad. Blood was collected from all animals by puncturing their ear vein before the first T C D D dose and just before the antigen inoculations. After an 8-w eek treatment period the animals w ere sacrificed by decapitation (under a C O 2 anethesia) and their blood and visceral organs collected. Tw elve days after the first and second antigen challenge, i.e., two days prior to second antigen and sacrifice, respectively, the animals were tested for skin reaction against tuberculin. Tuberculin (jensen-Salsbury Laboratory, Kan sas City, M O ) was injected intradermaily, 0.1 ml, in a shaven flank area and the diam eter o f skin reaction measured 24 and 48 hr after this injection. 18364 DOH2I 60501 A ETAL id also ' hand. :e was vity of itivity, trophy ted by mice imune ire, as sted a :edan resent lature I fora ,M I), /ided r one i and >oral Tent ent g 5.0 lied) cals, uted ons. if ail vere Com plete necropsy was performed on animals after sacrifice and their selected organs weighed. The organs (thymus, liver, kidney, spleen and pop liteal lymph node) were stored in formalin for histological examinations. The tissues were routinely processed and examined microscopically. Part of the spleen was saved in sterilized saline for splenic cultures as described below. O n e popliteal lymph node was frozen for evaluating the antibody secreting cells. The blood at all sampling times was used for total erythrocyte and total and differential leukocyte counts. Serum separated from blood was analyzed for serum IgG by quantitative immunoelectrophoresis using the method mod ified from Bjerrum et al. (1973), details of which have been described else w here (Sharma and Gehring, 1979). In serum samples obtained after the antigen inoculation, the antitoxin titers were determined by s ^ jijfrin g sheep erythrocytes (SRBC) with a tetanus toxoid vaccine (1 :200) and uslrtythese cells as indicator for a hemagglutination reaction. The highest dilution of serum causing complete hemagglutination in microtitration plates (V bottom. Cook Laboratories, Alexandria, VA) was considered an end point. The splenic lymphocytes were cultured in triplicate either without or with the presence of an optimal amount of either phytohemagglutinin (PHA) or pokew eed mitogen (PW M ). The culture technique has been described previ ously (Sharma and Gehring, 1979). The incorporation of thymidine per million lymphocytes was determined for each animal and the stimulation index for each mitogen calculated by dividing the disintegrations per minute (dpm) of cultures in the presence of the respective mitogen by the dpm of an identical culture from the same spleen but without any mitogen. Frozen sections of popliteal lymph nodes, 6-8 /im thick, were evaluated for the number of globulin secreting cells as described before (Street and Sharma, 1975). The number of fluorescent cells were counted in 10 random fields and averaged to determine a fluorescence score for each animal. All values are presented as means and their standard deviations or stan dard error of-means. The parameters of treatment groups were compared with the control group using a t-test. The body weight and organ weights were com pared using a D unnetfs t (Steel and Torrie, 1960). RESULTS T C D D produced toxicologic manifestations in rabbits at the highest dose, i.e., 10 /ig /kg /w k. During the experiment two animals died in this group with clinical signs attributable to TC DD toxicity. These animals had accumulations of ascitic fluid and a gradual wasting syndrome leading to death. The deaths of five other animals (one receiving 10 ig/kg/wk and 2 each from groups receiving 1 and 0.2 n g/kg/wk) were caused by the deposition of dosing materials into their lungs. Table 1 illustrates the body and organ weights of animals surviving the 8-week experimental period. Whereas no effect of T C D D treatment is apparent on the body weights of surviving animals, the size of their liver was increased in animals receiving 1 or 10 *g TC DD /kg/w k. A significant reduction in the size of thymus, adrenals and popliteal lymph nodes was also seen in the animals e x p e n d to 10 ptg TC DD /kg/w k. 18365 324 SHARMAET AL TABLE 1. Body and Organ Weights of Rabbits Treated with TCDO (dr 8 Weeks Weekly Dose of TCDO. pg/kg 0 (Control) 0.01 0.1 1 10 Body Weight (kg) 3.170 SO. 193' 3.388 = 0.369 No. of Animals Surviving Organ Weight, g. S 5 (organ/body wt. g/IOOg) for Liver 89.64 - 19.99 7 8 .3 8 = 10.13 (2.82 = 0.S1) (2.31 = 0.13) Kidneys 16.82 = 1.71 15.64 = 2.43 (0.53 =0.03) (0.46 = 0.04) Brain 9.17 = 0.42 9.52 = 0.26 Heart (0.29 = 0.02) 6.10 = 0.86 (0.28 = 0.03) 6.87 = 0.55 Thymus (0.19 = 0.04) 4.51 = 1.83 (0.20 = 0.01) 5.11 = 1.28 (0.14 = 0.05) (0.15 = 0.03) Spleen 1.38 = 0.34 1.91 = 0 .85 (0.04 = 0.01) (0.06 = 0.03) Adrenals 0.57 = 0.14 0.68 = 0.10 (0.02 = 0.01) (0.02 = 0.00) Popliteal Lymph Nodes 0.58 = 0.11 (0.02 = 0.00) 0.64 = 0.12 <0.02 = 0.00) Testes 5.86 = 0.55 5.99 = 0.58 (0.19 = 0.02) (0.18 = 0.01) 3.4S5 =0.152 2.787 = 0.387 3.158 = 0.106 3 32 87.14 = 5.14 (2.53 =0.25) 16.51 = 1.10 (0.48 = 0.01) 9.20 = 0.40 (0.27 = 0.02) 6.51 =0.86 (0.19 = 0.02) 6.47 = 1.37 (0.19 = 0.03) 1.89 = 0.50 (0.05 = 0.01) 0.70 = 0.25 (0.02 = 0.01) 0.69 = 0.13 (0.02 = 0.00) 7.30 = 0.81 (0.21 =0.03) 111.98= 16.87 152.04 = 8.35 * (4.01 =0.1 IP (4.82 = 0.43P 16.61 = 2.11 16.52 = 2.74 (0.60 = 0.04) (0.52 = 0.07) 8.86 = 0.30 9.87 = 1.20 (0.32 = 0.05) (0.31 =0.03) 4 83 = 0.87 5.30 = 0.25 (0.17 = 0.01) (0.17 = 0.00) 3.80 = 0.60 1.77 = 0.36' (0.14 = 0.02) (0.06 = 0.01)' 1.81 =0.94 1.16 = 0.44 (0.06 = 0.03) (0.04 = 0.01) 0.40 = 0.07 0.25 =0.17* (0.01 =0.00) (0.01 =0.01) 0.52 = 0.10 0.41 = 0 .0 2 ' <0.02 = 0.00) 10.01 =0.00)' 4.76 = 0.98 5.18 = 2.16 (0.17 =0.03) (0.16 =0.06) 'Mean = SD. The number of animals surviving is not related to TCDO induced deaths. 'Indicate significant deviation from control at p<.05 (Dunnett's test). Histological evaluation of selected organs did not show any specific T C D D related lesions, except the atrophy of thymic cortex. The extensive hepatic lesions, usually noticed in rodents after the TC DO treatment were not observed in rabbits. The livers showed occasional mild to moderate degenera tion, presence o f m ultinudeated or pigmented hepatocytes, and occasional fibrosis and leukocytic perivascular infiltration. Dilated sinusoidal spaces were also observed but ail the lesions were neither consistent nor dose-related. A slight reduction in the total erythrocyte and leukocyte counts was observed in animals exposed to the highest dose of TC D D , although this change was statistically significant only at 6 weeks of exposure (data not shown!. Differential leukocyte counts revealed no consistent changes. Rabbits idjected with tuberculin after sensitization with Freund's adjuvant showed a dose-dependent decrease in the skin reactivity in two highest TCQO levels (Table 2). This effect was observed both after the primary and secondary- "' TCDOIMMUNOTOXIOTY IN RABBITS 32S 0H 2I 60505 TABLE 2 Skin Reactivity in Rabbits Exposed to Different Amounts of TCDO and Sensitized with Freund's Adjuvant Exposure Level of Primary Skin Response1 Secondary Skin Response TCDD pg.'kg/wk 24 hr 46 hr 24 hr 48 hr 0 0.01 0.1 1 10 17.6 2 .7 16.6 1.6 19.3 0.9 16.7 0 .7 14.6 2.4 23.2 2.1 18.4 0.8* 23.3 1.8 14.3 2.6' 4.3 3.4' 23.4 1.3 18.2 1.9> 19.3 2.3 14.3 1.5* 10.0 5.0* 17.2 2.6 13.6 2.3 16.7 2.6 12.3 6 .4 10.S S.S 'Primary skin response was measured 10 days after the First antigen injec tion whereas the secondary one was measured 10 days after the second antigen challenge. The values are millimeters of diameter of the skin reaction expressed as mean and its standard error. 'Indicate a significant difference from control at p<.05. sensitization, although a significant effect was noted only 48 hr after the first and 24 hr after the second tuberculin testing, respectively. In both instances a decrease at the 0.01 ig TC D D /kg/w k dose but no effect in the second high dose, i.e., 0.1 /zg/kg/wk, was observed. Table 3 presents the serum antitetanus titers in control and TC D D treated rabbits. A consistent decrease was seen only when the weekly dose of TC D D was 1 tg/kg or more. The fluorescence scores of popliteal lymph nodes in different groups of rabbits are also indicated in Table 3. An effect observed at the T C D D dose levels of 1 and 10 /zg/kg/wk corresponds well with the reduction in antitoxin titers in the serum. Serum IgG levels at different periods of T C D D treatment are given in Table 4. At the end of the experimental period, the group receiving 0.01 m8 T C D D /k g /w k had significantly higher IgG levels, whereas depressed IgG val- TABLE 3. Serum Antitetanus Hmagglutination Titers in Rabbits and the Evaluation of Plasma Cells in Rabbit Lymph Node in Relation to TCDD Exposure Exposure Level of TCDD pg/kg/wk Serum Antitetanus Titers* Primary Secondary Popliteal Lymph Node Fluorescence Score1 0 0.01 0.1 1 10 5.6 1.0 4.8 0.8 2.7 0.7* 2.0 0.0* 2.0 0.0* 11.2 2.0 11.2 2.0 8.0 0.0 5.3 1.3' 4.0 0.0* 15.0 1.1 12.8 2.S 12;0 3.2 9.0 1.2* 10.0 1.0*' 'The primary and secondary titers refer to the serum collected 2 weeks after the First and second antigen injection, respectively. 'Blind scoring okJO random fields in each lymph node under a micro- scope. See Methods section (or details. - "` 'Significantly different from the respective control at p<.05. v 326 SHAJIMA FT AL TABLE 4. Rabbit Serum IgC at Different Periods of TCDO Exposure Exposure Level IgG. mg/ml, at TCDD pg/kg/wk Pieexposuie' 4 Weeks 6 Weeks* 8 Weeks* 0 0.01 0.1 1 10 14.2 2 3.2 17.1 2 2.7 15.1 * 1.8 13.2 * 2.6 IS.2 2 2.9 16.0 --2.0 15.4 2 1.9 13.1 2 2.4 17.5 2 1.9 16.S 2 1.3 25.2 2 1.9* 16.5 2 3.8 13.4 2 2.3 24.6 2 6.4 16.7 2 2.7 14.2 2 3.9 12.6 2 5.0 13.0 2 4.5 8.7 2 2.0* 6.2 2 2.1* 'Values are means 2 S of 5 observations/group. except when the animals died during the experiment. *Two and 4 weeks after inoculation with a mixture ol tetanus toxoid and Freunds adjuvant. 'Significantly different horn control at pc.OS. ues w ere observed in the group receiving 1 and 10 /xg of TCDD/kg/wk for 6 and 8 weeks, respectively. The incorporation of thymidine in splenic lymphocyte cultures from the rabbits is given in Table 5. A relatively high degree of thymidine uptake, consistent w ith the challenge of animals with two doses of antigen mixture, was apparent in the control animals. Treatment of rabbits with T C D D enhanced this spontaneous blast formation in these cultures, these became statistically signif icant in group receiving 1 pig TC DD /kg/w k, in spite of the inherent large variation in different animals within a group. The splenic lymphocyte cultures were responsive to the mitogen induced blast formation in TCDD-treated groups. The reduction of stimulation indices (to nearly one-half) indicated in Table 5 is partly because of a nearly three-fold increase in the spontaneous blast formation in group receiving the highest dose of T C D D . TABLE 5. Incorporation of *H-Thymidine in Lymphocyte and Mitogen Stimulation Indices in Rabbit Splenic Cultures from Animals Challenged with an Antigen Mixture and Given Different Amounts of TCDD' TCDD Exposure pg/kg/wk 0 P M /10* Cells without arty Mitogen Stimulation Index' with PHA PWM 0 0.01 0.1 1 10 35.885 2 6.528 50.261 2 11,153 53,257 2 17,134 85.392 2 14,023* 90.276 2 50.081 7.99 2 1.36 6.01 2 1.95 7.09 2 2.95 4.33 2 1.77 4.07 2 1.32* 5.05 2 1.81 6.95 2 1.37 5.63 2 2.43 2.59 2 0 53 2.70 2 1.23 'The animals were given a mixture of tetanus toxoid and Freunds complete adjuvant. 2 and 4 weeks prior to their sacrifice. The values in - the Table are Mgan --SE. 'Stimulation index - dpm of cultureswith respective mitogen divided by - (he dpm of same cultures widtoul a mitogen. ` 'Indicate a significant difference at pc.OS.';. 328 SHARMA ETAL ln conclusion it may be stated that relatively high doses of TC O O (in the order o f 1 /ig /kg /w k or more) produced a consistent immunosuppression in adult rabbits. The T-dependent responses were influenced generally at a lower dose but an effect was also seen later on the humoral responses, which may have been caused by the interference with helper T-cells. Splenic lymphocytes from T C D D treated immunized rabbits showed a higher frequency of blast formation in culture, the exact implication of this finding is not clear. REFERENCES Bjeman O ), Ingild A, Lowenctein H, Weeke B. Carbamylated antibodies used forquantitation of human IgG. A routine method. In: Quantitative Immunoelectrophoresis. Axelsen NH, Krotl |, Week 8. eds. Oslo: Universitetsforiaget 1973. pp. 145-8 Buo-Hoi NP, Chaiui PH, Sesque C. Organs as targets o dioxin 12.3,7.8 rarachlorodibenzo-p-dioxin) intoxication. Neturwissenschaften 1972: 59:174-5 Ladmwnn P|. Immunologic tolerance and unresponsiveness. In: The Immune System. Hobart M|, McCon nell I. eds. Oxford: Blackwell Scientific. 1976. pp. 152-64 Sharma BP, ed itin g PJ_ Effects of 2.3,7,8-fetrachforodibenzo-p-dioxin (TCOO) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann NY Acad Sci 1979; 320:487-98 Steel BCD, Torrie HH. Principles and Procedures of Statistics. NY: McGtaw Hill Book Co., Inc. 1960 Street JC, Sharma BP. Alteration of induced cellular and humoral immune responses by pesticides and chemicals o environmental concern: Quantitative studies o immunosuppression by DOT, Arodor 1254, carbaryl, carbofotan and methyiparathion. Toxicol Appl Pharmacol 1975; 32:587-602 Thigpen |E, Faith RE, McConnell EE, Moot |A. Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7.8-tetrachforodibenzo-p-dioxin. Infect Immun 1975; 12:1319-24 Vot |G , Ktecftenbcrg |G , Engel HWB, Mtndsrhoud A, van Nooric-famen LM. Studies on 2,3,7,8-tetrachlorodiberuo-p-dioxin-induced immune suppression and decreased resistance to infection: Endotoxin hypersensitivity, serum zinc concentrations and effect o thymosin treatment Toxicology 1978; 9:75-86 Vos |G , Moot JA. Suppression o cellular immunity in rats and mice by maternal treatment with 2,3,7,8tetrachlordibenzo-p-dioxin. Inr Arch Allergy 1974; 47:777-94 Vos |G , f4nore |A, ZLaU |G. Effect o 2.3,7,B-retrachlorodibetuo-p-dioxin on the immune system of laboratory animals. Environ Health Perspect 1973; 5:149-62 MS 1442 v 00MZ160308 FI LETCDD- -K-66681-(l00)?^[l983] TCDF OR-Ol15--3259-- f'* 2,7-DCDD K-7453-f ^ 220 13S1 REGULATION OF EPIDERMAL GROWTH FACTOR BINDING AND UPTAKE IN CiM.rUED HUMAN EPIDERMAL CELLS BY 2 ,1,7,8-TETRACHLOROOIBENZOp-UlOXlN (TCDD). L.G. Hudson*. W.A. Toscano . Jr,*, and U.P. Greenlee* (SPON: R.A. Harcline). Harvard Sch. Pub. Hlch., Boston, MA 02115 and CIIT, Research Triangle Park, NC 27709. T h e proliferation of epidermal ceils 1$ regulated by several biochemical mediators Including cyclic nucleotides and epidermal erowth factor (ECP). We have examined the actions of TCDD, an gent that produces epiderral hvperplasia in humans and hair less mice on cell associated (l**I]-ECF in a buzan quacous OEll carcinoma line (SCC 12F). Cell associated ('**l]-ECF was determined by measuring the specific binding and uptake of P ^ I l - E G F at 37* after preincubat ing cells with TCDD. Treat ment of cells with TCOD resulted in a concentration dependent decrease in ceil associated (l25I]-ECF to a value of 35X of con trol with an apparent EC^q l nM. A decrease was detectable by 2 h and maximal after 72 h. The response of cells treated with 2,3,7, S-tetrachlorodIbenzofuran, an Isostereomer of TCDD, was equal to chat of TCDD. There was no significant alteration in cell associated ll2il]-EGF In cells exposed to the nonisostereomcrlc analog, 2,7-dichlorcdibenao-p-dloxin. The observed potency (EC5 0 h l nM) and scereospccif ic Icy suggest chat the de crease in cell associated (*2 *I]-EGF produced by TCDD la mediated by the dioxin receptor. Sustained refractoriness of SCC 12F cells to ECF may be an important mechanism by which TCDD alters regulated cell proliferation. Supported In part by NXEHS Crane ES-08266; CIIT Predoctoral Fellowship to ICH. Federal Proceedings, V. 42, N 7, 1988, 1983. 18370 I/*''.* ! Wi r id k ;:\i:-: !>:::. ;-a (::N. 22 i rc~ Q Q 2,1, - MCMgt.-j 11 : :I!' ! ' ; ' ' ' >.l* : ii i n - a v i r o : - : . - ^ ' h 0 0 Prolnctir-n Agcncy's Ai'- nn i-: -A- !><. :>, p.-c.- ;) t--*.m*; lor.y lioly as a candido "> Air cancellolimi. n< -n/*`sLni.'.t.'*< y<.itv-i Li>-1* j-:: .-nt01 thc s.iaipl:s (l* in n Lee! Ait nonituiiof | 1>*g;aai :1--.v.:..I nu t1L)b (*i 11 )-`t -nt detection Icvcls oi IO potts per billion. 1;IC rer.ininii!J 10 peie!ni -1!:;.:a;pL.'s, ; :m , '.v.;;lC '*i*t !*/ .p1-f:<a te- (;. ::1:;:r;'i-u:i(-,:i::iunr; 111 Lie l> 1 t !Lh'.>n<ni ..nnlytie.il .n!:,fii1 |..,.1... oPn:aining :i' .ionm !," --l-.u.i .n- Kit npi-.'.-Lefi'.i.-j'ial >ri.-r;i-n ions :iiiM IL- r-n1ip: ;ii< I0S08UNQQ ' The pan-'lalno note i: "Al -_!j::*-i*.:: :tiLiv:L^ (v/iih winch \v<- arc woiking) Micro arcintrinsic unc-.'iLiitiiier. v.*ith II- .malytieni a.-,thon." O ADMINISTRA'TOLi STILI, P.AlX.:; ii>X::'ASL.iiAMC!:.; IAW. TRAIN ALI lK.viS The /Vhn'.iiis'.ratjuil l i . ; : ; : : " !-. Protection Aoonry A.m.ini:-,: m ference -m IVUn is> r..; il-: lgislation eould Le :1o.a tr.:i `.inco:; 'egisiotion, nvi:o.'.mental t? Tm : .f::::r:o<ilisis v.*->:-,t .us press con-, i)ltil. !iV :e ; |<,,-, iu:intic estimale ', whc-n rs~ Notiiuj tnat Contres iv' -1: r`.lie y.-..: , it- mittee v/oui'i ptobnbly n 1.r>1- :,:Pi..;.Hi n ; a Page -) i.mit TeLiny o: *'.-:! rtprii.;. ' l\ Mning . to thc foli ('ninnicice ( in ::1-.S.-net-, ;k_-:: V/Q/ to r:o.'* iv~ 1ii.use Coramcree Comia.itu.-e- bill (Soc Dei l. us, liait th bill is v..t yet .'i^viuimly '.vu iiuvu .1 iong C P A bocks ino L i j C.'o liste: Lhi! in h--'js-, ".vitiiamendaient:;, " iie soid, siating tiwt the ogoncy*:; suggerite! nm- rAnioni:; v/o:e iCy to.go t.Vipitol liti! once CJongress cornes bock fresi ils rocess. Jutt bei<T< (.`onisiess k*i Son. Tonuey {!)-(." iif.) ici tlio.t "irro c o n c il b le d iffere n e e s 0.1 k'.-y pt uvisiuii:;' > th 1-r;tslulion ".m a :provento' ils miataient in t'- Lv.v." TU iiney .'M id, ". 'rti'n v j : > r mk A '-oriiiv/ -u'.v::'-! i::an a i::n!uto necessrt" to ju o to c t m e p u b lic :nm e n v i rom .u -nia 1 !ln`.n:;tn et trie t:;..*'.v.icn tiio cists ol -.citici a ie th c o w e s t ." S te tin g A '.:;'.' c -nt: ne le-v/i:;tai liie p i r r 'tiel stogo bccau:.. jobs h o v e not y e t b o eri coniaiilL-d n i ;1 -ipi.i.'nt not yn! puicivisod. Tutinay continu*, -i: " Llut e v e n a u s e i:npo>l.i::tl- , u iif .l ac tiiis s-o g e a v o id s ` lie p a in , a u n e r/ , .nd iu : *-mo c^.n.-n, i.-vcn d eatii, fliiTt 1P371 T O X IC O L O G Y A N D A P P L IE D P H A R M A C O L O G Y 7 4 . 91 -9 8 (1 9 8 4 ) R e v e rs ib ie inhibition o f in Vitro tp ith e iia i C ell P ro life ra tio n by 2 .3 .7 .8 -T e tra c h lo ro d ib e n zo -p -d io x in 1 Jo h n F. G ie r t h y a n d D ebora h C ra ne Center lor Laboratories and Research. Vt',, York State Department of Health. Albany, blew York 12201 Received November II. I98J: accepted January 12. 984 Reversible Inhibition of in Vitro Epithelial Cell Proliferation by 2.3.7.8-Tetrachlorodibenzop-dioxin. G ierthy, J. F.. and Crane. D. (1984). Toxicol. Appi. Pharmacol. 74, 91-98. Sub confluent cultures o f a mouse epithelial cell line, which after prolonged subculturing exhibited an clevaled saturation density as compared to ihe original cell line, were treated with 2,3.7.8ictrachlorodibenzo-p-dioxin (TCDD). Cultures of cells with or without TCDD grew at equal rates until conftuency was reached. At confluency. cultures treated with as little as 1 0 '" M TCDD showed a decline in cell proliferation relative to controls as demonstrated by cell enumeration and supported by reduced ('HJthymidine incorporation (both by liquid scintillation spectrometry of whole culture and autoradiography of individual cells). After 14 days of exposure, the salutation density of the treated culture was about 50% of the control culture. This TCDD-induced. increased sensitivity to density-dependent inhibition of replication (D D IR ) was accompanied by a change from a fusiform morphology in the high-saturation-density control cells to a flat cobblestone appearance in the treated low-saturation-density cells. The nondividing cultures treated for 14 days with 1 0 '" m TCDD had the same viability as control cultures. Upon trypsin suspension and reseeding, these formerly quiescent cultures were again capable ofgrowing to high cell density and of again showing susceptibility to TCDD-induced changes in cell growth and morphology. Evidence is presented to suggest that this reversible increase in sensitivity to DDIR and the morphological change are not a consequence of cell growth inhibition. This system may provide the basis for an in vitro model to study the effect of TCDD on the control of replication of these cells. 2.3,7,8-teirachlorodibenzo-p-dioxin (TC D D ) is ihe most toxic synthetic compound known (Poland and Kende, 1976; Poland and Knut son, 1982). Animal studies have demonstrated the pleotypic nature o f this toxicity, which is characterized mainly by a prolonged wasting syndrome leading to death but also includes embryotoxicity and/or teratogenicity, thymic and lymphoid involution, hyperkeratoses, edema, hyperplasia o f the epithelium o f the stomach, intestine, urinary bladder, and bile duct, and hepatocellular damage (Kimbrough, 1 Presented in part at the 186th National Meeting of the American Chemical Society. Washington. D.C., August 28 to September 2. 1983. 1974; H uff el a!.. 1980). The development o f acncform lesions is one of the most common toxic manifestations o f TC D D exposure seen in, but not confined to, humans (Kimbrough, 1974; H uff et at., 1980). The development o f this chlotacne appears to result from hyper keratoses o f the sebaceous glands leading to a cystic response (Kimbrough, 1974; Knutson and Poland, 1980b). The specific pathology and sensitivity to T C D D vary greatly among animal species. The LD50 for the species tested ranges from I pg/kg body weight in the guinea pig to 3000 jig/kg in the hamster (Schwetz et at.. 1973; Henck et at., 1981). The specific cause of death due to T C D D intoxication is unknown. 91 004I-008X/84 $3.00 Cop)10C (W4togAcademe t a , lac. 0 7 7 0 9 1 7 M0fl 92 GIERTHY AND CRANE Extensive studies o f T C D D in anim al and in in v itr o systems over the past decade have suggested that the toxic response elicited by TC D D isomers and congeners is associated with their binding to a specific cytosolic re ceptor (Poland and Glover, 1975: K ende et a i . 1974). The affinity of these com pounds for this receptor corresponds in rank order to their in v iv o toxicity, as well as to the induc tion of a battery of enzymes which include the cytochrome Pi-450-mediated microsomal monooxygenase aryl hydrocarbon hydroxylase (AHH) (Poland and Glover. 1977; Poland and Kende. 1976; Kende e t a l.. 1974). Additional evidence suggests that the induction of AHH is dependent upon the translocation o f the TCD D -receptor complex from the cytoplasm into the nucleus (Greenlee and Poland, 1979; Okey e t a l.. 1979, 1980), where evidence sug gests it induces genetic activation resulting in the production of cytochrome Pi-450 mRNA (Tukey e t a l.. 1982). Many cultured cell lines respond to TC D D exposure by inducing AHH. Despite this en zyme induction and the toxicity o f T C D D in animals, extensive studies have not dem on strated significant in v itro toxicity in cultured cell lines exposed to TCD D . No change in growth or morphology was seen in 23 cell lines exposed to TCDD (Knutson and Poland, 1980a). It was reported, however, that in v itr o keratinization could be induced in the cloned mouse-teratoma cell line. XB, by exposure to very low levels o f T C D D (K nutson and Po land, 1980b). These results suggested the use of this system as an in vitro model for the in vivo keratinization response. These experi ments also showed that this phenotype could change under conditions o f cell passage. We have also observed a change in phenotype and determined that, under our conditions o f cell culture, late-passage XB cells no longer ker atinize upon exposure to TCD D although they continue to proliferate and to grow to a higher density than the early-passage XB cells. The late-passage XB cells, which we designate XBF, have been found to respond to very low levels o f T C D D by an increased sensitivity to density-dependent inhibition of replication (D D IR ) and the appearance of a Hat epithelial morphology, as com pared to the fusiform morphology of high-density nonexposed rep licate cultures. We also present evidence that this TCDD-induced decrease in saturation density observed in these cells does not result from a general toxic response and is reversible. T he TCD D -induced D D IR seen in this cell line may provide the basis for an in v itro sys tem to study the effect of this com pound on the control o f cell replication in these cells. METHODS TCDD was obtained from Dow Chemical (Midland. Mich.). Purity was determined by mass spectrometry to be >99%. The XB teratoma-derived mouse epithelial cells and the 3T3 fibroblast feeder cells were the generous gift of Dr. Howard Green (Harvard University). Analytical grade dimethyl sulfoxide (DMSO) was obuined from the Aldrich Chemical Co. (Milwaukee. W'u.). Cell culture. XB cell stocks were grown in Costar plastic tissue culture flasks(75 cm ') in Dulbcccos Modified Eagle Medium (DM EM ; Gibco) supplemented with 20% fetal bovine serum (Flow. Rockville. Md.). 100 U of penicillin/ ml. and 100 tig of streptomycin/ml. This medium had been conditioned by exposure to a confluent culture of 3T3 feeder cells for 24 hr (25 ml of medium/75 cm1flask) and sterilized by filtration. The 3T3 feeder cell stocks were grown in DMEM supplemented with 10% calf serum (Row). The XB cells were routinely propagated every I to 2 weeks, when conflucncy was reached, by trypsinization (0.25%) and teplatint at a concentration of 2 x 10* cells per cm1. The culture medium was renewed once a week between p *ayx The confluent density of these early XB cultures was about 4 X 1 0 * cells per cm1 under these conditions. These cells, when seeded with Iclhaily irradiated 3T3 cells, were found to exhibit the TCDD-induced ke ratinization response described by Knutson and Poland (1980b). The magnitude of this keratinization response to TCDD exposure gradually declined aAcr passage seven, and by p3" 3! 13 this response was greatly reduced com pared to the early XB cells, although the sensitivity of this reduced response to TCDD was similar to that of the early cells. At this time the XB stock cultures, which were originally flat and polygonal, now had become mote fusiform in morphology and reached conflucncy earlier than the low passage XB cultures. This alteration in growth and morphology at high density progressed dur- i GO^ A - O i ii i77fiqi7imn Ni. CONTROL OF IS VITRO PROLIFERATION BY TCDD 93 ing the next five to seven p m " as the kentinization response to TCDD exposure disappeared. The confluent density o f the ceils at this d u x was about 15 X I04 cells per cm*. At this time it was discovered that medium con ditioned by exposure to 3T3 cells was no longer required for the growth of these cells, which were now designated XBF to indicate their divergence from the XB line. Sub sequently, stock cultures of X BF cellswere routinely grown in DM EM supplemented with 20% fetal bovine serum (Flow), lOOUofpenidllin/mLand 100 m( ofstreptomycin/ ml. The cultures were propagated by suspension in trypsin (0.23%), replaied at a density o f 3 X 10* cells per cm1 once or twice a week as conflucncy was reached, and incubated at 37' C in a humidified atmosphere of 5% CO ,. Exposure o fcells to TCDD. XBF cdb were suspended by trypsinizadon and seeded into 24-wdl plates (16-ramdiameter wdh. 5 X 10*cells per ml perwell) in conditioned medium with 3T3 cells (5 X 10* per ml-per well) which had been irradiated with 6000 rads from a cesium source. The cultures were incubated overnight and refed with a series comprising 10-fold dilutions o f a stock solution o f TCDO in DMSO or with DMSO alone in DM EM sup plemented with 20% fetal bovine senun. This refeediag was repeated every 3 or 4 days for the duration o f the experiment. The highest cumulative DMSO concentration was 0.1%. At the end of incubation the cultures were washed with phosphate-buffered saline (PBS), fixed with formalin in PBS, and stained with Giemsa. Alternatively, the cultures were suspended by trypsinizadon at varying times aAcr the initial exposure to TCD D . After thorough disaggre gation of the cells by trituration, the number o f cells per culture was determined with a Coulter Particle Counter (Coulter Instrument, Hialeah, Fla.). Complete disaggre gation was confirmed by microscopic examination of the cell suspension immediately before counting. [' Hpiiym idinc (New England Nuclear specific activity 0.67 jiCi/mm ol) incorporation at various times aAcr exposure to TCDD was determined by pulse-labeling the cultures for the indicated times and counting the abdprecipitabic radioactivity in a Packard Tricarb liquid adodilation spectrometer (Ellem and Gierthy, 1977% Auto radiography was performed asdescribed elsewhere (Elkm and Gierthy, 1977% Cell viability was determined by measuring the per centage o f trypsin-suspended cells capable o f c h iding 0.1 % trypan blue dye. Oouing efficiency was determined by seeding 200 cdb ia 3 mi o f medium into a 60-mm plastic lissue-cuhura Petri dish (Falcon) and incubating for 2 weeks. Cultures were then washed in PBS, fixed ia 10% formalin, sod stained with Giemsa, and colonira were counted. Absolute cloning eSococy was defined as the perernuge of ths cells seeded capable o f forming col onies. Safety considerations. Safe handling, containment, and disposal of the substances used in this study conformed to the general principles aod standards of biologic safety proposed by ihc Omce o f Biohazards and Environmental Control. National Cancer Institute, for class I hazards and to the guidelines o f the Laboratory Chemical Carcinogen Safety Standards Subcommittee o f the Department of Health and Human Services Committee to coordinate toxicology and related programs. RESULTS Induction o f Morphological Change in XBF Cells by Exposure to TCDD The XBF cells, cocultured with irradiated 3T3 cells for 14 days, grew to very dense cul tures characterized by apparent multilayering and the appearance of parallel growth patterns indicative ofa spindlelike morphology. Treat ment with 0.1% DMSO had no effect on this morphology (Fig. la). Cultures grown in the presence of 10- *m TCDD grew to confluency, and after 7 to 10 days began to develop into a cobblestonelike monolayer comprised of flat, evenly distributed cells very apparent by Day 14(Fig. 1b). Cultures ofinadiated 3T3 feederlayer cells, seeded from the same stock used for these experiments, did not show any in dication of growth or TCDD-induced mor phological change. By Day 14 these cultures had essentially disintegrated. This change in growth and morphology was induced by TCDD concentrations ranging from 10~" to I0~* m , the highest concentra tion tested. This dose-response effect was ap parent in unmagnified, stained preparations (Fig. 2), as well as in the number of cells in each culture after 14 days of exposure (Fig. 3% Solvent control cultures treated with DMSO at concentrations corresponding to those in the dilutions did not show this effect The presence of 0.1% DMSO did not affect the TCDD dose-response. The ability of TCDD to induce these changes in morphology and growth in XBF cells has remained stable for over a year of cell passage as described under Methods. Representative experiments are presented in the figures. 18375 94 GIERTHY AND CRANE ' V, v - vis: : i* ,^ 5 8 L .^:i .< ;;v-acm- i f f i H ' . `:'i 717 0 P I 7 M 0 Q Fla. I. Induction o f the flxt-cri] response byTCDDrtJ) The dense pecking and multiliyering ofcoatroi XBF/3T3 cultures, (b) The apparent inducboa of density-dependent inhifaitioa of cdl proliferatioa and Sat-cell morphology characteristic o f XBF/3T3 cultures after 14 days o fapoeure to 10"* M TCDD. Gicmsa sain and methods, as described in text. I00X magnification. Reduction in Cell Growth during Exposure to 2.3,7.8-TCDD Investigation of the kinetics of cell growth in exposed cultures revealed that the decline in cell proliferation after TCDD exposure was hot immediate. Cultures were seeded below confluency, as described under Methods, and after 24 hr were treated with 10~* M TCDD and DMSO (0.1%) or with DMSO alone. Fig ure 4 shows that both treated and untreated cultures accumulated cells at equal rates until Day 7, at about which time confluency was readied. After this time the solvent control cultures continued to increase in cell number, while the 2^,7,8-TCDD cultures did not This cessation of cell proliferation is apparently TCDD-induced, aapeflected in the TCDDconcentration-dependent decline in [3H]thymidine incorporation into the acid-insoluble fraction of treated cultures (Fig. S). This ex periment was done after 7 days of exposure to TCDD, at which time the cultures had reached confluency, and the cell numbers were the same per culture. The range of TCDD concentrations capable ofdiminishing (1H]thymidine incorporation corresponds to that which caused the morphological change and is supportive of the decline in DNA syn thesis which would be expected to occur with the observed inhibition ofcell growth. Further evidence that the lack of increase in cell num ber was due to a cessation of cell division, rather than to the TCDD-treated cells simply sloughing off resulting in a steady-state, was a decrease in the number ofcells incorporating [3H]thymidine into DNA, as shown by au toradiography. A 6-lufepulse with [3H]thymidine at Day 6, when the treated and un treated cultures showed equal growth rates. wm m m m m 18378 (7 7 p 0 7 Mf>n CONTROL OF IN VITRO PROLIFERATION BY TCDD 95 IO'9 IO'10 IO'" IO'12 IO'13 0 M TCDD FK>. 2. Dose-response relation of flat-cell induction by TCDD. XBF/3T3 cultures were exposed to various concentnnoas o fTC D D is described iu text. After 14 days the cultures were fixed and suined with Giemsa stain. The less intensely stained low-density cultures (10"* to 1 0 '" M) are similar to that shown in Fig. lb, while the intensely staining high-density cultures (K T 11, l< T '\ and 0 m ) are similar to that seen in Fig. la. The Qat-edl induction is flist evident with 1 0 '" M TCDD. resulted in 451 4.4% of the cells being labeled. By Day 13 of treatment the cultures exposed to 10"* M TCDD showed a reduction in la beling to 1.6 1.2%. The viability ofcells from these treated cul tures was tested by trypan blue exclusion, which indicated a high viability of 87 2.5% in the 10'* M TCDD-treated cultures, 85 4.2% in control cultures (0.1%DMSO), and 88 4.1% in untreated cultures. Cells from these cultures gave plating efficiencies of 33 3.6% for 10'* MTCDD-treated cultures, 37 6.3% for control cultures, and 34 4.5% for untreated cultures. When cells treated with 10"' M TCDD for 14days were suspended with trypsin, reseeded, and allowed to grow with or without added TCDD, again no difference in proliferation was seen until confluency was reached. At Fio. 3. Effect of I'COD on odl growth in XBF cultures. XBF cells wee seeded wills imdiased 3T3 feeder ceils and incubated described under Methods. Medium was replaced 24 hr ift Basing sail i m j 3 or4 daysthereafter with medium remaining the jndfcafed concmuations of TCDD in DMSO or DMSO sJooc. The cultures were trypsiaaed after 14 days, and ocfl aumber per culture well was tkm winrri The dam points represent the per centage of ccit in the TCDD-treated cultures compared to cootrai cahures and me the mean of four replicate cultures. Fvg. 4. Effect of TCDD on XBF ccft proiifcrarioo ki netics XBFcellswereseededend incuhesnd with inadimed 3T3 feeder odb as drerribrd under Methods. Medium was replaced after 24 hr and every 3 or 4 <feye thereafter with medium coalmining TCDD with DMSO or DMSO alooc. At the indiealed liases of exposure, the cultures were trypsinaad, and ccB aumber per culture weft was determined. ------ , XBF/3T3, 0 0 1 DMSO; ----- , XBF/3T3, IO ^ m T&XX 0-01% DMSO;data .points represent the mean of fear repiiemm SE. *f` T > 1837? 96 G1ERTHY AND CRANE the mitotic inhibitor cholchicine, were tec. in 11 10-fold dilutions starting with a cc centration o f 100 Mg/ml. The effects rang, from obvious toxicity, characterized by c. disintegration at the high concentratic through inhibition o f cell division, to no c servable effects at the low ranges. The indi tion o f a flat-cell morphology similar to 0 induced by T C D D in replicate XBF/3T3 c tures was not seen at any concentration these agents. Fig. 5. Efli... of TC D D on incorporation of ['H]thymidine into XBFceils. XBFcelb were seeded with irradiated 3T3 cells and incubated as described under Methods. Medium was replaced after 24 hr and after 4 dayswith medium containing tnc indicated concentration of TCDD. On Day 7 all cultures had reached confluency and had the same number o fcells: At this time the cultures were pulse-labeled with (' HJthymidine (0.3 iCi/ml. 6.7 Ci/mmoi) for I hr, fixed, and counted for radioactivity as described under Methods. A DMSO concentration of 0.1% was the highest used in inis experiment (O). Data points represent counts per minute X I O '1 per 16 mm culture well and are the mean o f four replicates SE. about this t i n : the TCDD-treated celb showed a decline in growth, while the untreated ceils continued to proliferate to a higher saturation density (Fig. 6). The reduction in cell growth o f these cultures o f pretreated cells had the same sensitivity to TCDD (10~" M ) as cells which were not pretreated as seen by micro scopic flat-cell evaluation and in the gross observation o f fixed and stained cultures (Fig.2). DISCUSSION The X BF cells used in this study evolve after serial passage, from the cloned XB c ithelial cell line derived from a mouse ter toma. The XBF cells no longer have the r quirement for a fibroblast feeder layer or f medium conditioned by a fibroblast cultu for growth, as did the original XB line. Th loss o f the ability o f the X BF line to be induce C C X r- cr C N t*<. Inhibition o f Cell Division and the Morpho logical Change Subconfluent XBF cells were treated with other inhibitors of proliferation, which have various mechanisms, to determine if the flatcell morphology was a consequence of a gen eral toxic effect related to inhibition ofspecific macromolecular synthesis. Inhibitors of DNA (hydroxyurea), RNA (actinomycin D), and protein (cycioheximide) synthesis, as well as Fig. 6. Effect o fT C D D on cell growth-in XBF culture of cells previously treated wiih TCDD. XBF cells, whkr had been exposed in the presence of irradiated 3T3 celb lo KT' m TC D D for 14 days and exhibited growth in hibition ind morphological changes, were seeded with fresh, noo-TCDD-treated irradiated 3T3 feeder cells anc incubaeed as described under Methods. Medium was re placed 24 hr after seeding and every 3 or 4 days thereafter with medium containing either I0~* m TC D D in 0.01% DMSO or 0.01% DMSO. The cultures were uyprinixed at the indicated times and tbs ceil number per culture wedwasdetermined. ------- . X B F /3T3,0.01% DMSO. ------ n , X B F /3 T 3 ,10"* m TCDD, 0.01% DMSO. D au points represent the mean o f four replicates SE. '18378 r T . i t e r irr ]-riiirtfir iaiiii CONTROL OF IN VITRO PROLIFERATION BY TCDO 97 by TCDD to keratinize is consistent with re ports ofothers describing a change in this phe notypic expression in some XB ceils after serial culture (Knutson and Poland, (980b; Rheinwald and Green, 1975). We have found that continued culturing of XBF cells resulted in an increased cell density at saturation and ac quisition of a fusiform morphology at high density, in contrast to the cobblestonelike ap pearance of the original XB parent cell line. This change in saturation density and mor phology may be the result of a spontaneous in vitro transformation and has been stable for over a year of twice weekly subculturing. The XBF cells exposed to TCDD have stopped replicating based on the absence of [}H]thymidine-labeled cells and the lack of increase in cell number. This is supported by the decline in [3H]thymidinc incorporation into acid-insoluble products. These effects were not immediate, as would have been ex pected ifa toxic response of the type seen with previously discussed inhibitors of macromolecular synthesis had occurred. The XBF cells were seeded at low density and divided in the presence of TCDD at a rate no different from that of control cultures, until confluency was reached. The cessation of cell division at that time, unlike that seen in the keratinized stra tum comeum of the epithelium was probably not a consequence of terminal differentiation which results in cell death. This conclusion is based on the observation that the quiescent XBF cells, when trypsinized and reseeded at low density, grew to high density unless treated with TCDD, in which case they again grew to a lower saturation density than the un treated cultures. The population of cells that regrew was probably representative of the en tire culture rather than of a selected resistant population, since trypan blue exclusion and cloning efficiency indicate the same degree of .viability in high-density control cultures, lowdensity TCDD-treated cultures, and untreated stock cultures of XBF cells. These results demonstrate the reversibility of this effect in response to TCDD. The fusiform nature of epithelial ceils at high density was probably a consequence of the high saturation density of the XBF ceiis. The change from the morphology and growth characteristics of the XB to the XBF cells may reflect a progression to a less-differentiated progeny, with loss of regulatory control of growth or a decreased sensitivity to contact inhibition of proliferation, as seen in other cell cultures as a spontaneous in vitro trans formation. Exposure to TCDD may reestab lish this regulation, resulting in a return to the parent-cell morphology and saturation density characteristics. The effect ofTCDD on other cells in culture has been described previously and, to a lesser extent, in this report In one of these studies (Knutson and Poland, 1980a) involving sus pension cultures and anchorage-dependent cells grown on a substrate, no toxic effects (altered morphology, decreased viability, or diminished growth rate) were seen in 23 cul tured cell types. Sparse cultures of the 16 an chorage-dependent cell types grown on a sub strate were unaffected by exposure to 10~7 M TCDD in regard to the previously mentioned end points from the time of seeding to confluency, at which time they were trypsinized and evaluated for viability and density. Our results for pteconfluence growth with the XBF line agree with these findings. However, the observed change in cell growth rates and mor phology in TCDD-treated XBF ceils was ap parent only after confluency was reached, as the untreated cultures continued to proliferate resulting in the observed high density. This effect was not due simply to inhibition of cell growth since inhibitors of macromolecular synthesis and mitosis did not induce this effect. TCDD is extremely potent for inducing certain gene products and keratinization. Further studies may indicate the usefulness of TCDD for inducing a specific gene product associated with the regulation of cell proliferation in the XBF system. The abilities of TCDD to induce keratini zation in the parent XB cell line and mor- t C r cr c r> c 1837S 9 7 7 0 0 I 7Mfin 98 G IERTHY A N D CRANE phologicai and proliferative change in the XBF cells may.be related. The existence of a highaffinity cytosolic receptor for TCDD in the XB cells has been demonstrated by others (Knutson and Poland. 1980b), and its inter action with TCDD has been postulated to be obligatory for a number of TCDD-induced responses (AHH induction, keratinization, etc.) (Poland and Knutson, 1982). Further studies of the XBF cell response to TCDD may determine if this receptor is pan of the mechanism of the induction of the flat-cell effect. The reduction of saturation density and morphological change seen in vitro may be analogous to the in vivo epithelial change from proliferating basal cells to nondividing cells of the stratum spinosum. Studies using ultrastructural and biochemical analyses forspecific markers of this differentiation may answer this question. The sensitivity of this effect suggests the use of this system as an assay for TCDD if specificity for this compound can be dem onstrated. ACKNOWLEDGMENTS The authors thank Ms. Victoria Laoibcnoo for prep aration of the typescript- This work was supported in part by National Institute of Occupational Safety aod Health Grant OHO 1333. REFERENCES Ft m , K.A.O-, and G ierthy. J. F. (1977). Mechanism of refutation of fibroblastic ceil repricatioa. IV. An analysis of the serum dependence of cell replication baaed on Michaciis-Mcnicn kinetics. J. C M Physiol. 92, 381-400. Gr e e n u i, W. F,, and Poland, A. (1979). Nudear up take of 23.7,8-tetradilorodibenzo-p-dioxm in CS7BL/ 6J and DBA/2J mice. J. Biol C W IS A 9814-9821. Henol. J. M,, New, M. a, Kocjia. R. J , and Rad, ' K. S. (1981). 2J,7,8-Tctracbloridibcazo-p~dioxia: acute oral toxidey in hamsters. Toxicol Appl Pharm. 59, 403-407. Huff, i . E_ M o o . J. a , Saraco. R , and Tomato, L. (1980). Loaf term hazards of polychlorinated dibenzodioxint and polychlorinated dibenzoAvans. En viron. Health Perspea. 36,221-240. Kjence, a . S., Wade, J. J.. Ridge. D.t and Poland, a . (1974). Synthesis and fourier transform carbon-13 nu clear magnetic resonance spectroscopy of new poly, hakxlibcnzo-p-dioxini. J. Org. Chem. 39. 931-937. Kimbrough. R. (1974). The toaiciiy of polychlorinated polycyclic compounds and related chemicals. CRC Cnt. Rev. ToxicoL 2, 443-498. Knutson, J. G , and Poland, a . (1980a). 2,3,7,8-Teiradtlorodibcnzo-p-dioxin: Failure to demonstrate tox icity in twenty-three cultured cell types. Toxicol. Applied Pharmacol 54, 377-383. Knutson, J. G , and Poland, A. (1980b). Ketatinization o f mouse teratoma cell line XB produced by 2.3,7.8tcrmchloridibenzo-p-dioxin: An in vitro model of toxkrity. CeU 22. 27-36. Okey, a . Ik, Bondy. G. P.. Mason. M. E , Kahl, G. F,, Eisen, H. J - Guenthner, T. M,, and Nebert, D. W. (|979)lRegulating gene product nf the Ah locus. Characterization of the cytosolic inducer-receptor com plex and evidence of iu nudear translocation. J. Biol. Chem. 254, 11636-11648. Okey, a . B-, Bondy, G. P,, Mason, M. E.. Nebert. D. W,, FOrster-Gibson. C. J , Muncan, J.. and Dufresne, M . J. (1980V Temperature-dependent cytosolto-nudeua translocation of the Ah receptor for 2J.7.8tetrachlorodibenzo-p-dioxia in continuous cell culture lines. J. Biol Chem. 255, 11418-11422. Poland, A-, and G lover. E. ( 1973V Genetic expression of aryl bydrocarboo hydroxylase by 2.3,7,8-ictrachloredibenzo-p-dioxin: Evidence for a receptor mutation in genetically non-responsive mice. Mol. Pharmacol. 11, 389-398. Fo u n d , A_. and Glover. E (1977). Chlorinated bi phenyl inductioo of aryl hydrocarbon hydroxylase ac tivity; A study of the structure-activity relationship. Mol. Pharmacol 13, 924-938. POLAND, A^ and KenDE, A. (1976V 2,3,7,8-Tcirachlorodibetuo-p-dioxin: environmental contaminant and molecular probe. Fed. Pmc. 35, 2404-2411. Poland, A_ and Knutson, J. ( 1982V 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related iulogenated aromatic hydrocarbons: Examination of the mechanism of toxidey. Arum. Xev. Pharmacol Toxicol. 22. 317-334. Rheinwald, 3. G.. and G reen, H. (1973V Formation o f a keratinizing epithelium in culture by a cloned cell line derived Dam a teratoma. Cell 6, 317-330. Sc h w e iz , B.a ., Norris, j . m ,, Starschu, G. L , Rowe. V . K , Gehrinq , P. J , Emerson, J. 1_ and Gerbio, G G. (1973V Toxicology of chlorinated dibenzo-pEnviron. Health Persp. 5, 87-99. Tu u y , R. H , Hannah, R. R-. Masahiko, N.. Nebert. D,, and EtSEN, H. j . (1982V The Ah Locus: Correlation of intranuclear appearance of inducer-receptor complex with induction of cytochrome PM 50 in mRNA. Cell 31,275-284. C- 231 CD O r'O 03 C3 (S 3 CO K -(lO) 9 8 2 0 9 1 ZM0Q TOXICOLOGY ANO APPLIED PHARMACOLOGY 73. 4 2 - 4 7 (1 9 8 4 ) Increased Epidermal Transglutaminase Activity following 2,3,7,8Tetrachlorodibenzo-p-dioxin: In Vivo and in Vitro Studies with Mouse Skin S. M . Pu h v e l, 1 D. C. Er tl, a n d C. a . Ly n berg Division o f Dermaioiogy. Department of Medicine. School o f Medicine. Centerfor Health Sciences. University o f California. Los Angeles. Las Angeles. California 90024 Received June IS. 1983: accepted October 25. 1983 Increased Epidermal Transglutaminase Activity following 2.3,7.8-Tetrachlorodibenzo-p-dioxin: In Vivo and in Vitro Studies with Mouse Skin. Puhvel. S. M .. Er tl, D. C . and Lynberg. C. A. (1984). Toxicol. Appl. Pharmacol. 73, 42-47. In previous studies it has been shown that topical treatment ofhairless mice with 2.3.7,8-tctnchlorodibenzo-jvdioxin (TCDD. dioxin) induces hyperproliferaiion and hypcrkeratinization in the epidermis of hairless mice. The present in vestigation demonstrated that such TCDD-induced morphological changes in skin in vivo are accompanied by increased levels in activity of epidermal transglutaminase (ETG ). the enzyme associated with terminal epidermal differentiation. Exposure of mouse epidermal cells in tissue culture to I0~* M TCDD also resulted in a significant increase in ETG activity, despite the fact that morphologically these cultures (grown at 0.07 mM ionic calcium concentrations) exhibited no signsof terminal differentiation. Thus one mechanism o faction o fTCDD in inducing cutaneous changes appears to relate to the stimulation o f increased ETG levels. In humans one o f the first signs o f expo sure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TC D D ) is the development o f a skin con dition known as chloracne (Moore, 1978). This syndrome involves hyperkeratinization of ductal epithelium of cutaneous sebaceous follicles, resulting in comedo formation which may or may not proceed to pustular and cystic chloracne. Experimental chloracne has been induced in skin of hairless mice by topical application o f TC D D (Puhvel et al., 1982; Knutson and Poland, 1982). Even though fol licular involvement in mouse skin is insig nificant, the hyperproliferative, hyperkeratinizing changes induced in interfollicular epi dermis are sufficiently well defined to permit the use of this animal model to monitor TCDD-induced enzymatic changes in skin. In this report we investigated the effect of TC DD on epidermal transglutaminase (ETG) activity first in skin o f hairless mice in vivo following topical application o f TC DD . Then, to verify our observations in a more defined system, changes in ETG activity of mouse epi dermal cells in tissue cultures were monitored following addition o f T C D D to culture media. METHODS Chemicals. 2.3,7,8-Tetrachlorodibenzo-p-dioxin was obtained from KOR, Incorporated (Boston, Mass.), and putrescinc dihydrcchloride, 2,3-JH (N ) (sp. act. 38.9 C i/ mmol) was obtained from New England Nuclear (Boston, Mass). Tissue culture Medium 199. calcium depleted Me dium 199. fetal bovine serum (FBS). and antibiotic-an timycotic mixtur were obtained from Grand Island Bio logical Co. (Grand Island, N.Y.y. puiresdnc dihydrochloridc. casein, dithiolhreitol (D TT ). and Trizma base were from Sigma Chemicals (St. Louis, Mo.); dimethylcasein was from Accurate Chemical and Scientific (Hicksville, N.Y.). In Vivo Studies ' To whom requests for reprints should be addressed. Animals. HRS/J strain of hairless female mice were obtained from the Jackson Laboratories (Bar Harbor. 004I-008X/84 13.00 CowmW C IW4 bf Academic Pent lac. A ll n g * u o f re p ro d u ctio n in any fo rm it i mrved. 18382 P0HZI6Q239 INCREASED EPIDERMAL TRANSGLUTAMINASE A C TIV ITY FOLLOW ING TCDD 43 Maine). Animals were 10 to 11 weeks old at the beginning ofthe experiment. They were ted Wayne Lablox (Universal Feeds. Inc., Colton. Calif.) laboratory chow ad libitum and were housed four to a cage in disposable plastic cages msidc a Biohazard safety containment box (Class III Glove box) for the duration of the experiments. Treatment. Three limes a week for 4 weeks animals were treated topically with 0 .1 yg of TCDD dissolved in 0.1 ml of acetone. Control animals received 0.1 ml of acetone alone. Groups of four mice were killed by cervical dislocation at 7. 14, 21. and 28 days after the beginning of treatment. Skins were removed by dissection, and the epidermis was separated from dermis by first immersing the skins in a S5*C waterbath, followed by immersion in ice water. Following this treatment, the epidermis was scraped from the dermis with a scalpel, while maintaining the skins at 4*C. Epidermal samples were homogenized with a Polytron homogenizer, and the tissue cytosol was separated by centrifugation at 30.000; for 30 min. Su pernatant fractions were used for the transglutaminase assays. Transglutaminase assay o fepidermal Homogenates. The methods described by De Young and Ballaron ( 1982) were used. Briefly. 0.1 ml of tissue supernatant fractions was added to 0.3 ml of assay mixture containing 0.0S M Tris buffer (pH 8.1), 0.01 m CaClj, 0.03 m D TT, 0.6 mg diroeihylcascin, and 0.37 nCi of (' HJputresdnc. Mixtures were incubated at 37*C for 60 min. and the reaction was supped by adding0.6 ml of 10 trichloroacetic add (TCA) and 5 ml o f5% TCA containing 0.1% unlabeled pufresdne. After 30 min at room temperature, incubation mixtures were filtered on 25-ram Whatman G F/A filter discs. Discs were washed once with 10 ml 3% TCA containing 0.1% unlabeled putrescinc and twice with 10 ml 100% ethanol. After drying, the discs were placed in Aquasoi II (New England Nuclear) and the radioactivity was counted. Sol uble proteins in the supernatant fractions were quantified by the Bio-Rad protein assay with bovine serum albumin as standard (Bradford. 1976). In Vitro Studies Tissue culture. Basal kratinocytecultures from neonatal BALB/c mice were established following the methods de scribed by Marcelo el at. (1978). Basal kratinocytes were separated by discontinuous Ficoll gradient centrifugation, and 1.5 x 10* cells were inoculated per 30-mm-diameter culture dish. Cultures were grown in parallel in the fol lowing media: one-third of the cells were plated in Medium 199 supplemented with antibiotics and 10% FBS; the re mainder were plated in calcium-depleted Medium 199 supplemented with antibiotics and 10% FBS (5% chelated, 5% normal). The final ionic concentrations of calcium in these media were 1.2 and 0.07 ism . respectively. TCDD treatment. On the third day of culture. TCDD dissolved in dimcthylsulfoxide (DMSO) was added in a final concentration of 10"* M to half the low calcium cultures. The remainder o f the low calcium cultures re- ccived DMSO alone in a final concentration of 0.1%. The medium was changed every other day and fresh TCDD and DMSO were added at every medium change. Cells were harvested from four dishes of each of the three media on Days 3. 5. 7, 10. and 12 by scraping the plates with rubber spatula and collecting all cells (i.e.. attached as well as free floating), by centrifugation. Cell pellets were washed twice in buffer and stored frozen at -6 0 *C until used for the transglutaminase assay. Transglutaminase assay of cell pellets. Cell lysates ob tained by thawing and freezing cell pellets three to four times were used for the transglutaminase assay recom mended by S. H. Yuspa and U. Lichti (personal com munication). This procedure was an adaptation of the method described by Ogawa and Goldsmith (1976). Cells were pelleted in a buffer containing 50 mM Tris (pH 7.5). 2.5 mM DTT. 0.13 M NaCL 0.83 mM EDTA, and 8.3 mM C aQ ], The reaction mixture consisted of 100 mI of cell lysate. 20 *1 o f casein (20 m g/m l). 10 yl o f ['Hlputrescine (5 uCi). and 20 ul of buffer. The mixture was incubated at 37*C for 10 min. Following incubation 50 ill of the mixtures was spotted on filter paper discs (Whatman 3M M ) and immediately precipitated by im mersion in 10% ice cold trichloroacetic acid (TCA) plus 0.1% putrescinc. The precipitates were washed in four separate 30-min washes of 5% TCA plus 0.1% putrescinc. tlien immersed in 100% ethanol, and dried. The radio activity of the precipitates on the filter paper discs was counted in a Beckman LS-7500 liquid santiilation counter. Results were corrected for background radioactivity (bund in lysate-free parallel assays. Soluble proteins in the cell lysates were determined by the Bio-Rad protein assay. To monitor the effect of 10"* M TCDD on epidermal cell proliferation, cell counts were performed at regular intervals in cultures grown in low calcium medium, with or without the presence o f TC D D in the medium. The effect of TCDD on terminal differentiation of epidermal cells in culture was investigated by transferring cultures grown in low calcium medium (with and without the presence of I0~* M TCDD) to high calcium medium on the seventh day ofculture, and monitoring the morphology of the cultures and the rate o f comificd cell envelope formation at 24 hr to 5 days after the change to the 1.2 mM Ca2* medium. Comificd envelope counts were per formed according to the methods described by Yuspa et at. ( 1982) with insolubility in 2% SDS and 20 mM D TT at 90*C for 5 min as the criterion for defining comified envelopes. RESULTS In V ivo S tu d ie s Hairless HRS/J mice treated topically with TC D D developed the same pattern of welldefined cutaneous changes which have been described in detail elsewhere (Puhvel el at.. 18383