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Tabic 0. Mortality of 2,4-U and 2,4,5-T exposed workers (hiring the follow-up period of 572-15/6. 'x Lxpocled numbers of deaths no based on the cloatb r.itus of the total Finnish male [opulation. CMuno or death (.ujo, yeirs) exposure started in the years 1950-1065 Observed number of deaths (O) . expected number of deaths (l~.) PoiraonyCcirs 1966 -1971 (0) IE) Personyear a Total (0) (b) I'lM'i'b'mycars ;i.i1i'in..r.l neoplasms )*>--2! 25-31 35-44 45-54 55-64 65-74 75 and over Total All natural causes of doatli 1 4 1 - 6 0.0 0.1 0.4 1 .5 4.2 3.8 0.2 10.2 52 720 9.1U 937 690 294 9 3628 - 0.1 - 0.3 - 0.5 - *2.0 2 4.1 - 0.8 -- 1635 1567 1256 1233 671 64 " _ 0.1 - 0.4 - 0.9 1 3.6 6 8.3 1 4.6 0.2 1037 2255 2174 2170 1361 353 9 2 7.8 5826 8 18.1 5454 IBBBSaBBaaBBBBBBUBBBCBIBSS SIS333=0 15-24 25-34 35-44 45-5 1 55-64 65-74 75 and over - 0.0 - 0.5 2 2.2 3 8.1 7 15.7 8 15.4 - 1.2 0.3 1 .0 1 3.1 6 10.6 6 15.3 1 3.3 " 0.3 1.5 3 5.3 9 18.7 13 31.0 9 10.7 1.2 Total 20 43.1 . ; ^ : . ': : s i i s c u ^ j i n B C S B a a a a B a E E j B U 3 B n i i if B ; ii 14 33.6 34 76.7 c.sarsuS3 000232 <1 ***oCO.. CO tlMl&faoa Table 6. Mortality of 2,4-0 an<l 2,4,5-T exposed workers by employer during the follow-up period of 1072-1976. Lxpeeled numbers of deaths arc based on the death rates of Lite total Finnish male population. Cause of death State hallways Observed expected deaths deaths (0) <L) Highway Authority (0) (Is) Forestry Authority (0) (b) Power COiT.iJriJV/ (O) (Ei All malignant noo|>J asms Hes|ji ratory cuncur All natural causes of death 7 4 15 7.2 3.1 29.8 1 3.6 - 1.5 9 15.6 V 7 6.4 2.6 27.7 0.9 - 0.3 3 3.6 0002324 rJ S Z 9 S i G M O a CoO 4*. Tahiti 7. Localisation of maliynant neoplasms amony 2,4-D and 2,4,5-T exposed workers. Observation period Localisation Luiiy Larynx Stomach Caecum Prostate Total 1950-1971 1972-1976 7 4 2 1 4 1 \ 1 1 13 0 00023 I ! J '^ 9 2 9 2 ie M o a 7306 0 DOW 129579 Strictly Confidential Chlorinated D iL e n zo -p -d io x in s and Dihiintof-jrans : Ruview o f T o x ic o lo g y and Cnryne Induction and Cons i d e r a tio n o f i'.echanisn o f T o x i c i t y . s 3- ,, IARC January 1973 Alan Poland -H e VJ\ I 73o-7 !-' ~>n^ Ll yCj\.}A.J I. The chenistry, toxicological and biochemical effects of the chlorinated dibenzo-p-dioxins and dibenzofurans have been extensively investigated in the past few years, and this information has been thoroughly reviewed in a number of symposia and reports. Despite these efforts, at present little Is known about how these compounds exert their toxicity. In this report I would like to briefly outline the najor toxic actions of these compounds, consider selected biochemical effects and propose a model for their mechanism of toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype of the group; it is the most potent toxin and the most thoroughly studied. The major toxic effects produced by TCDD compound are summarized below. 1) Chloracne ~ The most frequent sign of toxicity observed anong chemical workers exposed to TCDD has been chloracne, comedone formation, with or without cysts and pustules, often widespread In body distribution, and very persistent and refractory to treatment. Chloracne has been produced experimentally by TCDD In humans, monkeys, rabbits, and hairless mice. The lesion is characterized by hyperkeratosis, acanthosis, and keratinous material producing cystic dilitation of sebaceous glands. 2) Hepatotoxicity - The liver toxicity produced by TCDD varies greatly with the species. In the rabbit and the rat, there.is severe liver damage, with parenchymal cell necrosis, fatty infiltration, hyaline bodies, formation of multlnucleate giant cells and signs of hepatic dysfunction such as hyperbilirubinemia and.hypoalbuminernia. Hepatic damage is less extensive in-mice, and minimal and focal in guinea pigs, the species most sensitive to -the toxic effects nf TCDD.-- iJepat-i-e-porpliy~rirr accumulation and/or porphyria was observed reported in mice, rats, and humans exposed to TCDD. 0001581 DOW 129580 7308 2. 3) Thynus and lymphoid Organs - TCOD produces a dose related decrease In the mass of the thynus and a less dramatic change on the lynph nodes and lynphoid tissue of the spleen. There Is a rapid atrophy of the cortical cells (7-cells) of the thynus, and In the rat, mouse and guinea pig a suppression of the cell-mediated I m u n e response. 4) Teratogenesis - TCDD Is enbryotoxlc and teratogenic to fetal mice, rats, and guinea pigs. Fetal resorption, cleft palate, renal lesions, and edema of the soft tissue have been reported. 5) Chick edena - In newborn chicken, TCDD produces an edematous syndrome characterized by anasarca, and hydropericard!urn. 6) Other effects - TCDD and other dioxins have been reported to cause a number of other types of toxicity: neuronuscular effects, epithelial atrophy, metaplasia, and hyperplasia, hemorrhage In several tissues, gonadal atrophy, hyperlipidemia, depression of the blood forming elements in bone marrow; and lesions of the endothelium, myocardium, and kidney. Qualitatively and quantitatively the histopathology produced by TCDD differs with different species, and with acute versus chronic administration. ,,Following the administration of a lethal dose of TCDD, the animal loses weight, exhibits decreased motor activity, and eventually dies after a period of several weeks. Sensitivity to the lethal effects of TCDD varies greatly with species: the acute oral LD^g *s ' u9/^3 *n 9u Inca P'3S J 22 ug/kg in male rats; k 5 ug/kg in female rats; 1 15 ug/kg in the rabbit; 100-250 ug/kg in the mouse; and greater than 300 ug/kg In the dog. Pharmacokinetics * Following acute or chronic administration of TCDD to a rat, the drug Is accumulated primarily in the liver and to a lesser extent the fat, eliminated largely in the feces wi th a whole body half-life of about 21 days. Numerous attempts to demonstrate the netabolism of TCDD in vitro or Isolate a DOVV129581 CI mM o o 7309 3. D w 129582 metabolite In vivo have failed, however chronic feeding studies using radiolabeled TCDO suggest a more polar radiolabeled conpound appears In urine in amounts too snail to identify chenically. Direct demonstration of the metabolisn of TCDD is lacking, but if metabolism occurs, it proceeds very slowly. The acute toxicity of TCDD appears to be due to the parent conpound. M u t a g e n i c i t y and C a r c i n o g e n i c i t y There are two reports that TCDO Is mutagenic in bacteria, but no consensus has been reached on this. The data is anply reviewed in the C IARC report. Two recent reports Indicate that chronic life-time administration of low levels of TCDD to rats Is associated with an increased incidence of neoplasia. The study by the Dow Chemical Co. (as yet to be published) states: "In these rats receiving 0.1 ug/kg/day (but not 0.001 or 0.01 ug/kg/day) there were discernabie increases in the Incidences of hepatocellular carcinomas and squamous cell carcinomas of the lung, hard palatc/nasal turbinates or tongue". Van Miller, Laltch and Allen reported tumors In rats fed TCDD 5, 50, 500, 1000 and 5000 ppt in the diet. The low dose 5 ppt Is equal to 0.14 ng/kg/day or a life time dose of 95 ng/kg. In contrast to the Dow study, these authors found neoplasms at a 700 fold lower . concentration of TCDD in the diet, the neoplasms were of many types, and the incidence of neoplasia was not dose-related. ' j..-; : While these two reports suggest that the chronic administration of . TCDD is associated with an increase incidence of neoplasms, they do not indicate whether TCDD Is an Initiator or a promoter. This consideration is particularly Important because we lack unequivocal evidence that TCDD is ro C/J to o a mutagen and that TCDD is metabolized, and there Is no evidence that TCDD and/or its metabolite(s) covalently bind to macromolecules. 7310 I A. Induction of Aryl Hydrocarbon Hydroxylase Activity -- The administration of TCDD and other halogcnated dibenzo-p-dioxins and dibenzofurans stimulates a number of enzyme activities, most notably in the liver. I will confine my remarks primarily to the induction of hepatic aryl hydrocarbon hydroxylase (AHH) activity also called benzo(a)pyrene hydroxylase. AHH activity is one measure of microsomal monooxygenase function. The microsomal enzyme complex which consists of HADPH-cytochrome P-k50 reductase, and several cytochrome P - k $ Q subspecies (or Isoenzynes) , is responsible for tie metabolism of many lipophilic xenobiotlcs to more polar metabolites. ' Since many chlorinated lipophilic compounds which have long biological half-lives, such as DOT, dieldrin, lindane, and the polychlorinated biphenyls stimulate various hepatic microsomal monooxygenase activities, it comes as no great surprise that the chlorinated dibenzo-p-dioxins and dibenzofurans also have this capacity. However, rather than regarding this biochemical effect as common place, I wish to suggest that It is telling us something about the mechanism of toxicity of TCDD and its congeners. In the chicken embryo, TCDD produced a dose-related increase in hepatic rAHH activity, with an ED^g (dose that produced one half the maximal response) of 0.3 nmol/kg. In testing an initial series of 15 halogenated dlbenzo-pdioxin congeners, a well-defined structure-activity relationship was observed: The congeners which Induced AHH activity had 1) halogen atoms in at least three of the four lateral ring positions (2,3,7 and 8); a) at least, one .t. carbon atom was unsubstituted (octachlorodibenzo-p-dioxin was Inactive); and 3) the order of potency of substitution was B r > C l > F , NOj TCDD also produced a dose-related increase in hepatic -aminolevulinic acid synthetase DOW 129583 Cl O o 7311 5. (ALAS) activity in the chicken .nbryo, and the structure-activity relationship for the induction ALAS activity by the 15 halogenated congeners was the sane-- as that observed for Induction of AJiH activity. Many more halogenated . dibenzo-p-dioxins and dtbenzofurans have been studied for their capacity to Induce AHH activity in the chicken enbryo liver or in a hepatoma cell culture (Bradlaw and Firestone) and the structure-activity relationship outlined above has prevailed. The most significant finding was that there was an-excellent correlation between the potency of chlorinated dibcnzo-p-dloxin congeners to Induce AHH activity (and ALAS activity) and their tox'Ic potency. The work of Schwetz and his coworkers suggests that fey a given chlorinated dibenzo-p-dioxin which has a certain lethal potency (low L D ^ ) , it has a corresponding potency as a teratogen, acnegen, and potency In producing chick edema. The" recent study by McConnell et^ a k on the comparative toxicity of 13 chlorinated dibenzo-p-dioxins in mice and guinea pigs support thjs idea, that Is, the relative potency (or rank order) of a congener to produce one toxic response Is a good Indicator of Its relative potency (or rank order) to produce other toxlc man Ifestat Ions. Hepatic Cytosol Dindlnq Protein ^ ..v. Recently, a macronolecular binding species has been characterized In . the hepatic cytosol fraction of rat and mouse liver which has the In vitro binding properties predicted for the receptor for the Induction of AHH activity based on the In vivo biology. Namely: 1) JH-TCDD binds to this cytosol protein reversibly with a high affinity (Kd 0.27nM) comparable .to the EDgg hepatic AHH Induction (EDj.g In mlce'-wl nmole/kg); 2) the * binding affinity of halogenated dlbcnzo-p-dloxins and dlbenzofurans for this protein In vitro corresponds to their potency to Induce hepatic AHH activity In the chicken enbryo; 3) other compounds, such as the polycyclic aromatic hydrocarbons, which Induce AHH activity and cytochrome P| --^50 also conpcte for this cytosolic binding protein, but compounds which 129584 oo ! $ 1.0 CO m O ro 6. Induce other types of micrcsonal ronoaxyginase activities (e.g., phnobarbital) Q and steroids fail to bind. Thus It would appear.this cytosolic binding --^ 129585 protein is the receptor for the Induction of AHM activity. Structure-Activity Rclationship The structure-activity relationship for the Induction of hepatic AHM activity and for binding to the hepatic cytosol binding species has been extended to other classes of chlorinated aromatic compounds, and again there appears to be an excellent correlation between their capacity to induce /Mill activity and their potency to produce specific toxic effects similar to those produced by TCDD (e.g., chloracne, thymus atrophy, and chick edema). j- 3,4,3'4'-Tetrachloro azoxybenzene (TCAOB) and azobenzene (TCAB) are potent acnegens formed as trace contaninants in the synthesis of 3,4 dichloroani1Ine or herbicides based on this conpound. At high doses In animals, TCAB is reported to produce thymic Involution and liver damage similar to TCDD. Both TCAOB and TCAB arc potent inducers of hepatic AHM activity and bind to the hepatic cytosol binding protein with a high affinity. Congeners such as 3,5,3'5,-tetrachloro azoxybenzene and azobenzene fail to induce AHM activity, fail to bind to the hepatic C cytosol species, and fail to produce chloracne. Of 16 halogenatcd biphenyl tested, only 3,4 ^^'-tetrachloro,- a-- i 3,4i5,3'4,5' hexabrono-biphenyls induced hepatic AMH activity and bound ChLrO-- to the hepatic cytosol binding species. The 3.4,3,4 l-tetrachlorobiphenyl has been reported to produce chloracne. McKinney et al. found that of _.:five hexachlorobiphenyls tested In chickens, 3,4,5,3,4'5,"hexachlorobfphenyl wjs by far the most toxic, and the only one to produce significant chick edema and Involution of the thymus. C.1CJ Wft . 7313 98S62T From these studies we con generalize about the receptor site for binding halogenotcd aromatic compounds and initiating the Induction for AHH. 2 ,3,7,3-Tetrachlorodibcnzo-p-dioxin, 2,3,7,3 tetrachlorodibenzofuran, 3,4 ,3*4'-tetrachloroazoxybenzene and 3*4 ,3'4'-tet rachlorobIpheny 1 are approximate isosteroomers. 3 Te-FcWTel. c Cl J V/ ^v Ail of these compounds are planar or can assume a nearly planar - configuration with halogens at 4 corners of a rectangle approximately 3& o by 10A. We have synthesized a number of other tetrahaloheterocycl1cs - such as anthracene, phenanthrene, bipheny!ene, and phenoxazine - all of c ^ which bind to the hepatic cytosolic binding species and induce AHH activity. It remains to be proven whether all halogenated aromatics which '**' bind to this receptor and Induce'AHH activity do produce TCDD-like .1. ,. ' m * toxicity. At present to the extent the data Is available, I am not . * aware of any exceptions. However we must not forget many nonchlorinated ' polycyclic aromatic hydrocarbons bind to this site and induce AHH activity, but it has not been noted they elicit any TCDD-like toxicity. ! 00015S7 . 7314 8S6Zl^OCf 8. A IVjdcl for Mechanism of Toxicity __ The case I have tried to present is that there is a striking correlation for chlorinated dibenzo-p-dioxin, dibenzofurans and other chlorinated aromatic congeners between their potency to induce AHH activity and their toxic potency to produce specific lesions (chloracne, thymic involution, . chick edema). This correlation is an appropriate starting point in considering a molecular mechanism of toxi city. .* A) Older Theories - AHH Induction Per Say 1) It has been suggested that the chronic stimulation of microsomal monooxygenase activity produce by TCDD.might result in excessive inactivation of endogenous compounds (e.g., steroids) or produce excessive harmful metabolites of endogenous compounds. 2) It has been suggested that TCDD was metabolized to some reactive intermediate which covalently bound to some essential macronolecules analogous to the mechanism of hepatotoxicity proposed for bromobenzene, phenacetin and isoniazid. If the rate limiting step were the formation of the reactive metabolite of TCDD/then congeners which induce AHH activity, might stimulate their own metabolism and flood the system with an excess of reactive metabolites. Both of these hypotheses can probably be rejected ^on the weight of the available evidence. But while It doesn't appear that induction of AHH activity per se Is deleterious, we still must explain its correlation with toxicity. B) Coordinate Gene Expression ' ...So far we have focused only on the induction of AHH activity, but it is known that the administration of TCDD or one of the classical polycyclic aromatic hydrocarbon Inducers such as 3-nethylcholanthrene (MC) stimulates not only AHH activity but a number of other enzyme activities. 0 0 015 C8 DOW 129588 Doth TCDO and MC have been shown to Induce the following hepatic enzymes: _ cytochrome P |-^50 nediated nicrosonal ronooxygenase actlvi ty, *-aminolevul inic acid synthetase, aldehyde dehydrogenase, glutathlone-s-transfcrase 0, UDP' glucuronosyl transferase, and DT-dlaphorase. Very likely there are more enzymes which arc coordlnately expressed by one of these Inducers. From genetic studies In Inbred strains of nice (not reviewed here) it appears the Ah locus codes for the cytosolic receptor protein and this protein when combined with the Inducing compound (e.g. TC00) Initiates the coordinate expression of at least AHH, UDP-glucuronosyltransferase, and DT-diaphorase, and perhaps many more structural genes. We suggest that the cytosolic binding protein serves as the cellular recognition site for TCDO and like compounds, and the binding of these compounds to the receptor serves as a master switch which Initiates the expression (or repression) of numerous noncontiguous structuroigenes. This Is analogous to the battery of genes which are coordlnately expressed by the steroid hormones. Our hypothesis is that the sustained expression (or repression) of one or more of the genes controlled by the Ah locus (the TCDD-receptor '.complex) leads to the toxl c man fes tat Ions characteristically associated with the chlorinated dibenzo-p-dloxins. .Thus Induction of AHH activity is merely a signal that this gene battery is activated. This hypothesis: 1) appears to fit all the observable data, and while it does not indicate a specific biochemical lesion, 2) It does permit further Investigation to support or reject the proposal, and 3) It does suggest we might be able to Intervene at the level of the receptor (by a competitive antagonist) without knowing the ultimate biochemical lesion. 0 0 015 C f s . V X 7317 QOT onnn n n n TOXICOLOGY OF PHENOXY HERBICIDES AND H AZARD ASSESSMENT OF T H EIR USE IN REFORESTATIO N By Frank N. Dost, Ph. D. (Toxicologist, Associate Professor of Veterinary Medicine, Environmental Health Science Center, Oregon State University) U.S. Department of Agriculture Forest Service, California Region (Region 5) 630 Sansome Street San Francisco, California 94111 May 1978 7318 0000075 /.ox P o n o M O O TOXICOLOGY OF PHENOXY H ERBIC ID ES AMD HAZARD ASSESSM ENT OF TH EIR USE IN R E FO R EST A T IO N Frank N. Dost, Ph. D. FOREW ORD This report was prepared by Dr. Frank N. Dost (Toxicologist, Assoc. Prof. VetMedicine, Environmental Health Science Center, Oregon State University) under a contract with the U.S. Forest Service. The report was designed to provide a comprehensive evaluation of the hazard to human health of phenoxy herbicides, specifically to include 2,4-D, 2,4,5-T, and Silvex as used in forest, range, and brushland management on the National Forests in California. Dr. Dost reviewed cur present environmental statements on forest reestablish ment, rangeland enhancement, and brushland management and analyzed risks and provided suggestions on how to best minimize hazards to human health. He reviewed all available published and unpublished reports on research and studies, of herbicide effects. His work was reviewed by 16 scientists in related fields, .and their comments were considered in his final report This report is part of our current effort to do the best possible job in updating and, where appropriate, revising the Forest Service environmental statements for forest reestablishment, rangeland enhancement, and brushland management D O U GlL A S R. L E IS Z Regional Forester 7319 0000076 ACKNOWLEDGMENTS The author and Region 5 wish to thank the following individuals for their assistance and critical review of this paper. Dr. Hugh Black, Associate Professor Forest Wildlife Ecology Dept, of Forest Management Oregon State University Corvallis, Oregon 97531 Dr. R. K. Boutwell Professor of Oncology McArdle Laboratory for Cancer Research University of Wisconsin Madison, Wisconsin 53706 Thomas Milby, M.D. Stanford Research Institute 2150 Shattuck Avenue Berkeley, California 9^706 Donald Morgan, M.D. Agricultural Medical Institute Oakdale, Iova 52319 Dr. Logan Norris U.S. Forest Service, PNW 3200 Jefferson Way Corvallis, Oregon 97331 Dr. Don Crosby Dept, of Enviromental Toxicology University of California Davis, California 95616 Dr. Mike Newton Dept, of Forest Mgt. Oregon State University Corvallis, Oregon 97331 Ephraim Kahn, M. D., Chief Dept, of Public Health Epidemiological Studies Lab 2151 Berkeley Way Berkeley, California 94794 William Griffith California Dept, of Fish Game 987 Jedsmith Drive Sacramento, Ca. 95819 Dr. Steve Radosevich Dept, of Botany University of California Davis, California 95^16 Dr. Rojer Sandquist U.S. Forest Service Region 6 Fay Shon U.S. Forest Service, PNW 3200 Jefferson Way Corvallis, Oregon 97331 Dr. Keith Maddy California Department of Food 5 Agriculture 1220 N. Street Sacramento, California 95814 Dr. Wilbur McNulty^ Oregon Regional Primate Center 505 N. W. 18S Avenue Beaverton, Oregon 9700S Dr. Brian Sturgess U.S. Forest Service Region 5 Dr. James Witt, PH.D. 3223 N.V. Glyvood Cirdle Corvallis, Oregon 97330 7320 0000077 TOXICOLOGY OF PHLNOXY HERBICIDES AND HAZARD ASSESSMENT OF THEIR USF. IN REFORESTATION Introduction ........................................................ 1 Toxicology of T C D D .................................................. 3 History of Human Exposure ...................................... 3 General Toxicity in Laboratory Animals ..................... 7 Pathologic Morphology Resulting From TCDD Intoxication . . . 11 Effects of TCDD Upon Reproductive Functions ................. 15 Carcinogenic and Mutagenic Potential of TCDD ............... 18 Absorption, Distribution, Metabolism, and Excretion of TCDD . 20 Behavior of TCDD in the Physical Environment and in Submammalian Species . ...................................... 25 Induction of Microsomal Enzymes ............................... 32 2,4,5-T . ....................................................... 38 General Toxicity in Laboratory Animals ....................... Effects of 2,4,5-T on Reproduction ........................... Carcinogenic and Mutagenic Potential of 2,4,5-T ............. Absorption, Distribution, Metabolism, and Excretion of 2,4,5-T Behavior of 2,4J*5-T in the Environment and Effects on Submammalian Species ....................................... 38 43 47 55 2 , 4 - D ........................................ Toxicity to H u m a n s .................................. General Toxicity to Laboratory Animals ....................... Effect of 2,4-D on Reproductive Function ................... Carcinogenic and Mutagenic Potential of 2,4-D ............... Absorption, Metabolism, Tissue Distribution, and Excretion of 2 , 4 - D .................................................... Behavior of 2,4-D in the Environment and Effects on Submammalian Species ........................................ 61 65 67 68 71 S i l v e x ............................................................... 76 Biological Effects of Silvex and ItsDerivatives .............. 76 Effect of Silvex on Aquatic and Invertebrate Species . . . . 79 Metabolic Fate of Silvex in Mammals ......................... 83 Refere n c e s ........................................................... 85 Assessment of Hazards Associated with Use of 2,4,5-T, 2,4-D, and Silvex in Reforestation Practices ................................ 116 Problems Associated With Assessment of Human Health Hazard Associated With Use of Phcnoxy Herbicides .................. Assessment of Human Hazard Resulting from TCDD as a Contaminant of Phcnoxy Herbicides ..................... . . Hazard Assessment-- 2,4,5-T 116 120 7321 0000078 5 Hazard Assessn:cnt--2f4 - D .................................... 128 Hazard Assessment-- Silvcx . . ................................ 129 Recommendations of Steps Which Should be Considered to Assure That Exposure to TCDD is Minimized or P r e v e n t e d ................. 1.11 Research Needs to More Certainly Establish the Safety of Present Uses of Phcnoxy Herbicides . . '......................... .. 132 4 000^ i D O W 0008 171 TOXICOLOGY OF PHENOXY HERBICIDES AND HAZARD ASSESSMENT OF THEIR USE IN REFORESTATION . Introduction --\ The phenoxy herbicides 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), 2.4- dichlorophenoxyacetic\cid (2,4-D), and /T,4,5-trichlorophenox>Z)ro- y ?,*.5-t p -- * pionic acid (silvex) are important agents for reforestation site prepara tion and for release of conifer plantings. To be used, the compounds must be inserted into a segment of the environment that may be occupied by humans or may have a potential for migration to points of human con tact or consumption. This document is a review of the pertinent litera ture describing biological effects and environmental behavior of the three herbicides, and of 2,3,7,8-tetrachlorodiben20-p-dioxin, an extremely toxic contaminant found in -am a H quantities in 2,4,5-T and silvex. For various reasons, the greatest public attention has been focused on 2,4,5-T and its unique contaminant, TCDD. Since 1969 public and sci entific attention to TCDD has overshadowed the specific concerns about 2.4- D, which has no TCDD, and about 2,4,5-T and silvex, which are con taminated. The unusual nature of the TCDD contaminant has made a special case . of the herbicide 2,4,5-T, and has made it the subject of an unusually vehement public, political, and scientific controversy. An enormous scientific effort has evolved in an attempt to learn about the unique character of the compound, and to provide a rational basis for regula tory decisions. Hopefully, this effort to bring the existing literature into, focus will aid in the decision-making process. 0000080 7323 rN- H X O O O > O Q As matters presently stand, there is a division in the collective public and scientific minds on the safety and benefits of use of the phenoxy herbicides. As in any controversy, both views include unreason able people, but the bulk of the argument is among sincere citizens and competent scientists on either side. It is my hope that the reviews and assessments in this document are dispassionate and without bias; other wise it will be of limited utility. In preparing this review, emphasis has been placed on information about the toxic properties of the respective agents. An assessment of hazard necessarily considers along with biological effects, factors govern ing the probability of exposure, and the probability that the chemical once contacted will enter the organism. With the exception of the section on TCDD, I have chosen to touch only lightly upon the environmental behavior of the compounds. This information about phenoxy herbicides is well established and is much V* ;l - J less complex than the behavior of TCDD. A more thorough treatment of r the environmental behavior of TCDD is necessary here because: 1) this .^contaminant is clearly the most important of the four compounds under riSy* " ------ -- ' study; 2) the high toxicity demands thorough understanding of the com- ^ y ^ * p o u n d as it exists outside the target of toxic effect; 3) the environ ? mental chemistry of. TCDD is incompletely studied and subject to some controversy, whereas that of the herbicides themselves is well estab lished. The organization of this review and assessment includes sections re viewing the scientific literature on TCDD, 2,4,5-T, 2,4-D, and 2,4,5-TP - yy (silvex),.an assessment of the hazard potential of their distribution in the environment, recommendations on some aspects of methods of use of 7 2 000003V the herbicides and recommendations of needed research. A shortened ver sion of the review and assessment is also attached. Organization within the review differs slightly among the chemical sections because of differ ences in research emphasis or the character of the agent itself. In cerr tain instances, for example in considering the enzyme induction capacity of TCDD, an area may be dealt with that is not entirely germane to the issue of hazard but of considerable importance in understanding the biology of the compound. A number of useful reviews of interaction of phenoxy herbicides or TCDD with animal systems have been or are being published. The most recent one by Leng (1977), discussing metabolic fate of phenoxy acids in domestic animals, Mercier (1976) in response to the Seveso accident which spread large amounts of TCDD through several Italian communities, and McQueen et al. (1977) refuting alleged association between herbicide use and fetal abnormalities in humans. McKenzie et al. (1975), Norris et al. (1972), Buhler (1977), and the EPA position paper arising from the Dioxin Working Group (presently in draft) are also all of considerable value. TOXICOLOGY OF TCDD History of Human Exposure Probably the first clinical description of human TCDD exposure arose from the study by Kimmig and Schulz (1957) of workers in' chlorophenol factories in Germany. The principal symptom was a persistent skin lesion (chloracne; so-called because it is characteristic of a number of related compounds and was at one time thought to be caused by free chlorine.^ The active agent was established as TCDD, and animal studies showed / // that the same lesions appeared on rabbit ears after application of as 7325 0000082 DOWo008 17 % U ^ little as 0.002 TCDD (Kimmig and Schulz, 1957). High topical doses and oral doses of 50 pg/kg or greater caused liver necrosis. A similar in dustrial intoxication in France was reported by Dugois and Colomb (1957). 4 Pathology of the skin lesions is well described by Kimbrough (1974). A study of 29 phenoxy herbicide workers by Bleiberg et al. (1964) disclosed chloracne and a high frequency of tiroporphyrinuria, and tissue T deposition of porphyrins related with hyperpigmentation and skin fragility. This assemblage of symptoms is commonly called porphyria cutanea tarda. The excessive production and excretion of porphyrins apparently results from a hyperactivity of the enzyme 6-amino levulinic acid (ALA) synthe tase, the first and rate limiting step in porphyrin formation. Porphy rins are eventual components of heme proteins, such as hemoglobin and cytochromes. Effects on ALA synthetase and other enzyme systems will be A y ^ d i s c u s s e d in another section. Poland et al. (1971) examined the same l ^ f a c t o r i e s later, after somewhat more satisfactory industrial hygiene mea sures were established. Among th 73 male workers observed, ho clinical porphyria was apparent even though some degree of chloracne still per sisted in most of the subjects. There was also apparently a significant psychological effect as measured by the Minnesota Multiphasic Personality Inventory. The absence of porphyria and persistence of chloracne in the later study has led Poland et- al. (1971) to conclude that while both may be caused by TCDD, the mechanisms are probably * Several severe accidental exposures to TCDD have occurred. In several cases, runaway chlorophenol plant reactions caused widespread exposures within the factory or in the surrounding community. An accident in Germany in 1953 exposed a large number of workers^said to have suffered liver, kidney, splenic, eye, cardiovascular, and nervous symptoms. A 4 w,;1r** r/*)' .'. v. 'G worker who apparently was in extensive contact with a contaminated seg ment of the plant five years after the accident became ill and later died. An explosion in a Dutch plant in 1963 released 30-200 g of TCDD into the c cca main gallery of the factory exposing 50 people. In the subsequent two years, four died, but no clear association with TCDD intoxication could be.showrf^CHay, 1976). An explosion in a British plant in 1968 resulted V \ in 79 cases of chloracne (May, 1973). In Czechoslovakia a pentachloro- phenate production stream overheated under pressure, producing large amounts of TCDD (Jirazek et al., 1974). (It should be noted in this and some ear lier German work a different numbering convention was used. 2,3,6,7-TCDD and 2,3,7,8-TCDD are the same compound.) Of 80 exposed workers, 76 devel oped chloracne, porphyria cutanea tarda, disorders of plasma lipids, hepatic damage, neura-l lesions, and neurasthenia. reaction in a'plant at SeVeso, Italy, in 1976 scattered . .i . ^ i rv.*.' * - ,A * , / TCDD, apparently, in kilogram amounts, over a wide area that included sev eral communities. Details are still not widely disseminated but great A numbers of animals were killed and many people were made ill. A review of the incident was prepared by Mercier (1976) a few months after the incident but the slow onset and persistent effects did not permit a de tailed assessment of the damage. Since that time the Seveso disaster has been given unpre^ented publicity and as might be expected this tragedy has been sensationalized in press coverage, in part because there may have been an effort to minimize or cover up the extent of the problems' There were apparently defects in the administration of the plant that may have resulted in the accident itself, and management of public warn ing and information early after the accident was questionable. Further *y contributing to the extensive public attention and sensationalizing of /mc-vCiiLvvs. ^ 30 ) /V-O 7327^000084 A. Re<^UVv'>HA-*T -U m t the incident is a virtual absence of objective reporting in the scicn/ tific literature. As far as I am aware, there has been no scientific O . \ evaluation of the aftermath in publication since the review by Mercier Pv\ ^ ) (1976), shortly after the accident. O' L Q' j. In the U.S., TCDD extracted from hexachlorophene production was ^ stored in oil, then mistaken for waste lubricating oil and' sprayed on several horse arenas and farms in Missouri^ for dust control. The inci dents occurred in spring and summer of 1971. On one horse breeding farm, 62 of 85 horses exercized in the sprayed area became ill and 48 died, the last in January, 1974, two and a half years later. Hundreds of birds and rodents died, along with a number of dogs and cats. Several children were exposed through play in the arena soil. One developed a severe hemorrhagic cystitis and others showed clear evidence of chloracne. All recovered. The TCDD concentration in the soil was later established at more than 30 iO I** In # ppm, a truly massive amount. (This concentration amounts to 120 lb per acre per foot of depth, assuming 4 x 10** lb soil/acre-foot.) The details of the latter event are described in detail by Carter et al. (1975) and Kim- 1 10 ' s' tC<-1 brough et al. (1977). All of these exposures were to a mixture of TCDD with other agents. Possibly the only published account of exposure to pure TCDD is the des- o-' I, cription by Oliver (1975) of laboratory contact by three individuals as- sociated with synthesis experiments. The individuals all had utilized routine protective procedures but sufficient contact developed nonetheless, The exposures were not sufficient to cause porphyrinuria or liver damage but serum cholesterol was raised. In two cases personality changes and neural disorders developed two years after exposure. The* amount of TCDD contacted is not known. 7328 o00008 *If u u u u General Toxicity in Laboratory Animals c This section is a review of data on effects of TCDD other than repro duction and teratology, carcinogenesis, and its enzyme inducing properties upon mammals. These will all be considered independently, as will studies K " of tissue distribution and metabolism. Schwetz et al. (1975) have determined lethalities for several species. The guinea pig is exceedingly sensitive, with an LDgQ of only 0.6 pg/kg; rats are less sensitive; LD,.q for males is about 20 pg/kg and for females the LDgQ is in excess of 40 pg/kg. Rabbits are still less sensitive at more than 100 pg/kg. Dogs were found to require even higher effective doses. In these studies, responses of mice were highly erratic and were not included. McConnell et al. (1977a) have determined a single 30 day LDgg for mice of 284"pg/kg, and 2 pg/kg for guinea pigs. An important characteristic of TCDD is a very long delay before lethality, often 2-3 weeks and occasionally 7-8 weeks. The pathology associated with TCDD toxicity may include thymic atrophy, liver necrosis, and degeneration in the kidney and thyToid. The guinea pig is unique in that it suffers limited hepatic damage, even at doses that cause substantial thymus depletion (Gupta et al., 1973). The degree of thymic atrophy is generally dose dependent; the studies of Gupta et al. (1975) did not establish a( dose, but the lowest' dose in the \ rat of 1 pg/kg/day for 31 days caused moderate thymus and liver damage, and kidney and thyroid degeneration. Guinea pigs treated with 0.2 pg/kg weekly for eight weeks had limited thymus damage and no other pathology. Studies of guinea pigs at lower doses suggest a no observed effect level at 0.008 pg/kg/weekly for eight weeks, a total of 0.064 Pg/kg, although there was a slight decrease in lymphocytes at that dose (Vos et al., 7329 0000086. 7 T5DW9S-t78- % p 1975). Animals given tetanus toxoid at the fourth week to measure humoral V-0, n 4, immunity were found to have somewhat lower thymus weights than controls, & .r 'j'" although the difference was not significant. // According to the data of Kociba et al. (1976) the no observed effect level in rats given TCDD in the diet 5 days a week for 13 weeks is 0.001 lig/kg/ day, totaling 0.065 yg/kg, almost identical to the value found in \ ' guinea pigs. Again, thymus weight appeared to be the most sensitive param- y 0.*. ' iJ^ eter. The Kociba study dealt with a very broad range of clinical, chemical, and hematological measurements. At a dose of 1.0 yg/kg/day, 2 of 10 rats died and weight gain decreased sharply, erythrocyte numbers and volume ,V \^ A, yw ,v>' decreased, reticulocytes increased in males. In females thrombocytes decreased and total leukocytes increased. Differential counts were not CfUU^Guvwvi-*- altered. Urinary porphyrins, creatine* and 6 amino levulinic acid (ALA) p l all increased. Serum bilirubin, BUN and alkaline phosphatase^also were elevated. At an intake of 0.1 yg/kg/day red cell numbers and volume were still slightly decreased in males and porphyrin, ALA, bilirubin, and al- kaline phosphatase^was elevated in females. No changes in blood parameters occurred below these levels. fZ&L Lr* In a similar study Zinkl et al. (1973) found no changes in SGP^, * bilirubin, or erythrocytes after 31 days of treatment at 1.0 yg/kg/day and J l ^ | * ^ ' no c^ian8* SGOI^, cholesterol, or serum protein at 0.1 yg/kg/day. There was a modest depression in blood glucose at the lower dose rate. Mice treated with 1.0 yg/kg/week for six weeks were found to have depleted thymus weight (Vos et al., 1974) but no changes were evident at t the lowest dose, 0.2 yg/wteek. Hematological changes occurred only at a dose of 25 yg/k^/week, for six weeks. Some changes in serum proteins did JSO occur at an intake of 0.2 yg/kg/weekly. Unfortunately, there>^"no data) ^ u , 3 ?suggesting the magnitude of the no-effect single dose in any species 8 V 0000087 Studies in primates have.been limited. McNulty administered TCDD at a rate of 20 parts per billion in the diet to a single monkey and caused ^eat^ *n a ^ew days. A 10-fold lower concentration permitted survival t f1 & 9 Pttil eZ for about two months. Total intake was estimated as about 6 yg/kg (McNulty, 1977). A'study of eight female Rhesus monkeys fed 500 parts TCDD per trillion has recently been completed in the laboratory of J.R. Allen. The foo pp * an^ma^s showed dermatitis and menstrual irregularities w i t h i n six months z o.ozS after initiating treatment. One monkey died prior to the seventh month and *& d . ^ by nine months of exposure, all survivors experienced a severe anemia and leukopenia. Food intake was normal but all of the animals lost weight. After 11 months, five of the eight monkeys died, all with severe anemia and ^ 4 lo ft tf.2. destruction of blood forming tissues. The total dose was estimated at ? about three yg/kg (Alien et al., 1977a, 1977b) or about .3 yg/month. |) /rrta. A number of questions arise from these data. The monkey studies are >*** limited, but they do suggest the possibility that the lethal dose is g a *** similar regardless of the time over which it is administered. The im- piications are quite interesting. If it is true that TCDD dosage is time independent in the monkey, then the effect is truly cumulative. No cherai-. cal is known to have such properties, and if TCDD behaves in this fashion, any estimate of acceptable dose must assume additive effects over long periods. TCDD does not remain in the body of monkeys (Van Miller et al., 1976) or other species (Van Miller et al., 1976; Fries and Marrow, 1975; Rcse et al., 1976). A cumulative effect, if it exists, must then entail irreversible effects remaining after the molecule leaves active sites. TCDD is apparently not metabolized to a significant extent (Rose et al., 1976; Vinopal and Casida, 1973; Van Miller et al., 1976) so it is conceiv able as well th3t a single molecule could interact several times before 0000088 9 7331  - in.t*ir*ii..K.,'.i A...-,^ -- i.-^..... leaving the body, A possible mechanism may be intercalation of the planar . P TCDD molecule into DNA (Hussain, 1973), production of misinformation and 1^ departure of the agent to interact at another DNA site. Data in other species, especially the guinea pig do not support the ^ 2 idea of extensive cumulative effect. A dose rate of 0.2 yg/kg/weekly }.L for eight weeks provided a total dose almost three times the LD^q , but no deaths occurred and organ changes, while measurable, were not severe (Vos et al., 1973). Rats also accepted about three times the LDgQ of 20 ug/Kg over 13 weeks, without lethality. Some mice, on the oth er hand died at a total dose less than the acute LD,.n , distributed ^ over a four week period (Vos et al., 1974). There are a few other biological responses to TCDD that appear after high doses of from 10-25 ug/kg. One of the more interesting is a de pressed capacity for biliary excretion. TCDD sharply decreases removal o & of PCBs from the liver via the bile (Yang et al., 1977) and decreases rl plasma clearance of ouabain into bile (Hamada and Peterson, 1977). In O the latter work, steroidal inducers of microsomal enzymes reversed the TCDD effect. Whether this effect has potential significance in the f response to secondary toxicity can only be speculated upon. In any case the doses required for effect are quite high and probably not attainable .under realistic environmental conditions. Because TCDD causes increased microsomal foreign compound metabolizing activity in the kidney cortex (Fowler et al., 1975, 1977), Pegg et al. 1 (1976) evaluated proximal tubular function in intoxicated rats. They 'i i! found little change at any but large doses (25 and 50 pg/kg). A y . There have been few observations of metabolic changes caused by TCDD. j? Incorporation of ^H-sodium acetate into liver lipid fractions seems to be ? O *-/'0* h * qQ0&0 10 00 Jfc.h.'t.Hiii-, T f iT Q f io n AAr>n impaired at TCDD doses above 0.1 ugAj: (Cunningham and Williams, 1972), but the significance of this effect has apparently not been explored fur ther. Evidence of thymic atrophy after TCDD treatment has stimulated more direct study of effects upon the immune responses. Vos and Moore (1974) and Vos et al. (1973) found lymphocytic depletion in the cortex of the thymus and depressed cellular immunity in rats and mice exposed during gestation and the post-natal period. Guinea pigs were similarly affected (Vos et al., 1973). Thigpen et al. (1975) attempted to translate the effect into terms of infectivity and found that doses of 1 yg/kg or more increased mortality and time to death__of mice infected with Salmonella b e m . Course and severity of disease due to pseudorabies virus was not altered by TCDD. With all the study of TCDD effects, no pattern of pathology, tissue distribution, and biochemical or physiological change has emerged that is consistent enough to suggest a mechanism of lethal effect. McConnell et al. (1977a) quote papers in preparation by Van Logten et al. which show no association of death in TCDD intoxication with adrenal or pituitary hormones or malnutrition. Pathologic Morphology Resulting From TCDD Intoxication | Much of the more pronounced tissue change resulting from TCDD has been observed following high acute doses of the compound. There is | some question whether the acute damage seen represents that observed in a low level longer term experiment or in a field exposure. j Before TCDD as such became an issue Norback and Allen (1969) studied * the membrane changes in liver following ingestion of hexachlorohexahydrophenanthrene', a constituent of so-called "toxic fat." The lesions found under electron and light microscopy anticipated the damage later to be 7333 ->: ,, 0 0 0 0 0 9 0 Cs 00 tH 00 ooo o Q i I 3 *5! 1 ;. found after TCDD. Most striking was a great increase in the smooth endo plasmic reticulum (SER), with reorganization into a concentric arrange ment, and the rough ER became folded and rolled over a period of days to form dense arrays of concentric separate, not spiraling, paired membranes. The authors speculate that SER proliferation in response to intoxication 'is related to acceptance of enzymes synthesized by the RER. The in creased membrane mass may also be a means of sequestering chlorinated hy drocarbon in the lipid phase of the membrane in close proximity to foreign compound metabolizing enzymes. (This paper contains very useful schematic representations of the membrane development sequence.) The same authors later reported similar lesions in rats following administration of 1 yg TCDD/kg/day for 21 days (Norback and Allen, 1973). They found hypoplasia of lymph tissue and bone marrow with progressive anemia and leukopenia. The resultant reduced resistance caused substantial losses due to infec tion. Monkeys and chickens in the same study were much more sensitive in the latter parameters than rats. They also developed extensive fluid accumulation in all body cavities, probably due to decreased osmotic activity of blood due in turn to serum protein decreases. (Relative sensitivity of chickens has also been noted by Greig et al. (1973).) Monkeys were subject to extensive skin lesions, apparently corresponding to the chloracne of humans, but rats and chickens did not suffer skin damage. Monkeys and "chickens also exhibited degeneration of the testes and decreased activity of seminiferous tubules (see also Allen et al., 1975). Large doses of TCDD (single doses of 50 or 100 yg/kg; 16 to 31 daily doses of 10 yg/kg) to the rat caused severe liver and thymus damage, with / -- ---icterus and disseminated hemorrhages, especially in the myocardium. The 7334 12 q o o o q 9 V fcJL Lf - .-CUi, L py^-4--.i.A J s\*~-i Sc-Oj\ liver cells increase in sire and cellular regenerative attempts occur. t*ai Renal tubule cells became vacuolated. No pathological change occurred at doses of 0.1 Ug/kg/day over 31 days, at 1.0 ug/kg weekly for 6 weeks or 5 T Mg/kg in a single dose (Gupta et al., 1975). Fowler et al. (1973) studied" /i * "tl'H the progressive changes in rats over 28 days following a single dose of 5 or 25 yg TCDD/kg. Increased SER, roughly dose responsive, was evident by day three, especially in cells adjacent to biliary drainage. Both RER and SER were greatly increased by day 9, SER was almost at normal levels by day . 16, and both were indistinguishable from those of control animals by day 28. Jones and Butler (1974) carried out a similar experiment with rats 0 X LQSd given a single 200 yg/kg treatment. (A massive dose, considering the mini- , - , mum effective dose.) Most change was found in centrilobular cells, with D U t*****.1*7 ` degeneration and necrosis of parenchymal cells evident within a week after treatment. They suggest that the multinucleate cells seen in in- : creasing numbers through two months after treatment are formed through r tDV- membrane disturbance and coalescence of hepatocytes. By 10 weeks some recovery was evident but fibrosis of central veins and sinus dilation still prevailed. No mention of lethality was made, although the dose was on the order of five (female) to ten (male) times the LD5 Q . Longer term treatment of rats (0.001-1.0 yg/kg/day, S days weekly x 13) caused L*6 P *ot it y almost complete thymus involution at a dose rate of 1.0 yg/kg/day, somewhat ^ {*ecrease<* cortical thymocyte and lymphoid cell numbers after 0.1 yg/kg/ day. Splenic morphology was essentially unchanged at 1.0 yg/kg/day. The latter dose also caused some gross edema, and one of five males had decreased spermatogenic activity. Females at the highest dose tended toward formation of cuboidal epithelium in the uterus and decreased size and numbers of corpora lutea. Ovarian tissue was also abnormal. At all 13 7335 0000092 r *- ___ " lower doses no effects were observed (Kociba et al., 1976). Two year HlZl*-- \Q continuous feeding studies at TCDD intakes of 0.001 to 0.1 pg/kg/day have D *'73 r< just been terminated and histopathologic evaluation is not complete. *></ ^f ^J -kG r o s s examination showed that in some rats given 0.1 or 0.01 pg TCDD/kg/ V ' day there were increased numbers of nodules in the liver (R.J. Kociba, <A Dow Chemical Company, preliminary status report; personal communication). r2i o9 o r o J Lesions generally similar to those seen in rats were found in mice LD given single oral doses up to 200 pg/kg and weekly doses up to 2S pg (Vos et al., 1974). At a dose rateCtl pg/kg/week for 6 weeks decreased thymus ^ weight occurred. At 25 pg/kg/week, serum globulins were decreased, and porphyria and bile duct epithelial proliferation occurred. Similar changes were also reported following treatment of mice with an LD^q (30 day) TCDD (McConnell et al., 1977a). McConnell et al. (1977b) treated rhesus monkeys with heavy single doses of TCDD (in excess of 70 pg/kg) which were fatal in every case. Integumental changes were pronounced, anemia and lymphopenia and an in creased neutrophil count followed treatment. Liver, adrenal and kidney appeared to increase in weight relative to body weight, but this effect may have been due to general wasting. Kith the skin hyperplasia, similar changes occurred in the epithelium of some hollow organs. The major dif ference from effects in similarly treated Tats was the absence of sig nificant liver lesions. Chronic low level intoxication (500 ppt in diets, about 0.01 pg/ kg/day) of monkeys until death at 7-12 months caused anemia and pancyto- \ penia, with extensive hemorrhage. Bone marrow and lymphatic tissues fra 3.1 & were hypoplastic, and epithelial structures were hyperplastic (Allen et al., 1977). 0000093 14 ( 7336 .....i-t - ,gi,ti, fZl C-j. >3 'U The pathological changes resulting from absorption of TCDD by `. tV v horses and cats during the Missouri horse arena episode have been recently Qftnn described in detail by Kimbrough et al. (1977). The most pronounced and r consistent findings were the generalized hepatic fibrosis and necrosis and gastric ulceration. There was an appreciable incidence of tubular degeneration and abscesses in the kidney and capsular thickening of the :o spleen. Lesions in cats were generally similar. L. Effects of TCDD Upon Reproductive Functions Of the many toxic responses to TCDD, the potential for teratogenicity has received perhaps the greatest notoriety. The early findings of 2,4,5-T teratogenicity raised a considerable public consciousness and the finding ) that the herbicide was contaminated by high levels of TCDD, also found to cause fetal malformation, focused additional attention on the contaminant. In evaluating this aspect of TCDD hazard it should be noted that the teratogenic potential of a toxicant can be considered in two ways. The absolute teratogenic dose of TCDD to the pregnant animal on a body weight basis is very low, as is true of all TCDD effects. A more realistic way of relating the teratogenic potential, however, is by comparison with the dose required to cause general toxic effects in the mother. For example, in the guinea pig, TCDD teratogenesis has not been studied because the teratogenic dose is apparently higher than the lethal dose. U ' tf.6'1 &O ** * This section is limited to studies of relatively pure TCDD upon reproductive function in experimental animals. There is reference else where to reproductive studies of 2,4,5-T and silvex, which contain at least t some TCDD. Teratogenic defect resulting from TCDD are rather specific in experi r *1 mental animals; the principal effects are increased frequency of cleft 7337 js 0000094 CD palate (reported only in mice) and an abnormality in which the central 9 collection region of the kidney becomes enlarged, with an associated fluid o accumulation (N'eubert et al., 1975). Limited limb deformation may be seen o o in a few animals (Sparschu et al., 1971). Intestinal hemorrhage is also seen; this effect is not teratogenic but rather is direct toxic consequence o of fetal exposure to TCDD. Fatty degeneration of fetal livers is in the a same category (Becker, 1974) as is subcutaneous edema and delayed ossi- fication. In teratogenicity studies of rodents the usual procedure is to ad minister intoxicant at some point during, or throughout days 6-15 of preg I nancy, which covers the organ forming period. Sparschu et al. (1971) found that the lowest effective dose of 1.2S Pg/kg/day caused some fetal mortality, resorptions, and intestinal bleeding. Skeletal abnormalities were limited to delayed ossification. Renal defects tended to occur at about the same frequency regardless of dose. These changes are all a result of direct toxicity to the fetus and are not developmental. In- testinal hemorrhage has been reported in rat fetuses at maternal dose i rates Of 0.25 Pg/kg/day (Khera and Ruddick, 1973). At higher doses up to 8 pg/kg/day the same.kind of effects occurred with greater frequency; .iM AH1 0.5 pg/kg/day caused decreased maternal weight gain and higher doses 6A resulted in severe toxicity. Courtney and Moore (1971) examined rats and three mouse strains and ^ found that a dose of more than 1 pg/kg/day was required to produce kidney 10 defects in rats, and the cleft palate and kidney changes in mice appeared at about 3 pg/kg/day, over days 6-15 of gestation. The C57B1 mouse strain was much more sensitive than the others tested. A somewhat larger group * *S of NMRI mice showed a cleft palate frequency of about 3% (Neubcrt and N-- ^ JU Os. ^ . 5 SSL- f bp* zri-if"' i 133B QQQQQ95 T V .1 0 0. & 3o I* * Dillman, 1972), similar to the low frequency groups of Courtney and Moore. The period of maximum sensitivity for TCDD is at the eleventh day of gestation, as distinguished from dexair.ethasone, for example, which is) 1 X'- most effective at day 13 (Neubert et al., 1973). x Continuous administration of 0.001 Pg/kg/day to male and female rats for 90 days prior to mating and through weaning of offspring produced no effect on fertility (Murray et al., 1977). The study continued through three generations, with each preceeding generation sacrificed at weaning of its offspring;the third generation was maintained until two years om the beginning of treatment of the fg generation^ Toxicity and poor litter survival dictated termination of feeding at 0.1 Pg/kg/day; 0.01 pg/kg/day caused decreased fertility in generation, f^ and ^ but not ^g* fj and fj litters were smaller and growth and survival were decreased after treatment at 0.01 Pg/kg/day, but no pathological change was observed in liver, kidney, or thymus. /K o ^ r e p ^ n p l 5 ^ Smith et al. (1976) have treated CF-1 mice at doses ranging from 0.001 pg to 3.0 pg/kg/day through days 6-15 of gestation. --- There was no maternal toxicity at any dose. Cleft palate appeared at 1 pg, renal dilatation appeared at 3 pg/kg/day. No significant effects occurred at 0.1 pg. A ^fi^ni^report by Courtney (1976) described results of treating CD-I mice with high doses of TCDD (25-400 pg/kg/day) on days 7-16 of gestation. These very high doses caused no maternal deaths during the gestation period, but resulted in high fetal mortality. The nature of observed terata were typical of TCDD intoxication, but the enormous doses render the study useless for hazard evaluation. However, the 7 3 3 `J 17 0000096 . bovyoo9'lS8 substantially greater incidence of tevata after subcutaneous administra tion, compared with that following oral TCrD suggests that hepatic metabo lism may affect TCDD more than has been believed. A dose rate of 2S yg/ kg/day orally caused 3% incidence cleft palate; the same dose subcutane ously caused 82% incidence of cleft palate. ~&t*l - S S O f'&y, There has been little research on effects of TCDD on primate repro duction. Experimental animals are in limited supply and the numbers of ] offspring are so low that a satisfactory study is almost impossible. i Allen et al. (1977) fed TCDD to eight female rhesus monkeys at a con 0. o\ centration of 500 ppt in the diet, which provided an intake of about 0.3 Ug/kg/month. Skin lesions, endocrine and menstrual changes occurred in Util all of the subjects by 3 months. Six females of the eight were bred after 7 months of treatment; one monkey died before mating and another was ex cluded from the reproductive study. Three of the animals conceived; two aborted, and one completed a normal pregnancy. Two of the three barren animals were bred four times, the other was bred twice. This dose rate is on the'order of 0.01 yg/kg/day and comparison with the rat study of Murray et al. (1977) suggests that reproductive toxicity in monkeys may be some what greater than in rats, but is by no means proportional to the lethal dose. ^ / ^Carcinogenic and Mutagenic Potential of TCDD 7340 Van Miller and Allen (1977) have evaluated pathological changes in J r jy / i *t*MJ^** ftp/r \ ^\jJrats fed TCDD in dietary concentrations ranging from 1 ppt to 1000 ppb, Jr beginning at animal weights of about 60 g. Estimated Weekly intakes of. - A M j o A t r<xu_ z 0 . 0 0 0 0 1 to , TCDD ranged, fyom 0.0003 yg/kgpit'a dietary level of 1 ppt to 2 yg/kg atr fSOOO ft* C,OCO Ut, ftOootoot> ftpZ 5 ppb. Actual intake of TCDD at dietary concentrations of 50-1000 ppb "iitu a was not clear. At 65 weeks the abdominal viscera of surviving animals 1,0 * % < oXfiO ^ ^^ . .^ / r t 02 8000 M O Q an increase in de novo synthesis of enzyme, rather than activation of preexisting protein or decreased degradation of protein (Haugen et al., 1976). Whether the effect of TCDD on inducible enzymes has implications on other aspects of protein synthesis can only be speculated upon. Poland and Glover (1975) showed about a 10-fold difference in the dose necessary to cause induction. Through the finding that heterozygous offspring of C57B1/6J (responsive) and DBA/2J (non-responsive) strains are intermediate in sensitivity, they support the contention of Poland and Glover (1975) that the difference is a receptor site mutation. Later studies have strengthened that premise in finding that hepatic accumula- 14 tion of C-TCDD administered intraperitoneally was greater in respon sive than non-responsive strains (Poland et al., 1976). In the same study, in^vitro examination of binding in the soluble fraction of liver cells showed that specific binding sites exist, and that their affinity correlates with the .genetic capability for induction. Furthermore, tests of an extended series of halogenated dioxins and dibenzofurans showed that cytosolic binding corresponded closely with the induction potency of the chemical. Niwa et al. (1975) have measured AHH induction by TCDD in a variety of cell cultures. They used 10 established cell lines, human lympho cytes, and primary fetal cultures from chick, rat, rabbit, hamster, and four strains of mice. Generally, kinetics of TCDD induction in each cell type is similar to that of 3-MC, and the induction was inhibited by actinomycin-D and cyclohexamide, which are inhibitors of protein syn thesis. They found no relation between inducibility and cytotoxicity of TCDD, which is further -evidence that TCDD itself is not metabolized. The sensitivity*, of some cell lines is such that the authors suggest use 37 0000116 DOW 0008 208 of H-4-II-E cells (derived from Reuber hepatoma H-35 in rats) as a bio assay, with a suggested sensitivity below 1 pMole ('o 0.0003 ug) in 3 ml of culture medium. The same kind of genetic differences have been studied in human lymphocyte cultures, which represent the genetic background of the cell donor and are therefore useful in making relatively non-invasive studies of potential human response- to intoxication. The sensitivity to TCDD induction of AHH is about 50-fold greater than to 3-MC induction, con siderably less than the 30,000-fold difference in responsive mice (Kouri et al., 1974). Atlas et al. (1976) have carried these studies forward with lymphocytes from human tissues, but have as yet dealt only with 3-MC, not TCDD. Part of the stimulus for examining human cells lies in the sug gestion that extent of induction at contact sites (i.e., skin, lung) may relate to the probability of cancer initiation by activated carcinogens (Kellerman et al., 1973). Although TCDD is as yet not established as a carcinogen such research may provide suggestions about the extent of expected variations in other TCDD responses in humans. 2,4,5-T General Toxicity in Laboratory Animals Apparently the first published account of 2,4,5-T acute toxicity was that of Drill and Hiratzka (1953). The L D ^ for dogs was about 100 mg/kg; the. principal symptom was a mild incoordination. When fed five days weekly for 90 days, doses up to 10 mg/kg/daily were without evident effect, but 20 mg/kg/day was lethal to all 4 treated animals between 11 and 75 days after the first dose. Symptoms were limited to muscle twitching and imoaired swallowing. A field study by Grigsby and Farwell 38 (1950) [as quoted by Rowe and Hymas (l`.)54)] exposed a variety of stock on pasture immediately after spraying 2,4,5-T at 2-4 times usual levels, with no effect. Rowe and Hymas (1954) summarized the available data at that time and estimated the acute oral median lethal dose for male rats to be about c C c -c cc cc: r c u 300 mg/kg, male mice 389 rag/kg, and guinea pigs 381 mg/kg. LDggS for various esters were higher than for the acid. A sheep fed the propyl glycol butyl ester of 2,4,5-T died after 369 daily doses of 100 mg/kg and another sheep and a cow died after seven daily doses of 250 mg/kg (Palmer and Radeleff, 1964). The triethylamine salt of 2,4,5-T at 100 mg/kg caused no observable effect after 481 days of treatment at 100 mg daily. As single animal observations these can be considered rough estimates only, but they convey the" generally high doses necessary to cause harm in rumi nants. 2,4,5-T has been shewn to cause decreased volatile fatty acid production in vitro at concentrations o f 500 yg/ml oV greater (Kutches et al., 1970); this concentration is probably greater than can be main- tained by a survivable daily dose of 2,4,5-T. ? Subchronic (90 day) feeding of male and female rats caused no ef fects below 30 mg/kg/day, but body weight and food intake were depressed after treatment at 100 mg/kg/day. Alkaline phosphatase and SGPT were elevated and erythrocyte count and hemoglobin.'iJK^ecreased. The his- topathologic findings were limited and inconsistent (Mo/Tol lister and- 7 -Kocibit 1-070}-. Rats were able to tolerate 186 mg/kg/day treatment with mixed mono-, di-, and tripropylene glycol butyl ether esters of 2,4,5-T t over 90 days but developed some indications of toxicity. At a dose of 18,6 mg/kg no evidence of toxicity was observed (Dow Chemical Company, 1961). 39 0000118 DOW 0008 210 A two year study of rats given 3 to 30 mg 2,4,5-T/kg/day has very recently been completed by Dow Chemical Company. Much of the analysis has yet to be completed, including morphologic pathology. Animals that received 30 mg 2,4,5-T/kg/day were found to have increased urinary por phyrin excretion after 4 months of treatment, and this change continued through the entire 2 year period. No changes were found in any other hematologic, urinary, or clinical chemistry measurement at that rate of intake, and the increased porphyrin excretion did not occur at 10 or 3 mg/kg/day (R.J. Kociba, Dow Chemical Company, preliminary status report; personal communication). A feeding study of reindeer was prompted by allegations that a high incidence of death and abortion occurred in 1970 in a herd after use of phenoxy herbicides the previous year. Fifteen of thirty pregnant rein deer were fed birch leaves which had been sprayed with a 2,4-D/2,4,5-T mixture, through a 1.5 month period late in gestation. . The daily dose of phenoxy acid was about 1 mg/kg/day. No clinical chemical changes could ^ b e detected, nor did any changes appear at autopsy either in the adult animal or the full term fetus (Erne, 1976). The toxic effects of 2,4,5-T in rodents have been studied in much more detail than the gross toxicity studies already described. Highman et al. (1976a, 1976b) established that lethal doses in pregnant or nonpreg nant nice caused myocardial lesions, bone marrow aplasia, and lymphocytic depletion in thymus, spleen, and lymph nodes, and that animals which re mained apparently healthy despite similar doses did not- suffer the same severity of lesions. A mild hemolytic anemia did occur in such animals. Pregnancy did not augment the toxic effect. It was also clear that major differences exist in the response of specific strains. Many NCTR mice were t 7344 40 0 0 0' 0 ' T 7 Z 8000 M Q seriously affected at doses below 60 ng 2,4,5-T/kg/day (6-9 days of treat ment while most CRBL mice remained unaffected at doses as high as 90-120 ' mg/kg/day at the same time. The histopathology, hematology, and blood chemistry changes in treated mice were also studied. In many monitored '; animals, some myocardial fibers were found to be pale and swollen, with longitudinal striations. The condition was rarely seen in treated ap parently non-intoxicated animals. Necrotic changes were also often seen in the outer myocardium. High doses caused thymic atrophy, with almost no lymphocytes in the cortex and increased numbers in the medulla (this ef fect was enhanced in late pregnancy by the normal tendency to cortical involution in pregnant mice). Spleens were often atrophied, and thyroid follicles were enlarged with enlarged epithelial cell. In the liver, glycogen was often depleted. Blood chemistry changes appeared not to be marked. 2,4,5-T has been found to cause increased liver weight in rats at doses of 167-334 mg/kg over a two-day period, apparently through stimu lation of protein and RNA synthesis, but not as newly formed 2,4,5-T metabolizing enzymes. The change reverses after withdrawal (Chang et ai., 1974; Rip and Cherry, 1976). Liver nuclei isolated from treated animals were more active in RNA synthesis than those from controls. 2,4,5-T at ijih* a concentration of 4 mraolar is capable of sharply inhibiting in vitro in^ -------- 14 corporation of mevalonate- C into non-saponifiable lipids by rat liver (Olson et al., 1974). If data obtained in mice by Nony et al. (1976) can be applied, this concentration is approachable at single doses of SO to ? 100 mg/kg, which are survivable by most species. Koschier and B e m d t (1976a, 1976b, 1976c) have described the effect of 2,4,5-T upon renal physiology. Large doses of 2,4,5-T appear to 41 0000120 2 T 2 BOQOfrOCL impair secretion of itself by decreasing the activity of the organic acid transport system in the proximal tubule. Organic base transport was also depressed. Small doses of 2,4,5-T were excreted very rapidly, but large daily doses led to renal depression and retention of the compound. The authors consider the data to support the conclusion that transport of phcr.cxy herbicides is an active process. The psychopharmacology of 2,4,5-T does not seem to be a popular field of study. In the one study found, single doses of up to 100 mg/kg were given on day 7, 8, or 9 of pregnancy. The male offspring of females given the highest dose exhibited more exploratory open field behavior, but no difference was found in females. The highest doses caused de creased litter sire but no increase in malformations (Sjoden and Soderberg, 1972). Poultry seem relatively insensitive to 2,4,5-T. Whitehead and Pettigrew (1972) found that a single oral dose o f ^ f T m g / k g to 4 week old chicks caused 40% lethality. However, feeding of 1000 eg 2,4,5-T per kg diJi, diet^dsy:-for three weeks to chicks, beginning at one day of age caused only some slowing of growth; 5000 diet was lethal. At levels not causing gross toxicity, plasma "calcium and magnesium were not affected. Given a choice, the birds rejected the treated diet in favor of non- contaminated food. Turkeys fed 2,4,5-T at a rate equivalent to 62 mg 2,4,5-T acid daily for 11 days were unaffected (Roberts'and Rogers, 1957). Bjorklund and Erne (1971) introduced various 2,4,5-T derivatives into water ana feed of chickens, quail, pheasants, and ducks, In water the LCjo o the triethanolamine salt for chickens over a 29 week period was 1000 ppm acid equiv. (about 200 mg/kg). In the diet of other species over 42 o o o o i * j)ow ooo S^ t3 a 7-day period, the LC^g was in excess of 2000 ppm. Kenaga (1975) has extensively reviewed avian toxicity and safety of birds in areas treated with herbicides and concludes that the no observed effect levels are substantially above amounts that might be contacted in a field application Effects of 2,4,5-T on Reproduction The teratogenic potential of 2,4,5-T has received wide publicity since 1969 when allegations were made that its use as a military defoli ant had caused fetal malformations in the Vietnamese population. A succession of studies over the next three years confirmed the terato genicity of 2,4,5-T, but the implication of the herbicide in any increase in birth defects has not been supported. The initial report of 2,4,5-T teratogenic effect {Courtney et al., 1970; Bionetics Research Laboratories, 1970) described cleft palate and cystic kidneys at doses of 46 and 113 mg/kg/day on days 6 through 14 of gestation. Of the two lesions, cystic kidney appeared to be a more sensitive indicator. The 2,4,5-T used in the study was found to contain 30 ppm 2,3,7,8-tetrachlorodibenzo-_-dioxin (TCDD), and the respective toxicities of the two agents were still unclear at that time. The ijD^ also criticized for an unusually high and variable incidence of embryotoxicity in control animals (Neubert and Di liman, 1972). Emerson et al. (1971) evaluated a commercial 2,4,5-T with less than 0.5 ppm TCDD and found that 24 and 40 mg/kg/day through days 6-15 of gestation caused no teratogenesis in rats. Sparschu et al. (1971) found that 50 mg was also nonteratogenic, although there was a slight increase in incidence of delayed skull ossification. (Such alteration in development is not considered teratologic, because the abnormality dis appears with age. One criterion of teratogenic effect is irreversibility.) 43 7347 0000122 DOW0008 214 Treatment at ICO ir.g/day for days 6-10 caused generalized maternal to x i c it y , with only 4 survivors of 25 treated animals. In testin al hemorrhage, considered a / perron f e t o t o x i c but not teratologic lesion, was found in */ S only one pup in this study. Khera and McKinley (1972) found a similar dose response!. In-nice the effective dose is similar to that in rats. At 50 mg 2,4,5-T/kg/day, through days 6-15 of gestation, the frequency of cleft palates was increased from 4.7% to 20% and resorption frequency increased. The incidence was raised to 73% by a dose of 110 mg/kg/day with a decrease in fetal weight (Bage et al., 1973). The high dose also caused a two-fold increase in rib and vertebral malformation, but only marginal increase in dilated renal pelvis. N'eubert and Dillman (1972) found increased cleft palate in NMRI mice at doses over 200 mg/kg/day through days 6-15, increased embryo lethality at doses over 45 mg/kg/day reduction in fetal weight above 15 mg/kg/day. Strain differences in mice are significant. The dose causing at least one cleft palate in 50% of litters was 25 mg/ kg/day in the most sensitive of 5 strains tested and 105 mg/kg/day in the least sensitive (Gaines et a 1., 1975). Roll (1971) found detectable teTatogencsis at doses above 35 mg/kg/day through days 6-15 of pregnancy. The no-effect level with respect to teratogenesis was established as 20 mg/kg/day. Hamsters are less sensitive to cleft palate, but do have a somewhat higher incidence of delayed head ossification which is not generally defined as a teratologic manifestation (Collins and Williams, 1971). The doses ranged from 40-100 mg/kg and there was a considerable difference among samples 6f 2,4,5-T of different sources and levels of TCDD contami nation. r'r-v n/."/>>uri Fetal' rabbits are apparently unaffected by maternal doses up to 40 mg 2,4,5-T/kg/day through days 6-18. Ruminants are apparently also quite resistant to teratogenic effects of 2,4,5-T. Binns and Balls (1971) fed 100 mg 2,4,5-T/kg to 11 ewes from' the 14th to 36th day of gestation, and fed 100 mg 2,4,5-T(PGBE)kg to 'another group during the same period. No evidence of deformity appeared in any of the lambs. The TCDD content was 1 ppm. The no apparent effect levels in rodents reported in the preceeding studies contrast sharply with data in abstracts of reports by Konstantinova (1974a, 1974b), who is said to have found that 4.2 mg/kg/day of the butyl ester of 2,4,5-T through the entire gestation period caused embryotoxicity, nervous and hematologic change and histopathology in the mother. A dose rate of 0.42 mg/kg affected growth and development, nervous, liver and kidney functions and lowered fertility. The succeeding generation was found to have minor changes in organ weights. (Direct translations of these papers are not available; data quoted is obtained from Chemical ! Abstracts.) There has been limited study of the teratogenic effect of 2,4,5-T in primates. Dougherty et al. (1975) administered 0.5, 1.0, and 10 mg/ kg/day to groups of 10 pregnant rhesus monkeys from day 22 through 38 of gestation. The high dose was established after finding that 12 mg or more 2,4,5-T/kg daily for 18 days caused vomiting and weight loss in monkeys of both sexes. No teratogenesis occurred, although there were 1 or 2 abortions, premature births, or neonatal deaths in all groups, */o 4 o> owe. imp0rtance 0f TCDD ih the teratogenicity of 2,4,5-T was examined soon after the contamination was recognized. Courtney and Moore (1971) tested 2,4,5-T with 0.5 and 0.05 ppm TCDD, 2,4,5-T and TCDD together and ) ^ T-coz, if Cfy*. & srrufr-t. 000124 9T2 80kMOd V a TCDD alone in mice. It was evident that the concentrations used or addi tion o f 1 pg TCDD/kg/day through days 6-15 did not a lte r the apparent teratogenicity of 2,4 ,5-T . Neubert and Dillman (1972) reached a sim ilar conclusion, suggesting th3t to potentiate 2,4,5-T e ffe c t on pregnant mice igr j at least 1.5 ppm TCDD is necessary. Collins and Williams (1971) observed 0 ^ i^, additive effects of 2.9 and 45 ppm TCDD contamination of 2,4',5-T adminis- tered to hamsters, but many of the changes appeared characteristic of pri- ,, nary TCDD intoxication. f1 4*. K/K*. Nutritional status seems to have little influence on the effect of rfr* 2,4,5-T on reproductive functions. Hall (1972) fed 250 and 1000 ppm 2,4,5-T with diets containing 20% and 60% casein. Resorption and still birth incidence and litter size were similar in all groups, although fetal organ and placenta weights were decreased in the high 2,4,5-T, high pro tein group. Effects of low protein intake were augmented by the high concentration of herbicide. Incubating eggs have been considered to be particularly vulnerable to herbicides because they remain in one place, and because the eggshell is porous. A number of studies of 2,4-D effect on eggs have appeared since 1967, with conflicting results (see section on 2,4-D), but 2,4,5-T has not received much attention. Somers et al. (1973) sprayed hen eggs with a formulation containing 2,4,5-T and 2,4-D in a treatment equivalent to 11.2 kg/hectare (10 times normal field application rates) prior to incubation without effect on hatchability or early survivability of chicks. They' then treated eggs of the pheasant, as a genetically more heterogeneous species, in the same way, without adverse effect. Entry of herbicide into the egg y was verified analytically. The treatment did, however, increase weight gain in male chicks during the first four weeks of life (Somers et al.. 0000125 46 j U >'J 1974). The LD^q of 2,4,5-T in D'iSO, injected directly into the air space, of chicken eggs at doses up to 125 mg/kg has been calculated to be 62 mg/ kg, and in acetone carrier, 153 mg/kg (Strange et al., 1976). No tera togenic effect was evident. The solvent toxicity was shown to be substan tial, but it seems clear that acquisition of an effective dose of 2,4,5-T by an egg in the field is highly unlikely. In fact, immersion of hen eggs in 1% 2,4,5-T was ineffective, and a 5% solution was only moderately effective. The eggs were immersed for 10 seconds, then returned to incubation (Gyrd-Hansen and Dalgaard-Mikkelsen, 1974). Insects may be more sensitive; Davring and Sunner (1971) and Davring (1975) have shown that while Drosophila are highly resistant to lethal effects of 2,4,5-T ester (LDS g - 4700 ppm in the diet), 1 ppm caused disturbed egg follicle development and chromosomal defects in developed oocytes. < A study on a species of killifish has shown that concentrations of 20 ppm have substantial teratogenic effect. The embryos developed several cardiovascular anomalies, and occasional eye and splenic defects. The 20 ppm treatment reduced hatchability to less than 50%, and .25 ppm allowed less than 5% to hatch. No anomalies were found after treatment with 14 ppm 2,4,5-T, and the hatch was reduced only 4% (Schreiweis and Murray,. 1976). Carcinogenic and Mutagenic Potential of 2,4,5-T Probably the most important issue about any chemical introduced into the environment by human activity is the possibility that it may increase the incidence of cancer in the human population. The probability of muta- 7351 47 0000126 genic activity is of almost equivalent concern., botli because of the possi bility of genetic alteration and because mutagenesis may be a useful pre dictor of carcinogenic activity. The relationship is by no means constant. However, McCann et al. (1975) have assembled data from a number of labo ratories using microbial systems ("Ames test") and find that 85% of known carcinogens are positive, 10% of non-carcinogens arc active. This is not to say that almost every agent identified as a mutagen will be carcinogenic, tut the test should become an aid in deciding priorities for direct screen ing. Only a limited number of experimental studies of 2,4,5-T carcinogeni city have been made. A screening program for 120 pesticides and industrial chemicals was reported by Innes et al. (1969). Eleven of the agents caused an elevated incidence of'tumors; none of the phenoxy herbicides, including 2,4,5-T, caused increased tumor formation at a dose previously determined to be the maximum tolerable daily dose over 20 days, without lethality (21.5 mg/kg/day for 2,4,5-T). Muranyi-Kovacs et al. (1976) treated two strains of mice with 80 ppm 2,4,5-T (TCDD content 0.05 ppm) in the diet for more than 500 days. Daily dosage was about 12-15 mg/kg. In one strain survival time was significantly decreased in males, in the other significantly increased in females. An apparently significant small increase in tumor incidence occurred with the increase in life span. The authors are concerned that altered life span may confound the analysis, but consider that 2,4,5-T can not yet confidently be considered non-carcinogenic, and requires fur ther analysis. A two year study of rats given up to 30 mg 2,4,5-T/kg/day has just been terminated by Dow Chemical Company, but the pathological evaluation had not been completed at the time of this preparation. 48 One epidemiological study has been made of tumor incidence in a group of Swedish railroad employees. It was possible to isolate groups who had handled phenoxy acids exclusively, and a cohort of 207 persons with at least 45 days of exposure, totaling 1747 person-years. No in- crease in tumor incidence could be detected in these workers although increased incidence appeared in groups exposed to other herbicides (Axelson and Sundell, 1974). Mutagenic assessments have been carried out on a number of systems. Buselmaier et al. (1973) were not able to show 2,4,5-T induced mutations in Salmonella typhimurium G46 his-, and Serrati^marcescens a21 Leu- and a31 his- in a host mediated assay in mice. Serum from animals treated orally with 2,4,5-T also did not induce mutants in S. typhimurium his(Styles, 1973). Anderson et al. (1972) were unable to demonstrate any mutagenic properties of 2,4,5-T or other phenoxy herbicides when tested against eight histidine requiring mutants of S. typhimirium. Sex linked lethal tests of adult male Drosophila showed 2,4,5-T to be negative, although fertility was decreased (Vogel and Chandler, 1974), Majumdar and Golia (1974) were able to show an increase in sex linked recessive lethal nutations after IS day feeding on 1000 ppm 2,4,5-T. A non-significant increase was apparent after feeding -250 ppm. In gerbils six-day feeding of up to 150 mg 2,4,5-T/kg produced no increase in chlorosomal abnormalities, but higher doses did cause some increase (Majumdar and Hall, 1973). Jenssen and Renberg (1976) tested 2,4,5-T for mutagenicity by seeking micronuclei in erythrocytes of mouse bone marrow, which is a high resolution detection system. The system was cytogenetically negative, although some mitotic depression was de tected. It was noted that only about St of circulating 2,4,5-T found its 7353 I 9 49 0 0 0 0 1 2 8 : O ' M W o00 o o oa way into the cells, which was suggested as indicating a limited hatard due to lack of access. A limited survey of pesticide applicators who had been in contact with a variety of agents showed some increase in chromatid gaps and breaks during the spray, season (Yoder et al.f 1975). Recovery in the off season reduced frequency below that of controls, suggesting an augmented repair system. 2,4,5-T was the least frequently used agent, and it is not likely that any observed effect can be attributed to it. Dow Chemical Company has maintained a program of continuing health monitoring of 2,4,5-T production workers. Cytogenetic evaluations of 52 men in early 1970 were reported to be negative (Johnson, 1971), and a'second analysis in 1974 also found no evidence of abnormality (Kilian, 1975). An interesting finding, however, was that the fraction of each exposed group found to have no abnormal cells was above 80%, while of the control group only 74% had no abnormal cells. Absorption, Distribution, Metabolism, and Excretion of 2,4,5-T 2,4,5-T has a rather short residence time in all species. A cow given 450 mg of 2,4,5-T acid in four daily doses excreted the entire amount in urine in six days (St. John et al., 1964). Erne (1966a, 1966b) administered 100 mg of the amine salt/kg to rats and swine and found plasma half tine for the rat to be three hours, and 10 hours for pigs. Kidney, liver, lungs, and spleen concentrations of 2,4,5-T could occa sionally be forced above plasma levels, but apparently none of the material entered the brain or adipose tissue. Tissue half times ranged from 5-30 hours. When the agent was administered repeatedly plasma levels tended to lessen, with increased excretion. Of the 2,4,5-T in blood, about 50 20\ was in erythrocytes. 2,4,5-T is excreted more slowly by mice than by rats, at a rate o f 1-4*4 o f the original dose per hour (Zielin ski and Fishbein, 1967). By overloading sheep (four 250 ng/dtfscs) i t is possible to produce . residues in tissues. Maximum accumulations were about 100 ppm in fat and c < \ muscle. In each case residues were found in the acid form regardless of the form in which it was fed (Clark et al., 1970). The latter finding is at some variance with Clark et al. (1971), in which the PGBE ester of 2,4,5-T was reported to remain as the ester in urine and tissues. Cattle fed 0.15 and 0.75 mg 2,4,5-T propylene glycol/fy butyl ether esters/kg/day for 32 weeks, produced no increase over background residue. Clark et al. (1971) later reported feeding sheep 2000 ppm 2,4,5-T in the diet and finding 1.0 ppm 2,4,5-T in muscle. No residue could be detected 7 days later. Deer browsing on land treated for reforestation were found not to contain sig nificant tissue residues; measurable 2,4,5-T was found in stomach contents, urine, and feces up to 43 days after herbicide application (Newton and Norris, 1968). The implication, again, is that sustained field intake will not cause detectable tissue residues. Grunow et al. (1971) found about 50-70% of administered 2,4,5-T in urine, and found that a substantial fraction emerged as derivatives, one of which was identified as N(2,4,S-tricnlorophenoxy acetyl) glycine. The taurine conjugate and 2,4,5-trichlorophenol have also been identified (Grunow and Bohme, 1974). Nony et al. (1976) have measured 2,4,5-T, its glycine amide and alkaline hydrolyzable conjugates in blood, urine, and feces. The proportion of metabolites is quite low in blood, and increases substantially in urine from 24-72 hours after administration of the herbi cide. In the feces the glycine amide is found in only limited amounts, but other conjugates may account for as much as 25% of the total recovery. SI ' 7355 0000130 DOW0008 222 Research with labeled 2,4,5-T in rats showed that the half-tiriv (T/2) of plasma clearance was about 4.5 hours and essentially dose inde pendent below doses of 50 mg/kg but increased as dosage increased to 19.4 hrs at ICO mg/kg and 25.2 hr at 200 mg/kg. The T/2 of a 5 mg/kg dose in dogs was ..inch longer, 77 hours. About 90% of the 2,4,5-T in blood was reversibly bound to plasma protein, over a wide range of concentrations. It was found also that as the dose was elevated, a small amount of fecal excretion took place. Detectable, minute amounts of C emerged in the res piratory gases (Piper et al., 1973). Fang et al. (1973) also showed a non-dose dependent T/2 at lower doses. Nony et al. (1976) found in excess of 10% of the total excreted material in feces. The extent of protein binding has been further defined by Kolbcrg et al. .(1973); bovine serum albumin binds 13-14 moles' 2,4,5-T/molyBSA. As suggested by the slower T/2, the percentage of dose excreted/day decreased sharply at the higher doses, indicating a limit in excretory capacity by the kidney. Rat kidney tissue slices can concentrate 2,4,5-T about'15 times, slices from dogs can concentrate about 9 times. B e m d t and Koschier (1975) found an energy dependent uptake of pnenoxy herbicides by renal cortical tissue. Metabolic inhibitors depress the process. Acetate and lactate as substrates enhance the accumulation. Increased 2,4,5-T in the medium slows the concentrating capacity of the kidney. The above work was later carried forward by resorting to intravenous injection of 2,4,5-T in order to take advantage of useful pharmacokinetic models (Sauerhoff et al., 1976). Doses of S mg/kg and 100 mg/kg were used, and samples were taken through 36 hours (5 mg) and 72 hours (100 mg). S After 5 mg/kg rats excreted about 50% of the body burden every 12 hours, in spite of a 4.3 hour plasma T/2, further illustrating the concentrating f 7O' C, O iJ j- 52 \ capacity of the kidney. At 100 ng/kg the plasma T/2 was 23.1 hours for the fir s t 36.hours, then was comparable to the animals given a lower dose. V. In humans (Gehring et al.^197 3) found that a 5 mg/kg dose was ex- / *. creted with a T/2 of 23 hours for both plasma and whole body clearance. Essentially a ll of the administered material emerged in urine unchanged. ^ The phenoxy acids are relatively^strong organic acids, which is one ' of the reasons they are excreted in large part as the parent molecule. The possibility of hydrolysis by rumen flora or by tissue enzymatic pro cesses was first considered by Wright et al. (1970) who found that after feeding of 2(2,4,5-trichlorophenoxy) ethyl-2 ,2-dichloropropionate to sheep, 2,4,5-trichlorophenol residues could be detected in tissues. Clark et al. (1975) measured phenoxy acid and phenol residues in muscle, fat, liver, and kidney of sheep fed .2000 ppm 2,4,5-T for 28 days, and found 1.00, 0.27, 2.29, and 27.2 ppm 2,4,5-T and 0.13, <0.05, 6.1, and 0.9 ppm 2,4,5-tri chlorophenol, respectively. Seven `days after withdrawal, 2,4,5-T residues were well below 0.1 ppm or below the detection limit of 0.05 ppm. The phenol remained at significant levels in both liver (4.4 ppm) and kidney (0.81 ppm). According to Koschier and Berndt (1976c) the rate of excretion rises . almost immediately to about 80% of input in animals treated daily, then continues to rise for 7-9 days until excretion approximately equals input, where it remains through the duration of treatment. The rate of adminis tration was 20 mg/kg/day. As dose rate increased the initial percentage excreted was less, but 7-9 days was still required to reach a rate equal to input. An apparent over-shoot then took place for 5-6 days. The markedly different rates of 2,4,5-T excretion by rats and dogs was studied in vitro by Hook et al, (1974). They found that kidney of 53 0000132 7357 DOWOOOS 224 both species actively accumulated 2,4,5-T by a saturable, oxygen dependent process which was depressed by other organic anions. Rat kidney had a greater dependence on potassium, and dog kidney increased accumulation of 2,4,5-T in the presence of acetate. The capacity of kidney to trans port the standard^test anion, para-amino luppuric acid (PAH), can be com petitively inhibited by 2,4,5-T, indicating that both are transported by the same mechanism. The authors concluded that the greater capacity for PAH transport by rat tissues accounts for the difference in retention between rats and dogs. Apparently lower renal excretion is also responsible for the extended half-time of 2,4,5-T in new-born rats observed by Fang et al. (1973); Hook et al. (1974) have found that kidneys of 10 day old rats are much less efficient than adults. Subsequent work by the sane group (Hook et al., 1976) indicated 'that plasma binding in dogs was more tenacious than in rats. The high specificity of 2,4,5-T in causing cleft palate has been investigated by Dencker (1976) as a distribution phenomenon. The sensitive peribd "has been shown to be late in fetal development, during days 12-13 (Neubert and Dillman, 1972). Palatal closure occurs very late in organo genesis and is complete at about day 15. Dencker (1976) points out that no unusual uptake of 2,4,5-T in these structures occurs, but overall uptake in the fetus is greatly increased between days 11 and 18. It may well be that this increase, at a time when formation of most other structures have progressed far enough that they are not sensitive, is responsible for cleft palate. , It is possible for 2,4,5-T to reach milk if a sufficient level is / included in the diet. At 30 ppm in the diet for two weeks,-fullowed by jyif nr w no 2,4,5-T or trichlorophenol was found at 0 0 0 C V `-'J 54 V'c >r-P a detection level of 0.05 ppm; at 100 ppm trichlorppnenol was barely detec table. At 1000 ppn 0.31-0.54 ppm 2,4,5-T and 0.16-0.27 ppm trichlorophenol was found in milk, and somewhat less was found ini cream (Bjerke et al., 1972). These figures can be put in the perspect/ve of forage consumption. s' " " -------------- 1 i U. Residues on grass are on the order of(jLOO-150 ppm/lb/acre^Morton et al. 1967).immediately after application. These amounts drop sharply to about 20% of initial levels during the initial two weeks post-application. This factor, with the rapid excretion characteristic of 2,4,5-T make even limited human exposure through dairy products or meat highly unlikely unless prod ucts are obtained immediately after heavy spraying. J- Behavior of 2,4,5-T in the Environment and Effects on Submammalian Species One factor in .the hazard potential of an herbicide is determined by its persistence after application and its movement and reactions in soil, water, or in plants. Appearance of 2,4,5-T in forest streams has been shown to result only if the herbicide was introduced directly to the stream, and diminished "from about 0.1 ppm to less than 0.01 ppm within one day after treatment (Norris, 1967). Norris (1968) also found that heavy rains six months after treatment did not move detectable 2,4,5-T into streams. The herbicide is usually degraded on the forest floor quite rapidly. Norris (1969) found that nearly 90% disappeared in two months, although cold weather, sterile soil, and lack of moisture may extend the period of degradation over as much as 9 months. The trichlorophenol hydrolysis pro duct of 2,4,5-T degrades faster than the parent compound (Alexander and Aleem, 1961). Evaluation of several soils from South Vietnam indicates / that concentrations on the order of those expected after a typical forest application should be virtually gone in seven weeks (Byast and Hance, 1975). tf o' o <o 55 0000134 DOWO008 C'c C J More than 20'a of the radioactive carbon of labeled 2,4,5-T had become in corporated in the soil, but actual residues of herbicide were usually about 1%. 2,4,5-T has also been found short lived in various Texas soil by Bovey and Baur (1972). Altom and Stritzke (1975) studied 2,4,5-T in three Oklahoma soils and found half-times of 24, 14, and 21 days. The shorter tine was on grass lands, the others were forests. Leaching tests show that 2,4,5-T bound in soil remained in the upper 6 inches of test columns even after application of 4.5 inches of water (Wiese and Davis, 1964). The various esters of 2,4,5-T are often intrinsically more toxic than the parent acid, probably because they are more fat soluble. In an aqueous system, however, they hydrolyze rapidly to the acid, and the rate is enhanced by soil-and microorganisms (Teasley and Williams, 1970, as quoted by Kenaga, 1974). It seems reasonable to expect a fairly rapid flJhfi fin hydrolysis in any system with some water present, In essence, 2,4,5-T disappears relatively rapidly after application, probably within two months in a forest environment.. The evidence also seems firm that 2,4,5-T deposited on the ground will not move into a water course, and will not leach into deeper layers. It is difficult to separate physical behavioT after application from effects on lower organisms, because there is a very close interface be tween an agent bound to soil or in water, and insects or fish. Lower organisms are of direct importance in assessing environmental impact of any introduced chemical. Food chain effects may predict repercussions at higher levels, and specifically sensitive organisms may be useful in dicators of pollution. Ecological displacement of any kind must be ac cepted only after careful examination, whether secondary to the intended ir?c *5 r*. 56 t o -/ W OOO impact of the chemical on plant species or as a result of direct toxic effect of the applied chemical. The various derivatives of 2,4,5-T differ in toxicity to fish. The parent herbicide, the amine salts and isooctyl esters are relatively limit in toxicity, but the propylene glycol butyl ether and butoxy ethanol esters are quite toxic to some species. There is a large number of gross effect studies on various aquatic species; these generally use an end point of death or immobilization. Shellfish studies measure shell growth as an index of effect. Standardization of methods has not received as much attention as seems justified. Study periods vary from 24-96 hours, water temperatures sometimes appear outside the normal acceptable range for the species, and most evaluations seem to be carried on under static conditions. Nonethe less, the relative gross effects can be useful in reference to a given application in the field. As a very rough approximation, a 2 kg/hectare application over a water course would produce a concentration of about 2 mg/L or 2 ppm, if an assumption of dilution in the upper 10 cm of water is accepted. Flowing water will diminish the concentration below detectable limits in a few hundred yards (Evans and Duseja, 1973), and application over deeper water will dilute accordingly. Concentrations of 2,4,5-T or its derivatives found to have no ef fect are listed below as acid equivalents: Na salt Species killifish mullet sea lamprey bluegill No Effect Exposure Acid Equivalent Period Concentration ppm Reference 43 h 48 h 72 h 50 50 2 Butler, 1963 Butler, 1963 Applegate et al., 1957 12 d 46 Hiltibran, 1967 S7 0000136 s z z eooofAoa 2,4,5-T butyl ester trout blupgill lamprey 24 h 24 hr 24 h 2,4,5-T isooctyl ester bluegill green sunfish bluegill 8d 8d 12 d 4.1 Applegate et al. , 1937 4.1 Applegate ct al., 1957 4.1 Applegate et al., 1957 7 Hiltibran, 1967 7 Hiltibran, 1967 0.7 Hiltibran, 1967 Median lethal concentrations of 2,4,5-T derivatives are similarly tabulated: Species Exposure Period (hours) LC50 Acid Equivalent Concentration ppm Reference 2,4,5-T DMA salt bluegill 48 144 Hughes and Davis (1963) 2,4,5-T bluegill ..24 54 Davis and Hughes (1963) TEA salt 2,4,5-T Oleic-1,3propylene diamine salt bluegill 48 2.9 Davis and Hughes (1963) 2,4,5-T . isopropyl ester bluegill 48 1.7 Davis and Hughes (1963) 2,4,5-T bluegill 48 31 Hughes and Davis (1963) isooctyl ester 2.4,S-T butoxyethanol ester bluegill 48 1.4 Hughes and Davis 1963 2,4,5-T PGBE esters bluegill spot harlequin .48 24 48 17 0.21 1.0 Hughes and Davis, 1963 Cope, 1965 Alabaster, 1969 While the more complex esters of 2,4,5-T tend to greater toxicity in water at pH 6.5, they hydrolyze to the parent acid in about a day (Teasley and Williams, 1970, as quoted by Kenaga (1974). 58 +1 00 oaaa o CEffects of 2,4,S-T on other aquatic organisms have also been measured. In flowing salt water 1 ppm 2,4,S-T caused no mortality in brown shrimp and 2 ppm was without effect on oyster shell growth (Butler, 1963). The PGBE ester of 2,4,5-T at 0.14 ppm (0.09 ppm acid equivalent) caused a 50% decrease in oyster shell growth after 96 hour exposure (Butler, 1963). Studies of terrestrial non-mammals seem to have been limited to honey bees. Honey bees are an important component of the crop cycle and e. in many areas are systematically managed for pollzhation. Phenoxy herbi cides, including 2,4,S-T, fed in 60% sucrose-water solution at 10 ppm did not affect hatching, but reduced brood development when fed at 100 ppm (Morton and Moffett, 1972). When sprayed in a water carrier at field application rates, 2,4,5-T was non-toxic. (Petroleum solvents alone caused high mortality during the first day after spraying.) Aerial spraying did not result in herbicide accumulation in honey sacs or colonies (Moffett and Morton, 1972). The point was made that the primary damage would be failure of flowers and loss of nectar, rather than direct toxicity. When fed directly to newly emerged bees, the *phenoxy herbicides were essentially non-toxic at concentrations up to 1000 ppm in the diet (Morton et al., 1972). 2,4-D Toxicity to Humans For some reason there have been reported several incidents of 2,4-D poisoning in humans, but virtually no reports of human intoxication by A.2,4-T. Probably the best known was a suicide by a single dose of 2,4-D DMA salt of which more than 90 mg/kg was estimated to have been retained by the victim, after extensive vomiting (Nielsen et al., 1965). An 59 -73G-3 0000138 accidental poisoning with a formulation containing 49 S-ethyldipropyl- thiocarbamate, 91 kerosene, and 36.5% 2,4-D isoorty] ester caused synp- tems and laboratory findings attributable to 2,4-D intoxication. Among these were twitching and paralysis of intercostal muscles, hemoglobinuria, and myoglobinuria. Mo evidence of peripheral neurological damage was evident during 56 months of follow-up observations (Berwick, 1970). DOW C008 230 Goldstein et al. (1959) reported on three cases in which 2,4-D esters were absorbed through the skin, resulting in peripheral neurological changes. One patient spilled 60 ml of a 10% solution of 2,4-D ester on the forearms and did not wash. Unusual fatigue developed in a few hours, and over 10 days after exposure there was extended nausea and vomiting, with considerable weight loss. A second exposure caused similar symptoms, then pain and numbness in all digits, loss of skin from the palms, and within six weeks there was substantial general neural damage. Another patient eventually developed metacarpal' pain and swelling in both hands, and later became partially paralyzed. The third experienced gastrointes tinal disturbance and vertigo, then paresthesia in the arms and legs and persistent general muscle fasciculations. In two of the cases, there were second exposures which appeared to cause greater impact than the initial event. Similar cases have been described by Todd (1962), Berkeley and Magee (1963), and Wilson (1956). Seabury (1965) attempted use of 2,4-D in treatment of a terminal case of disseminated coccidoidomycosis, on the rationale that the 2,4-D. as a synthetic plant hormone might alter the course of the fungus infec tion. At the time of the treatment (1949) there was no therapeutic agent for the diseases In 24 treatments over a period of more than a month the dosage was raised to a final treatment of 3600 mg. No prior doses, up i to 2000 mg caused any response, but the final administration caused 7 extreme quiescence, and fibrillation of muscles in the face and hands, * followed by deep stupor and reflex failure. The patient recovered from the 2,4-D within 48 hours and died of the fungus disease about two weeks later. General Toxicity to Laboratory Animals t c C One of the earliest published studies of 2,4-D toxicity was by Bucher in 1948. She found an acute LD^q of 280 mg/kg in mice, and found that single doses of 150-200 mg/kg would produce a myotonia persisting several hours. The animals remained awake and alert, but when moved ex hibited gross incoordination. They were capable of working out of the syndrome with continued exercise, but if allowed to remain quiet the myo tonia would recur. The chemical also caused diarrhea in'mice at the higher doses, and gastrointestinal and upper, respiratory irritation in dogs. Mice were able to tolerate daily doses of 1/2 LDj q for three months. Hill and Carlisle (1947) established LD^q values for several species: mice, 375 mg/kg; rats, 666 mg/kg; rabbits 800 mg/kg; and guinea pigs, 1000 mg/kg. They also treated monkeys and were able to give up to 214 mg/kg without severe residual effect. Twice that dose caused vomiting, incoordination, and loss of muscle tone. Rowe and Hymas (1954) summarized lethality data on 2,4-D and five salts or esters; in general the lethal doses were Yery high, with the exception of a few dogs.given 2,4-D it self, for which the LDSQ was estimated at 100 mg/kg. The latter data j was apparently derived from the observations of Drill and Hiratzka (1953). Hansen et al. (1971) conducted a two-year feeding study on rats, at dosages t of 5 to 1250 ppm 2,4-D in the diet, without appreciable change in growth, ; hematologic values, or organ weight. In the same study dogs were main tained for two years with little effect at doses up to 500 ppm 2,4-D. 1 7 3 6 5 0000140 DOW0G08 232 Drill and Hiratzka (195.3) found that 20 ng/kg daily was fatal in 3 of 4 dogs within 49 days of a 90-day study. Grazing species of anir.als might be expected to suffer exposures in the field from eating treated vegetation. Palmer and Radeleff (1964) evaluated a series of herbicides at high doses in limited numbers of `ruminants. One sheep tolerated 481 daily doses of 100 mg 2,4-D alkanolamine salt, another was unaffected by the same treatment with 2,4-D propy lene glycol butyl ethyl ester. The first compound was lethal after 7-500 mg/kg doses; the second was lethal after 9-250 mg doses. A bovine tolerated 112 doses of 50 mg of the alkanolamine salt, but another de veloped digestive problems after 80 doses of 100 mg/kg. Palmer (1963) administered daily doses of 50-250 mg 2,4-D/kg as the alkanolamine salt to a group of steers. ``The steer given 100 mg/kg developed ruminal atony after 86 days, presumably as a result of the herbicide. Animals giver. 200 and 250 mg/kg became intoxicated in 34 and 15 days, respectively, with dessicated mucous membranes and tendency to nosebleed when restrained. * Ruminants on sprayed pasture or cows given 5.5 gm daily did not show clinical evidence of toxicity, nor did production decline. 2,4-D appeared in serum of a cow fed 5.5 gm/day for 106 days, but none was passed in milk (Mitchell et al., 1946). Chickens and other fowl also appear relatively insensitive. Bjorn and Northen (1948) found no impairment in weight gain at doses up to 28 mg 2,4-D alkanolamine/kg three times weekly for four weeks; at 280 mg/kg there was a marked decrease in gain. Single doses of 765 mg/kg were lethal, 380 mg/kg was not. 2,4-D did not cause decrease in growth rate -/ at dose rates up to 1000 ppm in the diet, but at 7500 ppm growth essen tially stopped (Whitehead and Pettigew, 1972). The acute LD^q was 6 P R Z Q f i n n AA n r estimated at 900 mg/kg. Whitehead (1973) tested dietary 2,4-D at concen trations up to 100 mg/kg diet, and found that at dietary levels of 10 mg/kg diet or greater, growth rate was depressed. Because food conver sion efficiency was not affected, it was suggested that palatability was affected resulting in decreased consumption. Chickens will discriminate against 2,4-D contaminated food (Whitehead and Pettigrew, 1972). The U.S. Fish and Wildlife service tested a number of chemicals by feeding to various game bird species in the early 1960s. 2,4-D acetamide, given to young quail at 2S00 ppm caused 72% mortality in 12 days, the butoxyethanol ester at 5000 ppm caused 28% mortality in 135 days, and 2500 ppm dimethylamine salt caused 12% death in 138 days. In older birds 2500 ppm was almost without effect after 50 days, and after 111 days of 1000 ppm feeding. Tests "on Coturnix indicated a similar tolerance (Stickel, 1964). More recently, Hill et al. (1975) have shown the LCj q for 2,4-D acetamide in the diet of bob white, coturnix, pheasant, and mallard to be in excess of 5000 ppm. The butoxy ethanol ester and DMA salt have a -similar low toxicity. A number of other biological effects of 2,4-D have been found ex perimentally, usually at very high doses. Dybing and Kolberg (1967) sug gested that 2,4-D was actively reabsorbed, and if so would conpetitively decrease the clearance of p-aminohippurate without interfering with crea tinine clearance. Studies with rabbits given 100 mg priming doses, then 2 mg 2,4-D/min produced decreased PAH clearance at doses of 62-115 mg/kg. Chang et al. (1974) studied a numbex of effects on rat liver following treatment with 2-5 g^/kg 2,4-D over 4-7 weeks. Glycogen content was 50100% higher, and liver nuclei synthesized RNA more actively in vitro than 7367 63 0 0 0 0 1 4 2 did controls. DN'A content per liver was decreased. In this work, 2,4-D 0\yoOQ9 234 was compared with 2,4,5-T; the latter compound caused increased liver weight and protein accumulation, while 2,4-D decreased liver weight and lessened RNA content, with no protein accumulation. Speculation is jus tified that TCDP in 2,4,5-T was responsible for the difference. A dietary supplement of 60 ppm 2,4-D to lambs had no effect on rumen liquor or sheep or serum proteins. Weight gain was slightly decreased over a 12 week feeding period (Abou-Akkada et al., 1975). The same authors (1973) had previously found that 2,4-D did not alter ruminal microbial function. 131 At fairly high doses (80 mg/kg/day, for seven days) 2,4-D increases I intake by the thyroid. The effect only occurs in a normally functioning thyroid gland; it was not seen after hypophysectomy or iodine depletion (Florsheim and Velcoff, 1962). The effect is apparently due to lowered thyroxine binding to serum protein (Florsheim et al., 1963) but whether this is secondary to 2,4-D binding or is a specific pharmacologic effect is not known. There has been limited in vitro study of 2,4-D. Weiss and Beckert found stimulated mitotic activity and increased chromatin after cultured monkey kidney cells, Girardi heart cells, and trout gonad cells were exposed to 10 or 50 ppm 2,4-D for 72 hours. 2,4-D inhibits mevalonate incorporation into non-saponifiable lipids of liver, but only at concen trations in excess of 1 mM (Olson et al., 1974). Oxidative phosphory lation in rat mitochondria appears to be sensitive to concentrations as low as 10"4 M; at 10~^ M respiration was normal but (P/0 decreased to 20% of normal (Brody, 1952). In L929 cells in monolayer culture, 2,4-D J / causes triglyceride accumulation when present at a concentration of 500 V'g/ml (^olberg et al., 1972) and inhibits all growth at 50 yg/ml (Kolberg et al., 1971) 64 . 2,4-D at high doses is used also as a chemical inducer of experi I mental myotonia, as a model of the congenital disease (Iyer et al., 1977; ^ Eyzaguirre et al., 194S). The action is apparently due to an increase in OOcy membrane resistance and reduced chloride transport. Electrical activity ... of the brain .was found to be reversibly inhibited by doses of 200 mg *2,4-D/kg, in rats (Desi et al., 1962). The chemical also produces a primary myopathy that is useful in study of the family of diseases which includes white muscle disease in lambs (Heene, 1969). These effects are not of toxicologic significance except in cases of massive acute intake resulting in myoneural symptoms. Effect of 2,4-D on Reproductive Function As with 2,4,5-1, the possibility that 2,4-D has teratogenic poten tial was first studied by the Bionetics Research Laboratories study (Bionetics Research Laboratories, Inc., 1970). The data were suggestive that incidence of failed lower jaw formation was somewhat greater than that resulting from the DMSO carrier. Schwetz et a l . (1971) measured teratogenic and fetotoxic effects of 2,4-D and two esters on rats. The o higher daily doses (75 mg 2,4-D/kg; 75 mg propylene glycol butyl ester of 2,4-D/kg; 87.5 mg isooctyl ester of 2,4-D/kg, each just below the maternal toxic dose) caused decreased fetal weight, subcutaneous edema, delayed bone ossification and wavy ribs. T h e s e changes are fetotoxic rather than teratogenic. The last two are developmental ef fects but have no effect on survivability. No teratogenic responses were found at any dose. There jis some difference in definition of teratogenic response among authors in the field. A variety of skeletal defects that do not interfere 7389 0000144 DOWTOOS 236 with postnatal survival were found by Khr-ra and McKinley (1972); ir.ost ef fects observed were wavy ribs or fused sternum. The increased incidence of-these changes was evident at doses as low as 25 mg/kg/day. 2,4-D Vteratogenesis was studied by Bage et'ai. (1973) but only in presence of 2,4,5-T. The mixture caused some teratogenesis, but was less effective 'than 2,4,5-T alone, so the impact of 2,4-D in the system was difficult J to evaluate. Hamsters are subject to a teratogenic effect of high doses of 2,4-D. Collins and Williams (1971) found that 2,4-D from three different sources caused a low incidence of anomalies, usually fused ribs, at doses of 100 mg/kg/day through days 6-10 of gestation. There was no satisfactory dose response relationship. Dietary 2,4-D at 500 and 1000 ppm did not alter reproductive function in a three-generation, 6 litter study with rats. Percentage of pups surviving to weaning and weanling weight was decreased at 1500 ppm, however (Hansen et al., 1971). Sheep are apparently not subject to 2,4-D induced teratogenesis. Birins and Johnson (1970) administered 2 grams daily for 30, 60, and 90 5 *iDi' * days following breeding and caused no malformation. There were presuraably 6 sheep per group but the number in this specific experiment was not stated. Eggs are peculiarly vulnerable to exposure to herbicides and several studies have been directed toward defining the hazard of 2,4-D to chicken or game bird eggs. An injected dose of 10 mg 2,4-D/egg caused 50*# mor tality, 5 mg/egg resulted in 3G% loss, and at 0.5 mg/egg 90% of the eggs hatched. No deformities occurred at any dose (Dunachie and Fletcher,. r Ay 1967). A later study by Dunachie and Fletcher (1970) confirmed these findings for 2,4-D and 2,4-DB (2,4-dichlcrophcnoxybutyric acid). i o> 66 0 0 0 0 1 ^ 5 / ^ ^uy/~ 0*cst*ptZ~ty* were examined surgically and any observed tumors were biopsied. The diet with TCDD was then continued through the 7Sth week and a l l surviving animals sacrificed at 95 weeks. T o tz t cU * z a . 0 0 0 3 X - ? 9 Z. o. o - 7 ? v ^ t J i i 7 ? : / S C ^ 4.^ /-Aj 7S /' oThere were 10 animals at each dose range; a ll those at 50-1000 ppb ^ TCDD died by the fourth week. In the group given 1 ppt there were no tumors, but of those fed 5, 50, 500, 1000, and 5000 ppt 23 of 50 had developed tumors of several types. No control animals for this or a o o o GG parallel experiment developed neoplasms. The great variety of tumors 00 suggested to the authors the possibility that TCDD is a promoter rather ------------- ... ^ 5 ^ than inducer of neoplastic activity, in view of the usual narrow spec- trum of tumors caused by many chemical carcinogens. General pathologic r.j- ' changes have been described in a previous section. /a Two studies of-carcinogenesis in rats have utilized continuous /tr\^r,UL^ feeding of TCDD over a two-year period. One program has been completed but the analysis of histopathology and gross tumor incidence have not been completed. The other study, by Dow Chemical Company, has been completed and a preliminary report filed with EPA. Dietary levels were 21, 210, and 2200 ppt TCDD. The maximum dose caused increased tumor incidence in some organs and decreased incidence in others. There was substantial mortality resulting from general toxic effects. The inter mediate dose caused moderate systemic toxicity but no tumors and the lowest dose was without effect other than slight liver changes such as induction of increased drug metabolizing capability. These latter changes were seen only in females (J. Davidson, personal communication). The reasons for differences between the Dow and Van Miller studies is not apparent; it is paradoxical that the Dow work had a h igher-incidence of tumors in control animals, while showing a lower incidence of effect * treated animals^ ^ UXAA-<\ "VN. -fi-Vf h ^^ i9 ^^ *s7l 0000098 /- - Cytogenetic studies of TCDD by Green and Moreland (1975) suggested that the dioxin does not have potential for producing chromosomal ab- normalities in bone marrow. The dose schedule provided up to 15 pg/kg/ daily for 5 days, with sacrifice on day 5. A single injection experi ment with sacrifice at four weeks was also negative. Khera and Ruddick O '(1973) were also imable to find mutagenic activity of 2,4,5-1 cr> Since it is nots possible to obtain an absolutely TCDD-free prepara CD SQ tion of 2,4,5-T, the studies of the herbicide may also be of value in considering TCDD effects. Effects of 2,4,5-T and TCDD on dividing Afri *o can blood lily endosperm cells are not explicitly pertinent to animal a studies, but the relation shown between herbicide and contaminant is of 4 interest. Highly purified 2,4,5-T at 10 M was not effective, but 0.2 tf n pg TCDD/L with or without 2,4,3-T, .and commercial 2,4,5-T all caused t D.o . |^dramatic mitotic inhibition and chromosomal aberations (Jackson, 1972) c0.2200 '7oDt> C0-2 yg/L = 200 w t ) . Hussain (1972) found that TCDD was somewhat mutagenic using the procedure at doses that were intrinsically very toxic to the or ganisms. The authors suggested that the limited mutagenic activity was by intercalation of TCDD into DNA. Seiler (1973) has also obtained data suggesting that TCDD is mutagenic in the . typhimurium TA 1532 strain. There was no indication of the concentrations necessary to i 3 produce effects. Absorption, Distribution, Metabolism, and Excretion of TCDD The biological action of a foreign compound is .highly dependent on its logistics. A chemical must obviously be absorbed from the environment -/ in order to interact, and it must be carried in blood to cells and move A 00000" 20 to or across cell membranes to sites of reaction. The agent in its origi nal form may interact with sensitive systems, or it may be converted to an active derivative after entering an organism. Such conversion usually takes place in the liver. A given organ or cell type may have a particular ' affinity or sensitivity for a toxicant and thereby be singled out for at- ! tack. The chemical nature of the agent or its product will also dictate the extent and rate of excretion in urine or bile or respiratory gases. Even with an active excretion mechanism such factors as sensitivity of . bladder epithelium or an active enterohepatic circulation may result in toxic effects after elimination seems to have been accomplished. In spite of the enormous capacity for induction of mixed function K\ oxidases (discussed below) TCDD is metabolized to a very limited extent, w v 14*" ^ -- -- " ! ^ 0 6 0 Rats given a single-*dose of 50 yg/TQ^D-U- C/kg^by stomach tube excreted jp/ about 30% in feces during the two days following treatment, and then 1-2% 14 daily over the 19 days following, to a total of 53%. Urinary- C was | about 13% and respiratory excretion totalled 3.2%. After the initial 2 i days, total clearance half-time (T/2) was about 17 days. Residual TCDD at 1 ; 3, 7, and 21 days was greatest in liver and fat (Piper et al., 1973). The ' 'L u long residence time and limited respiratory and urinary excretion suggested ' that TCDD was essentially unchanged hut th-tc was not verified. As a gen- eral rule lipophilic compounds are either sequestered in fat, eliminated as conjugates in bile, or converted to a water soluble -form and excreted by kidneys. Absence of urinary excretion therefore implies limited converZcU -AijA sion. In a later study of rats given a lower single dose of l yg/kg, TCDD was identified in feces but not urine, and again, liver and fat contained the highest concentrations. T/2 was 31 days (Rose et al., 1976). In the same investigation, repeated doses 5 days a week for 7 weeks resulted in . . .. . some urinary loss, but most TCDD was excreted into feces. In this program 21 7 /S'7'> 0000100 of essentially continuous intake, T/2 was calculated as 24 days. The residual radioactivity in the liver was identified as unchanged TCDD. The report of Vinopal and Casida (1973) is suggestive that TCDD does not y L D 5 o metabolize only because after giving a dos of 130 pg TCDD-3H/kg to mice, no mention was made of tritium in the urine. Allen et al. (1975) found CNI that over a 25 day period about 4.5% of a dose of 50 pg TCDD/kg appeared in 9 urine. The daily fraction of total intake excreted gradually increased, o which seems indicative of an unusually stable molecule, because at least a modest exchange of tritium with body water should be expected. O The variety of indirect evidence indicating that TCDD is not metaboli- Q cally altered must be considered in the perspective of other indirect evi dence that metabolic change may occur. Beatty and Neal (1975) have shown that pretreatment with phnobarbital decreases and castration increases Y TCDD toxicity. The former treatment increases and the latter diminishes Sp drug metabolizing capacity. The teratology study of Courtney (1976) also V' x tf ,is suggestive that liver metabolism of TCDD occurs. An oral dose of 25 f s a V ----------------J* <r.JJg/kg/day through the organogenesis period caused only 3% cleft palate rxut -- ---- 3* ^ among the experimental fetuses, but subcutaneous administration of that dpse resulted in 8 2 % -cleft palates. In view of the apparent affinity of P r TCDD for the increased endoplasmic reticulum following induction (Van Miller et al., 1976; Poland et al., 1976), it is possible that induction merely increases the capacity for sequestration of TCDD in a temporarily non-active system, but the remarkable difference seen by Courtney is \ difficult to ascribe to distribution differences only. h**' ^ The selective distribution.of TCDD into liver and fat has also been shown by Fries and Marrow (1975), Allen et al. (1975), and Van Miller et al. (1976) in rats and by Van Miller et al. (1976) in primates. The <rjl*' 22 0000L01 AAOCl o p '*' liver TCDP appeared to be sequestered on smooth endoplasmic reticulum (SER), principally that induced by presence of the toxicant (Allen et al.. While liver storage of TCDD in monkeys was much lower than in rats, . .A^ ^ jyi^Y the difference appeared to be due to the characteristic lesser prolifera-' ' 1 tion of SER in the monkey that has been described by Van Miller et al. ' i i*"1 " (1976). Considering the difference between species in total induced ER sty' o membrane, TCDD appeared to bind to endoplasmic reticulum to about the same \ extent in both species (Van Miller et al., 1976). Unlike the behavior of other chlorinated hydrocarbons in fasted or underfed animals, TCDD is not lost from adipose tissue with fat mobiliza tion (Allen et al., 1975). In the case of PCB, for example, the fat . soluble chlorinated hydrocarbon is liberated with mobilized fat and even tually may reaccumulate in the liver or other target organs. The reason for the difference is not clear, but it seems possible that TCDD may non- j specifically partition into fat, then selectively bind to adipose cell membrane protein. Liver TCDD residues do decrease during fasting, pos- 1 -----------------sibly because increased gluconeogenesis makes demands on available labile protein including the endoplasmic reticulum to which the TCDD may be bound. y Hepatic subcellular distribution has been further defined by Poland and his associates (Poland and Glover, 1974, 1975; Poland et al., 1976; Nebert et i 2 j jjvJ* al., 1975); and by Chhabra et al. (1974) in studies in which the geneti cally determined susceptibility for TCDD induced benzpyrene hydroxylase induction was found to correlate directly with the extent of TCDD binding X n ei * pjrix -jv* in liver. Vct> i AM fit** The T/2 for TCDD residence in the body has ranged from about 16-20 days for single dose experiments (Piper et al., 1973; Allen et al., 1975), v A although Rose et al. (1976) arrived at a figure of 31 days. When TCDD was 3 7 5 23 0 0 0 0 1 0 2 A 'a /j a>%u^ ^ 'fffC^l'' /------- s. administered in the diet ovqu- 12 days^nt dose rates of about 0.5 and 1.5 t j y \ Ug/kg/day, T/2 was found to be 12 days for males and 15 for females (Fries and Marrow, 1975). Rose et a l . (1976) calculated a half-tim e o f 24 days after a seven week feeding at dose rates o f 1-0.01 Ug/kg/day. :r The concentration of TCDD reaches a maximum at a given dose rate. Fries and Marrow (1975) found that after 6 weeks of administration reten 0 H tion approached a steady state which would be 10.5 times the daily intake. 00 1 0 o Rose et al. (1976) found similarly in 7 weeks that a steady state con centration of a little more than 10 times the daily intake could be pre dicted, agreeing remarkably well with the Fries and Marrow estimate. The difference in distribution between primates and rats is of in- 3 terest. Seven days after equivalent doses of TCDD- H, livers of adult monkeys contained 0.09% of total dose/gjil tissue, rats contained 4.54% ^ (VanMiller et al:, 1976). In adipose tissue the relationship was monkey, 0.16, rat 3.46, suggesting that storage or retention is not strictly re lated to binding on inducible membranes. In terras of total body distri bution, the rat liver contained 40% of the dose and that of the monkey, * 10%. This is a much closer ratio (4/1) than that of liver concentrations (4.54/.09). Skin as a whole stored a much higher percentage of adminis I fyj' tered TCDD, because of the much higher amount of adipose tissue, in spite of a concentration ratio (3.5/0.16) that was also quite unfavorable. Whether these differences bear any relation to differences in sensi- & tivity to toxic effects is not known. The difference in liver is at least ^ \ y r^ associated in amount and time with the much greater capacity for induction i r in the rat. There;is no data available to indicate whether the thymus of ^ ^ 0oN species like the guinea pig, which is subject to more extensive TCDD in- ' duced thymus damage, has a selective affinity for the.toxicant as does the t 73V O r' 24 0000103 liver in the rat. The thymus of infant monkeys is expected to be highly active, but appeared less retentive compared with other tissues, while the adult rat thymus accumulated more TCDP/unit weight than most organs (Van Miller et al., 1976), further confusing this issue because liver damage is thought to be the primary lesion in the rat. /fA* o Each of the single administration experiments utilized heavy doses of TCDD, taking advantage of the. long delay in onset of symptoms and death to obtain improvement in radiochemical detection capability. Studies*in which 400 ug/kg (about 20 x LD,-n) were administered (Van Miller et al., ~ '~ ' "Stico,oaayyaiT 1976) produced liver concentrations of about 3.6 ppm. Kociba et al. (1976) c c e Q o O discernable but minor hepatic lesions at a total dose of 0.65 yg/kg given over seven weeks. The expected concentration of TCDD in liver at this intake is on the order of 20 ppb (Rose et al., 1976). A ten-fold lower dose caused no histopathologic damage. It is not surprising there fore that the beach mice studied by Young et al. (1974) which accumulated 300-500 ppt TCDD in their livers, did not show liver morphologic lesions. -- * -- <t-f*<--L< '-- g / r m i ' ' "_ j Im . r, ,, , f ,, J4 M 4 K X J-4 . ZSt Behavior of TCDD in the Physical Environment and in Submammalian Species ) The extremely high toxicity of TCDD and the very small amounts of the agent present in 2,4,5-T and in the environment present a problem that is ti 'in different less in philosophy than in scale. There is a tendency on the one hand to dismiss TCDD as a pollutant because the amount available at /ft'*' jr > . $ 1jr" any one point or even sector any amount in excess of true is minute, and on the other to assume zero residue is catastrophic, because that of pJ / IT $ r the enormous toxicity of the material. The amount of TCDD distributed on a vegetation after a typical treatment is so small as to defy direct analysis and the quest for residues in biological materials has forced analytical 7377 0000104 25 C> (S.) rH - 00 Q O a O Qj sensitivity closer to zero than has been the case with any other pollutant. In spite of these unique characteristics, TCPD must, be considered in the same way as any other chemical: How much is present in the human environment? How persistent is it? Can it move to unintended targets? If so, will the amount assimilated by humans or other organisms reach harmful proportions? Pr e s e n ^ ^ n u f a c t u r V 2,4,S-T contains about 0.02 parts TCDD per million parts 2,4,5-T, although manufacturers claim only about 0.OS ppm. Some lots contain only about 0.01 ppm. A two pound per acre application of 2,4,5-T will, therefore, distribute a little less'than 20 yg TCDD/acre. It is necessary to predict what may be expected to happen to that material by examining existing laboratory data. The environmental persistence of TCDD is highly dependent on the conditions to which it is exposed. When mixed into soil, degradation of was found to be very slow, with a half- J & e^ of one year (Kearney, 1972). 0J I , tp ^ i O Because the material was introduced as an acetone solution there has X been some question about possible crystallization and limited access of PY" "io TCDD to soil organisms. However, TCDD in 2,4,5-T deposited on leaves and j< 5 exposed to direct sunlight was found to degrade with a half-^iAe of a ^little more than an hour (Crosby et al., 1977). The latter study shed a (A ^ ^ g o o d deal of light on earlier studies in which pure TCDD was carefully coated on clean glass slides, water, or dry sterile soil for exposure to O' y ft* .. sunlight, and found to be almost insensitive to photodecomposition (Plimmer 1 j4^ et al., 1973; Kearney et al., 1972). Crosby et al. noted that reduction would proceed in methanol, and that the reaction slowed significantly in highly purified reagent. The finding that benzene accelerated the degra dation of TCDD in aqueous systems (Plimmer et al., 1973) suggested that a d 'V7" S 26 0000105 \ kO & gcod hydrogen donor was essential to phntodecon-,position. This conclusion has been verified in more recent work (Crosby ct al., 1077). In tiiat stu.'.v n)o TCDD with 2,4,S-T was also applied on_ soil, and a half-J-te of 50-55 hours could be inferred from the data. This finding provides some suggestion about the reaction time in indirect light, because the rough surface will provide shaded areas where reaction must be slower. As yet, studies of the effect of various parts of the spectrum emerging from light trans mitted through leaves, or reflected from various surfaces has not been done. The absorption maximum for TCDD is known (Crosby et al., 1973) but the relative sensitivity to degradation at that wave length apparently is not ` , Accumulation of TCDD in the biota has been studied both in the labo ratory and in the field, but there are still many gaps in the needed data. In spite of the limited solubility of TCDD (0.2 p"pbj, it can accumu late in aquatic organisms to the extent it is available on sediments or other reservoir (Matsumura and Benezet, 1973). In their experiments, / ; TCDD was deposited on sand, in solvent which was evaporated to leave the yA dioxin as a film on sand particles. The sand was placed in a small con- ^ jfK. fined static aqueous system and various aquatic organisms added. Brine X- shrimp reached a concentration of 157 ppb, mosquito larvae concentrated TCDD to .4150 ppb and silverside fish accumulated virtually no TCDD. It may be assumed that the concentration of TCDD in water was maintained throughout the experiment by the excess dioxin residue on sand. jb u Jt 0% 2 fiy a 3 / \A^ * /r^ u When sufficient TCDD to represent 162 ppb in the aquarium was ingested `& --------------- by algae which were then placed in an aquarium with Daphnia and with Os.- tracods, the latter species accumulated TCDD to concentrations of 879 and & cLlu.to 0? 279 ppb, respectively^ ihey also showed that mosquite fish concentrate ............ 2277 * 7379 0000106 TCDD to a lesser extent than do the bottom feeding larvae on which they feed. It is clear that accumulation does occur, but depends on maintaining saturation of the ambient water.. 7 3 ? Matsumura and Benezet (1975) also examined movement of TCDD which had been bound to sand, to a sandy loam soil (inorganic to organic soil). Very 'little TCDD was translocated by a slow leaching with water. Subsequently, a more complex food chain system of soil, water, algae, duckweed, snails, 00 daphnids, gambusia, and catfish was assembled (Isensee and Jones, 1975). H 14 C-TCDD bound to soil in concentrations varying from 0.0001 to 7.45 ppm 00 O was placed in aquaria. The residues found in the various species after O o 33 days exposure tended to peak at the Daphnia stage. Gambusia were added O at the end of the exposure period with access to water and other organisms D for 3 days. Catfish fingerlings were exposed for 6 days, beginning after all other organisms were removed. The catfish accumulated TCDD to roughly the concentration found in the first biological stage, the algae. None theless, at a water concentration of 7 ppt TCDD a terminal accumulation iS r - W- ratio of more than 10,000 was found in catfish, representing TCDD concentrations of about lOf/ppb ^ s e n s e e and Jones, 1975). The work also shows, however, that as soil or water concentrations decrease, so also \/f/ S * io concentrations in the organisms of the system. Toxicity was not studied ' although the higher tissue accumulations exceeded mammalian toxic doses, assuming uniform distribution of dosage. The authors point out that with the long latency of TCDD toxicity, the fish may not have had time to develop lesions and die. 73 Ward (1976) examined the persistence of TCDD in lake water and sedi ments. As expected, TCDD is almost entirely bound to lake sediments ; and is subject to very limited but nonetheless real metabolic degradation M when microorganisms were present. The slow microbial activity is enhanced r "'T it ) trJU. XU 7-ceo x v A 0 0 ij t ' S ' /--.. . /.< J I ifIil-lrA *rf by presence of nutrients. Ward suggested that water-mediated evaporation 6/ A OOO of TCDD occurred to a limited extent. Toxicity of TCDD to coho salmon has been assayed by Miller et al. (1973). TCDD was administered in the diet and dosage expressed as ng TCDD per gram organism mass. At 13.1 ng/g only about 30% of the experi mental group survived for 80 days past exposure, and at 5.4 ng/g almost half the fish did not survive. Deaths often did not occur until 10 days after the end of exposure, and some lethally affected fish survived 60 days or more. In studies of rainbow trout there were no apparent effects at intakes of 6.3 ng TCDD/g or greater. Mosquito larvae pupated at a nor mal rate in water containing 200 ppt TCDD; snails reproduced normally and oligochaete worms suffered some reproductive deficit at the same concentration. Beatty et al. (1976) found Rana catesbiana tadpoles to be remarkably resistant, with no apparent lethal effect by intraperi toneal doses of 1000 pg TCDD/kg. Doses up to 500 pg/kg did not affect adult frogs, and no histopathological lesions appeared. Field studies of fish and mammals have provided evidence both sup porting and contradicting claims of TCDD accumulation in higher animals. An evaluation of biota in the area used by USAF for training bomber crews in application of military defoliants showed little accumulation of TCDD in fish living in the streams of the area. The area was liter ally saturated with 2,4,5-T, receiving on the order of 1000 lb/acre during the peak year, (containing up to 2-10 ppm TCDD. Mice in the area accumu- lated tissue burdens of several hundred ppt as measured at the end of the spray program, when the sandy soil had accumulated residues up to 700 ppt. These animals demonstrated no pathological changes (Young et al., 1974), which is not surprising when extrapolating the data of Rose et al. Z .,4 ,5 --T ^ o ^ ^ 29 7381 0000108 (1976) and Kociba et a l . (1976) to relate tissue levels and e f fe c t iv e dose rates in rats. Given the extended period of exposure, i t seems possible that the mouse population could adapt through survival of non-responding S' strains over the estimated 30 generations of exposures. I t may be also n^ -~**T that fish strains have evolved similarly as non-accumulators of TCDD, al- r : though such adaptation seems unlikely. Fish have also been analyzed in drainages from areas in Texas and y Arkansas which had been extensively treated with 2,4,5-T (Shadoff et a l ., 1977). The Texas samples were from a pond which collected run-off from a watershed on which the herbicide was used for brush control. The Arkansas site was a pond into which adjacent rice fields were drained and from which irrigation water was obtained, in essence recycling any contaminants. Treatment was 1.25 lb 2,4,5-T/acre, 4-8 weeks prior to flooding. The cycle had been in use for 18 years. Fish from the Texas site did not contain TCDD, at detection limits of 5-7 ppt. Mud at a detection limit of 3 ppt and water at 0.1 ppt were also negative. Six human milk samples obtained from the San Angelo area near the Texas site were also negative at a detection limit of 3 ppt. No TCDD was found in the Arkansas fish samples. Meselson and O 'Keefe (correspondence to Rep resentative James Weaver, 1977) have presented preliminary findings of about 1-2 ppt TCDD in human milk from the San Angelo area, and from an area in western Oregon. j / ^A y . ,/ . A*. Cows milk samples from 2,4,5-T-treated areas of Oklahoma, Arkansas, and Missouri were found to be negative for TCDD by Mahle et al. (1977) with detection limits on the order of 1 ppt. TCDD in beef fat was un detectable in two series of animals which had grazed on 2,4,5-T treated pastures in Oklahoma, Texas, and Missouri. In a third group of seven 0000109 30 /oi^s o o o / ^ o a animals confined in an entirely sprayed pasture, three samples were posi tive at the detection limit of 3-4 ppt.(Kocher et al., 1977). TCDD has been found, however, in several beef cattle maintained in a field experi ment on fields which had received 1, 2, 3, or 4 lbs 2,4,5-T/acre. Average TCDD in fat was 20.3 ppt in the 4 lb/acre group, but there were only three samples, one of which was extremely high. At 3 lb/acre the average was 12.8 ppt, again derived from.some high values and many negatives. Inter- pretation ofJAixs data by EPA is still in process, and is subject to some Hr iS*' argument. . A number of wild animal samples were obtained in treated forest areas t* * of western Oregon, several of which contained unusually high concentrations of TCDD. The unique character of the data raises some questions, since there seems to be few or no samples containing TCDD concentrations in termediate between the low background level and the 100-200 ppt values in a few animals. An issue has been raised about formation of TCDD during combustion of 2,4,5-T, and subsequent distribution in air. Stehl and Lamparski (1977) ignited grass treated with 2,4,5-T in a system where combustion was self supported and found less than 0,0002% converted to TCDD. Combustion of 2,4,5-T and trichlorophenol on filter paper led to production of even less TCDD. If the conversion of a field application were at this rate, about 2 parts of available 2,4,5-T per million would be converted, which is con siderably more than the residual contaminant level. It should be remem bered, however, that burning would not be done until foliage dries, and with a 1-2 week half time it is doubtful whether much herbicide would be available for conversion. Even this maximum level of conversion is probably not of great concern, since the product would be diluted with combustion gases and air in millions of cubic meters of atmosphere. The 31 0000110 7383 o W 30 oo 0 iD i :j effect of exposure to light on decomposition while in the atmosphere is unknown. Masking or wavelength shifts may have an inhibitory influence on photodegradation, but the duration of atmospheric suspension and light exposure should enhance the reaction. Induction of Microsomal Enzymes TCDD has been found to be a remarkably potent inducer of many enzymes which act on foreign chemicals. The effect is apparently dependent upon synthesis of additional enzyme, rather than activation of partially syn thesized or otherwise inactive complete protein. TCDD also increases activity of 6-aminolevulinic acid synthetase in some species, resulting in an accumulation of porphyrins in tissues and excretory routes. The potency of TCDD for inducing some enzymes is between four and five orders of mag nitude greater than that of such classical inducing agents as 3-methyl cholanthrene or phnobarbital. According to Hook et al. (1975a) TCDD induces cytochrome P-4S0 at doses of fewer molecules of TCDD than there are of P-450 in the liver. This property is important because the induced enzymes may increase conversion of other organic intoxicants to non-toxic products, or conversely may increase conversion of non-toxic materials to highly reactive intermediates which exert profound toxic effects. Many carcinogens are activated in this way. The changes in "drug-metabolizing'' enzymes also have been used to describe profound genetic differences among animal strains, and their respective response to intoxication. Enzyme induction may also serve as a predictor of general toxicity, since the induction potency seems well correlated with lethal, acnegenic, and teratologic potential (Schwetz et al., 1973; Poland and Glover, 1973a). e 7384 V32 u \j * rotrooo nod The enzymes which oxidize foreign chemicals have evolved in response to reactive groupings on molecules found in nature. While natural and synthetic chemicals may represent an almost infinite variety of struc tures, the building blocks are the same, and they exist in relatively limited numbers. Consequently, organisms have not had to develop an ` infinite number of enzymes to survive, but rather have developed en zymes specific for reactive groups. The effects on these systems are among the more sensitive changes found to be caused by TCDD. To simplify presentation the following table identifies specific enzymes and the TCDD doses at which responses have been observed. Unfortunately, few explorations of the entire dose response curve have been made; the ''no observed effect" intake has usually not been determined, nor has there been an adequate study of the impact of low level chronic studies on the microsomal enzymes. Most of the data in the table refers to rats. Relatively few studies of mice have been made, and in the guinea pig the effective doses approach the lethal dose, and observed changes- are minor. Hook et al. (1975b) administered 0.175 yg/kg to guinea pigs and found a slight increase in liver biphenyl-4-hydroxylase and biphenyl-2-hydroxylase, and no change in aryl hydrocarbon hydroxylase or UDP glucuronyl t r a n s f e r a s e / Several generalizations appear applicable tb the enzyme inductive properties of TCDD. While most microsomal enzymes re increased after TCDD, the enzymes identified as dcmethylating aminopyrine, benzphetamine and morphine are decreased (Lucier et al., 1973). Females seem to be more sensitive to t h e inducing effects of TCDD (Lucier et al., 1975b). While effects on UDP glucuronyl transferases are substantial, steroid glucuronyl transferases are not changed. The 33 < 7385 0000112 *.**>vf JrA m.++ >u*ktit v a . ^ J L S i ^ k t t .W t o \ill.fr * a ; II) `Wl'bi >--ia 5 a-yC(C<yt DOW 0008 204 Induct of Foreign Compound Metabolizing Enzymes by TCDD (1) Enzyme TCDD Dose Extent of Initial Induction (x normal) Duration of Increased p-Nitrophenol-UDP glucuronyl transferase 3 3 25 5 0.2 14 7 6 5 2.5 >34d >34d 73d >28d *-- References Lucier et al., 1975a Lucier et al., 1975a Lucier et al,, 1975b Hook et al., 197Sb Lucier et al., 1973 Lucier et al., 1973 Remarks (liver unless other pregnant rats (2) neonate, (3) male rar Benzpyrene (aryl hydrocarbon) hydroxylase 4 3 3 2.5 2.5 0.2 0.2 1.0 25 25 10 10 m ____ ^ .08 90 Aniline hydroxylase '5 0.2 Cytochrome P-450 Cytochrome CT> iyl-2-hydroxylase o -vl o'1 to 1.0 1.0 25 25 14 2 14 100 7 8 1.5 3 >70 180 10 100 3 13 2 1.2 1.6 1.4 13 18 >21d ---- --------- >15d >15d -- -- -- Lucier et al., 1975a Lucier et al., 1975a Berry et al., 1976 Berry et al., 1976 Lucier, 1973 Hook et al., 1975b Hook et al., 1975b Hook et al., 197Sb Hook et al., 1975b Poland and Glover, 1974 Poland and Glover, 1974 Poland and Glover, 1974 Poland and Glover, 1974 Beatty and Neal, 1976 Lucier et al., 1973 Lucier et al., 1973 Lucier et al., 1973 Lucier, 1977 Hook et al., 197Sb Hook et al., 1975b pregnant rats (2) neonate, (4) pregnant rat, (5) fetal liver male rats (6) female rats (7) male (7j male rat liver male rat kidney (8) rat kidney rat lung rat intestine C31l/HcN mouse male rat liver (9) male, 3 day male, 3 day rat liver rat kidney (11) y** t i I Induction of Foreign Compound Metabolizing Enzymes by TCDD (cont) Enzyme TCDD Dose (Pe A k ) Extent of Initial Induction (x normal) Duration of Increased Activity References Biphenyl-4-hydroxylase Amino levulinic acid synthetase 25 25 25 25 1.5 ng/egg 2 40 none 2 2 Hook et al., 1975b Hook et a l ., 1975b . Woods, 1973 Goldstein, 1973 Poland and Glover, 1973c Remarks (liver unless other tissue specified) rat liver rat kidney mouse (11) chick embryo *This table is selective and is intended to show the variety .of work done, the dose ranges studied , and duration tin* of effects. In most cases the lowest dose producing effect has been shown and data for higher doses omitted. The references noted should be consulted for greater detail. 2 Pregnant rats, treated on day 10 of 23 day gestation, sacrificed 21 days post partum. ^Mother treated at day 5 of gestation; activity measured at day 21 postnatal. parturition. 4 Same as 2 except measured at day 8 postnatal. No increase in activity before "'TCDD at day 17 of gestation, sacrificed at day 20. CO '00 6Male rats* sacrificed 3 days after TCDD. <1 ^Male rats did not respond at this dose; there was a small but significant (P < 0.05) increase in amino pyrine demethylase and cytochrome P-450 in females at 0.2 pg TCDD/kg. O Resting activity below detection limit. 0000114 ^Maximal activity in cytosol at. 7 days, still rising in microsomes at 15 days, pg/kg produced no effect. ^Resting activity very low in these tissues; factor of increase approximate. SOS 8000MOQ bulk of induction occurs in liver but a similar but usually lesser in crease can occur in the kidney and some other tissues (see table). In kidney, activity seems concentrated in. the ''outer stripe" of the medulla and in the cortex. This region contains the terminal straight segments of the proximal tubules, which appear to be the only significantly in ducible cells in the kidney (Fowler et al., 1977). The induction of aryl hydrocarbon hydroxylase (AHH) by TCDD has been very useful in explaining genetic variability in receptor and effector 4 mechanisms. TCDD is about 3 x 10 times more potent than 3-methylcholan- threne, producing half maximal stimulation in the rat liver at 0.85 1 nmoles/kg and in the C3H/HeN mouse (female) at a dose of 0.42 nmoles TCDD per kg (Poland and Glover, 1974). While the susceptibility to AHH induc tion is about the same in the species tested (ED^ = 0.4-1.2 nmole/kg),- and the LD,.q S differ by about two orders of magnitude, this difference does not detract from the value of the system in studying the genetics of drug response and the great problems of non-homogeneity of exposed populations. There is known to be a profound difference in the responses of various strains of mice to AHH induction by 3-methylcholanthrene (3-MC), leading to a designation of "responsive" :and "non-responsive" strains. Even at high doses, 3-MC fails to cause induction in the non-responsive animals. TCDD, however, will elicit an amplified enzyme activity in such mice (Poland f: V :3 : and Glover, 1975; Chhabra et al., 1974). . Poland and Glover (1975) and N'eubert et al. (1975) have shown that the 1' genetically non-responsive mice have the genetic apparatus necessary for expression of the inducible activities and suggest that the difference arises from a mutant form of inducer-binding receptor with a diminished- affinity for aromatic hydrocarbons. The induction process is apparently 36 In contrast, Lutz-Ostertag and Lutz (1970) sprayed pheasant and grouse eggs with 2,4-D at rates commonly used in the field and caused embryonic mortality and terata. Somers et al. (1973, 1974) were not able * to support these findings after spraying mixtures of 2,4-D and picloram at usual field rates (2.S kg/ha) and 2,4-D and 2,4,5-T on hen eggs (1973) and pheasant eggs (1974) at 10 times field concentrations (11.2 kg/ha). They found no "adverse effect on hatching success, incidence of malformed embryo or subsequent chick mortality". Kopischke (1972) also found no effect on pheasant eggs sprayed at field concentrations, but found that diesel fuel as a carrier blocked hatching completely. Dipping hen eggs in 1% 2,4-D for 10 seconds, then continuing incubation was similarly ineffective (Gyrd-Hansen and Dalgaard-Mikkelsen, 1974). An interesting observation of 2,4-D distribution in mouse fetuses has been made by Lindquist and Ullbcrg (1971). Labeled 2,4-D given late in gestation accumulated early in the yolk sac, passed on to the fetus and was almost completely eliminated by 24 hours after administration. Distribution among tissues was non-selective, and concentrations tended to parallel those of the dam, perhaps explaining in part the lack of teratogenic effect. Carcinogenic and Mutagenic Potential of 2,4-D Innes et al, (1969) screened 120 compounds for tumorigenic properties in mice. 2,4-D and several of its esters were included; none caused in creased tumor incidence. Hansen et al. (1971) carried out a carcino genesis study in rats and concluded that although tumors were found, the observed incidence did not support a finding that 2,4-D is carcino genic. There seemed to be some inconsistencies in interpretation that may have confused the issue. Apparently no other evaluations of cancer 67 DOW 0008 238 potential of 2,4-D have been made. A number of mutagenic screens have included 2,4-D, however. Jcnsscn and Real erg (1976) found that 2,4-D would not induce increased micronuclei in mouse bone marrow erythrocyte, but th$icompound did s lig h tly depress mitotic a c t iv it y . Sex-linked le th a lity assay of 2,4-D in rale Drosophila was also negative for mutagenic a c tiv ity '(Vogel and Chandler, 1974). Styles (1975) treated rats with 2,4-D, then used serum from the animals in a host mediated assay with histidine-requiring / S. ^ p h i m u r i u m mutants. No effect of 2,4-D was evident. In a screen of 110 compounds with the ''Ames test," using eight histidine-requiring mutant strains, Anderson et al. (1972) was unable to detect mutagenic activity Absorption, Metabolism, Tissue Distribution, and Excretion of 2,4-D 2,4-D was absorbed rapidly from the lung of rats in which the herbicide was injected intratracheally as 0.1 ml of 0.01-10 mM solution. The animals were sacrificed from one half to 120 minutes later. The half time for 2,4-D absorption was 1.4 minutes, and concentration had little influence' on the rate (Burton et al., 1974). When ingested, 2,4-D is absorbed primarily by the portal circulation; the lymphatic drainage accounts for very' little 2,4-D absorption, as might be expected from the limited fat solubility of the herbicide (Sieber, 1976). Blood concen- 14 tration of C from radiolabeled 2,4-D administered orally to sheep has been shown to rise very rapidly (Clark et al., 1964). The rate of ab sorption of 2,4-D from the rumen is not known; the immediate elevation in'blood 14C suggests that at least some material was removed direct ly from the rumen. However, Gutenmann et al. (1963) fed 5 ppm 2,4-D to y cattle and found that the concentration in the rumen contents decreased from 3.5 to 0.5 pm over a 24 hour period, which does not suggest rapid 68 0000W7 6EZ 80nnN\a absorption, but rather dilution with passage of rumen content. The chemical did not disappear in an a r t i f i c i a l runen. Kohli ct a l . (1974) gave 5 mg orally to six human volunteers and found peak plasma concentrations by seven hours; the average plasma concentration one week later was about 10" of the peak concentration. , J _1 - ---- N Half time for.excretion was calculated at 33 _________________" ^ \<J ^ Clinical observations of human patients who had extensive skin contact with 2,4-D showed systemic toxic responses within a few hours (Goldstein et al., 19S9; Todd, 1962), indicating ready absorption through the skin. Application of 2,4-D as the DMA salt, isooctyl ester and butyl ester to the body surface of rabbits was relatively ineffective (Kay et al., 1965). Treatment was with 15 ml of aqueous or oil solution applied on a 4 x 3 gauze patch, covered tightly with plastic film. Contact was seven hours daily, five days a week for three weeks; concentrations of 2,4-D were 0.626 and 3.13% acid equivalent. Some animals were treated on abraded skin. A few animals died during the experiment but the pathology and symptoms were not typical of 2,4-D poisoning; most of the fatalities were among the animals with skin damage. No neurological lesions were found, nor were any other observed parameters changed. The only lesions observed weTe at the site of application. There seems to be general agreement, other than the report by Kohli et al. (1974), that significant amounts of 2,4-D do not remain in an-animal for much more than one or two days. Zeilinski and Fishbein (1967) com pared whole body residence times of two esters of 2,4-D and the 2,4-D acid after subcutaneous injection of 100 mg/kg to mice. Of the butyl // ester, 95% was gone after 6 hours, and in animals that had been pre- 69 0000148 DOWGOQ8 240 treated with five similar daily doses, the rate of disappearance was fur ther enhanced. The isooctyl ester was considerably slower to disappear, with a half time of somewhat less than four hours, and the acid itself was retained slightly longer. The authors did not specifically note whether hydrolysis of esters to 2,4-D acid was identified as part of the metabolic process, but the method appeared to account for the disappear ance of all forms of the compound. As a comparison, the disappearance half-time of 2,4,5-T acid was on the order of 20 hours. 14 Khanna and Fang (1966) administered 2,4-D- C to male and female rats ?t very high (80 mg/rat) and relatively low (1 mg/rat) doses and found tissue residues to be greatest at about 6 hours, and almost undetectable by 30 hours after treatment. Extremely high concentrations were found in the stomach but there was no information about separation of stomach content from the tissue proper. No label was found in respiratory gases. A very small fraction of urinary label was found to be an unidentified metabolite, but the data indicate that almost all 2,4-D is excreted with out change by rats. Although 2,4-D binds reversibly to bovine serum albumin (Kolberg et al., 1973) and therefore probably to other plasma proteins, there seem to be no specific tissue binding sites as are found in plants. Hrarre (1974) has examined fish muscle and rat liver after exposure to 2,4-D, seeking specific binding sites and has concluded that none exist. 2,4-D apparently does not move into milk of lactating animals in significant amounts, whether treated by direct administration of the herbicide or by grazing on treated land (Bjerke et al.,! 1972; Gutenmann et al., 1963; Bache et al., 1964; St. John et al., 1964; Klingjnan et al., 1966). Under forcing conditions, however, (1000 ppm 2,4-D in diet), the 70 0000 U9  X i, - ~-i^&~ X L 0 _A^- level of 2,4-D residue m cows milk wns forced up to 0.06 ppm, and were 0. s t i l l barclv detectable seven days later. A substantial capacity for conjugation of 2,4,5-T has been des- ti cribed elsewhere in this report (N'ony et al., 1976). Grunow and Bohme (1974) have found that the ability to conjugate 2,4-D with glycine and taurine exists but is considerably less than that for 2,4,5-T. In dog fish and flounder, however, a major fraction of urinary 2,4-D is excreted as the taurine conjugate (James and Bend, 1976), and in three of the four dogfish studied 10-20% of the label was found in bile after 48 hours. In spite of the rapid loss of 2,4-D by fish, persistence of the herbicide or its products may be prolonged. Treatment of pond weeds with up to 9 kg/ha usually left no detectable residues in fish by 28 days post-application, even at the highest rates (Schultz and Harman, 1974). Schultz (1973) has found, however, that some unidentified products may be present in fish 2-3 months after treatment. ' j.r y The rapid appearance of intact 2,4-D in urine is apparently mediated * a presumably active and saturable tubular excretion mechanism (Erne and Sperber, 1974). The extensive excretion of 2,4-D in urine has been noted in steers (Lisk et al., 1963), sheep (Clark et al., 1964), rats (Sieber, 1976), and humans (Kohli et al., 1974), and apparently is rapid enough to remove all but massive exposures before significant damage can occur. l u w - tkiU - U Behavior of 2,4-D in the Environment and Effects on Submammalian Species This report is primarily concerned with effects of herbicides on non-plant species, but a cursory qualitative survey of literature on field persistence is useful in judging how long a given amount of herbi cide may remain available. 71 0000150 1 DOV*J008 24 I Katcr concentrations arc a major concern, in relation both to aqua tic species and to water supplies for the human population. 2,4-D ap !W plied in a watershed area appears in streams to a very limited extent (White et al., 1976; Kramr.es and Willetts, 1964). Movement into sub- 'surface water flows also appears negligible (White et al., 1976). In .-j aerated lake water 2,4-D persists up to 120 days, but in lake mud 2,4-D j i l hydrolyzes very rapidly, because of microbial activity (Aly and Faust, 1964). Half-time of 2,4-D disappearance seems overall to be much less than two weeks in aqueous systems. For some applications on water, relatively massive applications of up to 40 lbjli per acre are necessary. Two reservoirs on the Tennessee 1/ River were treated at 20 and 40 lb^'/acre depending on the nature of the water milfoil infestation.- The treatments did not affect fish or other fauna, and had little adverse effect on most other plants. Plankton did retain significant amounts of herbicide over extended periods, and 2,4-D was detectable on occasion in finished domestic water from the reser voirs (Wojtalik et al., 1971). In soils, 2,4-D has been found to have a half-time of 4-5 days by Altom and Stritzke (1973). White et al. (1976) found that 95%. of 2,4-D in the top 0.5 cm of soil wouid disappear in 7 days. However, an in n vitro study by Alexander and Aleem (1961) indicated that 2,4-D would be .detectable for as much as 94 days in one type of soil and only 23 days in another. The preparation included 100 ml of nutrient medium and 4 mis of soil as an inoculum; whether such a system represents actual break down conditions in soil is possibly questionable. Sufficient material to cause detectable phytotoxicity persists for about a month, according to Mullison (1972). In areas where 2,4-D is used annually, the break down may be more rapid in the later years than following the first ' ( 7394 I DOW008 <24-3 treatment (Hurle and Radenacher, 1970). Wiersma ct al. (1972) reported a 1969 national program for soil monitoring in which three samples from a total of 28 which had been treated with 2,4-D were found to have from 0.01 to 0.03 ppm 2,4-D present. It seems unlikely that 2,4-D would per sist in either water or soil for more than a month. 2,4-D has been used extensively in control of aquatic weeds, par ticularly water hyacinth and water milfoil, and has probably been sub jected to more study of aquatic toxicity than any other herbicide. A number of short term screens have been conducted with fish. Alabaster (1969) reported on 164 compounds, including 2,4-D sodium salt, which was found to have a 24 hour LC^q of 1160 ppm for harlequin fish, and the butoxyethy1ester of 2,4-D which was much more toxic with a 24 hour LD^q of 1 ppm, for the same species. The 24-96 hr LC^q of PGBE esters of 2,4-D for rainbow trout were slightly above 1 ppm (Cope, 1964). For mullet and killifish the LC^q (24 hr) was 5 ppm for both the PGBE and butoxyethanol esters. The parent acid was ineffective at 50 ppm (Butler, 1963). Oyster shell growth was 50% inhibited by 3.75 ppm butoxyethanol ester, but the acid and dimethylamine salt were not effective at 2 ppm (Butler, 1963). Exposure to the DMA salt for 24 hrs at 2 ppm caused no mor tality in shrimp; 48 hour exposure caused 10% lethality. The butoxy ethanol and PGBE esters caused no mortality to shrimp at 1 ppm x 48 hour. The ethyl hexyl ester of 2,4-D caused 38% decrease in oyster shell growth at 5 ppm over 96 hrs (Butler, 1965). The considerable differences in 2.4- D effect among various species were illustrated by Sanders (1970a) 2.4- D acid hds a 48 hour median response concentration (TL^q ) of 100 ' 7395 73 0 0 0 0 1 5 2 mg/L (100 ppm) for Daphnia and 3.2 mg/L for scud. (Crosby and Tucker (1966) found the same value for Daphnia.) The PGBE ester TL^q varies from 0.1 for Daphnia to 2.6 for scud to more than 100 ppm for crayfish. The DMA salt TL^q is more than 100 ppm in bluegill but T L ^ for the butyl ether ester in bluegill is 1.1 ppm, and 100 ppm for crayfish. Stor.efly naiads are less sensitive to 2,4-D itself (LC^q x 96 hr * 15 ppm) than the butoxy ethanol ester (iC^Q x 96 hr = 1.6 ppm) (Sanders and Cope, 1968). Sanders (1970b) has also reported a 96 hr TL,-q for 2,4-D amine of 100 ppm for tadpoles. The lethal effect on tadpoles is apparently limited, but Euslovich and Borushko (1976) found that 2,4-D sodium salt would inhibit metamor phosis of Rana temporaria tadpoles, and blocked thyroidin stimulation of the process. The DMA salt was effective at 2 ppm. The authors specu late that 2,4-D antagonizes thyroid hormone. Few studies of truly chronic exposure of fish to 2,4-D have been made. Mount and Stephan (1967) compared the 96 hour LC^q for 2,4-D butoxyethanol' ester (5.6 ppm) with various 10 month exposures, and found that 1/lSth of the LCgg, or 0.3 ppm could be tolerated by fathead minnows without effect on growth or reproduction. Eggs were much more sensitive than adults over 4S hour exposures. Schultz (1973) placed bluegill, channel catfish, and largemouth bass in solution of 2,4-D DMA. salt, labeled with 14C, at concentrations of 0.S, 1.0, or 2.0 mg/L (ppm). Fish and 14 water samples were removed at intervals up to 84 days for analysis of C and 2,4-D content. t Considerable radioactivity remained in fish tissues but apparently / none was associated with 2,4-D, suggesting extensive metabo44sir- The ( 7396 74 f i r s t tissue in which radioactivity appeared was the gall bladder of cat fish and b lu e g ills , and eventually appeared in every tissue analyzed. In both c a tfish and b lu e g iil, the amount of radioactivity in muscle tended to increase through the collection p e r io d ^ N o t o x ic ity was evi- dent in any of- the exposed f is h . To relate concentrations of one ppn to fie ld condition, an applica- tion of 2 kg/hectare (10000 M ) to water will give a concentration of 2 rag/L or 2 ppn if the water is 10 cm deep. Deeper water will result in further dilution. If applied to flowing water, water movement will dilute the herbicide quite rapidly, and that material distributed on soil and vegetation will tend to remain in place. Obviously a spill or other accident presents different problems. Among insects, the effects of 2,4-D on honey bees has received the most attention. Palmer-Jones (1964) has listed a number of investiga tors who concluded that 2,4-D is safe for bees, as well as others who have shown toxic effects, some of which may depend on the species of plant on which the herbicide is deposited. In a field investigation of a three lb/acre application Palmer-Jones found that a 22% mortality oc curred in 48 hours after dusting. However, when bees at the hive were heavily dusted directly, there was no mortality, leading to the conclusion that the bees in the field were acquiring intoxicant by some mechanism other than surface contact. Moffett et al. (1972) sprayed caged bees diTectly with various phenoxy herbicides at a one pound/acre rate with very little effect. When included in the diet at concentrations up to 100 ppm, 2,4-D did not decrease lifetime of bees and the ester used was ineffective it 1000 ppm (Morton et al., 1972). The herbicide docs cause decreased brood development when fed at 100 ppm but has no reproductive effect at 10 ppm (Morton and Moffett, 1972). 75 * 7t* ~ w. '' 7i 0000154 DOW0008 246 i i Treatment of Coccir.cllid beetle larvae with 2,4-P at a rate equal to 8 oz. acid cquivalent/acre caused greatly increased m ortality, re gardless of the age of the larvae through day 12 at time of spraying (Adams, 1960). SILVEX There appears to be a much more limited accumulation of data on bio logical effects of silvex and related compounds than is available for other phenoxy herbicides. The descriptions of biological effects of silvex will be categorized in two broader sections rather than the more detailed entries prepared for the other agents. Biological Effects of Silvex and Its Derivatives The acute median lethal dose of silvex and its esters is quite high, and falls in a relatively narrow range among species and among the deri vatives. Rowe and Hynas (1954) summarized data which had been established at that time. Silvex and its mono-, di-, and tri-propylene glycol ether esters were of almost identical LD^q in guinea pig of 1200 and 1550 mg/kg. The lethalities to rats were also similar at about 650 mg/kg. The rat appears to be most sensitive, and chicks and mice require doses similar to the guinea pig. Subchronic (90 day) studies of rats fed 10-600 mg/kg/day of the PGBE ester resulted in more than 50% lethality at the highest dose. The time *i of death ranged from 15-85 days. Growth was depressed in rats fed 500 l i mg/kg. There is somewhat of a paradox in the findings, because pathology i ! j in the dead animals indicated malnutrition rather than herbicide toxicity. j i A paired feeding/Study at 300 and 600 mg/kg/day indicates that depressed i 1I growth was not entirely due to inadequate food consumption. Dose rates of l 76 \ 30 and 100 mg/kg/day caused an increase in liver weight but 10 mg/kg/day caused no chang^^ (Mullison, 1966). The study was followed by a 90-day feeding of up to 10000 ppn silvex (sodium salt) in the diet. 10000 ppm is l 1% of the diet, and if the animals consumed 10 gm daily and weigh 300 gm, the dose rate would be on the order of 300 mg/kg/day. That dose rate was highly lethal and was abandoned, but did produce swelling and granular degeneration of hepatocytes and ultimate cellular necrosis. Renal tubule cells were swelled and vacuolated, and seminiferous tubules were degenera ted. At levels above 10 ppm (about 3 mg/kg/day) some growth depression a, occurred. Mullison (1966) fed Kuros^l (potassium salt of silvex, containing 53.3% silvex acid) for two years at concentrations up to 300 ppm. The highest dose caused a slightly^increased kidney/body weight ratio in males, but TOO ppm and lower dose rates caused no change in food consumption, growth, gross and microscopic morphology hematology or bone marrow. 100 ppm was stated by the author to be equivalent to 2.6 mg/ kg/day of silver. A* Kurospl was without effect on beagles at a feeding rate of 56 ppm (about 19 mg/kg/day) over 2 years. Females fed 190 ppm suffered some hepatic degeneration and necrosis after a year of feeding, but at the end of two years no damage could be found. At the 56 ppm intake, there were no changes over a very broad spectrum of hematologic, clinical chemistry and morphologic analyses. The liver damage at higher levels included hepatocyte necrosis, bile duct proliferation, and bile pigment deposition throughout the liver and t in the epithelium of kidney tubules. In a subcljrcnic study in cattle by Palmer et al. (1964), one yearling Brahma-cross fed 100 mg silvex/kg/daily died after 29 days. Two other 77 7399 00ooi56 DOWDGOfi 248 animals given 2S and 50 ng/kg for 73 days showed no evidence of to x ic it y . A 90-day experiment with 50 mg/kg/day, cither by drench or by injection into a rumen f i s t u l a resulted in death of one of the la tte r group of three animals. Two of those treated orally developed severe inflammation in the parotid area, apparently due to local ir r it a t io n . ^jik^sp'fyey .fc^'Innes et al. (1961) sjrcefeped silvex Against two strains of mice for 18 months. The concentration of silvex was 121 ppm, which was the maximum tolerable dose rate, and there was no increase in tumors in either strain. The mutagenic screen by Anderson et al. (1972) did not detect point mutations resulting from silvex treatment in either T^ phage or S. typhimurium (histidine requiring). Silvex is teratogenic at high doses. Courtney (1975) found that 398 mg/kg/day, on days 12-15 of gestation, caused_3% cleft palate in the group given silvex in DKSO subcutaneously, and 7% where given orally in c o m oil. Fetal mortality increased to 25% in the group treated subcuta neously. The Dow Chemical Co. has also conducted a series of teratology studies with silvex. Dose rates of 75, 100 and 150 mg/kg/day from day 6 to day 15 caused several cardiovascular anomalies; 50 mg/kg/day caused retarded ossification in the sternum and skull.(Dow Chemical Co., 1972). The no . adverse effect level was considered to be 25 mg/kg/day. Tne PCBE ester --- ------- - /.Mm / caused skeletal changes at an intake of 50 mg/kg/daily but no changes were found after 35 mg/kg/day (23 mg/kg silvex acid equivalent). Birds seem peculiarly resistant to the herbicides. For example, DeWitt et al. (1963) fed 5000 ppm BEE ester of silvex to young bob white quail, pheasant and mallard ducks. The average lethal intakes were 9350, 9240 and 21,000 mg/kg, and time of death varied from 10 to 100 days. 7 *00 78 r.C-.vWY 3 6 * 2 8Q00JA Mallards were able to consume 100 ppm for 100 days with no lethality, but reproduction was impaired. Silvex acid at 5000 ppm in the diet was tolerated for 5 days with no mortality by Coturnix. The 5 day LD-Q for pheasants was 4500 ppm of silvex BEE ester, for bobwhite quail the L D ^ was 30 ppm over 5 days, for Coturnix it was in excess of 5000 ppn, for pheasants LP-q was less than 3000, and no mallards died-after 5 days on 5000 ppm. All birds were two weeks old at the initiation of the study (Heath et al., 1972). Stickel (1964) reported US Fish and Wildlife studies in which bobwhite were fed 1000 ppm of an unspecified silvex ester; 50% were dead by day 34 with average total dose of 17000 mg/kg. Of a group fed 5000 ppm, the average daily dose was 2300 mg/kg. Half of the birds survived more than 4 days, 40% survived more than 10 days, and an additional 4% died during the remaining 14 days. The average dose to birds that survived 24 days was 54,500 mg/kg. These findings indicate that wildfowl should not be adversely af fected by any field application of silvex, or for that matter even a gross Effect of Silvex on Aouatic and Invertebrate Species Silvex has become an herbicide of choice for control of surface and underwater water needs. As a consequence, there has perhaps been more attention paid to its impact on aquatic species than any of the other . phenoxy herbicides. There is an unfortunate diversity of experimental methods and conventions for expressing concentration and other factors which sometimes makes comparison of data difficult. Table 1 has been constructed to simplify consideration of the various reports of toxicity to aquatic species. 7 4 0 1 0 0 0 0 158 1 79 DOWOOgl 250 if Tabl 1. Gross Toxicity of Silvcx and Its Derivatives to Fish Species Form of Silvex Concentration (Acid Equiv.) ppm Effect Bluegill (young) (fry) (eggs) (fry) Stoneroller (eggs) Chorus frog (tadpole) Fowler's toad (tadpole) BEE ester BEF. ester Isooctyl ester K salt liquid X salt granular PGBE ester K salt, liquid X salt, granular PCBE ester PGBE ester PGBE ester PGBE ester K salt K salt K salt PGBE ester BEF. DEE BEE 0.36 1.7 1.4 83 100 0.3 100 150 10 5 1 3 10 75 25 5 20 10 22 LC50 LC50 LCso LCS0 LCSO tolerated tolerated tolerated no effect on hatch 100-i lethal no effect livor degen. no effect 35* lethal 95* hatch 45* hatch LC50 LC50 LCSO Period 48 h 48 h 48 h 48 h 48 h 96 h 96 h 96 h 36 h 80 days 2 weeks 2 1/2 mo 2 1/2 mo 72 h 72 h 24 h 96 h 24 h Reference Cope, 1964 Hughes and Davis, 1966 Hughes and Davis, 1905 Hughes and Davis, 1965 Jones, 1962 Jones, 1962 Jones, 1962 Wilber and Whitney, 1973 Wilber and Whitney, Cope, 1964 Cope, 1964 Cope, 1964 Cope, 1964 Hiltibran, 1967 Hiltibran, 1967 Sanders, 1970b Sanders, 1970b Sanders, 1970b 1973 TS3 8Q 00M Q Among factors that influence to x icity to fish are the chemical form of the agent, whether the formulation is granular or liq u id , and water hardness. Generally the esters are more to x ic , and liquid formulations are more to x ic . Data on water hardness is contradictory; but the ten dency to greater to x ic ity in soft water seems to be associated with the sa lt rather than the esters (Surber and Pickering, 1962). Distribution rates for silvex treatment of aquatic weeds may be as / high as 40 lb^acre. An assumption of dilution throughout an 8 foot depth is apparently conventional, which would give a concentration of 1.8-1.9 ppm. If this concentration of an ester fomuilation were to hold in a given area, it would clearly be lethal to many fish. The salt should have up to a 100 fold safety factor for fish, depending on mineral content of the water. Such application is not a consideration in forest operation, but accidental overwater dilution could occur. Terrestrial application is at rates at least 10 fold less than 40 lb/acre but many water courses are much shallower than 8 feet, thereby leaving a similar hazard. If flowing, dilution will be rapid and periods of exposure should be limited. In static water the agent will diffuse away from the application site with time except in small shallow ponds, where some damage might possibly occur Lower aquatic organisms have also been well studied because of silvex use on water weeds. Representative though not comprehensive data is compiled in Table 2. Again, it appears that the salts of silvex are much less toxic than the more complex esters. The most vivid contrast is in the effect on Daphnia magna, which is 50% immobilized by 100 ppm silver potassium salt, and by only 0.18 ppm silvex PGBE ester. Some species, however, are particularly resistant to even the PGBE ester of 7 4 0 3 0000160 81 silv ex . The crayfish LC^g is in excess of 100 ppm; for a number of other organisms this factor is less than 1 ppm. I t is not necessary to cat. Icgue the entire literature on silvex effects on either fis h or other aquatic species. The range o f e ffe c tiv e concentrations. is well illu stra ted as well as the extent, i f not d e ta ils , of species differences. Effects of silvex on terrestrial insect life have been evaluated in honey bees by USDA workers at Tucson. All of the phenoxy herbicides including silvex were found to be relatively non-toxic to bees when applied in water at field concentration (Morton et al., 1972; Moffett et al., 1972). When fed at 100 or 1000 ppm, silvex reduced brood pro duction, but at 10 ppm, no adverse effect was found. In each case, when the toxicant was removed, the colonies regained their original reproduc tive efficiency (Morton and Moffett, 1972). 1 O C <ac K) Crt CO Metabolic Fate of Silvex in Mammals There have been few studies of the disposition of silvex after ab'ty&r+ZZ* m. s<SZ4^ y ^ sorption by mammals. Bjerke et al. (1972j fed silvex at 1000 ppm in feed and foundfO.12 pom 3-t4,5 tfai Ugohonol (?P)lIff>milk and 0.16 ppm i i 'f^ . in cream. In one week off contaminated feed the concentrations decreased blw 0*05 ppnw Leng (1972) in a review, r d p ^ f e T ' w o r k ^ ^ ^ i i c h ^ j ^ l ^ T C P ( -h/VJ ' . Lvl jir was not detected^ however, residues of silvex were substantial in the ^ liver. After feeding 300 ppm silvex 28 days, 4 ppm were found in liver, A , 18 in kidney, 0.6 in muscle and 0.9 in fat. After 2000 ppm in the a a .K' ' diet, 12, 30, 2 and 4 ppm were found in the respective tissues. In a S*m **ar exF9r*ment Clark et al. (1975) found roughly similar values, with V. traces of 2,4,5-TCP in each tissue. In a single cow experiment, 5 ppm 1U f V) W f /> ff / . S3 0000162 REF HRliNf:r.s Abou Akkada, A.R., M.A. Hassan, and M.A. Naga (1973). Effect of some pesticides and herbicides on the rumen micro-organisms of sheep. J. Agr. Sci. 81:231-256. Abou Akkada, A.R., M.A. Hassan, and M. Naga (1975). A note on the effect of herbicides on the metabolism and performance of lambs. Anim. Prod. 20:311-313. Adams, J.3. (1960). Effects of spraying 2,4-D amine on coccinellid larvae. Can. Jour. Zool. 38:285-288. Alabaster, J.S. (1969). Survival of fish in 164 herbicides, insecticides, fungicides, wetting agents, and miscellaneous substances. Internat. Pest. Control 11:29-35. Alexander, M . , and M.'l.H. Aleem (1961). Effect of chemical structure on microbial decomposition of aromatic herbicides. J. Agr. Food Chem. 9:44-47. Allen, J.R., J.P. Van Miller, and D.H. Norback (1975). Tissue distribu14 tion, excretion and biological effects of [ C]tetrachlorodibenzop-dioxin in rats. Fd. Cosmet. Toxicol. 13:501-505. Allen, J.R., D.A. Barsotti, and J.P. Van Miller (1977a). Reproductive dysfunction in non-human primates exposed to dioxins. Presented at 16th Annual Meeting, Soc. Toxicol., Toronto, 1977. Abstract 111. Allen, J.R., D.A. Barsott, J.P. Van Miller, L.J. Abrahamson, and J.J. Lalich (1977b). Morphological changes in monkeys consuming a diet containing low levels of 2,3,7,8-tetrachlorodiber.zo-p-dioxin. Food and Cosmetics Toxicol, (in press). Altom, J.D.,/and J.F. Stritzke (1973). Degradation of dicamba, picloram, and four phenoxy herbicides in soils. Weed Science 21:556-560. 0000164 85 Beatty, P . a n d R.A. Neal (1976). Induction of DT-diaphorasc by 2,3,7,8- tctrachlorodibenzo-p-dioxin (TCDD). Biochem. Biophys. Res. Conn. . 68:197-204. Beatty, P.W., M.A. Holscher, and R.A. Neal (1976). Toxicity of 2,3,7,8- tetra'chlorodibenzo-p-dioxin in larval and adult forms of rana catesbeiana. Bull. Environ. Contam. Toxicol. 16:578-581. Becker, B.A. (1975). Teratogens. In: Toxicology. The Basic Science of Poisons. L.J. Casarett and J. Doull (eds). MacMillan, New L York. pp. 331-532. Berkeley, M.C., and K.R. Magee (1963). Neuropathy following exposure to a dimethylamine salt of 2,4-D. Arch. Int. Med. 111:351-352. Berndt, W.O., and F. Koschier (1973). In vitro uptake of 2,4-dichloro- phcnoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by renal cortical tissue of rabbits and rats. Toxicol. Appl. Pharmacol. 26:559-570. Berry, D.L., P.K. Zachariah, M.J. Narakung, and M.R. Juchau (1976). Trans placental induction of carcinogen-hydroxylating systems with 2,3,7,8- tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 36:569-584. Berwick, P. (1970). 2,4-Dichlorophenoxyacetic acid poisoning in man. J. Am. Med. Assoc. 214:1114-1117. Binns, W., and A.E. Johnson (1970). Chronic and teratogenic effects of 2,4-D (2,4-dichlorophenoxyacetic acid) and atrazine (2-chloro-4- ethylamino-6-isopropyl amino-s-triazine) to sheep. North Central Weed Control Conference Proc. 1970:100. Binns, K . , and L.. Balls (1'971). Nonteratogenic effects of 2,4,5-tri- chloroph^noxyacetic acid and 2,4,S-T propylene glycol butyl esters herbicides in sheep. Teratology 4:245. 87 ' 74060000166 r*n Butler, P.A. (1963). Pesticide-wild]ifo effects. U.S. Fish and Wildlife Service Circular 167. pp. 11-26. Butler, P.A. (196S). Effects of pesticides on fish and wildlife. U.S. Fish and Wildlife Circular 226. pp. 65-77. Byast, T.II., and R.J. Hance (1975). Degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Bull. Environ. Contain. Toxicol. 14:71-76. Carter, C.D., R.D. Kimbrough, J.A. Liddle, R.E. Cline, M.M. Zack, Jr., W.R. Barthel, R.E. Koehler, and P.E. Phillips (1975). Tetrachloro- dibenzodioxin: An accidental poisoning episode in horse arenas. Science 118:738-740. Chang, K-C., J.W. Rip, and J.H. Cherry (1974). Effects of phenoxyacetic acids on rat liver tissues. J. Agr. Food Chem. 22:62-65. Chhabra, R.S., J.M. Tredger, R.M. Philpot, and J.R. Fouts (1974). Selec tion of inducers: An important factor in characterizing genetic differences to induction of aryl hydrocarbon hydroxylase in strains of mice. Life Sci. 15:123-130. Clark, D.E., J.E. Young, R.L. Younger, L.M. Hunt, and J.K. McLaran (1964). The fate of 2,4-dichlorophenoxyacetic acid in sheep. J. Agr. Food Chem. 12:43-45. Clark, D.E., J.S. Palmer, and C.H. Ayala (1970). Residual and toxicolo gical aspects of 2,4,5-T and an ester in sheep and cattle. Presented at meeting of Am. Chem. Soc. Pesticide Division, and Chemical Inst, of Canada. Toronto, 1970. Clark, D.E., and J.S. Palmer (1971). ! Residual aspects of 2,4,5-T and an ester in^sheep and cattle with observations on concomitant toxicolo gical effects. J. Agr. Food Chem. 19:761-764. 00001&8 1 (89 7407 O Cunningham, H.M., and D.T. Williams (1072). Effect of tetrachlorodibenzo-p-dioxin on growth rate and the synthesis of lipids and proteins in rats. Bull. Environ. Contam. Toxicol. 4:45-51. c c QC Davis, J.T., and J.S. Hughes (1963). Further observations on the toxicity of commercial herbicides to bluegill sunfish. Proc. Southern Weed Conference 16:337-340. Davring, L. (1975). Effects of.a 2,4,5-T ester on early oogenesis, fertility and development in Drosophila melanogaster. Hereditas 80:255-262. Davring, L., and M. Sunner (1971). Cytogenetic effects of 2,4,5- trichlorophenoxyacetic acid on oogenesis and early embryogenesis in Drosophila melanogaster. Hereditas 68:115-122. Dencker, L. (1976). The herbicide 2,4,5-T: Early placental barrier and accelerated fetal uptake with advancing gestation. In: Tissue Localization of Some Teratogens at Early and Late Gestation Related to Fetal Effects. Acta Pharnu Toxicol, (suppl.) 39:59-72. Desi, I., J. Sos, J. Olasz, F. Buie, and V. Markus (1962). Nervous system effects of a chemical herbicide. Arch. Environ. Health 4:95-102. DeWitt, J.B., W.H. Stickel, and P.F. Springer (1963). Pesticide-wildlife studies. U.S. Fish and Wildlife Service Circular 167. pp. 74-105. Dougherty, W.J., M. Herbst, and F. Coulston (1975). The non-teratogenicity of 2,4,5-trichlorophenoxyacetic acid in the rhesus monkey (Macaca mulatta). Bull. Environ. Contain. Toxicol. 13:477-482. Dow Chemical Company, Internal Communication (1961). Results of 90-day dietary feeding studies on Dowanol 9713 ester 2,4,5-T in rats. 0000170 191 7408 C92 80Q0/AQ Evans, J.O., and O.R. Duscja (1973). ilc-rbicide contamination of surface runoff waters. USEPA Report 2-75-206. Eyzaguirrc, C., B.P. Folk, K.L. Zierler, and J.L. Lilienthal, Jr. (1948). Experimental myotonia and repetitive phenomena: The veratrinic ef fects of 2,4-D in the rat. An. Jour. Physiol. 155:69-77. Fang, S.C., E. Fallin, M.L. Montgomery, and V.H. Freed (1973). The metabolism and distribution of 2,4,5-trichlorophenoxyacetic acid in female rats. Toxicol. Appl. Pharmacol. 24:555-563. Florsheim, W.H., and S.M. Velcoff (1962). Some effects of 2,4-dichlorophenoxy acetic acid on thyroid function m the rat: Effects of iodine accumulation. Endo. 71:1-6, Florsheim, W.H., S.M. Velcoff, and A.D. Williams (1963). Some effects of 2,4-dichlorophefioxy acetic acid on thyroid function in the rat: Effects on peripheral thyroxine. Endo. 72:327-333. Fowler, B.A., G.W. Lucier, H.W. Brown, and O.S. McDaniel (1973). Ultrastructural changes in rat liver cells following a single oral dose .of TCDD. Environ. Health Perspec. 5:141-148. Fowler, B.A., G.E.R. Hook, and G.W. Lucier (1977). Tetrachlorodibenzo- p-dioxin induction of renal microsomal enzyme systems: ultrastruc tural effects on pars recta (Sj) proximal tubule cells. Jour. Pharmacol. Exp. Therap. (in press). Fries, G.F., and G.S. Marrow (1975). Retention and excretion of 2,3,7,8tetrachlorodibenzo-p-dioxin by rats. J. Agr. Food Chem. 23:265-269. Gaines, T.B., J.F. Holson, Jr., C.J. Nelson, and H.J. Schumacher (1975). Analysis of strain differences in sensitivity and reproducibility of results in assessing 2,4,5-T teratogenicity in mice. Toxicol. AppJ. Pharmacol. 33:174-175. 0000172 93 DOWu(jW 265 3 Mall, S.M. (1972). Effects on pregnant rats and their progeny of ade quate or low protein diets containing 2,4,5-Trichlorophenoxy acetic acid (2,4,5-T) or p,p'-DDT. Fed. Proc. 31:726. Hamada, Nr., and R.E. Feterson (1977). Reversal of 2,3,7,8-tetrachlorodibcnzo-p-dioxin-induced depression in biliary excretion of ouabain by pregnenolone-16a-carbonitrile and spironolactone in rats. Int. Cong. Toxicol., Toronto. Abstracts p. 25. Hansen, W.H., M.L. Quaifc, R.T. Haberman, and O.G. Fitzhugh (1971). Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs. Toxicol. Appl. Pharmacol. 20:122-129. Haugen, D.A., M.J. Coon, and D.W. Nebert (1976). Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled protein synthesis in response to treatment with B naphthaflavone or phnobarbital. Jour. Biol. Chem. 251:1817-1827. Hay, A. (1976). Toxic cloud over Seveso (Editorial, no references). Nature 262:636-638. Heene, R. (1969). Redeutung experimentell-histochemischer Befunde fiir die pathogenese von muskeldystrophien. Klin, h'sehr. 47:389-390. Heath, R.G., J.W. Spann, E.F. Hill, and J.F. Kreitzer (1972). Compara tive dietary toxicities of pesticides to birds. Bur. Spt. Fisheries and Wildlife, Spec. Scientific Report (Wildlife) 152. Highman, B., T.B. Gaines, and H.J. Schumacher (1976a). Sequential histopathologic, hematologic, and blood chemistry changed induced in mice by a technical and a purified preparation of 2,4,5-trichlorophenoxyacetic acid. J. Toxicol. Environ. Health 1:469-484. Highman, B., 'p.B. Gaines, H.J. Schumacher, and T.J. Haley (1976b). Strain differences in histopathologic, hematologic, and blood chemistry 95 0000174 7110 Hughes, J.S., and J.T. Davis (1965 (lD'-Z conference)). Comparative toxi city to blucgill sunfish of granular and liquid herbicides. Proc. Conf. Southeast Assoc, of Cane and Fish Comm. 16:319-322, Hughes, J.S., and J.T. Davis (1966). Toxicity of pesticides to bluegill sunfish tested during 1961-1966. Louisiana Wildlife and Fisheries Comm. 8 pp_. Hurle, K., and B. Rademacher (1970). Studies on the effect of long-term application of DMOC and 2,4-D on their breakdown in the soil. Weed Research 10:159-164. Hussain, S., L. Ehrenberg, G. Lofroth, and T. Gejvall (1972). Mutagenic effects of TCDD on bacterial systems, Ambio 1:32-33. Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell, and J. Peters (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Nat. Cancer Inst. 42:1101-1114. Isensee, A.R., and G.E. Jones (1975). Distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic model ecosystem. Environ. Sci. Technol. 9:668-672. Iyer, V., M. Whiting, and G. Fenichel (1977). Neural influence on ex perimental myotonia. Neurology 27:73-76. Jackson, W.T. (1972). Regulation of mitosis. III. Cytological effects of 2,4,5-trichlorophcnoxy acetic acid and of dioxin contaminants in 2,4;5-T formulations. Jour. Ce.. Sci. 10:15-25. James, M.O., and J.R. Bend (1976). Taurine conjugation of 2,4-dichlorophenoxyacctic acid and phcnylacetic acid in two marine species. Xenobiotica jS:393-398. ,* 97 000017 ro*v r>r\r>r\ i n H Khanna, S., and S.C. Fang (1P66). Metabolism of C^-labclcd 2,4-dichlorophcnoxyacetic acid in rats. J. Agr. Food Chem. 14:500-503. Khera, K.S., and J.A. Ruddick (1973). Poly(ch]oro)dibenzo-p-dioxins. Perinatal effects and the dominant lethal test in Wistar rats. ACS Adv. Che:n. Series 120:70-84. Khera, K.S., and W.P. McKinley (1972). Pre- and postnatal studies on 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and their derivatives in rats. Toxicol. Appl. Pharmacol. 22:14-28, Kilian, D.J., M.C. Benge, R.V. Johnston, and E.B. Whorton, Jr. (1977). (Personal communication, unpublished manuscript). Cytogenetic studies of personnel who manufacture 2,4,5-T. Kimbrough, R.D. (1974). The t-oxicity of polychlorinated polycyclic com pounds and related chemicals. CRC Crit. Rev. in Toxicol. 2:445498. Kimbrough, R.D., C.D. Carter, J.A. Liddle, R.E. Cline, and P.E. Phillips (1977). Epidemiology and pathology of a tetrachlorodibenzo-dioxin poisoning episode. Arch. Environ. Health 32:77-85. Kimmig, J., and K.H. Schulz (1957). Berufliche Akne (sogenannten Chlorakne) durch chlorierte aromatische zyclische ther. Dermatologica U S : 540-546. Klingman, D.L., C.H. Gordon, G. Yip, and H.P. Burchfield (1966). Residues in the forage and in milk from cows grazing forage treated with esters of 2,4-D. Weeds 14:164-167. Kocher, C.W., N.H. Mahle, R.A. Hummel, and L.A. Shadoff (1977). A search for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in beef fat. Bull. Environ. Contam. Toxicol, (in press). ^* 000C17S 99 7412 Koschier, F.J., and h'.O. Berndt (197(,b). Specificity of 2,4,5-trichloro- c TS& QnnnAAn phcnoxyacetatc on tetracthvlanmonium transport. Toxicol. Appl. Pharmacol. 38:297-306. Koschier, F.J., and W.O. Berndt (1976c). In vitro uptake of organic ions by renal cortical tissue of rats treated acutely with 2,4,5- trichlorophenoxyacetic acid. Toxicol. Appl. Pharmacol. 35:355-364. Kouri, R.E., H. Ratrie III, S.A. Atlas, A. Niwa, and D.W. Nebert (1974). Ar>*l hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Life Sciences 15:1585-1595. Krammes, J.W., and D.B. Willets (1964). Effect of 2,4-D and 2,4,5-T on water quality after a spraying treatment. U.S. Forest Service Res. Note PSW-52, Pacific SW Forest and Range Experiment Station, Berkeley. 4 p. Kutches, A.J., D.C. Church, and F.L. Duryee (1970). Toxicological effects of pesticides on rumen function in vitro. J. Agr. Food Chern. 18: 430-433. Leng, M. L. (1972). Residues in milk and meat and safety to livestock from the use of phenoxy herbicides in pasture and rangeland. Down to Earth 28(1):12-20. Leng, M.L. (1977). Comparative metabolism of phenoxy herbicides in ani mals. In: Fate of Pesticides in Large Animals. G.W. Ivie and H.W. Dorough (eds.). Academic Press, New York. Lindquist, N.G., and S. Ullberg (1971). Distribution of the herbicides 2,4,5-T and 2,4-D in pregnant mice. Accumulation in the yolk sac epithelium. Expericntia 27:1439-1441. t Lisk, D.J., W,'H. Gutenmann, C.A. Bachs, R.G. Warner, and D.G. Wagner (1963). Elimination of 2,4-D in the urine of steers fed 4-(2,4-DB) or 2,4-D. J. Dairy Sci. 46:1435-1437. 101 00001.30 7413 Majumdar, S.K., and J.K. Golia (1974). Mutation test of 2,4,5-trichlorophcnoxyacetic acid on Drosophila mclanogastcr. Can. J. Genet. Cytol. 16:465-456. Matsumura, F., and H.J. Eenezet (1973). Studies on the bioaccunulation and microbial degradation of 2,S,7,S-tetrachlorodibenzo-p-dioxin. Environ. Health Persp. 5:255-258. May, G. (1973). Chloracne from the accidental production of tetrachlorodibenzodioxin. Brit. Jour. Indust. Med. 30:276-283. McCann, J., E. Choi, E. Yamasaki, and B.N. Ames (1975). Detection of carcinogens as mutagens in the Salmanella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. 72:5135-5139. McCollistcr, S.B., and F.J. Kociba (1970). Results of 90-day dietary feeding study on 2-,4,5-trichlorophenoxyacetic acid (2,4,5-T) in rats. Dow Chemical Co. Internal Report. McConnell, E.E., J.A. Moore, J.K. Haseman, and M.W. Harris (1977a). The comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs. Toxicol. Appl. Pharmacol, (in press). McConnell, E.E., J.A. Moore, and D.W. Dalgard (1977b). Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in rhesus monkeys (macaca mulatta) following a single oral dose. Toxicol. Appl. Pharmacol, (in press). McNulty, K.D. (1977). Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin for rhesus monkeys: brief report. Bull. Environ. Contam. Toxicol. 18:18-19. McQueen, E.G., A.M.O. Veale, W.S. Alexander, and M.N. .Bates (1977). 2,4,5-T and human birth defects. Report of the Division of Public - S Health, New Zealand Department of Health. 0CGC182 103 DoW 0008 275 Muranyi-Kovacs, I., G. Rudali, and J. Imbert (1976). Bioassay of 2,4,5trichlorophcnoxyacctic acid for carcinogenicity in mice. Brit. Jour. Cancer 33:626-633. Murray, F.J., F.A. Smith, K.D. Nitschkc, C.G. Humiston, R.J. Kociba, and B.A. Schwetz (1977). Three-generation reproduction study of rats ingesting 2,3,7,8-tetrachlorodibenzo-p-dioxin. Presented at 16th Annual Meeting, Soc. of Toxicol., Toronto. Abstract 166. National Academy of Sciences and National Research Council (1977). Drink ing water and health. (In press). Nebert, D.W., J.R. Robinson, A. Niwa, K. Kumaki, and A.P. Poland (1975). Genetic expression of aryl hydrocarbon hydroxylase activity in the . mouse. J. Cell Physiology 85:393-414. Neubcrt, D., and I. Dillmann (1972). Embryotoxic effects in mice treated with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzop-dioxin. Naunyn-Schmiedeberg's Arch. Pharmacol. 272:243-264. Neubert, D., P. Zens, A. Rothenwallner, and H-J. Merker (1973). A survey of the embryotoxic effects of TCDD in mammalian species. Environ. Health Persp. 5:67-79. Newton, M., and L.A. Norris (1968). Herbicide residues in blacktail deer from forests treated with 2,4,5-T and atrazine. Proc. West. Soc. Weed Sci. 32-34. Nielsen, K., B. Kaempe, and J. Jenscn-Holm (1965). Fatal poisoning in man by 2,4-dichlorophenoxyacetic acid (2,4-D): Determination of the agent in forensic materials. Acta Pharmacol, et Toxicol. 22: 224-234. Niwa, A., K. Kuiqaki, and D.K. Nebert (1975). Induction of aryl hydro carbon hydroxylase activity in various cell cultures by 2,3,7,8tetrnchlorodibcn:o-p-dioxin. Molec. Pharmacol. 11:399-408. 105 OOOC1S4 2 8000 palmer, J.S. (1965). Chronic toxicity of 2,4-D alknnolamine salts to cattle. Jour. Air.. Vet. Med. Assoc. 143:398-399. Palmer, J.S., and R.D. .Radeleff (1964). The toxicologic effects of cer tain fungicides and herbicides on sheep and cattle. Ann. N.Y. Acad. Sci. 111:729-736. Palmer, J.S., D.E. Clark, and L.M. Hunt (1964). Toxicologic effects of silvex on yearling cattle. J. Amer. Vet. Med. Assoc. 144:750-755. Palmer-Jones, T. (1964). Effect on honey bees of 2,4-D. N.Z. Agric. Res. 7:339-342. Pegg, D.G., W.R. Hewitt, K.M. McCormack, and J.B. Hook (1976). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on renal function in the rat. Toxicol. Environ. Health 2:55-65. Piper, W.N., J.Q. Rose, and P.J. Gehring (1973). Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Environ. Health Persp. 5:241-244. Piper, W.N., J.Q. Rose, M.L. Leng, and P.J. Gehring (1973). The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral adminis tration to rats and dogs. Toxicol. Appl. Pharmacol. 26:339-551. Plimmer, J.R., V.I. Klingebiel, D.G. Croshy, and A.S. Wong (1973). Photo chemistry of dibenzo-p-dioxins. ACS Advances in Chemistry Series 120:44-54. Poland, A.P., D. Smith, G. Metter, and P. Possick (1971). A health sur vey of workers in a 2,4-D and 2,4,5-T plant. Arch. Environ. Health 22:316-327. Poland, A., and E. Glover (1973a). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ. Health Persp. 5:245251. 107 0000130 Rose, J.Q., J.C. Ramsey, T.H. Wentzler, R.A. Hummel, and P.J. Gehring (1976). The fate of 2,3,7,S-tctrachlorodibcnzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36:209-226. Rowe, V.R., and T.A. Mynas (1954). 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. Jour. Vet. Res. 15: 622-629. Sanders, H.O. (1970a). Toxicities of some herbicides to six species of freshwater crustaceans. J. Water Pollut. Cont. Fed. 42:1544-1550. Sanders, H.O. (1970b). Pesticide toxicities to tadpoles of the western chorus frog Pseudacris triseriata and Fowler's toad Bufo woodhousii fowleri. Copeia 19-70:246-251. Sanders, H.O., and O.B. Cope (1966). Toxicities of several pesticides to two species of Cladocerans. Trans'. Amer. Fisheries Soc. 95:165-169. Sanders, H.O., and O.B. Cope (1968). The relative toxicities of several pesticides to naiads of three species of stoneflies. Limnol. Oceanogr. 13:112-117. Sauerhoff, M.W., W.H. Braun, G.E. Blau, and P.J. Gehring (1976). The dosedependent pharmacokinetic profile of 2,4.,5-trichlorophenoxy acetic acid following intravenous administration to rats. Toxicol. Appl. Pharmacol. 36:491-501. Sauerhoff, M.W., K.H. Braun, and J.E. LeBeau (1977a). Dose-dcpcndcnt pharmacokinetic profile of silvex following intravenous administration in rats. Jour. Toxicol. Environ. Health 2:605-61^. Sauerhoff, M.W., K.B. Chenoweth, ILL. Gordon, W.H. Braun, G.E. Blau, and P.J. Gehring (1977b). The fate of silvex following oral administra- 109 000lS8 Sjoden, P-0., and U. Soderbcrg (1972). Sex dependent effects of prenatal 2.4.5-trichlorophcnoxy acetic acid on rats open-field behavior. Physiology and Behavior 9:357-360. Smith, F.A., B.A. Schwetz, and K.D. Nitschke (1976). Teratogenicity of 2.3.7.5- tetrachlcrodibenzo-p-dioxin in CF-1 mice. Toxicol. Appl. Pharmacol. 58:517-523. Somers, J., E.T. Moran, Jr., B.S. Reinhart, and G.R. Stephenson (1973). Effect of external application of pesticides to the fertile egg on hatching success and early chick performance. 1. Pre-incubation spraying with DDT and commercial mixtures of 2,4-D: Picloram and 2.4- D:2,4,5-T. Bull. Environ. Contam. Toxicol. 11:33-38. Somers, J., E.T. Moran, Jr., and B.S. Reinhart (1974). Effect of exter nal application of pesticides to the fertile egg on hatching success and early chick performance. 2. Commercial-herbicide mixtures of 2.4-D with picloram or 2,4,5-T using the pheasant. Bull. Environ. Contam. Toxicol. 11:339-342. Sparschu, G.L., F.L. Dunn, R.W. Lisowe, and V.K. Rowe (1971). Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid on fetal development in the rat. Food Cosmet. Toxicol. 9:527-530. Sparschu, G.L., F.L. Dunn, and V.K. Rowe (1970). Study of the terato genicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Food Cosmet. Toxicol. 9:405-412. ----- Stehl, R.H., and L.L. Lamparski (1977). 4 ---------Combustion of several 2,4,5- trichlorophenoxy compounds: Formation of 2,3,7,8-tetrachlorodibenzo- p-dioxin. Science 197:1008-1009. St. John, L.E.,/Jr., D.G. Wagner, and D.J. Lisk (1964). Fate of atrazine, kuron, silvex, and 2,4,5-T in the dairy cow. J. Dairy Sci. 47:1267- ( ( .I * g o.A/. _ 0Q0S190 /L<jC-Lui+. .ly. L u no. Jiiy. / ` C 8 Z 8000 A\0 Vos, G . J . , J . A . Moore, and J . G . Z i n k l (1973). Effect of 2 ,3 ,7 ,8 -te tra chlorodibcnto-p-dioxin on the immune system of laboratory animals. Environ. Health Persp. 5:149-162. Vos, G . J . , J . A . Moore, and J . G . Zinkl (1974). Toxicity of 2 ,3 ,7 ,8 -tetrachloridibcnco-p-dioxin (TCDD) in C57B1/6 nice. Toxicol. Appl. Pharmacol. 29:229-241. Vos, J.G., and J.A. Moore (1974). Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin. Int. Arch. Allergy Appl. Immunol. 47:777-794. i jf X /itZCatt m t , . ( ) . F~m~Ct ef* 7 .S -- w-- Ward, C.T. (1976). F f r li " ? thff d^ rn ^riffn of ? . .~)7J 8 tr-traThlnm-- t * ty? xfr a ***^*LeJ. a . f - + ^ \' jf dibenzo-p-dioxin (TCDD) in lake wa-cex-and sediaeft^g. M.S. Thesis, '/M - University of Wisconsin, Madison, Wisconsin. Weiss, S.V., and W.H. Beckert (1975). Herbicide effects on cultured & animal cells. Jour. Cell. Biol. 67:451a. White, A.W., Jr., L.E. Asmussen, E.W. Hauser, and J.W. Turnbull (1976). Loss of 2,4-D in runoff from plots receiving simulated rainfall and from a small agricultural watershed. J. Environ. Qual. 5:487-490. Whitehead, C.C., and R.J. Pettigrew (1972). The subacute toxicity of 2j4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid to chicks. Toxicol. Appl. Pharmacol. 21:348-354. Whitehead, C.C. (1973). Growth depression of broilers fed on low levels of 2,4-dichlorophenoxyacetic acid. Br. Poult. Sci. 14:425-427. Wiersma, G.B., H. Tai, and P.F. Sand (1972). Pesticide residue levels in sails, FY 1969. National soils monitoring program. Pesticide Monit. Jour. 6:194-228. Wiese, A.F., and R.G. Davis (1964). Herbicides movement in soil with various amounts of water. Weeds 12:101-103. 113 C0CV97 0 ink 1, J.G., J.G. Vos, J.A. Moore, and B.N. Gupta (1973). Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibonzo-p-dioxin in laboratory animals. Environ. Health Persp. 5:111-118. % r>. o Q OD cQn0 r / 115 0GCCI3! pO^OGOS 287 This issue is no nore clouded by those viewpoints than is any other del ate over c:.c::.icaI us.-.ge, ar.d wc are best served by dealing with the problem without those extremes. Without. question, rite issue of phenoxy herbicid^/^usc hns evolved to concerns ab^ut the cent.rair.ant TC!d> in 2,4,;>-T and s ilv e x . Though 2,4-i) has no such impurity, i t has become tarred with the sane brush, and i t is net unrealistic to devote more space to the contaminant than any o f the primary chemicals. Because of the extremely low environmental levels and enormous in trinsic toxicity of TCDD, the most critical technical question about that chemical centers on analytical methodology. There are few labora tories with the sensitivity to deal with concentrations on the order of 10 opt or less, and there is real disagreement about reliability of detec tion or measurement at such low levels. When the data are considered, some samples in which TCDD was undetectable had high detection limits, some areas with no spray history were found to have measurable TCDD. Also, many samples of similar origin differed widely. Nonetheless, the ?data appear to be telling us clearly that some TCDD is present in some segments of the environment, that the amounts are uncertain, and that existing residue information must be augmented if we are to understand the behavior of TCDD. With any effort presently imaginable, however, it will probably not be possible to directly monitor the amount of TCDD in the physical environment. A proper analysis of hazard requires consideration of chemical be havior and ambient levels of the potential intoxicant, but in the case of TCDD wc must jely on indirect assessment. The extent of uptake in 7421 117 c r 0 ' 196 D O W U 0 8 283 The most d i f f i c u l t problem of the several that are a part of the t:i<V of assert'.or.t i^, af.a.ip., part of the decision pattern for any cl'.e:.:ical, the risk-benefit analysis. Risk benefit analysis is an excellent basis for cor.vorsnt ion bat nearly useless in decision making. In the present case.there seems to be a general conviction that herbicide appli cation is a highly useful tool in forest production. At the same tine, estimates of differences in actual relative costs of tree production or retail materials, either near-term or long-term, are d i f f i c u l t to find. Technically, economic benefit should be an accessible fa c to r , but i t seems elusive. Risk, on the other hand, is not quantifiable unless major and obvious effects occur; such situations make the decisions for us. If we are able to show any human illness resulting from environmental practices of chemi cals, almost certainly they will be disqualified. An exception may be in effects which are certainly reversible. As a society we have yet to learn a usable method of determining whether, or how much, human injury can be considered acceptable. The assessments of hazard in this report are therefore opinions by one person about the potential for human harm resulting from distribution of the chemicals at minimum levels that will achieve the forestry objec tive. This document should be taken as a working base enabling incor poration of other opinions, and it should be subject to periodic updating, based on new findings, existing papers inadvertently missed in review, and existing papers which may become more pertinent in the light of other new findings. ,/ j . *. 119 0G0 0i experiments^-.; nor produced a "no ohscrvc-d e ffe c t " dose because the detailed analyses available only through interim s a crific e have not been ir-ulc in pri-.ates. The group of monkeys studied in Allen's laboratories fir s t showed clin ica l syv-ptor.s after three months of exposure to about J.iil TCI P/ky/Viny, or a total dose of somewhat less than 1 pp/kp, but c O cC GD CD H* detected chanr.cs at the more tl'.an 10-fold lower dose found ineffective in the rat and guinea pig. The existing literature also does not provide access to a useful single dose no observable effect level. There is no useful data that relates specifically to human effects. It therefore seems reasonable to accept 0.06 pg/kg as a subchronic total dose over a short tern, say, one year below which r.o effect can be detected. From this' point it follows that some attempt must be made to judge potential human exposure levels. It is clearly impossible to directly evaluate acquisition by surface contact with treated foliage. Direct analytical methods for TCDD are probably more sensitive than for any other organic compound, but measurement of foliar distribution can not be made at field application levels. In my opinion significant TCDD exposure by inhalation is highly im probable. An intake of 0.02 pg of TCDD would require inhalation of one g 2,4,5-T containing 0.02 ppm TCDD, along with 10 g of diluent at usual dilution rates. The volume of air in which the spray is distributed is very large. Such an intake for a 50 kg person would constitute 1/150 of the assumed no observable effect dose. The potential for inhalation of TCDD formed during combustion of 2,4,5-T is infinitely less, because of the enormous dilution in air. 121 0000200 assumption of perfect retention of TCl'D or its effect. In point of fact, the high TC!'U concentrations arc in liver and fat and such a hypo thetical whole ar.ir.al concentration would not be approached. A slightly different approach by Hr. George Streisinger makes as cO GC CO CO sumptions about fat concentration and the percentage of fat in, as an n. ft ^ `yarple, rroun.l rent. He has concluded that cither 78 or 4OS (depending ,'* /)X Uri-L&vS'*/-{j**C >< /O O - /ooo y on different no-effect assumptions) ^half-poi'ind trials would exceed the "ft**. dangesbarrier. Dr. Streisinger included a 100/1 safety factor, however, and the specified number of meals would bring a person to within l/100th -t/u asrrt*6C<-~r of a possibly hnaurnm.fnuml xiinitLraik/veU.. -- ~~u* -- ` > . 2,4,5-T is applied on a given forest area only 1-3 times in a timber growth cycle. iVhile it is possible for a deer to acquire significant residues in a single year*, the likelihood of a long-term continuous ex posure of deer would require migration among sprayed plots, or repeated treatment of a single area, and is therefore remote. Even range land is not usually treated annually. The significance of TCPD in mothers milk is considerably greater than S consumption in meat. The analyses by Dr. Meselson's group indicate the if* possibility that TCDD on the order of 1-2 ppt may be present in human ^ milk. Dr. Meselson has been careful to state that while he has great confidence in the individual analyses, they are cise-to detection limits, and the data really asks rather than answers the question. /I For the sake of discussion, a sustained output of 1 ppt in human milk ~ could provide an infant dose approaching the no observed effect level. Assuming a liter of milk consumed daily at 100 days, total intake would 74 2 4 be 0.1 yg. If, the average weight of the infant were 6 kg, the 100 day 0 0 G J 3 2 0 2 I /vi a ft a/'*it>itA. K t / ff a t M iy o e l/ / til O.f T cl>> -k. A+s. +* ett^C J-.,\ 1,1r Jot Jf 6 J Z jl ******* ( n o A .* < . TCf ,/> f*.? = * * '.i f* r DUW UUOfaj .295 .'.12 ppt at fiO-'.iQ day?, exposure of a nursing infant will therefore be :.ir.lv.nl. It appear? to me therefore that the potential for harmful exposure / k'\ infants through human :;:iIk should he negligible. This route is none- / thclcis the .-.os? probable of the several potential means of contact, and .'.rands a t'-.crrr:;!'. analysis of human milk in exposed populations. 4 . The data on TCDD carcinogenic potential suggest that it is not a carcinogen. It has been suggested that TCDD is a promoter of carcinogenic activity on the basis of a wide variety of tumor types developed over a long exposure period by the Wisconsin group. The recently completed Dow / Chemical Co. study indicates that tumor incidence changed only at doses that were lethal to many animals over the two-year test period. The data base exists for one and probably several human epidemiological studies which could answer the question of influence on human cancer incidence. A recommendation is made later for such studies. j- Of the toxicological data evaluated, perhaps the most disturbing is . fi t- u that of Allen et al. (1977) suggesting that the lethal dose in monkeys is similar whether the agent is administered over a short period or over t\r *several months. Time independence of dosage has never been demonstrated \J.* * I .J ^ for a chemical. If it truly exists for TCDD, there is implication that the compound leaves permanent damage, even though it has .departed from the body. In this situation, continual low level impact would cause gradually accumulating injury until clinical illness would prevail. In that sense, the idea of a no-effect level would not be applicable, and no exposure would be permissible. As with radiation, however, a small intake is possible without endangering health over a lifetime. In the case of TCDD this amount is not known, but it is finite. In view of a 125 0000204 M'y ariosi ifnr:ircl A - S " ''-rT--2 , ,5-T Tbe acute toxicity of 2,-1,5-T is quite low and in itself is not a factor in the environment:1.1 hazard potential of the herbicide. This seg ment is concerned only with 2,-i,5-T; the implications of the dioxin con- tar,inr.nt have beer, considered earlier. A wide variety of specific toxic chmes have been found in experiments with acute or short-torn repeated administration of high doses of 2,4,5-T. 2,4,5-T is teratogenic, but again, only at high doses. The dose response to 2,4,3-T is suciy^i large fraction of the maternal lethal dose is required to produce birth defects. There are substantial differences in effective teratogenic doses ar.o`ng strains of mice. The.lowest effective dose is still substantial, however, and the kind of"*teratogenic response remains the same. The dif ferences among the genetically very specific mouse strains do raise the possibility of high individual sensitivity in the heterogeneous human population. This question applies to any potential effect in humans by any chemical and really constitutes a common social question that we have not learned to handle. The excretion of 2,4,5-T by mammals is rapid, with relatively little conversion to other compounds. Initial residues after application are high enough to possibly cause some temporary slight tissue deposition of 2,4,5-T in tissues of grazing animals, if sustained for a period of more than two weeks, but environmental degradation of 2,4,5-T is rapid and the chemical does not migrate extensively, once deposited, ror these reasons and because general toxic responses to the herbicide only occur at high doses, I do not consider that 2,4,5-T as used properly in forestry proce,/ dures represents a general toxic or teratogenic hazard to the human popu lation. 127 0000206 DOWOOOS 299 The reproductive effects of 2,4-P do not appear until doses approach ing. the lethal level are reached, and there seems to be no reason for concern in this province. As a potential carcinogen, 2,4-D has had very little study. The only evaluation of which I am aware was negative, in one species, which indicates that the compound is not highly carcinogenic. This finding of course can give no confidence about low level carcinogenicity. Sev eral studies of mutagenic potential have been negative, adding some confidence. Perhaps the key information lies in the rapid excretion of intact 2,4-D in the urine. This process is rapid and relatively complete. In some species a portion of the compound is converted to other forms for excretion, but the proces.s is rapid. Rapid excretion probably is the factor that prevents 2,4-D or its products from reaching the detectable margin in milk after field exposure. It' is possible experimentally to overload an animal to the extent that the material will appear in milk. Because of its short persistence in the environment and the absence of toxic responses at any doses that might be found in the field, I be lieve the usejt of 2,4-D in practices presently considered standard is not hazardous. There are, as with any chemical, some open questions, which are addressed in the section on recommendations. These relate primarily to epidemiological needs and clinical surveillance. Hazard Assessment-- Silvex The general considerations applicable to 2,4,5-T may also be applied to silvex. There are some minor differences between the compounds, but the ranges of toxicity, the manner m biological disposition, and the 129 0000208 DOV/0008 301 A jVo ^ i r < t^ <> > RrCGT-tr-KPATIONS OF STEPS WHICH SHOULD BP. CONSIDERED TO ASSWRP. THAT EXPOSURE TO TCPD IS MINIMIZED OR PRr.VT.NTPD I have stated my opinion that 2,l,5-T can be used safely, with cer tain additional safeguards. I believe that opinion should be reviewed as new data emerge/. A number of clinical questions now being asked have accessible answers, and these mist be obtained. In addition, the toxic nature of TCDI) and our inability to measure it satisfactorily in the environment dictate^ a very conservative attitude about its distribu tion. I sec no inconsistency in accepting its presence in small quanti ties and recommending unique steps to prevent those quantities from reaching people. To assure public protection and a more compatible relation with the public, I suggest the following considerations in designing a spray pro gram. 1. With the consideration of health hazard, there is a political reality that needs more attention. Every treatment operation must be designed to prevent intrusion of spray onto premises not under control of the agency or firm using the chemical, as a matter of principle. I would not be surprised if Forest Service policy includes that concept, but I would also be surprised if its application is somewhat less than perfect. Personal rights are becoming more and more clearly defined, and it seems to be time to decide where such rights begin and end, and take a visible public position on the issue. 2. As a means of preventing any involuntary exposure, there should be assurances that every residence is identified, with any water sources that may be in ,the Forest. Non-legal residents should also be identified. 131 0000210 u o W > V 0 8 303 in ry mind that people complair.ipr. of physical injury arc in fact affected. I an s'f.eprica 1 that the application of herbicide causes these effects in more than a few incidents, but it is possible. Of more importance, there ray he some other public health hacard operant in the area which causes the syrrt-:;ns. It is oven possible that other illness is brought to the attention of the victims by their immediate concern about improper her bicide intrusion or. their private property. 2. The opinion of Van Miller et al. (1977) that TCDD is a promoter A $ ry of carcinogenic effect, by other compounds seems reasonable according to their evidence. If the forest application of 2,4,5-T containing TCDD is inserting effective levels of TCPD into the human environment, epidemio logical evaluation should disclose higher than normal incidence of various f> At forms of cancer in 2,4,5-T use areas. Lane County, Oregon, has a very 3? effective tumor registry program, with virtually all pathology or human I y jj /r ' cancer examined bv a single consortium of specialists. These data should ^ -J* provide evidence of any existing difference in cancer patterns from other areas. A more definitive study should be possible in rangeland or rice- growing areas where the herbicide is used annually. Epidemiological studies such as these may settle the persistent question of cancer latency, because on an area basis, the pattern of past use of herbicides should be reasonably accessible. 3. A thorough health surveillance should be made of all herbicide applicators and others with industrial or agricultural exposure to 2,4,S-T or silvex. Cytogenetic evaluation through several seasons of the year should be an integral part of the study. Design of the medical components of the survey should be by a nationally constituted panel. 133 0000212 VIO LANO A N A LY T IC A L LABORATORIES W. B. -Crummett, 574 E. L. Garfield, 607 M. 3. Chenoweth, 607 T O L. P`. McCarty, 17 01 M. L. Leng, 9008 ccn'5,'2z y ~ :/1 . z \ r ^Cv z . D . AM. TO r . R0 NOT COPY RESTRICTED REPORT, COPIES MAY BE OBTAINED FROM E.L. Garfield, Medical Dept, 607 or R.H. Stehl, 574 Building written recruest only. D O W CHEMCAL U.S.A. AL K L'M ^ P AL-5C439 RESTRICTED Acer, n u m b e r U S FUNCTION 8004 L A S . NO, P R O B L E M NUMBER DATE June 16 , 1978 A PERSONNEL INTRODUCTION Human blood samples were submitted by Bud Garfield, Medical / Department, for a determination of phenoxy herbicides - 2,4dichlorcphenoxyacetic acid (2,4-D), 2-(2,4,5-trichlorophenoxy) propionic acid (Silvex), and 2,4,5-trichlorophenoxvacetic acid (2,4,5-T). The samples were obtained from personnel working at the 2,4,5-T production facility. a_ a -3 EXPERIMENTAL The samples were prepared by extraction, hydrolysis, mthyl ation with diazomethane, and silica gel column clean-up prior to analysis by gas chromatography with electron capture detection. The experimental procedure used is detailed in report I1L-AL 78-50252. The silica gel column clean-up was changed slightly. The column used was packed with 1.5 gram dry silica gel (^ll cm x 6 mm i.d., glass). The column was eluted with 60/40 methylene chloride/hexane. The first 2 ml of eluent were collected and discarded (waste fraction). The next 13 ml of eluent were collected for analysis. Then an additional two ml were collected to check for recoveries (extra fraction). The collected fractions were evaporated to dryness. Prior to analysis, the sample residue was dissolved in 1 ml benzene. The waste fraction and extra fraction were also analyzed by dissolving the residue in 200 Pi benzene. Validation data for this procedure are also obtained in ML-AL 78-50252. '3 SIGNATURE 'I A TM. L. Langhors' 0000778 RHONE 6-6207 bu ilo in c 574 fftftO A T S FIN ISH E S 1HOURS (PRHEOSUNLETSS REVIEWS BY 6/14/73 16 |. June 16, 1S78 -2- ML-AL 78-50489 RESULTS AND DISCUSSION Results are detailed in Table I. Figure 1 shows a typical standard chromatogram and chromatographic conditions used. Figure 2 shows a typical chromatogram for a blood sample from an employee at the 2,4,5-T plant. Figure 3 shows the analyzed waste fraction and extra fraction. Figure 4 shows a reagent blank. REFERENCES 1. Langhorst, M. L., "Determination of Phenoxy Herbicides in Human Urine and Blood Samples - (Validation Datal", . `ML-AL 78-50252. . 2. Langhorst, M. L., "Determination of Phenoxy Herbicides in Human Urine and Blood Samples" (Restricted Report), ML-AL 78-50405. 3. Langhorst, M. L., "Determination of Phenoxy Herbicides in Goat Tissues", ML-AL 78-50080. jdc 2 0000779 June 16, 1978 -3- ML-AL 78-50489 TABLE I PHENOXY HERBICIDES IN BLOOD FROM 2,4,5-T PLANT PERSONNEL Sample CONCENTRATION FOUND (ng/gm blood) 2,4-D Silvex 2,4,5-T CO CO 028736-75638 028737-75631 028996-J. Schaefer Reagent Blank ND (3) ND (3) ND (3) ND (3) 1.5 ND (0.5) ND (0.5) ND (0.5) 1.2 1.8 ND (1) Notes : 1. N.D. = not detectable with the minimum detection limit shown in parenthesis in ng/gm blood. 2. Minimum detection limits (MDL's) by GC-EC are: Compound 2,4-D Silvex 2,4,5-T M.D.L. (ng/ml) 17 2.3 4.7 M.D.L. (ng/gm blood) 3 0.5 1. (assuming 5 gram blood sample) 3. Any phenoxy herbicides present in the samples as esters are hydrolyzed to acids and methylated for analysis as methyl esters. Results are reported as phenoxy herbicide acids, although the compounds may have been present in the original sample as esters or acids. 0000780 F1&UZ5 I TYPICAL STANPAR& " CM R A M A T O R A M Operator' Date 6-I 4- -7R COLUMN lin g * Mating S P - 2 4 Q I Support S u p e lfo ^ r f__________ _ M^Lr0o/l2c TEMP: Cat IL _ j L Z _ --C final___________ C Rate_________ClimsL DL. 3 SO C.lnj. 2 ) 0 . C O RRIS OS JUJuaaoa. Raie___ 2Q_ jnL/niiL Pressarsi: nisi. ___Outlet____ Hydrogen. ml.ftma.ir_ DPTSTOP. Eliit*TMfiyihirft. NL6*3., jna. jcLjniii. ____toits Sryreaqw Rata__ jnL/mia. Spas- 8 K I O_______ Ret- range I______________ mr. SAMPLE STANDA gP V ? ' Sae . 2 id .__________ Soirent 'ntxant.Ai^zunt, Cents.J&> --------- 0000731 II It I1I DOW 4777 F i w e a. T Y P IC A L CHROMATOGRAM B l O D S A M P L E F R O M PLANT M PitfY6E &c. coNPrn^MS Sawt CL5 Baure* I. IGUtes 3 DOW04778Q IO ln 'W O / b * V./AST& F A C T IO N A n d e X n Z A FfLACPOM f=^M 'SiUCA GeL. CLAN-Vp w G c COUPtnCN-S S A As FiGUHB 1. T S i i l 'O i N O 0 rl&UfiS 4 RgB<r6>lT &LANK. G r .C . C N l p i n o N i S A M S Ai plGrUAS A ! 0000784 \ IL 3 7438 n -V T CLiir ' i OCCUPATIONAL EXPOSURE MEASUREMENT OF AN AERIAL SPRAY CREW TO A MIXED SPRAY OF 2,4-D AND 2,4,5-T by K. Yoshida, Senior Research Scientist K. Wallace, Junior Research Scientist Physics Division Q r. v '/ n U 1 1A '-.l \> Z V-* ^ J i Lj\i . .i"*i i V*` July 1373 Mpn lioil 39 0003543 9/err?30M oo .1 OCCUPATIONAL EXPOSURE MEASUREMENT OF AN AERIAL SPRAY CREW TO A MIXED SPRAY OF 2,4-D AND 2.A.5-T by K. Yoshida, Senior Research Scientist K. Wallace, Junior Research Scientist Physics Division July 1978 P 78-5 7440 0003544 ozccaooMon PREFACE This investigation was carried out in response to an urgent request from, Dow Chemical (Canada) Ltd. at Sarnia, Ontario, for such information particularly as it pertained to 2,A,5"T. A research contract with Dow was signed, and the completion of this report by SRC for them is the culmination of the present contractual arrangement. The report itself must be considered of a preliminary nature as the time scale did not permit a full meteorological analysis of the airflow and drift characteristics to be undertaken. J. Maybank, Head Physics Division 7 003545 OCCUPATIONAL EXPOSURE MEASUREMENT OF AN AERIAL SPRAY CREW TO A MIXED SPRAY OF 2 , h -D and 2,lf,5-T K. Yoshida and K. Wallace INTRODUCTION Aerial spray application of pesticide accounts for only 10?i of ' the total spray operations in Saskatchewan, but it does include large acreages of bush and community pasture spraying. For these a mixture of 2,^-D and 2,4,5-T is commonly used. The operation from which exposure samples were collected was by a local aerial applicator who was well acquainted with the topography of the pasture and experienced in aerial spraying. The two flagmen were beginners, locally hired. While it is sometimes difficult to pursuade such crews to cooperate in exposure sampling, in the present case they agreed. The entire operation followed routine procedures commonly practised by average aerial applicators, with the aircraft and other equipment being used in an adequately maintained condition. The sampling team tried to avoid disturbing the spray operation so the exposure samples collected should be unbiased by sampling procedure. Standardized methods of sampling and analysis were used (see Appendix l). The results obtained through analysis are presented, without interpretation, for the absorption of active ingredient by the human body. O \ CC CO 05 Si 03S46 / -2- DESCR!P71ON OF SPRAY APPLICATION 1) Aircraft Two Cessna Agtruck aircraft were used, one a recent model, the other about 10 years old. Nozzle settings of these two seemed adequate for 3GPA application, except that on the older plane the straight back nozzle orientation under the belly caused excessive deposition of spray i to the centre rear. On the newer aircraft the belly nozzles were in the straight down position. There was no noticeable leak of spray liquid except occasionally while refilling. Details are given in Table 1. 2) Spray formulation The application rate set by the owner of the pasture was 24 oz/acre (acid equivalent) of 2,4-0 butyl ester and 8 oz/acre (a.e.) of 2,4,5-T iso-octyl ester. For improved deposition, 15 oz/500 gal. of adjuvant (Amway) and 0.5 SPA of diesel oil were mixed with the water which was pumped from a local slough. 3) Field conditions The area sprayed was well developed pasture 'with shrubs scattered over a gently rolling terrain. The size of pasture sprayed tor the day was approximately 1,3C0 acre, being roughly 2 miles in the east to west direction. The estimated number of swaths from the geometry of the pasture would be 80. Each aircraft landed for refilling around 24 tires. ^ --' : *^0 v.; , 1^ OCO3 5 4 7 6 /CCOOOMOfl -3- k) General weather Conditions The operation lasted from 5:30 p.m. to 10:30 p.m. during which time the air temperature remained near 15C and the relative humidity around ^0-^5%. The wind was from the northwest, initially varying between 8 and 15 km/h, and decreasing toward sunset. k The operation was controlled by a field supervisor who could decide start or stop spray operation due to the weather conditions. He also supervised the action of two flagmen in the pasture, and checked the deposition pattern of spray chemical occasionally. \ 0003548 -4- 1 DESCRIPTICN Or EXPOSURE SUBJECTS 1) Pilot The pilot A (with recent model of aircraft) has been doing aerial spray for the past 20 years and seemed to be an experienced person. He agreed to wear a Gas badge and film badge on his lapel j throughout the operation. He was spraying two days before the sampling day at another field, but did not spray 2,4,5~T. Pilot B has been doing aerial spraying for the past 10 years and agreed to wear gas badge. Due to nis-orientation of belly nozzles, pilot B might have been exposed to drifting chemical from the belly of aircraft (Table 2). ^ O C7-; CC ^ C CO 2) Loading and Mixing Crew The loading attendant has several year's experience in aerial spray operation. He refused to wear the charcoal tube sampler or to provide urine specimen, but did agree to wear the badge. His duty was to open the 45 gal drums (2,4-D or 2,4,5-T concentrate) and by a hand pump to transfer the concentrate to a mixing tank, then to proceed in mixing and loading the aircraft. While the aircraft was being loaded, he stood on the upwind side of the liquid hose. On disconnecting the hose from the aircraft, there was usually a small amount of spillage. 3) Water truck operator This person stayed with the loading attendant except when he was loading the aircraft or when she drove the water truck to the nearby slough. She also kept the records of chemical consumption and aircraft 7445 refills. She agreed to have one of the charcoal tubes in her truck and to wear gas badge and film badge. No urine sample was collected 0 0 3549 " 3" 0 Loading Assistant This person carried a charcoal tube at his lapel position, gas badge and film badge and almost every time followed the loading attendant and so likely had an equivalent degree of exposure. He had not been exposed to any kind of herbicide spray for the past one year. Urine samples were collected (Table 2). ^ 5) Flagmen Flagman A was locally-hired temporary help (a university student) inexperienced in aerial spray operation. He was exposed to other herbicidal operations ^0 hours before the date of sampling (for 6 hours operation). He agreed to provide urine samples, and to wear gas badge, film badge and charcoal tube. Flagman B was also a locally hired worker with some pilot training. He v/as also exposed in the same way as flagman A in the previous operation, however he has no previous experience of aerial spray operation before he came to Canada -from Europe (Table 2). From the observation of the pasture (field) supervisor who drove through the field under spraying, these two flagmen were directly sprayed at the swath ends before retreating to the adjacent position, due to their inexperience in field operation. 6) Field Supervisor This person has been supervising bush spray operations each year for the past 5 years. He stayed most of the time near the mixing station except for occasional driving to the field to observe the operation. He agreed to wear gas and film badges. No urine sample was c o 11ected (Table 2). \ G G o CCO Co C3 0003550 -6- 7) Observers Two observers were always stationed upwind of the loading site and helped to maintain the landing strip. They never touched spray liquid and wore gas and film badges (Table 2). d C o Cc C~GJ C-T I 7447 0003551 -7- % SAMPLING AND ANALYTICAL M.ETHCDS 1) Dermal Exposure Film badges were worn on clothing lapels. A 47 mm glass fiber filter (Gelman type A-E) was wetted with 1.0 ml of ethyleneglycol by pipetting and then mounted in a rectangular-based culture dish (MI 11ipor PDi5Cn700). The filter paper was retained in the cell by an o-ring.l The purpose of wetting the surface of the filter paper is to increase the retention of chemical. The film badges were kept with lids in an air-tight container with the lids being removed prior to the beginning of the exposure period. Afterwards the lids were replaced and the air-tight badges were transported in a cold box to the lab. ci ^ O (-- ; C^ CO CO 2) Respiratory Exposure Standard size charcoal tubes (Environmental Compliance Corp. Cat. No. 226-01, NIOSH approved) were used by the two flagmen, the loading assistant, the mixer (water carrier), and by one of the pilots. Tubes were aspirated at 1.7 L.P.M..by personal sampler pumps (MSA model G ) , and attached at the lapel position which is within the breathing zone (60 cm radium about the nose). The flow rate of each pump was registered before and after the sampling period to obtain the mean value of flow rate. Gas badge organic dosimeters (Abcor. Inc.) were worn by every member of the crew and by the supervisor and observers. Badges were used strictly according to the instructions supplied by the manufacturer. The expiring date of the activated carbon collection element set by the manufacturer was 10 days after the date of use (Abcor. Inc. 1977). 000 -8- 3) General Background The degree cf air contamination at the mixing station was monitored fay using two sets of midget impingers at downwind side of the station 10 meters from the edge of chemical wagon. The inlet of impingers was maintained at 1 meter above the ground level. The impingers were filled with 15 ml of ethyleneglycol and aspirated at 2.8 L.P.M. forf the entire spray operation. b ) Urine samples Immediately after the spray operation started, the two flagmen and one loading assistant started collecting uring in a plastic bottle. Two flagmen completed 2 b hour collection with nighttime passing uncollected. At exactly 2 b hours after the beginning of spray operation, the last passing was collected. The loading assistant collected the entire passings of urine for the next 24 hours, and then to 43 hours, and one passing at 72 hours. 5) Chemical Analysis The draft shield and charcoal element of the gas badge as w e l 1 as / the filter badge and charcoal tube were placed in 20 ml or methanol for desorption in a teflon stoppered test tube. The samples were poured through a pad of glass wool to remove any traces of charcoal, then evaporated down to 1 ml in a round bottom flask using a rotary evaporator. Samples were then methylated using 14;; BF^-CH^OH (5 ml) at 70C in a water bath for twenty minutes; after cooling, 15 ml of saturated sodium chloride and 15 ml of hexane were added and the sample shaken. A suitable aliquot (usually 2-4 ul of the hexane) was injected into CC Co CO CO 744 9 003553 -3- For the urine samples, 100 ml of urine were passed through a column of XAD-2 resin for cleanup prior to methylation. The urine was acidified and elutriated with 50 ml of 203 acetonitrile in 0.1 M sodium bicarbonate and extracted with two 25 ml portions of . di-ethyl ether. The ether was dried with ^ 3 0 ^ (anhydrous) and evaporated to dryness then methylated with SF -CH,0H for 20 min. at 70C. Hexane and saturated sodium chloride were added and 2 or 3 ul^ were ingected for quantitation of 2,^-D methyl ester and 2,ii,5-T methyl ester against standards. To confirm the presence of these two esters the samples of methyl esters were butylated and the corresponding butyl esters of 2,4-D and 2,^,5-T were found and compared to standards. The procedure is given in greater detail in appendix I. CO Co CJ! C99OM0n 0003554 -10- RESULTS 1) Dermal Exposure From analysis of film badges (Table 3) it is obvious that the two flagmen were exposed to both droplet and vapor forms of the mixed spray 2 liquid. They are the highest in density (up to 11 mg/cm ) among the goepnanwofi crew and observers. The values found for the 2,A-D/2,k ,5-T r^Jtic are closer to 2:1 than to the nominal 3:1 of the solution mix; this may be due to preferential evaporation in the field of the higher volitile component. The loading attendant who was in most cases exposed to the vapor form of the chemicals received the next highest dosage, which averages close to the expected 3:1 ratio of the two chemicals. It would be primarily received in the form cf vapour and not spray droplets as for the flagmen. The exposures of other members of the crew and of the observers were considerably lower, at least as measured by the badges; they too would tend to be primarily vapour.(Table 3). There are two major sources of dermal exposure, one being direct contact with the spray solution and the ether being exposure to air borne droplets or vapor. The film badge cannot discriminate between vapour airborne and droplets as the source of contamination. Therefore these values must be considered as measures cf general exposure to airborne active ingredients (assuming that there was no accidental contact with the film surface by contaminated hands or clothing. (Hays, 1977). fy /tv OJ i >.J 3555 -n - 2) Respiratory Exposure nceiiooMon Both the gasbacges and the charcoal-filled tubes provided a treas ure of the inhalation amounts; values found for the individuals wearing each are given in Tables 4 and 5 respectively. It is possible to. convert the latter into resonably equivaletn concentration values but such is not generally feasible with the former. It can be seen that gas badge exposures were in the 2-4 mg;.f range for 2,4-D near the threshold limit and generally at "Trace" for 2,4,5T (i.e. below the sensitivity limit for this chemical and technique). The charcoal tube is considered to provide somewhat more reliable data, and in Table 5 more of the samples lie above the threshold limit. Again it is seen that one flagman at least received a considerably greater dosage than did the other crew members. A surprising feature is that the ratio of 2,4,5-T to 2,4-0 is close to unity, not 3:1 as in the spray mix. The reason for this is not clear, but could be due to preferent ial absorption, or greater ease of subsequent desorption of the former chemical . 3) Urine Excretion The concentration of the two active ingredients in the 24 hour excretion was up to 5-11 p.p.m. (2,4-0) and 1.16 p.p.m. (2,4,5-T) with flagman A, and roughly one half these amounts with Flagman S. Tnis is somewhat surprising inasmuch as both the dermal exposure data (Table 3) snd the inhalation amounts (Table 5)- indicated higher values in the vicinity of B than around A. It may be due to uncertainties in the collection of the urine samples and the inability to ensure, under 7452 0003556 - 12- the circumstances, total collection from each individual. The amounts of 2,4-D and 2,4,5-T found in the urine for the loading assistant were generally much lower, would be expected from his exposure data. They shewed a diminution with time out to 72 *ho'jrs as would be expected. The metabolism of 2,4-D and 2,4,5-T has been reported elsewhere. (Kutz et a l . 1977) 4) General Remarks I One of the most difficult problems encountered in the field work was how to persuade the spray crew to be volunteer exposure subjects. This is possible only once because some of them happened to be aware of the exposure problem and they tend to become suspicious on a second O o Cc CO Co attempt. They are also exposed to spray chemicals intermittently, which in turn makes the continuous sampling of urine extremely difficult. The collection and storage or extraction must be done immediately after each passing or at least immediately after every 12 hours. This was impossible with the flagmen who travel a great distance from the filling station, where sampling team was located. The degree of exposure of flagmen is highly dependent on the nature of drifting of spray solution from an aircraft. Had the spraying been done under significantly hotter conditions higher vapour ingestions would have been likely. This problem has been studied in previous years (Maybank, et al 1972). However, further study is needed to delineate the behavior of drifting spray droplets and vapor in view of exposure determinations. (Druham and Wolf, 1962, 193)* Spray application of 2,4,5-T in the Saskatchewan pasture will continue in years to come, there 5 3 fore this could be practically one of the very few of large ope ra ticns,*\i^ Q ^ North America where exposure samples for 2,4,5-T could be obtained. - 13- acknowledgments This study was conducted under a short-term contract with Dow Chemical Canada Ltd. The authors would like to acknowledge the assis tance provided by the following persons: Dr. A. E. Smith of Agriculture Canada Research Station at Regina for help*on the analytical methods for urine, Dr. F. Kelada of Occupational Health and Safety Divisi Qfl of the Saskatchewan Department of Labour for general planning of exposure study and M. Peters and J. Ross of the Physics Division of SRC for gen eral instrumentation in sampling and analysis. d Z<E C CD C= CO CO C-> CO e 7454 0003558 OOCCOQOMOfl -11- r REFERENCES AND SHORT BIBLIOGRAPHY 1) Abcor, Inc. : Grasbadge organic vapor dosimeter, use and analysis instructions. PP 15. 1977. 3) Durham, W.F. and Wolfe, H.R.: Measurement of the exposure of workers to pesticides. Bui. World Health Org. Vol. 26: 75 - 91, 1962. 2) Durham, W.F. and Wolf, H.R.: An additional note regarding measurement of the exposure of workers to pesticides. Bui. World Health Org. Vol. 29: 279 - 281, 1963. 5) Hayes, Jr. W.J.: Toxicology of pesticides, Chapter 2. General principles, dosage and other factors influencing toxicity. Williams Wilkins, 1975. 6) Lee, Jr. R.E. Ed.: Air pollution from pesticides and agricultural processes. Chapter 5-Field exposure to airborne pesticides. Chapter 6. Human health hazards of respiratory exposure to pesticides CRC press, 1976. 8) Maybank, J. Yoshida, K. Shewchuk, S.R. and Grover, R.: Spray drift behavior of aerially-applied herbicide: 1977 field trials. Sask. Research Council, Report P73 - 2. 7) Kutz, F.W. etd: Survey of pesticide residues and their metabolites in humans. In pesticide management and insecticide resistance. D.L. Watson and A.W.A. Brown Eds, Academic press. PP523 - 539, 1977- 7455 000355 Table 1 Description of Spray Operation 1) Aircraft Model: Cessna Agtruck X2 Cruising speed: 110 m.p.h. Height: 4-8 ft. Tank capacity: 200 gal Spray nozzles: D6-45 and D10-45 mixed, horizontal Estimated swath width: 55 ft. 1 2) Spray Formulation Application rate (liquid) set: 3 G.P.A. Active ingredients: 2,4-D butyl ester 128, 2,4,5"T iso octyl 112. Application rate (active) (oz/a): 2,4-D (24), 2,4,5-T (8) Adjuvants used: Amway (15 oz/500 gal.) Diesel Oil (0.5 G.P.A.) 5) Field Condi tions Area sprayed: approximately 1,300 acres Size of field: 1 mile x 2 mile Estimated number of swaths: 30 Number of aircraft refill: 24 x 2 aircrafts 4) General Condition for spraying: Moderate to good condition Air temperature: steady 15 deg. (C) Relative humidity: 40-45S Wind: NW 5"10 (m.p.h.) Sky: partly cloudy Operation period: 5:30 p.m. to 10:30 p.m. G' G G G Cc Co 05 CO 1 7458 0003560 TA3LE 2 Crew Pilot A Pilot B Loading Attendant Water truck operator Load ing Ass istant Flagman A Flagman B Field Supervisor Observer A Observer B 2 GC C 9 9 0 M0 fl Phys icai Sex Age Appearance C 1oth ing Smoking Alcohol M Hid 1*0 Fat Coverai 1 Moderate No hat M Mid Moderate to Coverai 1 Moderate>! Moderate 30 slim hat Mid Slim M 20 Covera11 mi tten no hat Heavy Heavy Low F 20 Moderate Coverai 1 No no hat Moderate Mid Moderate Fu 11 s 1eeve'No Light M AO jacket no hat M Low Slim 20 Coveral1 No no hat No M Low Slim 20 Coveral1 No no hat Heavy Mid SI im M 20 M H igh Slim 20 M H igh Slim 20 Fu1Is lee ve Moderate Mode rate jacket hat Short sleeve No sh irt no hat Moderate Short sleeve Moderate sh irt no hat Light / V O >ty 0003561 Table 3 Analysis of Dermal Exposure (film badges) Mass Exposure Subject Methyl ester form (jjg) 2 ,'i-D 2,'1,5-T Corrected for extraction eff (MS-) 2,1|-D 2,'1,5-T Load ing Attendant 56.0 19-0 7*.'1 25.5 V/ater truck 6.6 2.5 8.8 Operator 3.'* Load ing 1.2 15.0 1.6 20.0 Assis tant Flagman A 65.6 31-9 87.2 'i2 .7 F 1agman B 131.3 Field Supcrvi sor 1.0 65.6 I7't.6 0.60 1.3 87.9 0.8 Observer A Observer B 0.6 O.'i 0.75 0.19 0.8 0.5 1.0 2.5 Total mass in sample (t,g) Density in badgi jig/cm ) in ester 2,'i-D 2,'1,5-T 2,'t-D 2,1,5-T 87.8 30.0 5.08 1.73 10.'l <i.O 0.60 0.23 1.9 27.0 0.11 1.56 103.0 58.1 206.0 119.5 1.5 1.1 6.0 3 -36 11.9.1 6.91 0.08 0.06 0.9 l.'i 0.6 3.' 0.05 0.08 0.03 0.20 Correction Factor (x) 1.33 1 .3^* 1.18 1.36 0.058 0.058 CRC390M00 Z9SCOOO Table ^ Analysis of Gasbadgcs Hass Exposure subject Pi lot A Watertruck Operator Loading Assistant Methyl ester form (pg ) 2,i<-D 2.0 2.0 2.0 2 .I'.S-T T 0.3 T Corrected for extraction ett w 2,'i-D 2 2.08 2.88 T 0.57 2.08 T Flagman A 2.3 T 3.31 T Flagman D Field Superviser Observer A Correction Factor (x) 1.5 2.0 1.0 NA T T T HA 2.16 2.88 I.M l.M T T T 1.90 I Total mass in s amp 1e (ug) 2,'i-D 2, <t,5-T 3.*i0 T 3.'i0 0.78 3. T 3.91 T 2.55 3 -f<0 1.70 T T T 1. 18 1.56 * Total mass in nominal formulations, 2 --D butly ester and 2,^,5-T isocctyl ester. 0003563 ^eecmoMnn * Table 5 Analysis of Charcoal Tubes Mass Methyl ester form (jig) Total mass in sample (yg) Exposure Subject Pilot A Loading Assistant 2 ,*t-D 0.21 0. 17 Watertruck Operator 0.25 Flagman A F 1agman B Correction Factor (x) 0.25 0.60 - 2,1,5-T T o. Wi T 0.20 0.k5 - 2,i-D 0.25 0.20 0.30 0.30 0.71 1.18 2,1,5-T T 0 .19 T 0.27 0.61 1.36 Concent rat ion (pg/ni ) 2,li-D 0.57 2,i,5-T r 0.1i5 0.i3 0.67 0.68 1 .61 2.26 T 0.61 2.26 0r0o /' j 1 o1 fr Extraction efficiency is assumed to be 100%. 1 Total mass In sample In nominal forms of active ingredients. 2,^-0 butyl ester and 2 r h ,5~T iso octyl ester. ! t Air volume sampled during the total operation Is 1.7 L.P.M. x 260 = k h 2 i . ^9SCo *9 seccfnoM oo CD o ...... Hass Exposure Subject Flagman A Flaqman B 2k hr. No. 1 No. 2 Mean Mo. 1 No. 2 Mean Table 6 Analysis of Urine Excretion (Part l) Methyl ester form (pg) 2!,*(-D 2,i,5-T Total mass in 100 ml (pg) Concentration (PPM) 2,1-D 2.'i,5-T 2 ,1|-D 2 ,(,5" T m 113 360 83 -225 50 165 35 -- 652.01 133.37 1(89.63 - 97.9' - 306.02 22 1.1(2 - 59.01 1(1.31 - 6.52 4.90 5.71 3.06 2.2k 2.65 1.33 0.98 1. 16 0.59 0.il 0.50 Total Urine Vol (ml) Total mass in total urine (pg) 2,(-D 2.1|.5-T - -- - -- 830 6880 1398 - -- -- h1o 550 1(818 909 . Total mass in 100 ml is in nominal forms of ester , 2 ,i-D butyl ester , 2,1(,5-T Isoocytl ester. . Urine collected from fi rst 2-V-3 passings and final 2 i hour pass ing. I i; 0003565 9serfnoMnn M 05 sf1 t Tab le 6 Analysis of Urine Excretion (Part II) Loading Assistant Methyl es ter form (pg) Total mass in 100 ml (pg) Concentrt ion (PPM) Total Urine Time c 1lapsed 2 ,*-D 2 ,,5*T 2'i hr. No. 1 3.0 1.13 2 ,'i-0 2,4,5-T J.08 1.33 2,1<-D 2,i,5-T (ml) O.O'i 0.01 - No.2 il.o 1.7 5. Mi 2.01 0.05 0.02 - Mean - - -- 0 .0k 5 0.015 1720 *i8 hr. No. 1 2.8 0.6 3.81 0.71 0 .0k 0.007 - No. 2 3.3 0.75 h .h s 0.89 0 .0k 0.009 - (lean - - -- O.O'l 0.008 2300 72 hr. No. 1 1. k 0.6 1.90 0.71 0.02 0.007 - No. 2 1 0.6 1.90 0.71 , 0.02 0.007 - Mean - - -- 0.02 0.007 220 i Total mass in Total urine (pg) 2 ,i|-D 2.'i.5-T 26.2 17.* 0.91 - 8.7 - 1 M N> 3.5 0.32 2 2,, Urine collected as total oF k hours and the next k hours and then one pass ing at 72 hours after the be- ginning of operation. O a w -v* cm ^ cn cr_j o> /esccooHon / 96CfSOOMOn -23- APPENDIX I Determination of 2.4-D in urine 1. Scooe*24 . This method is suitable for the quantitative determination of 2 D in urine at the 0.C5 ppm level. 2. P rinci ole *^ 100 ml urine sample is cleaned up using a column of XAD-2 resin. Following elution with 20% acetonitrile in 0.1 M sodium bicarbonate and acidification of the eluate, the 2,4-D is ether extracted from the acidic solution. The ether extract is evaporated to dryness and the residue is methylated using diazomethane or boron trif1uride-methanol reagent. The 2,4-D methyl ester can then be quintitated using electron-capture gas chromatography. 3- Special Ecuioment a) Gas chromatograph, Hewlett-Packard, Tracor, etc., equipped with 3n elect ron-cap ture detector and glass columns. b) Chromatography columns, 14 mm i.d. x 400 mm long with stopcock. 4. Reagents a) Acetonitrile, benzene, n-hexane, methanol, Glass distilled, (Caledon Laboratories Ltd., Georgetown, Cnt.). b) Hydrochloric acid, sulphuric acid, sodi um bi carbonate, (reagent grade), n-butanol , (certified), (Fisher Scientific). c) Amber lite XAD-2 resin, Mal 1inckrodt, (North American Scientific Co.). d) Di-ethyl ether (Caledon Laboratories Ltd., Georgetown, Ontario). -2*1- 5. G.C. Condi11 crs " [For H-P 5713'Awith nickel-63 detector) Column . . . . . 1.5 m x 6 ran 0. d. (Amm i.d.) glass column (on column injection) packed with Ultrabond 20M {The R F R- Corp., 1 Main Street Hope, R. I. 02831.) Gas........ Argon-methane 3 5 : 5 , a t bO ml/min Temp....... Column and injector 150C for methyl ester R.T. 3-5 min i l65C for butyl ester R.T. 4.0 min Detector 300C 6. Standard Curve Prepare standard solutions of 2,4-D methyl and butyl esters in n-hexane Concentration may range from 0.1 to 1.0 ng/ul. Prepare standard curve of peak heights _vs_ concentration. 7. Analysis Place a small plug of glass wool in the bottom of the chromatography column and add 5 gm XAD-2 resin. Wash resin down onto glass woll with dis tilled water until the resin in suspended in the water. Allow resin to settle to the bottom again. Wash resin with 50 ml -of 20* acetonitrile in 0.1 M sodium bicarbonate. Rinse again with distilled water. Acidify 100 ml urine with 10 ml cone. HC1 and add this to the column. Allow urine to pass through at about 5 ml/min. Wash resin to neutrality wi th distilled water. Elute with 50 mi of 20',' acetonitrile in C. 1 M sodium bicoarcnate 2 ml/min. Collect eluate in a beaker containing 5 ml. cone. HC1. Wash column with a further 25 ml distilled water after eluate has passed through and collect with eluate. Transfer eluate and wash to a separator/ funnel and extract nesesnoMnn 7464 0003568 \-25- 1 with 2 x 50 ml oi-ethyl ether. Check that aqueous phase is still acidic and discard. Evaporate ether to dryness cn raotry evaporater. Traces of water are removed from the flask by azeotroping with equal portions of methanol and benzene. Quantitatively transfer residue to a suitable con tainer and methylate with diazomethane or boron tri f 1unde-methanol reagent. After irethy 1at io n , take up ester in 25 ml of n-hexane. Inject into gas o o '/ C o i o M o n chromatograph. 8. Confi rmation, Confirmation of 2,4-D presence is obtained by either buytlation of the residue after evaporation of the ether extract or by trans-buty1aticn of the methyl ester after GLC quantitation. In the first case, if enough urine sample is available, the column clean-up and ether extraction of a second 100 ml urine is carried out as in Section 7. Following the evaporation of the ether extract to dryness, the residue is transferred to a test tube using a small amount of di-ethyl ether. The ether is evaporated to dryness and 2 ml n-butanpl and 5 drops of concentrated sulphuric acid are added to the tube. The tube is heated in an oil bath at 100C for one hour. Add 50 ml distilled water and 25 ml n-hexane and shake well. Ory the hexane layer over sodium chloride and inject into the gas chromatograph using 2,i-D butylester as a standard. If urine sample is insufficient for a second extraction, the confirm ation may be done by trans butylatics of the methyl ester from the initial extraction after GLC. A portion of hexane (5 ml) is transferred to tube containing 2 ml of n-butancl and the hexane evaporated off using a rotary 0003569 * \J I 0 ;4G890MOn -26- evaporator. 5 drops of concentrated sulphuric acid are added and the tube heated 1 hr at 100C as before. Add 50 ml distilled water and 5 ml n-hexane and shake. Dry hexane over sodium chloride and inject into the GLC. 9. Recovery Determination Blank urine samples are spiked at 0.1 ppm with 2,t-D acid. Urine is then cleaned up and extracted as described in Section ~. Recoveries should be in excess of 90%. 10. Comments a) Recoveries from male urine samples ages 25-50 years have been found to be 95*. b) Butylation and trans-butyl at ion yields have been found to be 20-95*. c) Urine blanks, when subjected to this clean-up and extraction, gave no interfering peaks on GLC trace. d) In the procedure for confirmation of 2,4-D by butylation or trsns- butylation it may be necessary to shake the hexane layer with a second 50 ml of water to remove all the n-butanol before drying hexane for injection into the GCL. 0003570 /< /3 /V 3 JiW.j,( / I NALYTiCAL REPORT s_ ',.a n _ o analytical laboratories ,________________ M. B. Chenoweth, 607 kj. H. Saunders, 607 E. L. Garfield, 607 W. B. Crummett, 574 TO M. L. Leng, 9008 W n . o l a U n ^ l o U j L. P. McCarty 1701 & 607 DOW CHEMICAL U.S.A iA*uu NP2STRICTED-- --R--EPO--R' ___AL 73-- 50663 Add T. NUWdER SUB PUNC TlON 8004 LA8 NO. PROBLEM NUM9SA OATE July 28, 1 S 7 8 W -E DETERMINATION OF PHSNOXY HERBICIDES IN URINE AND 3LQOLT"SAMPLES ~O' FROM 2.4-5-T -p t .a m t P E R S O N N E L - IT.________________________________________________________________________ O INTRODUCTION Human urine and whole blood samples were obtained from personnel working at the 2,4,5-T production facility for a determination of 2,4-dichlorophenoxy acetic acid (2,4-D), 2- (2,4,5-trichlorophenoxy) propionic acid (Silyex) , and 2,4,5:trichlorophenoxy acetic acid (2,4,5-T). EXPERIMENTAL The samples were prepared by extraction, hydrolysis, mthylation with diazomethane, and silica gel column clean-up prior to analysis by gas chromatography^-mass spectrometry (GC-MS) . The experimental procedure used is detailed in Report ML-AL 78-50252. The silica gel column used was changed slightly. The column used was packed with 1.5 grams dry silica gel (A- 11 cm x 5 mm i.d, glass) . The column was eluted with 60/40 methylene chloride/ hexane. The first 2 ml were collected and discarded (waste fraction). The next 13 ml of eluent were collected for analysis. Then an additional two ml were collected to check for recoveries, (extra fraction). The collected fractions were evaporated to dryness. Prior to analysis, the sample residue was dissolved in 50 vl benzene. Validation data for this procedure are detailed in ML-AL 73-50252. CONFIDENTIAL - SU3JECT TO INJUNCTION D.C., E.D. Ml. 4-4-73; DOW/EPA AGREEMENT 9-79 RESULTS AND DISCUSSION Results are detailed in Table I. Figure 1 shows a typical standard ion chromatogram and GC-MS conditions. Figure 2 shews '4 typical GC-MS data for a urine sample from a 2,4,5-T plant w o r k e r _ 0 0 \j ( o o * t* Marsha L. Landhorst Data 3ook AL 1207 o. 118 p c RM v* s : 3U1LOINS OATE PINIdHCO HOURS PREhSOUnLSTOS 6-6207 574 7/13/78 37 & 7 nW4781J July 28, 1978 - 2 - AL 78-50668 Figure 3 shows typical GC-MS data for a blood sample from a 2,4,5-T plant worker. The presence of Silvex was asked to be confirmed. It was questioned whether Silvex could have an interfering com pound by GC-MS six-ion monitoring or whether Silvex or an interference could be created from 2,4,5-T during the sample preparation or analysis. To check the creation of an interference during sample preparation, a urine and blood sample were spiked with high levels (1.85 yg/gm sample) of 2,4,5-T. The sample was pro cessed through the usual procedure and analyzed. No peaks were found other than 2,4,5-T. See Figure 4. To check if the ethyl ester of 2,4,5-T might interfere with the methyl ester of Silvex, (both have parent ions by MS at m/e =282) by GC-MS. A sample of 2,4,5-T was ethylated with ethanolic KC1. The sample was analyzed by GC with electron capture detection and found to elute at a different retention time than Silvex. Therefore, it could not inter fere by GC-MS and produce a response interpreted as Silvex. In addition, to check if any ethyl ester of 2,4,5-T is formed during methylaticn, a relatively high concentration of 2,4,5-T was methylated. No peaks were detectable except the methyl ester of 2,4,5-T. This evidence is shown in Figures 5 and 6. The identify of Silvex in urine and blood samples is confirmed by retention time, ions in the mass spectra, and ion abundance ratios (282/198). / 9 */ _ Q V 000 07 G6 DOW 047814 July 28, 1978 -3- AL 78-50668 REFERENCES 1. Marsha L. Langhorst, "Determination of Phenoxy Herbicides in Human Urine and Blood Samples - (Validation Data)", ML-AL 78-50252. 2. Marsha L. Langhorst, "Determination of Phenoxy Herbicides in Human Urine and Blood Samples (RESTRICTED REPORT)" ML-AL 78-50405. 3. Marsha L. Langhorst, "Determination of Phenoxy Herbicides in Human Urine and Blood Samples from 2,4,5^-T Plant Personnel, .(RESTRICTED REPORT)" ML-AL 78-50489-.. . td <! 0 0 007 G7 July 28, 1978 AL 78-50668 TABLE I RESULTS PHENOXY HERBICIDES IN 2,4,5-T PLANT PERSONNEL Sample Concentration found (ng/gm Sample} No. Person Type 2,4-D Silvex 2,4,5-T U-l Urine 380 21 540 U-2 Urine 520 4.1 220 U-3 Urine 510 23 310 U-4 Urine 270 11 170 B-l Blood - 2.0 N.D.(l) '*8.7 B-2 Blood 8.6 N.D. (1) 5.6 B-3 Blood 9.3 N.D. (1) 7.9 B-4 Blood 3.8 N.D. (1) 2.4 C *** o C SPIKED SAMPLES FOR RECOVERIES Sample No. Type Spiked with 2,4,5-T Cone found (ng/gm) % 2,4-D Silvex 2,4,5-T Recovery U-l Urine 1850 ng/gm urine N.D. (1) N.D. (0.5) B-l Blood 1850 ng/gm blood N.D. (2) N.D.(1) 1540 1400 83 76 NOTES: N.D. = Not detected with minimum detection limits shown in parentheses. 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S3 1734. 42 263 00 230 73- . 2f - " C:jr T I f7jCTE D = 2.00 :, .i1 0 !r" :> l'J ! z. ~3i2.C0 FS I I N =233. 0 3 = 1 ;'3 F = I3 4 - 3 .;;,,0 r- .- , T* a.. r -2:- 'I ; ; ?: n "L : * r! `u / ' : 0 0 0 0 7 9 2 :: "; ;i 2 iISIIi1 .i .""t-.yA' h' * - r"' i iS 3 Fl&uftc ' R fcltN rjoN r \ h e s o f S t a n d a r d c m ^ o m a t d &r /\m s a n d 6C CoN P iTiO N S . PUe^oxT H(3R.13IClpe STANDARD - PHGN0X7 HERfllC.iP&S f i b nernvL esruRs lK|SngQMfNf Varittn 3700 6 os. Ctlirt) 0|.eralor.Wl-;..UN.6j.l<>R:r....n.ilc..Z:.:;i.:.Zfi. ....... Column No..ClI.4.^!i......Lcni;Ui...fl..fKh..........Dia...?rtKn...fl.... Coallnj;....... ........................................ Concn.....-3.%..................... Suppoit....'icpC.lu0.pE.<'.t?.......................... Mcsh.!f?9./l2.Ct.. TEMP: Col: l.iit.......1.75?................C Final.....................C Italo...............C/min. Dcl.,`iflO ....C lnj....& Q ..........C CARRIER GAS.../sJilyi).cj(*.n......Rato..... 3 0 .....................ml./mln. Pressures: lolul............................... Gullet.................................... IlyJfocnn ...................... ml./mln. Air..........................ml./mln. OETCClOUtcxYtAi 9.)........t.C ......................F.I.D............................. Scavenger............................... Rale...................................ml./mln. Scus.il>'. l.yW.lfii...............Rcc.Rjnue........... I........................mv. SAMI-1 E...A>. JoA-d rri..........................................SIic.AaMk."}.... Solvent...V'v'AIrAr.W:............... Concn.............................................. y >-.j)' O i r y <c to A (S' X o O C-i O CD 0 4 8 -i C.D rh'nU-feS -- > OJ SfANDARP * l , i S - T a s entyi. Esrei? >J' O'n I OSRil'fS'0 0 O Ocr rc VV) $ s: *I o t 0000794 n&U/gg <o C M r o h ATOG-RAMs O F 2 ^ 5 - r AFrtT. ^ g T H Y tA T 7 0 M " ^ ^ lU,N&' E-STfZ 15 F M BP S>U%\Kj(r MET7I Y LA T IO N vtfiiT 5THYU 1HH 1HH i 7S -n 7 Schweizerische Medizinische Wochenschrift 108(42), 1617-25 (1978) JOURNAL OF SWISS MEDICINE 21 OCTOBER 1978 Pathological-embryological investigations in cases of abortion related to the Seveso accident by Kelga Rehder, Laura Sanchioni, F. Cefis, and A. Gropp O O Correspondence Dr. Helga Rehder or Prof. A Gropp, Dept, for Pathology of the Medical Hochschule, Ratzeburger Allee 160, D-2400 Lbeck. Dr. Laura Sanchioni or Prof F. Cefis, Laboratorio di ricerche cliniche, anatomia ed istologie patologica, Istituti elinici di perfezionamento, Via San Barnaba 8, Milano, Italy. 'S 385330 0002518 Summary. After the explosion accident on July 10, 1976 in Seveso (Italy), material from 30 interrupted pregnan cies and from k spontaneous abortions was investigated by embryological and histomorphological studies. No in dications of mutagenic, teratogenic or fetotoxic effects of TCDD could be found. The cases of spontaneous abortion, albeit more suspect for dioxin damage, showed different morphological alterations due obviously to a variety of causative factors independent of TCDD. On the other hand it is not possible to exclude entirely an embryotoxic effect of TCDD because in the majority of cases the fetal tissues were incomplete. O o CO 00 cn CO CO The explosion in a chemical factory of ICMESA on July 10, 1976 in Seveso, northern Italy, by which the factory and large surrounding inhabited areas were exposed to the especially toxic 2,378-Tetrachlorodibenzo-p-dioxin (TCDD) and Trichlorphenol (TCP), has aroused the attention and interest of world publicity above all on account of threatened health injuries to the inhabitants. Factory accidents with the release of dioxin gases were already known from earlier years. In 1953* 5^ workers from a chemical factory in Ludwigshafen suffered the effects of such an accident [15]. To a large degree ' 7 4 .n 0002519 DOW 385332 2 their illnesses were longstanding chloracne and injuries to the inner organs [9.36]. In an explosion in 1963 in a chemical factory in Amsterdam 50 workers were ex posed to injurious dioxin gases [12]. In 1968 79 men, after an explosion in a chemical factory in Bolsover, England were affected [233 Similar accidents occurred in chemical factories in the USA [2,28] as well as in Czechoslovakia [17], Austria [31] France [6,7], Russia [35] and in 1955 in Hamburg [19 ]. In contrast to these accidents, which are limited to the factories affected and remain essentially a work and accident medicine problem, the explosion in Seveso took on the dimension of an environmental catastrophe because the toxic dioxin gases were carried from the factory area by wind conditions and exposed bordering localities. A health danger to large groups of people was therefore possible, adults as well as children, which was already evident shortly after the accident with the first chloracne cases. In addition, exposure of animals and plants must be reckoned with and also contamination of the ground in an extensive land area for many years to" come [1 2 ,1 3 ,1 *0 0002520 DOW 385333 Although at the time of the accident positive, defineable embryotoxic injuries from dioxin combinations in humans were not known and in animal experiments as well [525 33,3*0 were only little known, very quickly assumptions on possible damages to pregnant females and to unborn children with regard to the great toxicity of dioxin combinations appeared. According to one estimate, at the time of the explosion accident in the area affected, ca. 150 women were in the first trimester of pregnancy. Additional women appeared endangered in as far as preg nancy had already begun or was in the meantime begun, by delayed evacuation and insufficient separation mea sures. The public discussions, carried out on political, world view and religous levels, about the risks of these women bringing malformed children into the world because of the effects of dioxin, were very lively in the weeks and months after the explosion accident in Italy and outside the country as well with the general discussion about termination of the pregnancies. From this arose for the pregnant women, doctors and other affected per sons and institutions, special situations of conflict. 125 women from -the exposed area as well as the bordering regions, in the time up to October, 1976 decided on termination. In 30 cases Interruptio was approved 0002521 580^0 4 and carried out. Place of living and working of the applicant as well as other contacts with dioxin were taken into consideration. According to the measureable degree of ground contamination with dioxin, the district administration divided the southeast area bordering on Seveso into a heavily exposed Zone A and a less exposed Zone B (diagram 1). The naming of an R Zone (restrict area) came later, when small to nearly no dioxin content in the ground - and very much later than in the A and B Zones- was recognized as a biological exposure by cases of chloracne appearing or domestic animals dying. As "contact with dioxin" was also considered transit through the heavily infected area (superstrada) and the consumption of regionally produced, possibly dioxincontaining vegetables or meat. The fetuses removed by Interruptio as well as b spontaneous abortions from the Seveso region, which were all regis tered in the Gynecological Clinic in Milan, were also examined embryologically and pathologically-anatomically in the Department for Pathology of the Medical Hochschule in Lubeck in co-operation with the Laborat'oria di ricerche cliniche, anatomia ed istologia patologica, Instituti clinici di perfezionalmento, Milan. The results of these investigations were conveyed in a 0002522 SEES8E 5 summary to the Health Officials of the Lombardy region on February 25, 1977* A detailed compilation and ob servation appeared also after a long interval on the explosion accident in Seveso, especially with regard to additional untested reports on congenital malformations by dioxin. The completeness of the documention is required by the far-reaching significance of the results and of public discussion. O Material Investigations (see Table 1) Of the 30 removed fetuses by Interruptio, 3 came from the A Zone, 5 from the B Zone and 13 from the R Zone. In the remaining 9 cases the mother's place of living was outside of the danger zones, but in 7 cases could be made valid in connection with place of work, visit, travel and nutrition. In 2 cases the Interruptio occurred for psychic reasons. The fetuses represented very different gestational ages/ The smallest, of a few millimeters in size, was judged to be 5 weeks, the largest, with a crown-heel length of 17.5cm, 20 weeks gestational age. In the majority of the cases the- developmental ages were between 10 and 12 weeks. 0002523 6 DOW 385336 In the 25 cases with gestational age of 5 to 12 weeks the Interruptio was by instrument curettage. There was an unavoidable dismembering of the fetus and a loss of fetal tissue during the suction during the action. As a result there were, in these cases only a few fetal organs available for pathological-anatomical examination. In one case only a foot and a small piece of liver, in another only a head segment and a vitelline sac were present. In the five larger fetuses of a total length of 13-17*5cm the Interruptio was introduced by intraamial prostaglandin instillation and in some of these ended by extraction. Therefore here also were artificial lesions on the fetuses, for example, widespread skin defects or opening of the cranium cavity with loss of the brain. The 4 spontaneous abortions had occurred in the ll-24th week of pregnancy in the A and R Zones. In three of these cases fetuses of ll-19cm crown-heel length were present, in the fourth case there was merely an empty amniotic sac with placenta tissue. A majority of the Interruptio and abortion material was, at the end of September, 1976, in formalin, Bouinsic solution or Carnoy solution sent to us. Thereby the useable examination techniques were limited from the beginning to the paraffin histology. Histochemical or 7485 0002524 7 .electron optical investigations could not "be carried out since they require fresh material or a differentiated fixation technique. DOV/385337 In the cases in which only incomplete fetal tissue frag ments were available, they were examined with the Lupen microscope* for form and for the position of the organs or organ segments within the organ assemblage. Each piece of tissue was photographed for documentation, measured and described. The material was finally im bedded in paraffin and in stages - or a series of cutscompletely worked up. Many times well over one hundred cuts were made. For the histological examination the following dyes were used; Hematoxylin-eosin, connective tissue-, Elastic- and PAS- dye, aldehyde-fuchsin dye, Esterase proof and Berlin blue reaction. The larger, more intact fetuses were examined by X-rays for skeleton changes. This was however, unsuccessful in the 2 Carnoy-fixated cases, since the lime from the bones was dissolved by the fixative. A regular obduction Lupe means magnifying glass, whether this is a special type of microscope the translator does not know. 7486 0002525 8 DOW385333 with organ section followed with help of the Lupen microscope. Each individual step was documented photo graphically, the organs were weighed, worked up for the paraffin histology and as described above, dyed. The brains were, in as far as they were intact, sent to Professor GULLOTTA,. Neuropathological Institute of the University of Bonn (Dir. Prof. Kersting) for examination. In a to a high degree autolytic fetus of 4cm in length, cross sections were taken and examined macroscopically and histologically in series cuts. Findings and Evaluation A. In a first group, to which the majority belong, namely 23 of a total of 30 Interruptio cases (Table 1) the macroscopic and histological examinations revealed no malformations; likewise no tissue mis-differentiations or degenerative organ changes were shown. The tissue differentiation degree and the size of the fetuses were largely compatible with the length of pregnancy. Small differences of 1-2 weeks were not counted since the exact conception date is only seldom possible and also the pathological and anatomical criteria allow only an approximate estimate of the developmental age. Normally individual variations exist in the course of development. i 7487 0002526 9 DOW 385339 Determination of sex was possible only in cases in which the material contained differentiated gonadal tissue or differentiated Wolffian or Muller ducts allowed an opinion (see Table 1). B. In a second group of 6 interruption cases, changes of a different kind were morphologically detectable (see Table 1). They can be identified as artifacts, development retardation to a slight degree or as within the range of variation of normal development. So in case 6 there was a secondary obliterated duodenum, through epithelium bridges, a condition which can appear in this age of development as a temporary appearance [10]. In case 15 over the coccygeal bone there was a subepiderman cyst, whose lumen was no longer in contact with the ventriculus terminales above it of the spinal cord. The cyst consisted of a so-called "coccygeal medullary vestige" which, after formation of the Filum terminale on whose distal end, therefore in the area of the rear neuroporus, which in the meantime has closed, persists sometimes for a while [21], In the same case there was a defect of the ventricle septum underneath the 7488 000252? 10* septum membranaceum. Because of the parietal tissue tearing and the missing endothelium lining it was under stood as artifact. A prolapse of the retina in the area of a lateral rup ture of the sclera was seen in cases 8 and_12 after localization by the presence of fresh bleeding and the lack of a tissue reaction in the surrounding area as artificial, occurring at the earliest, during the curettage. O O CO 00 CJl CO o Case 27 showed, in a male decapitated fetus of 16.5cm crown-heel length, a rotation of the liver in a counter clockwise direction to the left with fixation lacking on the right diaphragm. Displacement of the cecum in the right upper abdomen as well as partial intrahepatic gall bladder. In addi tion there were widespread bleedings in the lumen and wall of individual intestinal loops in the sense of a beginning hemorraging infarction. This together with the decapitation, is seen as an indication of trau matization during Interruption which also led to rupture of the Lig. trigonum hepatis and to dislocation of the liver and secondarily of the intestine. The fact is thus considered that a congenital misplacement of the liver is hardly isolated in appearance either it takes 002528 11 DOW 385341 place primarily within the "boundaries of a syndrome, as in Ivemark-syndrome, pr it is observed secondarily in connection with a stomach wall or diaphragm defect, an omphalitis or hernia. The partly intrahepatic posi tion of the gall bladder was also a normally frequent anomaly with no essential significance. Case 18 showed, in comparison to the size of the fetus a still widened kidney pelvis calyx system. This can be seen as physiological up to the 10-llth week of gestation. Here it is a result of a slight development retardation. Also the differentation of the elastic fibers in the trachea seemed retarded. C . A third group contains the 30th case as well as the 4 spontaneous abortions (cases 13*29*31 and 34i Table 1), in which with regard to a possible dioxin damage, the question of the cause of the spontaneous fetus deaths is especially urgently asked. In Case 30 there was a great discrepancy in the anamestic data. The Interruptio took place in the 2?th week of pregnancy. The female fetus, decapitated by the manipu lation during the action showed a nape of the neck-heel length of only 4cin. It was to a high degree autolytic, shrunken and mummified, the placenta tissue clearly regressively changed, so that in this case a spontaneous previous death of the fetus with retention in utero a "missed abortion" must be assumed. At the time of the explosion accident the mother was in the 9th week of pregnancy. The fetus death occurred surely shortly thereafter, without morphological proof of its cause. 7490 02529 12 In case 13 was a female fetus of 17.5cm length from the 15th week of pregnancy, whose good condition of preservation points to a fetus death shortly before or during the Abortus. The numerous fresh areas of inner bleeding spoke for hypoxia as the cause of death. Widespread hematoma of the cranial soft parts obviously as a result of labor pains following amniotic sac rup ture, had developed before the fetal death; the intra cellular hemosiderine deposits in the region spoke for this. An indication for the cause of the early labor or the sac rupture was not to be determined from the morphological picture of fetus and placenta. In case 29 a male fetus of 19.5cm length from the clini cal 24th week of pregnancy, an infarct placenta was seen as the cause of death. The placenta was small and 4/5 of its volume was interspersed with widespread older and fresh infarctions. The fetus seemed, with regard to the given length of pregnancy, underdeveloped. This can be traced back to lack of sufficient oxygen as a result of the older placenta changes. A "missed abortion" can be excluded because of lacking maceration signs. The pronounced autolytic changes of the inner organs in this case were the result of a misplaced fixation. Case 31 was a female fetus 11cm in length from the 12th week of pregnancy, which already had definite maceration. Here there was an anomaly in the region of the heart with an aorta valve defect as well as a longitudinal swelling of the aorta wall next to the pulmonary artery. This began directly above the aorta valve commisure and reached to the aorta curvature. Histologically there was a circumscribed texture disturbance of the elastic fibers of the aorta wall. The fetus showed, in addition, a serration anomaly of the right lung as well as a peculiar Facies with inclined lids, flat nose, a large capped on the point, tongue, as well as a receding chin. The skull was stretched, the occiput flat with a neck edema above a questionable strangu lation ridge. It was to be assumed that the interpre tation of the outer phanotype was injured by the advanced autolysis, so that the characteristics named can not be evaluated alone. In connection with the inner anomalies however- with all retention- it can be seen as an indica tion for the presence of chromosomal aberration. The fetus was strangulated by the umbilical cord and retained in utero. The placenta showed regressive changes as m s8 E M a 7491 00025:0 GKS8Moa 13 well as fresh chorioamnionitis, which apparently developed after the fetal death. Case 34 has to do with an abortion in the clinical 14th week of pregnancy. The abortus material contained, in addition to decidua tissue, however, only a small hazel nut sized amniotic sac, without content, that is without a fetus. The scanty placenta villi were hardly vascularized, hydropically swollen and showed only slight trophoblast proliferation on the surface area as well as individually inflated and vacuolized histiositic giant cells in the villi. The histological picture spoke in favor of an early abortion with resorption of the embryo as well as degeneration of the placental tissue by primary tissue unworthiness of the embryo placement. FHILLIPPE and BOUE [27] described such changes of the placenta during chromosomal aberration in early abortion so that in this case the assumption of a chromosomal anomaly of the embryo placement seems possible. Discussion The attempt to evaluate the described findings and the discussion of a possible embryotoxic effect of the dioxin, especially with regard to conclusions made in fear, zeal and emotion, require cool consideration. The epidemiological as well as the anamnestic and clini cal data must be taken into consideration, which are partially stated in Table 1. So, in 4 of 5 cases (cases 29,30* 313*0 with spontaneous fetal death, it is shown that the mothers came from the *A case of Interruption in which, however, an intra uterine fetal death had occurred earlier. 7492 0 0253l DOW 385344 14 so-called R zone (restrict area) or had visited them. Although the question is asked especially urgently in these cases about dioxin damage, they are of a zone which, to be sure showed a biological infection; in which however, the chloracne cases appeared in isolated instances and very much later than in the primary exposed zones A and B. In addition, the women affected could give no information on possible contacts with dioxin, namely neither consumption of regional vegetables or meat nor actual time spent in the heavily exposed regions. Only in Case 13 belonging to the group of spontaneous abortions, had the mother resided until the evacuation on July 27, 1976 in the most heavily exposed A zone. The abortus occurred 4 weeks later and was attributed to the lack of older morphological changes in the pla centa and fetus causing an early rupture of membranes. Information on symptoms as possible clinical indication of an intoxication of the mother was not present in these cases of spontaneous abortion. In case 31 a hyperemesis during the pregnancy as well as fever at the time of the abortion were present. This complaint * coincides with the pathological-anatomical finding of chorioaranionitis. A slight rise of the y -glutamyltransferase in case 30 can be seen in connection with 0002532 15 the long retention in the range of a "missed abortion". As a whole the causes for abortion seem very hetero genous. If it is taken into consideration, that normally the comprehensible abortion rate of all conceptions is about 10# [8], even if the majority are early abortions, one must consider that some of these miscarriages would also have occurred without dioxin damage. DOW38534 The Committee for the Organization of Health Affairs of Brianza Seveso [**] has, in a situation report on CTT abortion frequency in the area around Seveso, taken the position, that proceeding from a general spontaneous abortion rate of 10#, in the pregnancies with terms in July to September, 1976 in some areas around Seveso, a slight but clear rise of the spontaneous abortion rate occurred, in Seveso itself nearly 20#. For the pregnancies with term in October-December, 1976 the increase of the abortion frequency became still more clear and reached almost 23#, which was also true outside of Seveso. The absolute figures are, to be sure -corresponding to the individual areas- relatively small, so that smaller devia tions already cause large relation variations. Neverthe less the distribution pattern of the abortion frequency, with a rise above all in Seveso, can be seen as a possible causal connection with dioxin injury, without final conclusions being drawn at this time. 7494 0002533 16 In the larger group of those cases, in which contact with dioxin was to a greater extent, there were no morphological changes in the accesible Interruptio material for examination. Also, in case 19, in which the mother had shown mild symptoms of chloracne, the fetus seemed normal by embryological-morphological criteria. Certainly the evaluation of the negative pathological-anatomical findings can be given only with the reservation that the Interruptio material, for the most part was incomplete and that the methodology of the paraffin histology imposed certain restrictions of interpretation. ao 00 00 on oo Ci A toxic substance can, depending on the time of its becoming effective during the pregnancy, exhibit different injurious patterns. It can, as a mutagen, in the embryo cells, induce a gene change or cause structural chromo some aberrations or division defects, from which chromo some anomalies of the embryo arise. An embryo cell injury of this or that type by dioxin can however, be discussed only in the cases in which conception' occurred after the explosion. This excludes a case such as the one in December, 1976 where a child with Ichthyo sis congenita (on which, as on many others under similar circumstances, many cases have been reported in the press) 7495 0002534 17 'DOW 335317 was said to have a series of possible gene mutations caused by dioxin. In both abortion-cases 31 and 34, in which a chromosomal aberration is regarded as possible to probable, conception did not occur until September. One can, however, on the other hand, not fail to take into consideration that chromosomal aberrations in spon taneous abortions are also normally frequent. Among early abortions- case 34 would fall in this category- they can make up as much as 6ofi [21]. In animal experiments TCDD has produced, in addition to a positive mutation test on bacteria [32] only in one plant [16] and mammal species [24] chrosome aberrations. A dominant lethal test on rats was negative [18]. TCDD is therefore seen only as a potential mutagen [30] with high species specificity, whose mutagenic effect in humans seems questionable.- An injury by a toxic substance can be called teratogenic which affects embryo development in the early stages and can lead to the lack of certain developments. Since in humans the formation of most organs is completed' by the 6th week of gestation, a teratogenic effect of dioxin would be possible only in cases in which at the time of the accident the pregnancy was not older than 5-6 weeks. A child that was born in January, 1977 with 0002535 18 a rectum atresia in the region around Seveso can therefore only with difficulty "be seen as as a valid victim of dioxin contamination, since the teratogenic determi nation period for the rectum atresia is before the 7th week of gestation. To be sure, for TCDD in animal experiments and also with regard to its teratogenic effect, a high species specificity seems to exist. Cleft palate as well as hydronephrosis could only be produced in the mouse [5* 25] but not in rats [5t333^], rabbits 37] and sheep [1], while only in the hamster were eye deformities brought by TCDD [3*26]. In the cases we observed there were no deformities of a comparable type, a statement which, to be sure, is again an indication of the in completeness of most of the embryos examined. The slight broadening of the kidney pelvis in case 18, which is still seen as physiological up to the 10-11 week of gestation, was identified as a slight developmental retardation and not as a malformation. ,o( D CO CO cn co GB Only the anomaly of the aorta valve with the valve defect and texture disturbance of the aorta wall in case 31 is understood as 'a deformity in the narrow 0002536 19 DOW 385349 sense. The aorta wall lesion could occur as a result of a disturbed division of the arterial heart cham ber or secondarily as brought on by the mechanical overload because of the valve anomaly. Within the range of chromosomal syndrome, aorta valve changes occur especially frequently. The additional degenerative signs present axe regarded as proof for chromosomal symptom complex and the heart anomaly therefore not seen as caused by a teratogen, or at least not definitely caused by a teratogen. A fetoxic injury is finally to be assumed if a substance influences a fetal organism after the 6th gestation week, therefore during tissue differentiation and the growth of the organs. To be expected are growth retardation, that is organ hyplasis or degenerative injuries to inner organs with cell necroses. These changes are especially difficult to identify in the fetus since they happen without reaction. Only in widespread necroses are clear defect formations, especially as observed in the brain, the result. Although in the material examined from Seveso, no morphologically tangible proof for such injuries existed, the difficulty lies in recognizing the real and essential limitation in the early or middle fetal stages, which works against a definitive interpre- ! 7498 002537 20 tation or even a complete exclusion of fetoxic injury. In the animal experiments TCDD in mice produced a toxic liver cell fatty degeneration as well as liver cell necroses, bile duct proliferation and a raised iron content, without histologically tangible siderosis [22,39]* These changes stand in connection with a hepatic porphyry, which is seen by POLAND and GLOVER [29] as the result of a strengthened induction of the a-Aminolavalin acid synthetase by TCDD. In addition, there was an atrophy of the thymus and of the lymphatic system within the picture of a "lymphocytic depletion" [11,38]. With high TCDD doses in rat fetuses intestinal bleeding was caused [3^]* In the material present, degenerative changes in the liver and thymus, also compared to fetuses of the same age not exposed to TCDD, could not be proven. It is difficult, the present findings in hand, to make a statement on the embryo or fetotoxic effect of dioxin in humans. To be sure it can be clearly stated that an actual proof of such an effect was not accomplished; however a mutagenic, teratogenic or fetotoxic effect of TCDD can not, on the grounds of the reservations named- incompleteness of the material and limited exami nation techniques- be completely eliminated. This is b C00O cn co Cl o ' 7499 0002538 DOW 385351 21 valid especially for the increased spontaneous abortion rate which is reported from the region around Seveso (see above) for whose cause however, on the few examined fetuses of spontaneous abortion in this report, no uniform morphological substrata could be found. Nevertheless it can be shown, with the present investi gation, which aspects are to be considered in the evalua tion of malformations in the cases observed in the region of Seveso, before a connection with a dioxin injury can be substantiated. In addition it must be considered that statements without knowledge of the deformationstatistical and epidemiological situation in this region before the 10th of July, 1976 can cause a false judgment on those affected as can publicity. 500 0002539 Schweiz,mcd. \V%el>f. MS. Nr. 42 (1473) dia-gram 1. TCDD contaminated zones in the Seveso region [4]. A zone-up to 5400 TCDD/m2 B zone-up to 50 /g TCDD/m R zone a )mora than ^ TCDD/m2 b) positive TCDD proof in animal organs c) chloracne' cases O 50 certain chloracne cases (1-18-77) 1 certain chloracne case a 1 suspected case W* /0 0 0 2 5 4 pW 385353 S3 % -5* -J H3% ii a 3U3. si la n III 1ii i ii III i - 1 t*- itS S W * 2 12 2 |22 f. S l i U S1 J r HI i I i! 3 3 1 2 {! H|t I i Hit fit lilt!' lit: si; is a in ii n 1m 3* iiif 1 J. > *** Met-/. cirsMUtarcva * i i n 4 i n 4 i n u4ivlu* m * u 9 -* ; 3i2 T.T. JC I *? a O I3 a a; 7S*Ci3** : c :M! o!3 a ;i! i *li *. v 'V nn n i i. 5 *V T. . V t ii aft A A ft a a l! f ! i l"i37 Hi?! 5^3 i-* a I S* t S *.3 il**-M*a 3 sss'l s.s; I ui*n lll s> I* i S } ! s n t * 7 V ** ! see ss ili. Tt i i >. Va jj ii |3 i : p i 5 - ii & a 22 2 i SX B i * B 6 B * R B ti U`ti* I li I* i ! ss -2 ,,t SS : M*>Ic 1 s sa B b & BS i T. Ti 2B A4 & 4 I C S' 4 I 5 f 1 Binns W., Balls L. : Nonteratogenic effects of 2,4,5- Trichlorophenoxyacetic acid and 2,4,5-T propylene glycol butal esters herbicedes in sheep. Teratology 4, 245(1971). 2 Bleiberg J., Wallen M., Brodkin R., Applebauro I.L.s Industrially acquired porphyria, Arch. Derm. 89. 793-797(1964). 3 Collins T.F.X..Williams C.H.: Teratogenic studies with 2,4,5-T and 2,4-D in the hamster. Teratology 4, 229(1971). 4 Comitato di Coordinamento dei Consorci Sanitari di Brianza Seveso l-2-3(Sede: Ospedale Desio): La situa zione sanitaria e di rischio - Informazioni. Autoizzasione: Tribunale di Monza, Nov. 1977* Direttore responsabile: Valerio Galimberti. 5 Courtney K.D., Moore J.A.: Teratology studies with 2,4,5-trichlorophenoxyacetic acid and 2,37.8-tetrachlorodibenzo-P-dioxin. Toxicol, appi, pharmacol. 20, 396-403(1971). 6 Dugois P. .Ambiard P., Aimard M., Deshors G.> Acne Chlorique collective et accidentelle d'un type nouveau. Bull. Soc. franc. Derm. Syph. 25 260-261(1968). 7 Dugois, P., Colomb. L,x Acne chlorique au 2,4,5trichlorophenol. Bull. Soc. franc. Derm. Syph. 62, 262-263(1956). 8 Fuchs F., Stakemann G.t Die Fehlgeburt. In: 0. Kaeser, V. Friedberg, K.G. Ober, K. Thomsen, J. Zander (Pubi)t Gynakologie and Geburtshilfe, V.. I, p. 733-759 Thieme, Stuttgart 1969. 9 Goldmann P.J.i Severest acute chloracne, a mass intoxication by 2,3*6,7-Tetrachlorodibenzo dioxin. Hautarzt 24, 149-152(1973). 10 Hamilton W.J., Mossman H.W.: Alimentary and respira tory systems, pleural and peritoneal cavities. In: Human embryology. Prenatal development of from and function, pub. by W.J. Hamilton and H.W. Mossman, Heifer and Sons,/Williams and Wilkins; Cambridge/ Baltimore 197.2. 11 Harris M.W. Moore J.A., Vos J.G.,Gupta B.N.: General biological effects of TCDD in laboratory animals. Environm. Hlth. Persp.. 5 , 101-110(1973 ). 385354 I C7 O * 7503 0002542 12 Hay Ai Sevesoi the aftermath. Nature (Lond) 263. 538-5W1977). 13 Hay A .t Toxic cloud over Seveso Nature (Lond.) 262, 538-540 (1977). 14 Hay A.t Seveso solicitude, Nature(Lond.) 267, 384385 U977). 15 Hofmann H. Th Neuere Erfahrungen mit hochtoxischen Chlorkohlenwasserstoffen. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 232. 228-230(1957). 16 Jackson W. T.i Regulation of mitosis. III. Cytological effects of. 2,4,5-trichlorophenoxyacetic acid and of dioxin contaminants in 2,4,5-T formulations. J. Cell. Sci. 10, 15-25 (1972). O Q 385355 17 Jirasek L . , Kalensky J., Dubec K . , Pazderova J., Lukas E. Chloracne, Porphyria cutanea tarda and other intoxications by herbicides. Hautrzt 27. 328-333 (1976). 18 Khera K.S., McKinley W.P. Pre and postnatal studies on 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and their derivatives in rats. Toxicol, appl. Pharmacol. 22, 14-28 (1972). 19 Kimmig J., Schulz, K.H. Occupational acne (Chlor acne) by chlorinated aromatic cylcic ether. Dermatolologica (Basel) 115, 5^0-546(1957). 20 Lazar P . , Gueguen S., Boue J., Boue A.i Epidemiologie avortements spontans prcoces a propos de 1469 avortements caryotypes. Symposium INSERM. Paris 12-14 sept. 1973. Pub. by A. Boue and Ch. Thibault. Proceedings, p. 317-332. 21 Lemire R.J.Loeser J.D.,Leech R.W. Alvord E.C.i Secondary caudal neural tube formation. In R. J. Lemire,J.D Loeser, R.W. Leech, E.C. Alvord jr. (Publ)1 Normal and abnormal development of the human nervous system, p. 7I-83. Harper and Row. Hagerstown, Maryland 1975 22 Lubet R.A., Brown D.Q., Kouri R.E.t The role of 3-0H-benzo(a)pyrene in mediating benzo(a)pyrene induced toxicity and transformation in cell culture. Res. Commun. Chem. Path. Pharmacol. 6, 929-942(1973). ^ ^ 7504 23 May G .1 Chloracne from the accidental production of tetrachlordibenzodioxin. Brit.J.industr. Med. 20, 276-283(1973). 0002542 D O W 385356 24 Moore J.A.i TCDD toxicity Symposium on chlorinated phenoxy acids and their dioxins. Genet. Workgroup. The Royal Academy of Sciences, Stockholm, Feb. 1977 25 Neubert, D.,Dillmann 1. Embryotoxic effects in mice treated with 2,4,5-trichlorophenoxyacetic acid and 2,3,78-tetrachlorodibenzo-p-dioxin. Naunyn-Schmiedebergs Arch. Pharmacol. 272. 243-264(1972). 26 Neubert D. Some remarks on embryotoxic resks induced by polychlorodibenzo-p-dioxins and similar coupounds 6th Conf. of the European Teratology. Gargnano, Sept. 1976. 27 Philippe E., Boue J.G.j Le placenta des aberrations chromosomiques letales. Ann. Anat. Path. 14, 249-266 (1969). 28 Poland A.P..Smith D . A health survey of workers in a 2,4-D and 2,4,5-T plant. With special attention to chloracne, porphyria cutanea tarda and psychologic parameters. Arch, environm. Hlth 22. 316-327(1971)* 29 Poland, A.P..Glover E . 2,3,7.8-Tetrachlorodibenzop-dioxin a potent inducer of alpha-aminovulinic acid synthetase, Science 179. 476-477(1973). t 30 Ramel C. TCDD toxicity: Symposium on chlorinated phenoxy acids and their dioxins. Genetic workgroup summary. The Royal Academy of Science, Stockholm 1977* 31 Reggiani G.i Medical problems raised by the TCDD contamination in Seveso/Italy. 5"th International Conf. on accupational health in the chemical industry (Medichem). San Francisco. Sept.,1977 32 Seiler, J.P. The mutagenicity of benzimidazole and benzimidazole derivatives. I. Forward and reverse mutations in Salmonella typhimurium caused by benzi midazole and some of its derivatives. Mutation Res. 15, 273-276(1972). 33 Sparschu G.L., Dunn F.L., Rowe V.K.t Teratogenic study of 2,3 718-tetrachlorodibenzo-p-dioxin in the rat. Toxicol, appl. Pharmacol. 17, 317-318(197D). 34 Sparschu G.L.,Dunn F.L., Rowe V.K^j, Study of the teratogenicity of 2 ,3 7 #8-tetrachlorodibenzo-pdioxin in the rat. Food Cosmet. Toxicol. ,404-412(1971). 7505 002544 DOW 385357 35 Telugina K.A. Affection of the follicular apparatus of the skin in workers occupied in production of buthyl ether of 2,4,5-trichlorophenoxyacetic acid. Vestn. Derm. Vener.(Moscow) 44, 35(1970). 36 Thiess A.M..Goldmann P.i Follow-up-report on the trichlorophenol dioxin accident in the BASF Co. on Nov. 13* 1953 IV. Medichem. Congress, Haifa, Sept.1976. 37 Thompson D.J. .Emerson J.L. .Sparschu G.L. Study of the effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on rat and rabbit fetal development. Teratology 4, 243(1971)* 38 Vos J.G.,Moore J.A.,Zinkl J.G. Effect of 2,37*8tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environm. Hlth Persp. 5 , 149162(1973). 39 Vos J.G.,Moore J.A.,Zinkl J.G.1 Toxicity of 2,378tetrachlorodibenzo-p-dioxin (TCDD) in C57Bl/6mice. Toxicol, appl. Pharmacol. 2, 229-241(1974). 7506 ' 0002545 \ MS' \ *4S'  * t\ r /tyj .. .ttsG-' ^~/^/77 m j j REC.D vtmoi*.'-' UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON. D.C. 20460 4 i.*AY 1977 SUBJECT: Dioxin: Position Document TO: FROM: Dioxin Implementation Task Force Edwin L. Johnson Deputy Assistant Administrator for Pesticide Programs w/ So - z o o - Wu oU >**' When we met last fall I agreed that the Agency would put toge t h e r a s ummary of what it had l e a r n e d s ince w i t h d r a w a l of the h e a rings in 1974. You have been c o o p e rating with E P A in the c o n d u c t of a m o n i t o r i n g p r o g r a m to d e t e r m i n e the e x t e n t a nd f r e q u e n c y of d i o x i n (TCDD) r e s i d u e s in the e n v i r o n m e n t as a result of the u s e of 2 , 4 , 5 , - T and r e l a t e d p e s t i c i d e s . T h e a t t a c h e d d o c u m e n t is a d r a f t of our z CO z CO o 00 U1 o CO VM C," o vn e v a l u a t i o n of phase I of the D i o x i n I m p l e m e n t a t i o n Plan a n d a s u m m a r y of our p l a n for p r o c e e d i n g w i t h p h a s e II of the program. As I indicated to you last fall, this document is being p r o v i d e d to you in a d v a n c e of a fo r m a l release for comment and suggestions. It has also been made available to the Administrators Pesticide Policy Advisory Committee for the sane purpose. We intend to make the document f inal and r e lease it generally in the l a t t e r part of M a y and w e t h e r e f o r e w o u l d a p p r e c i a t e you submitting any comments or suggestions you have for m o d i f y i n g this d o c u m e n t by M ay 19, 1977. A l l c o r r e s p o n d e n c e s h o u l d be f o r w a r d e d to Mr. W. T h o m a s H o l l a w a y WH-566, Office' of S p e c i a l Pesticide Reviews, E n v i r o n m e n t a l Protection Agency 401 M Stect S.W. Washington, D.C. 20460 (Telephone No. 202/755-9336). Your cooperation in this program in the past and y o u r sug g e s t i o n s for this document as w e l l as the future d i r e c t i o n s of the p r o g r a m w i l l be g r e a t l y a p p r e c i a t e d . ' Attachment : '//? 7508- 000256 /V5 7509 /V5 C9Z6T- WvOCl# December 13, 1978 PESTICIDE & TOPXaICgeC2H3EMICAL NEWS ENVIRONMENTALISTS ATTACK PROPOSAL TO REDUCE THE SCOPE OF RCRA wEgD\ enoonvuueilrgrd'oVant"somprMeosns.petarCalodogsusretocvluieenprgtsehlhaetthasvrseeeaatsthtionatlnodg 1hEu,ep0na0vloti0nrhokhnagamn/zmdeanorttdhnaoetlhuePsnorvwfoithraeoascntzteiamornredenoAgtu"ugslea(nwStcieaoyesnAtDsedsetmocfir.neoxi6mset,mrtahPpteoatrgreul2e0s). IEar(nRutntnCveaRmicrADoop)nnettImscrn.aetrotn5yhtmailtselootAmtdteichafrtyneiontroinenerCgtFeu"oonllustaentatolidveofae,nCtisttoohhnuneengaEpdrneeundrsvbsiCltrihacoiesntuimRzenexeepnnsprostorauteflersoccDsrteeeeaddCfeBIofnnrensotsRetmeeCrrFvcRuElaAennta.div"or,inrlEyoannhnmvadierz.noaRntredmscoaoeuivndsetarwtylhaAaAstccttteioann,d wmMauoscrhteeoavwseor1u,,l0dt0h0beyekadgr/imgspuoeondstehtdhoaoftfwbwyaisthrteeoluawtxoitunhlged btmheeenaernfuitltehosaftteo4n.ev6xirebomnilpmlitoegnnetnpaelorusanatdfoesrgsoupfarrhodadszu.acridnoguas szos vn haafTedsrrladeyhoeeaidemecdvietlne.ryidtngnoh.kmvpeAaeilnernadRtoddtCainwnRlftmsihigAtne.hewairnshnfechtaigdaacbiulnnhrimlscgiatcce,estaritdkoa.uaynlnslesesifFito*inonbrfrgaoeiesbnebcIhxraanjieialdunccmuasgtdsnee,ptdyd,rlmeeytnit,henooerewgufcyaturaeaarpssnnylatrdeidoupdpafsfr,irmeonoosssdioamasugmclhecatitethnroaiwecogseinanxeaw,soelntewmgedeclesotplhnrotafyiolrcdssposirmlaxccaoroaftoitulinnuhmnrcdregiaeinsuctreigis,ncat,dirctndwhuiidedyesosutieo,rrspsyltr,rleoficeattdohstbue*nercrytitacioinn^s'^ ArdFeiilgsnspuoaollaelsymtei,dopnthhhaae"syzsiazoarelrdedgloyubueysodnwthtthaehasetetenRbvwCaiRrshoiAisncdhmoo,feeetnshcntoea'ynltiosastmuastiiwdhco,acrsofiazntrehceeetxrhcnceoesEs.e"PtdsAs ottofhceelxeceaomnstipntogfinupdpruoispmterpriredospisferprolomysal. HUMAN BIRTH DEFECTS NOT CAUSED BY 2,4,5-T OR 2,4-D EXPOSURE, REPORT SAYS AShdaepCsfeeocccniotfssniuccaalltmulaldotyien,vdgethbtCheaaobCtuioen2suc,4inli,nc5ai-lp*thpTceoonoiYnrcatleru2rdd,a4meb-dDyditinehsxteirptisMocstriuenirpniesot1redt9r:i7d5on-f7oH6t ec(aSaleuthes,eDVaeiccc.tlou6rs,itaeP.raAogufesb5tirraathnlida,9). w"Aerneaclyasuisseodf bayll eixnpfoorsmuraetiotno a2v,4a-iDlabolre 2sh,4o,w5e-dT .no evidence that these birth defects "7 r-- hb"uiTrmhtheannaobbrnimrotrahml aaabglinrtioicerusmltiuanrlidatlioemus.seestoicf a2n,4im- Dalsanndor2i,s4 ,t5h-eTreheavsidneont cbeeeton csohnonwencttosuccahusuese io with al"enVthiacadtole.rq"iaualtaecksysstoermgaonfizseudrvreesilelaarncche oonf tthheoseepibdiretmhidoleofgeyctosfwbhiritchh daerefenctost raanpdidlalyck t)0026 I e3c-.eem2b4er 13, 1978 PESTICIDE & TOXIC CHEMICAL NEWS EcCononvnicgrloeunnsmiitoaennlstAaablnnPdorroombteasceltiritovienastAiiongnestnhceIynYotahfrefriacm"iRaDlespioscrtotruioclfdt,t"nhoetCboenrseualctahteidvefoCrocuonmcmileonnt on the Tfohoedroerpowrattsearids^upRpelgiueslairnsVamicpltiongr isahIotwcsotnhtaintu2e,d4:-D and 2,4,5-T do not.occur in wwapo"lrfBhhmooeitdcthnohhuescut2hgts3i,eaeo40nvdn-eeDyraoebarsaaleraneapsgndiudrwirbs2cielt,upit4hcloot,.5urnatr-ogeTTadrklhinceauhuoyraswleevthunecaorovndfaneoletctwhapererirmbssnoieedemeednucnitclowaatmuilriostpehnesofda.fupfeenieAcntrdtnyssVsyo.r"ionacenntcdooevtrrlheidareInsohefvoveoreealfrvlftseehedcatsisn the Iwuostnotfseaerbbttheiiewrsetntaahiroscerdadpecelloioaffrndfetteisic,orvitdnetishsens,i"rehcoaeinCpbasscloybicunwoenhitrttcwidhhgiileehnesengrniromattthieioenledasrtorhbotnherinecaurittdmdyeepipibtvoeeuihrsrsstasi.aoodgofnfecdotbehamifnarpendtcahrtpisnseed.artnihbnd"eiarHwttoahethlnhedcerdereeefaate2hntc,hedt4r,nerf5uoa-dumtTonebsedeasrontsrnhdaoitntty2p,att4hepw-epDoreear JTeiiUnnxhjcnet1eho9prete7eaed7pdrseotecdprtothtowntarhstottaaisiyIitndhnefeec,aidglrua"usdv0Irtewe.e2rdi.asa":sgpne.0Oop.lt0.beem6dvsee.rptlhvo.oaapftft.TimTooCnnC.DesDaDDsnoandimninhtpthuh2lamee,t4a(ftn,ooh5fre-emT2flf,eue4golc,afa5tftsle-iorTmonefad.axtcifhNmioedrou)pmsoreateswhlpeteoaircrisnitsde0aVde.m1siocptndpolei.rispnicha.umaVossi.svceetodrria nobhpc2"orobaoA,4ontvtsleae,etnew5hdnreo-bvotceuTieretaxgitedlohphnrl.nyyeeIrussTwetiisohedxheinlatpudaihtcsoeteessssitdueushoidbgenvnrsgeeaottfrepoagtosonrotrturhitsdecntiesssuaettitlhxtduotehpauuffditferreiscatlistytllhlaeloefeuydteffesedffaeirtfeanhrecnnoescvtgoftsiciersne2ormhwms,iwa4anavpl-ohreDtoafihecugbethnramhedingeectisdehnnulneialhm2tencaucr,okav4oratleame,l.o5ldpnpf-uIoooTi2tsnutp,a,niu4bsgdle,ae-set.Dnio.nor. waets"hpxliDmetrphaelocfoysoitesnthmriieowstpinmhvoisnueoavnlinilnodylullsvf-mraeeaecafdnltfnadueutirncefetdtaostcohtttfuoihenrtoahpisncgeeeogroncipacponflumlefealectpncIutontitroeusvadn.noldlwbvuIsetysidteaahirnpoiendp2faue,tt4tashh-rtgeerDsrimhaetaalheantraanedbtcruictfc2rhaiii,dcods4tekseu,en5srt.t-eh"csToa,onnfaenrt.eehgc.e.ted 419270 oc * JCP bUSPENSION REQUESTED BY HEALTH RESEARCH GROUP; NO RESPONSE FROM EPA bScthaeuenesnpcHeeaelnlnaaslsittowihonenRroeefhdseeaabalrlyricnuthghseeG(sSEroeonuefvpidDrl(obeHncrRmo.Gme6)no,twcahPhllaioPcgrhreoopa3tren5oct)pti.iaconinpTeahAte(gDeesDBnCehcPcya).vaiw8nt gapssurinserpsteeseqrnuvtsieeminsotoener.drseltaqasuttuesswteinheaktdhbenyot -'002520 \HV, , Of Mice and Men--The Troubles ofG, 4, 5-T V "\ 1 F ALL FORESTLAND COMrOVer- Against this background of pub ing phenoxy herbicides is like cur - iies, that over the use and abuse of licsuspicion and scientific progress ing a headache with a frontal - herbicides wins a questionable comes It is to forest pes lobotomy. In fact, not since prize for being the most enduring. ticides what Ali is to boxing-- the paraquat was used to ruin , .Now that public fears over clcar- meanest and the greatest. For the marijuana Helds in Mexico has 'cutting have abated, anxieties seem 10 have transferred nicely to the past thirty years or so T. as the chemical spraying been so roundly chemical isaffectionately known to attacked. practice of aerially spraying her applicators, has proven useful for Actually, it's not so much the bicides in forest management. For killing off brushy, broadleaf plants herbicide itself that is causing the this development, one must thank which often invade a forest site and outcry, but.the toxic impurity, newspapers and television for their then compete successfully with called dioxin (TCDD). that is con timely accounts of tumor-ridden commercially valuable conifers for tained in T. Dioxin sneaks into the mice and malformed fetuses, all sunlight and nutrients. T works, all compound accidentally in minute brought on by exposure to phenoxy right, but not always where in amounts, during the manufacturing Iherbicides of one kind or another. tended, and itcan be harmful when process. It isnot a necessary ingre 'Those who keep themselves cur- itdrifts onto non-target crops. One dient, and contributes nothing to )rent on the varied horrors we're spokesman for D o w Chemical the effectiveness of the herbicide. capable of inflicting on ourselves Company, which manufactures it, But it's extremely poisonous to will have surely heard of sleepy said, **You can stand in a vineyard humans; some say the most .Oregon valleys sprayed with chem- and think of 2,4,5-T and the leaves poisonous synthetic compound icals likened to Agent Orange, the will wither away." He isn't refer made. From Harvard to the Uni 1defoliating compound once used by ring to the power of. suggestion, versity of California at Berkeley, ;the Pentagon in Vietnam, diere -is either. .tests are confirming the draconian indeed something insidious about The use of T also happens to be characteristics of dioxin, and the ; Chemicals that cause plants, ani- relatively cheap. The reported associated consequences of giving 1mats, and even humans to drop costs of aerial application sound T to test animals in relatively mas Jead. It wasn't for nothing that suspiciously low -- under S50 an sive doses. Curiously, the astonish pone joined thalidomide as 'a- acre according to many users-- but ing number of tests run on the her nousehold word in our disaster lex aerial spraying is still considerably bicide has led to no consensus on icon. cheaper and perhaps more effective its long-run effects on humans, !. Aside from the irresistible appeal than manual attempts to control animals, or the environment. The j such stories have for reporters and vegetation. And even if the costs Hndings are sometimes contra ; broadcasters, there are good rea are somewhat higher than stated, dictory-- Texas Tech University sons we are hearing more lately the payoffs from a judicious use are scientists say dioxin isn't present in about forest herbicides. Appar- undeniable. According to accepted soils sprayed with T; others dis 4 eritlv the same people w h o give us estimates, chemical control of veg agree. A U.S. Air Force study says . poisonous compounds are refining etation can on average produce a 40 dioxin doesn't accumulate in water their techniques for measuring the percent increase in timber volume or animals, Berkeley says it does, . levels of poisons in them. Ten at the time ofharvest. Today, when and so on. If anything, tests seem years ago the state of the art was more than half the commercial to add to the confusion; what's safe fairly primitive. Biochemists.could forest is growing wood at less than to one scientist is sinister to detect, say, only one part of poison optimum rates, many argue that another. ' in a million parts of herbicide. But herbicide use becomes essential to Press and television accounts today, Harvard University scien meeting our long-term timber generally haven't served us very tists claim they can detect one part needs. " A major cause of this loss well. T w o major networks recently : in a trillion parts of herbicide, thus (of growing capacity] is competing dug up old Vietnam war footage to` . turning up truces where there once and undesirable vegetation," said a tighten the link between T and ! were thought to be none. It's called recent statement by the Society of Agent Orange, the military de-' t progress: science obliges us with American Foresters. foliant. (Orange was comprised of J lethal poisons, and finally with sophisticated means of measuring their toxicity, too. WencooNurDagFiRngFUtrele, gSAroYwCthRIbTyICsSp,rbayut about equal portions of T and 2,4-D, and applied in heavy doses.) Perhaps more than anything else. *1978 Society of American Foresters December 1978/Journal of F orestry/787 cited Jlie public outer) against her bicides. In the past two years, 'Senators Dale Bumpers (D-Ark.) and Gaylord Nelson (D-Wisc.) have proposed legislation to ban the use of phenoxy herbicides on public lands. The Environmental Protection Agency (EPA>-- which wrested herbicide jurisdiction from the Agriculture Department and now regulates such compounds under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA)-- has in the past moved to ban the use of T. changed its mind, but is n o w taking a second look at registration. With its testing procedures under fire, E P A initi ated on April 21 its Rebuttable Pre sumption Against Registration (RPAR) process for the phenoxy herbicides T, 2,4-D, and silvex, a mixture of T and D. By invoking this process, E P A permitted con tinued use but only until all in terested panics submitted evidence to rebut the agency's presumption against registering the herbicide. In plainer English, R P A R means that the continued use of T is in doubt. Meanwhile, the Agriculture De partment sought to head ofTa major 'onfrontation by imposing strict jidelines on the streamside use of~ T. It later liberalized them under pressure, but required each pro posed spraying on national forests to be cleared by Assistant Secre tary M. Rupert Cutler. ' ^ ^ hile policymakers debate the merits of more orless herbicide use, and their tireless counterparts in the laboratories decide just how toxic herbicides are, it's a sure bet that the media will exploit the con troversy for all it's worth, mixing creative new concoctions of fact and fantasy. Amid all this conjec ture and hairpulling, important but seldom reported facts tend to belie the impression created by the - doomsayers. For example, in the casually re ported link between T and Agent Orange the differences are greater than the similarities, yet never get equal time. Dioxin was of course sent in Agent Orange, but only . .ne sense that heat is present in both blast furnaces and body tem peratures. The amount of dioxin in production-grade.T is legally lim- ilcw . > ".I p.ti I liiiiiiiiii il liu- C 'de. winch runs to about 1ounce dioxin to 312 tons of T. And the Council for Agricultural Science and Technology (CAST) says that the dioxin amount in the T cur rently being manufactured is con siderably less than that: Moreover, the volume of T tised in Agent Orange applications was about 30 times greater per year per acre than that used today in forest manage ment. All in all, taking the dioxin levels and volume applied per acre, the dioxin level in Agent Orange was anywhere from 3,000 to 30.000 times greater than in domestic use. Even given the tremendous amounts of dioxin sprayed in Viet nam, where the eradication of veg etative cover and the destruction of the enrivonment were deliberate military management objectives, the environmental impact is still not clear. Phillip Handler, presi dent of the National Academy of Sciences, summed up the findings of a N A S study on herbicidal war fare in Vietnam in March 1974: " The volume of merchantable timber killed by herbicide spraying proved to be strikingly smaller than previously reported in preliminary estimates by others .... 2,4,5,-T and 2,4-D are rapidly excreted in unchanged form by most animals, and there is no evidence for ac cumulation in any tissues or in the food chain." As for reports of mal formed fetuses, the N A S report found " no conclusive evidence of association between exposure to herbicides and birth defects in hu mans." As more refined laboratory mea surements turn up traces of dioxin in humans and in the soil, it's useful to recall the remark social com mentator Irving Kristol made about class conflict in modern-day soci ety. " If you have to measure class conflict to find it." Kristol ad monished his fellow sociologists, " then there isn't any class con flict." Similarly, the reason dioxin is so difficult to measure in the soil is simply that there isn't much there. Studies suggest dioxin de grades fairly quicklyanyway, by as much as 50 percent in a year. Nor does it seem to appear in plant seeds, or readily dissolve in water. It disappears rapidly in vegetation too-- some tests show that 80 to 95 percent vanishes within a month. 788/Journal of FoRESTRY/December 1978 sJ O i ;uik is the amount of di pro ami sledgeha: aoxin x f that some experts have ..Jpects.-Bi concluded that only people with cent stu suicidal tendencies and enormous bicides.' appetites would ever become ill .. ricultural from T. A one-hundred-pound hu- 'ilfshould c man, say these experts, would have strained f (Theto quickly consume all the dioxin on one acre of sprayed forest to F sufferany illeffects. While thiswri .gust / ter is not anxious to lose 65 pounds o f the and test that claim, the example . Techm does dramatize the unlikelihood of /;! A dio.' harmful side effects from proper formed in applications of the herbicide. ..-/. t0 make T Caution should be exercised,' ^ cance of too, when taking lab test results at, years ago face value. It is obviously uncon-:*.vi centratior, scionable to dismiss or ignore such', sufficient results, but it is also hazardous to-if industrial infer from them that humans would,.eliminate suffer the same effects as animals.; (jon meth Despite the painful similarities; . routinely' men really are different from mice wjth and rats. don't develop the propensity to'-}jproductioi grow tumors that laboratory ani- ;|lj0 have r< mals often do when they're sub-- .^an impurit public anc doses of herbicides. In one test,75 .,3sufficient percent of the male mice injected --^ become ap with T developed tumors, but .so- chemicals did 78 percent of the mice who. ling the co: were given nothing at all. In fact,,y amount of the female mice which developed, ;|into the en tumors outlived those that did not,. ; T C D D These results are cited by Richard;' ajife, arid it Ellerby, president of the Oregon-. Joral LDso Society of Clinical Oncology, ,,to *v^|pigs to 115 allay the Orwellian speculationy are less sei often fostered by lab tests. Besides,;;*,*:; A m o n g lab tests also show that tumors;.^' hemorrhagi spring from steady diets of egg ^organisms. yolk, bracken fern, and even water, So the old Aristotelian maxim several wet isabsorbed " moderation in all things" applies,,.}; and elimina here, too. . -`ithrough the At any rate, organisms in the |is eliminate field never receive anywhere near W Birth defi the dosage ofeither T or dioxin that tered durini lab animals get. In terms of practi arc forming cal application, therefore, many;^ fetus rather experts do not consider T car-jjj-jismore ofa cinogenic at all. That conclusion) '<{death of the isn'tsurprising, given the legal limi-, of the narre tations on the amount of dioxin al-" $ level on the lowed in production-grade T, the T C D D is cl lower rates usually achieved in -;1 The evidt practice, and the fact..that most vijOO.OOO tim stands are sprayed intermittently, depending o say about once every 30 years. the Environi Whether these facts, or any. T C D D in cc others, influence E P A 's judgment in 10 million 7514on registering T next spring isany- about I part i one's guess. The arguments both dose of TCI ' ' * 20,000 toxic  *tml con vill be delivered with .`eehammer subtlety, one sus- .els. But the conclusions of a re fill study. " The Phcnoxy Herddes," by the Council for Ag.ultural Science and Technology oi'' check some of the unre al invective. C A S T has its d e t r a c t o r , w in - ;n>-,i;! to m e m-.:'.-, puise i f .dii'tri.il mune> behind its research program. But CAST's independent member-scientists choose their own research projects and count among their number some well-respected authorities in the herbicide field. The abridged p o i ln M l I C| 'I i i l t o l I'C RiA t.itions, whiuf"*I deleted) iaigel> concerns theV .ndings on the tox icity of T, probably the aspect most crucial to the continued use of the herbicide.-- L u k e P o p o v ic h 'The Phenoxy Herbicides, Second Edition, A u gu st I9 7 S, pa ges 20-22. R ep rin ted by perm ission o f th e C o u n cil fo r A g ricultural S c ie n c e and T e ch n o lo g y. A m e s. Io w a .) A dioxin contaminant referred to as T C D D is <rmcd in the manufacture of the trichlorophenol used >make 2,4.5-T and silvex. The presence and signifi.mce of this impurity first became known about 20 .-rs ago. 2,4,5-T formerly contained T C D D at conentrations of 1 to 70 ppm, the higher levels being ifiicicnt to cause skin eruptions called chloracne in idustrial workers. Although ithas not been feasible to 'iminate this contaminant entirely, present producun methods are able to reduce the dioxin level uitinely to less than 0.01 p p m in commercial 2,4,5-T ith occasional batches containing as much as 0.05 pm. The average level of dioxin in present U.S. oduction of 2.4,5-T appears to be about 0.01 ppm. ;>.have reduced to a thousandth of itsoriginal content n impurity in a chemical already widely used by the ublic and recognized as safe would appear to be a .ifiicieni solution of this problem. However, it has ecome apparent that T C D D is one of the most toxic V.-micals known. This poses the complexity ofassess:ig consequences of the presence ofa very small me of a very toxic substance in mixtures sprayed to the environment. T C D D is toxic to laboratory animals at all stages of ;fe. and it also is a weak teratogen in mice. The acute ral L D jo ranges from 0.0006 mg/kg in male guinea igs to 115 micrograms per kilogram in rabbits. Dogs :e less sensitive to T C D D than rabbits. A m o n g the effects are skin damage, liver damage, emorrhage and reduced ability to cope with disease -rganisms. Death from a lethal dose is often delayed everal weeks. Following ingestion by the rat, T C D D >absorbed from the gut, localized in the liverand fat, nd eliminated largely via the feces but to some extent trough the urine. About half of the ingested material >eliminated from the body in the first 17 days. Birth defects develop when the chemical is adminiscred during the time of pregnancy when fetal organs ~c forming. The effect is directly upon the developing ctus rather than on the genetics ofthe mother. T C D D smore of a toxicant than a teratogen. Itusually causes 'eath of the fetus rather than abnormalities. Because the narrow range of dosage between the no-effect :vel on the fetus and the lethal effect on the mother, 'C D D is classed as a weak teratogen. The evidence shows that T C D D is from 5,000 to ''0,000 times more toxic to mammals than 2,4,5-T. epending on species. Under current standards set by he ironmental Protection Agency, the content of 'C D D in commercial 2,4,5-T must be less than I part i 10 million parts of 2,4,5-T. lapractice the content is bout 1 part in 100 million. O n this basis, a single toxic ose of T C D D would be contained in from 200 to '0.000 toxic doses of 2,4.5-T, depending upon the test species. Thus, the current level of T C D D in 2.4,5-T does not contribute significantly to the toxicity of herbicidal preparations of 2,4,5-T. For a person or animal to be poisoned with T C D D without first being poisoned with 2.4,5-T would re quire the existence of a mechanism in nature that would separate T C D D from 2.4,5-T, greatly concen trate itand make itaccessible for consumption in food or feed. Any postulated mechanism must take into ac count what is known about the environmental fate of TCDD. The amount of T C D D distributed in the United States in 2,4.5-T and silvex isprobably no more than 1 ounce annually. This material is distributed over ap proximately 5 million acres at the rate of 5 micrograms. per acre. The most sensitive species known is the guinea pig. which has an LDso of 0.6 microgram per kilogram. Ifwe assume that we have a grazing animal about the size of a sheep or deer (175 lb or 80 kg) and the sensitivity of a guinea pig, this animal would have to consume, without excretion, all of the treated vege tation on more than 9 acres of land to get a lethal dose. Ifthe animal had the sensitivity of a rat, itwould have-, to consume, without excretion, all of the treated vege tation on more than 400 acres. T C D D fallingon foliage isnot absorbed appreciably by plants but israpidly decomposed by sunlight. W h e n T C D D on leaves is exposed to sunlight, most of the chemical decomposes the first day. Washing into the soil by rainfall plus additional slight losses by volatility serve further to dilute and disperse the remaining res idue. There is evidence also that T C D D isnot formed when 2.4,5-T issubject to ultraviolet radiation and that it is not formed in significant quantities from 2,4,5-T when treated vegetation is burned. Once in the soil, T C D D residues become firmly bound to soil particles and are not appreciably taken up by plants. The residues do not leach downward but remain localized in the surface soil. Microbial degrada tion then comes into play and decomposes most of the remaining chemical to basic materials over a period of probably a year or two. T C D D sprayed into waters rapidly disappears, due to vapor distillation into the atmosphere and to de composition by sunlight provided the waters contain small amounts of organic compounds. Its low solubil ity in water (0.0002 ppm) causes it to migrate rapidly out of solution and to adhere to any available surface on which it may be degraded in place. W h e n sufficient supplies of the chemical are present for a long enough time, T C D D can accumulate in algae, snails and-fish at concentrations exceeding those in the ambient w a ter. This property is not believed to be of practical concern, however, since herbicide spraying does not lead to a substantial amount of T C D D in the environ ment subject to accumulation. Analyses reveal that accumulation in food-chain organisms is not a prob- December 1978/Jo u r n a l of Forestry/789 I. i. I. ti l; \ i it ; . . . - * > > i ..* 3 ! I i( . ! .'ft !* ' i ? ! ; t i'' ' . 1. i.. *i)> .' ; l . iMt ;- o w a v Tnmnra rwrn? n jr v w y.. C O M P E N D IU M OF MflAJO R CABLE LOGGING SY ST E M S Cloogmgipnagrastyivsteemdesscbreiipntgiomn oanf uthfaecmtuarjeodr. cable dAInilrtleescrpntleayctiicfooincmaatpliaoSrnyasbstlaeerm.e goifvUenniintstahned are ' arvSevapparerdiceleiasfrbeisclneaatt.anitodinv1se6fcodrroifs5fse9-rsdeenicftftiecoranernroitafwgweinhscaghtievissi-ng a Sefoiugrmhcmtarmariraayjgotearsb.claebslgeiyvainrdgisnpgeccliafiscsaitfiiocnatsiofonrsthaend More than 60 descriptive illustrations. 1A1l2l opfatgheiss, iinnfoErnmgalitsiho.n is concentrated onto 5oA.p9euXrna8itq,i3uoeninsacnahsdeswc)oeilmsl adpseascuitgnbnioveoedkrsfio(t1rie5usXs.2e1incmfor--est Price: o61v----e5r10c10ocopcpioeipseiseUsUSUSSSS2S520e18aecaehcahch Pft&StrZsVMf^i piTiepL';M(o4.6B)joiKfl3C0rbsliia8lti'2grgidriintfBStStWemedenFi 3 E.S.P ACOvSaTilatbFl^eCCTtIovEallPOPEoSr;estWAimiaAnOaEg^eErNTs.0 E.S.S? 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PAAREOMRCIOEANLSTSHILPNYHGOTSOETGORRVIAPCRPEOHVYITOaHEA|TTHYCOOEULMEBCI1TNTREmOSNIPCRECDIATSAION 1?OpDAATTfhAeNhCTrOECaAmSUTLIaRLOZnIAOmNEfeFTa.GEEnyEtIRSCBN.YEpIDCThILEoUVEtTI.oPDIgUROLArFEILaPZpAEahQDSiEITcOTITEMHSoABRCoEORRcRUEEuGAGH*S*GOAc7EURAnTONtOls.C,S.S E.S.F HCATShAANOaSNOUtTHSAMEFUAiAFNCtOSAHRTGcOVEa!IRnuOSnCNiOGtFVeAdCeLCeAESNPTTD3AfASTNECES*1E*.DA.|NNYTAOTTHSEFIAOZREIPENRSDIITUNCSETcTRhYYnEOcUrs FCMORE INFORMATIONRITEORCALLt 0AYT0NAlARPB.BO0ERA.NCtH8.00*FATLA0O.1R3IDIANC.#32022 790'Journal of FoRESTRY/December 1978 hits been widely inve^-- ited using methods sensitive ; to as little as 10 partV. -*r trillion (ppt). Amounts of, T C D D sufficient to cause direct toxicity or birth de-fects. however, have never been found in food,or..; water as a consequence of proper herbicide spraying..^ In an Environmental Protection Agency-directed : test of 85 samples of fat from cattle grazed on pastures> sprayed with "2,4,5-T, one contained T C D D at a con-.',' centration of 60 ppt, two had approximately 20 ppt,*.( . five had 5 to 10 ppt (lower than the test's reliability),^ Timberk and the remainder had no detectable T C D D . The sig-; Decemh nificance of the positive values in this survey is ques-f* Continu tionable for various reasons including (a) inadequate^ sored b information on the sources of the samples, (b) the dif-IV Georgia ficulties of analyzing samples for such low concentre-j;] ia ,Kra! tions of T C D D in the presence of interfering sub^ Institute stances and (c) the failure to identify T C D D in anyof ?coorien the corresponding liver samples ( T C D D normally ^Georgia cumulates in the liver concurrently with accumulation.^ tion. Su in the fat). In any event, the average levels of T C D D :iifpiarinin; found in the fat and, indeed, even the highest Ievcl;U Manage represent concentrations well below the no-cffect level's :Will be 1 established by EPA. In the only formal experiment^ -with n0' designed to test the accumulation of T C D D in animals' t act : Dl in the wild, no detectable amounts of T C D D [were--, found] in tissues of mountain beaver at the end of45 to.': ^ "caii 60 days of feeding on vegetation that had been sprayedj " with 2 lb of 2,4,5-T per feeding period. acre at the beginning of the : ' V' NNoonrtghan Because the general public First became aware of ` T C D D as a very toxic contaminant in 2,4,5-T, recent * poisonings by T C D D from other sources have been^ improperly linked to herbicides. There have been sev-`"t eral accidents involving high-level exposure of persons to T C D D . One was an incident in Missouri involving waste oil used to keep down dust in stables. A recent; and more serious incident involving T C D D occurred WsMgFohbcooaaafiornilmbrrdnietknstechsafHeoettafs-li: in Seveso, Italy. Herbicides either of these poisonings. The higher levels of exposure of were not incidents m a n and involved in 3 involved far ': animals-;to\ ; tSAaecvrtve:i.c.Re.* . dustry.T C D D than are encountered in the field use of2,4,5-T;;\ and serve to document the safety rather than- the ^GCeonneferar hazard of 2,4,5-T. In the Seveso incident, forexample, T C D D was released in a village at rates per acre that were millions of times greater than those that occur. from 2.4,5-T treatments. People continued to live in the contaminated area for about 2 weeks after the ac-^-i cident. A number of animals were killed by the fall-out" '' tsSaepcaorRncesh<i ''lMinagd,isi of phenolics and possibly by other chemicals, and :.Aerialthere were numerous cases of human chloracne. No '.; 'terpretcases of severe human illness were attributable to e.X;, . posure to T C D D , however, nor was there an in- ; creased incidence of human birth defects. O- Evaluating the hazard of T C D D in the environment ; fwsUiocinreieidvdioeifi poses the problem of comprehending numerical values far removed from c o m m o n experience. One islikely to be so overwhelmed by the very great toxicity of' T C D D that he fails to comprehend the infinitesimal levels of exposure. One must recognize also the long history of safe use of 2,4,5-T containing a thousand or more times as much T C D D as at present. The evi dence indicates that the T C D D contaminant in 2,4,5-T and silvex is well below levels hazardous to humans and other organisms. `1[tsmfpsfsUtFerioicahermtaornacnoysnmrlitphsteea:vool;e,gsfee:1i 7516 A i t*V l Tn*ESlsdevtniecrtSocfietnhueATcotPaulbSliirsnhro*nmCeomm,p]a0ny(1,3A75m)st1e0rdJ-aUm3- Printed in The Netherlands CREAFRECRINENOCGEENTOICOrCRLCIAENSTORC4HGLOORFICNHEEPMEISCTAICLISDWESITH PARTICULAR, MELVIN 11014SwatsfieU Hood, Columhie,MD 21044 (VJj L) Received October 14th. JS77; in Anal form February 2nd, 1375) ABSTRACT The organochlorine chemicals comprise a large somber o f pesticides chat are used widely throughout the world. The organochlorine pestiddes gives in the diet to mice . ,, are eartinogenie for the Ever. They induce sot only cardnomas of the Ever, panic* uiariy at the higher doses, but also cardnomas and sarcomas in other organs in rats. They cause acute and chronic Ever and b'dney injury, which interferes with the devdopment o f cardnomas and sarcomas in rats. The testing o f chemicals for csrdnogenidty, with particular reference to organchlorine pestiddes, indudes discussions on the following topics: classification of hepatic lesions in mice and rats, diagnostic criteria for malignant tumors o f the Ever in mice and rats, toxidty versus cardnogsnidty in the testing of chemicals, a comparison of cardnomas and drrhosis of the Ever m experimental animals and humans, and the significance o f laboratory earrinogenidty findings to human health. .* INTRODUCTION The organochlorines comprise a large number of chemicals that have anesthetic and narcotic action, many ofwhich are highly toxic1 There has been wide distribu tion and use o f the organochlorines as agricultural pestiddes throughout the world. They are appEcd by all the conventional means using the common types of equipment on vegetables,firuit, forage,and fiber crops. Examination oflarge numbers of samples of specific food product classes coEeeted In m ail food stores bave.shcwn that they are contaminated by organochlorines. The organochlorines are absorbed into the system following inhalation, ingestion, or contact with the intact skin. After ingestion into the system they are stored to seme extent in the fat and Ever and to lesser extent in the kidney, rausde, and other organs. The foens in the toxidty and earrinogenidty studies has been primarily on the reaction of the Ever to the organochlcrine pestiddes. Certainly in the mouse, where we have thq_greatest amount of experimental data, this is the focal point of the * Preseci address: N O Frederick Canear Research Center. Frederick. MD 21701, U iA . 0002180 ooW 35764!) \ 107 distinguish between hyperplasia and carcinomas, rather than to use the term "hepat oma" as employed by some pathologists. The widespread and varying use o f this term over many years has left it, in my opinion, unacceptably vague. It should be noted in reports from feeding studies that metastases from carcinomas of the Hverto lung or to other organs are not reliable unless, among other things, serial sections are done. Serial sections were not done in any of the studies on the organo- chlorine pesticides. In addition, multiple sections of the Ever are also necessary to properly evaluate the lesions, pardenlariy local invasion of veins o f the Ever by carcinomas. Small carcnomas less than S mm, for example, might be overlooked on. pass examination. ^ THE UOORTANCZ OF BmXKASTJC NODOTJS OF THI UVfJt DOW 357651 Carcinomas develop in the Ever o f animals only after the cells pass through sages of hyperplasia. The earliest sage, referred to as "areas" of hyperplasia or as "hyperphuia" is seen during the early weeks and months of the administration o f certain chemicals. The change at this sage may not progress to that of earrinoma unless the chemical or "somult" continues to be administered. IT these hyperplastic ceils continue to grow, they form hyperplastic nodules. The cells appear much the same, hut have increased in number enough to form a nodule. This nodule is a mass which compresses the adjacent tissue and can be seen on careful gross examination o f the Ever. This hyperplastic nodule is precaxrinogeaic and has the capabiEty o f developing into a eardnoma even if the chemical is no longer given to the animal34~ 3*. Cells in hyperplastic nodules grow in sheeo. Additionally, the cells are also charac terized by the following: the ceils are increased in size, there are double nucleated cells, and there are mitotic figures. In nodules which progress to well-differentiated carcinomas, the cytoplasm tends to be eosinophilic; whereas in nodules which progress to poorly-differentiated carcinomas, the cytoplasm tends to be basophilic. The nxe at which the hyperplastic cells become hyperplastic nodules and carci nomas depends upon the potency o f the chemical as a carcinogen, the dose o f the chemical, the length of administration of the chemical, and the route o f administra tion. Growth also varies with the species of animal, the strain of animal, the age and sex of the animal, and hormones, as weO as lack of protein in the diet. This change of hyperplastic cells to nodules and o f nodules into oronom as is also dependent upon the health and nutritional sum s of the animal. If there are acute toxic effects, the animal will die from necrosis or insunt death of ceils and organs, or from acute infections diseases. The animal can also die as a result of malignant minors in organs other than the Ever, or from chronic diseases, other than maEgnant tumors, in the Ever or other organs. In the analysis of data, the number of carcinomas of the Ever is not the only indication that a chemical is carcinogenic. Animals may have hyperplastic nodules in the Ever or other changes of importance in other organs. A fuE evaluation, therefore, should involve an analysis of variants tzking into consideration ail available data1*. In the ease of the Ever, this indudes the most advanced lesions of the Ever, Le^ * U . oq 0002182 t 1 109 Hepatocellular carcinomas arise from parenchymal ceils and often retain the vesicular sudd and sometimes the eosinophilic cytoplasm. Poorly-differentiated hepatocellular cardnomas grow in sheets and generally can be fairly easily recognized. . The criteria are those for anaplasia; (1) size and shape of nudd and cdls; (2) presence of abnormal mitotic figures; (3) cytoplasm usually basophilic, but can be darkly eosinophilic; and (4) nudd vesicular with prominent nucleoli. Wdl-differentiated hepatocellular cardnomas are more difficult to recognize and often are incorrectly diagnosed. Histologically these cardnomas retain many of the characteristics o f normal liver cdls. Criteria for wdl*diiferendated hepatocellular carcinomas need not be those o f anaplasia, but are the following: (1) formation of cords, two or several layers in thickness, with the formation of canalicufi and often prominent sinusoids with fining cdls; (2) cytoplasm usually darkly eosinophilic, but - ' ean be basophilic; (3) cytoplasm ether decreased or increased in amount; (4) nudd vesicular with prominent nucleoli; and (5) absence of double nucleated cells or mitode figures. Formation of cords with canaficsfi can often be the most hdpful indication of weU-dlfferendated cardnomas. Occasional cardnomas may be made up predominantly of canaiicufi and can contain bile casts. Glycogen is rarely present. Cholangiocardaomas retain may of the characteristics of bile duet cells, Lo, columnar cells with lightly basophilic cytoplasm and oval nuclei. WeH-differentiated chclangiocardnomas are fairly easy to recognize. W dldiifereadated cholangtocardnomas: (1) form ducts sometimes containing muon in the cytoplasm or lumen; (2) cytoplasm is lightly basophilic; (3) "desmoplastic" back* ground of fibrous connective tissue; (4) nudd are oval with inconspicuous nucleoli; and (5) lack o f mitode figures. Pooriy-differendated cbolangiocardnomas grow in sheets and have: (-1) oval cells and nudd with some mitoses; (2) lightly basophilic cytoplasm; and (3) fibrous connective tissue stroma17. They appear to be attempting to form duets. Undifferentiated cardnomas grow in sheets. They are more anaplasde than poorly* differentiated cardnomas and have: (1) great variation in size and shape o f nudd and cells; (2) basophilic cytoplasm; (3) frequent abnormal mitoses; and (4) nucleoli prominent and often more than one. The most frequent sarcomas o f the fiver are hemangioeadothefial sarcomas. WdMiffereadated hemangiosareamas retain many o f thecharacteristics ofendothelial cdls1*. They are made up o f (1) spindle-shaped cells with spindle-shaped nudd; and (2) vascular spaces filled with red blood ceils. Lesser differentiated sarcomas are eadly diagnosed because of the anaplasde characterise. They also form some vascular spaces. More anaplasde sarcomas without vascular spaces can be refereed to as Kupffer ceil sarcomas. The fiver must be examined carefully for hemangioeadothelial sarcomas. They ean be small and grossly resemble an area of recent hemorrhage. Sometimes the cellular areas are aiso_cbscured by die red blood Us on the histologic section. Malignant lymphomas, particularly die redculum ceil types, are also seen in the fiver'4. Redculum cells are large with a large sudeus with prominent nudeoius and an ' DOW 357653 7520 ................................ 0002184 \ 111 from those observed is rats appeared is print. Before this statement could be corrobo rated, it was repeated with greater emphasis being placed upon it each time. Several years ago I carried out a transplant experiment in mice, repeating previous work in rats, to see what differences existed between these hepatic lesions in mice and rats*3. Carcinomas of the liver developing in mice are no different, histologically, biologi cally, and in other respects, than carcinomas o f the liver in rats and hamsters. Varying stages of hyperplasia precede the development of carcinomas of the liver in ail three species. Hyperplastic lesions cannot be transplanted; eardnomas can be transplanted. The histological patterns of the eardnomas are the same. They kill the animals with or without metastases. Cardnomas developing in the liver of animals dosely resemble those seen in humans*1. The gross and histologic patterns are the same. The well-differentiated hepatocellular cardnomas in animals and humans both metastasize to the pona hepatic lymph nodes and to the lungs. The poorly-differentiated hepatocellular eardnomas spread more widely throughoutthe body. Cardnomas in both humans and animals can cause death by rupturing and bleeding into the peritoneal cavity. Hyper plastic lesions are sometimes seen in humans. If humans, like animals, die from some other cause earlier in the course o f the disease, hyperplastic nodules can be observedin the liver. Terminally, though, hyperplastic nodules are rare in either animals or humans. The role o f cirrhosis in the development o f cardnoma o f the Ever in animals depends upon the chemical and the carcinogenic potency of that chemical. Chemicals that cause both cardnomas and drrhosis, such as aminofluorenes and carbon tetra chloride, need to be given in large doses, sometimes over a long period of time. If the dose is too large, the animals will die from drrhosis before they get-the chance to develop eardnomas. Cirrhosis often accompanies the development of cardnomas in humans. That situation then is similarto that in animals, except that the toxic agent in humans is usually alcohol or viruses. Chemicals that cause cardnomas only and not drrhosis generally are extremely potent and can be given in much smaller doses; the length of time depends upon the chemical. Here again, the animals die early from acute diseases, such as hepatic or renal necrosis, when larger doses are administered. The organochJorine pestiddes would appear to fall in the latter category with regard to animals. Chemicals such as carbon tetrachloride and mercuric chloride, when ingested in large doses by humans, cause death by either hepatic or renal necrosis. Chemicals in this group that cause cardnomas of the Bver only if given in the lowerdoses for longer periods of time in hnmans have not yet been denuded. StONinCANCE o r THE tABOJUTOJUf CAXCNOOSNIOTY JINDtKGS TO HUMAN HEALTH Cardnogesesis In man can be detemed or reliably predicted in either of two ways: (1) by direct observados of two groups of human beings, one exposed to a suspemed cardnegen mid the other protected from such agent; and (2) by one or more bioassays in experimental animals. The nrst type of study, labeled an epidemiological study, is o 357655 r r-^2.1 B t \ 113 jperies should be deemed to have relevases for other mammalian spes'es -- including mao. Quantitativdy, one must assess the relationship between the dcszge required to produce tumors in test animals and that required in man. This assessment, difficult as it is, is complicated by the fact th at44man" is much more variable in his physical characteristics than are most other species of mammals. One can state, however, that h is quantitatively impossible to establish any "safe level" of distribution of a caret* nogen in the environment. A safe level may exist for a particular human being, but because there is such a variation among human beings, it is impossible to establish such a level for a community. Furthermore, review o f the literature on carcinogenicity tests suggests that no such "null effect" threshold or "safe level" has beendetennined. . in test animals-- much less for man-- forany knowncarcinogen. Given the state of cardnogenidty testing and knowledge, there can be only one safe tolerarme for a carcinogen -- an absolute aero tolerance -- and the only way a substance shown to be carcinogenic in test animals will aot be a threat to human . health is amply to prevent tbarsubstanee from entering the environment. To the * extent that it does enter man's environment, it will constitute a very real threat to his health. Furthermore, acknowledging the difficulties involved in detecrisg carcinogens' in the human population, this threat may not he manifested for up to 30 or 40 years, given the long latent period of many known human carcinogens. Based upon this statement of principles of cardnegeaidty testing and the rignificance ofsuch tests for man, one must conclude that since the organcchlorine pesticides have been shows to be cardnogenic in mammalian test animals, they must be conrid* ered as potential cardnogeas for man. can q .tstoNs on Toxxary and o u u s N o c e n a rr o r oxganochlownx m u c o s The organocblorine pestiddes given in the diet to mice- are cardnogenic for the Over1*. These hepatic tumors are malignant, invade and metastasize, and grow on transplantation to isologous hosts. Oiddrin given in the diet induces significant numbers of malignant tumors in mice a every dose tested, induding the very lowest dose of 0.1 ppm4. The organocblorine pestiddes induce not only malignant tumors of the Ever in rats, particularly a the higher doses, but also carcinomas and sarcomas m other organs a doses as low as 0 J , 1 or 2 ppm in the diet*0? **. Rats at the lowest feeding levels also develop noticeably ealy hyperplasia, a weQ a hyperplastic nodules, and occasional cirrhosis and cardnem a of the liver. The organocblorine pestiddes, when given at high doses, cause considerable acute and ehrosie liver and kidney injury in rats4*-4 *. Necrosis of the liver and kidneys or chronic nephritis not only resulted in death of rats, but also interfered with the development of carcinomas and sarcomas. Syperpiatie nodules in die liver would most likely Have progressed to eardaom a of the liver, had the rats been healthier or survived for a longer period of time. The carcinomas of the liver and other organs and sarcom a observed in rats and o ^357657 000218 112 fi O fc tU338*JSi*fcfcfc M - fi&iaSiiafctBSWtf 65 66 DFM01MLJTMMRJMMgHACERMMMMMMMMMMMmMMMMMMMMMMMMMVC9.n.S.ai..R.am..7et..........Wo..................T.MppGnISi3DiXKJCODnOSDOOOODOODmdODODDD_DDDSDDOsatOD.i2eueo.d.tri.einn...e.....o...Pm....n........i.O..aundiF..ORnDrnRRtRRRsRRReRRRRRRRRRRRRRwRR.kRRldadoripRRUaoros.otuSauSianueseuueuauirusuuuuuuuuuuuuuuuireKmb.truuuduutafutaubbbVubblbbabbbbbbbibbbb,bbbt,,bAnuelbbbbbabttek,a,,JaaaaaaaaaUaaaaCaaaasnaaQHaa.dceaocriruara1rtT.rrrdSrrrrdrnrrYrrr.rrrrrrrrrhrI,),,,9er,,..,.r,,,,tT.,.,oE.arrSfano,aaLapaaa,a,F,*v7JfJLaHS7Sia3JJJ3CHilSGTnnl.a1nnnxnnlftntAlnd5a.cn..ua..f.i.eHe.r.*H.idd9paYd,edddetd,matiyn.isffHf.dijsfJGNfhr7at.a7anmWt.ii.)VipJnpairiyCutiamlJoHSVEandH..A4iaaEMeaC.iSneaaaoapapf.aisbn.aiSSi.d.do.nd,.Sittitit.hreia..ts..etf3rrirfLe.diitbroLC,ieOilKirWp5.i.LiaieL.kfLin..HL.uNo3otwp,HMdedcD9oiOitf.tnECeCCC7.eiCCrCdF.t5ni.tCrrt.^a1i.anasu.HnoneGxxsFo-O(H5iGpGSaaBaaaai.t0aiaia.mL91cisUea3etierdlasnsoninnefn0Ciininan(.9dTo7iVuhn0CiioneryYaoiah,e1oesrattxtodteat6tnpt.n1,aataaaaavaar,,aEni9sHai(vfaitalv3xrpH,9idrmmrra1frroferrraR2Le6inianntaH)Jiutm7SoHr9yAktr5,iI1bbr.9brIbriytn,f\ita45Hn,b.2..nap3o4nn,i,tay)1bcatay^aadpo(i0,(DHJam3fJCmMra4GaaJnepsan12ra1irrpe7uuptru9t.o)pa.4r/,.d(iut,.a99Mr,,iy^hta9,tbedi(ep1,p.ef,mHraG22,irJ.uf67r1Jpuirn.Atrli9pii.233Pe35epi3M3ie44d16E,..9o.iaate.a(2ct4ii3nrr6537d463i1r.w)51)orfr4eua,,SLnoa7tip0tmiCtii1Lo.i94xWtr0i5a5ins((d(t(f(.a(at().(ibeia.3-d1o1i17ia1(1a31o1)3f13.aii7dino1ra9Qm3CC99e95na99C9nQ.9rsn9.,f3i2aA39ni9(.66.ei)6ar676n6s7.Xteaa(Nua1aapiA.3.a3uN271no5546,Ctn9na0dnnC91naT7.7u.re))a9i)1b)a))))edla,i16tetada.eS)auweoafdb69bnamta9d93m39745nn7rad1mm.Cnxr677reltrK905i3b.a)43e7,d2Feinu.ita3b)6sfb1I1aa9q3ea75hr5IF3t53iofn.irhmt5m.nrr.rd..HD1...rat7toCe1aconr.tsyi.0ai.lDnd3iiuoi.oeua4sinte^npCR9ee..pdi1dn,r,,yo9eaufisnl(4caaaaa4d4m..i3tEbCoid(Wd,,Ln-4trnfr431/(ii1n,,aditef1oa9tna32p92(C(eSsc(ee9ams161197r19ausaami7r)ea(.399359ban.nEb(r6r()717.f6ps1tDd1stoT)(uel000it-7191iu9ahoanSer2))a)S169i6(aaTob.df.^n1f443fa3332emn1aiL9V31U15)cn)9tCris9xk7.s)a99n1tapa917iaanrat3.s.e.iir,1343itna)tsyeaa703D.ieU1adniLan157n.9Atff,it..o.Ei,sx7sde2rattt3A1niakiaors.H9d(retitn17aineriee9n3aLpSsnp6.in,nitAa4btouitaSnm)triodfoart5Mpctnmise5ttrDtsileaaa1,iu.pdIUiIrEninamDaCfsrtnjreeaetieaoasvftnCenitpiiinnteeanrniseiayrmatat,,t,i DOW 357651 0 0 0 2 V 90 ( ma IS S 7 7o  H mr:tuomipeeeinspcfdiolxemas1pucea[.tt7or8inanhIove]r1lnddeesf-tirdevedm.se[elt9sahn1dcainee)t miielegaefettofkrpshenetiuaroiirmenrzene-esdnne*ts, wuelcearnad- i i !i i 1 *col., 17 d,, 146 t7y6.P i , 5 (1975) acol., 19 armacol. TOoExiUcotTlowgry/.N9or(t1h9-H78o)ll7a6nd--6S6cientific Publiahara Ltd. *>>-) 11 m < ) ! SICINMTOFUMNEDUCCIENETNESIOTOSNRUNA:PE2TPN,IR3ODE,7NSO^SST-iTAOOENXNTDIRNAEA>HFCfFYtHEPLCECORTRRSO&OEFNmATSSBHIEETYDNIVMZRIOOTESY*SIpI,NS.SDTETIARORNUEXCAMINETZJMTNIONEDCNUTCED JN.GO.OVROLES,JAJ.GNS. EKNREEFTENBERG, H.W J. ENGEL, A. MINDERHOUD and LJd. V, National InatituU of Public Health, P.O. Box 1, Biltbovra (Tha Nathcrianda) ((RAaccctehprtaeddAOucgtuoabte2r 33rrdd,, 11997777)) UNO 7020218*30686133 SUMMARY dsdstAatlmwsrlsbzultbFyyrihiehiteoniiknoiosieuilmdoldilyelndtsSentceikaidxdhomutohucealutprnnyeiyy,cfmtzlmnebochouiolseT,isrvytLrnlifwsdodsteue,noe(-ee,deicncTcutclicnagasripathnpyeetteeteusClte,tleoliaceotrrneleamlvfwslerrseDsldootf-vfedeisesomoefdpiaeaeddDaiaoatfcrbomrlrhuhzenlostestt)meterlioecychdddserenssTtctxwereoeatoiibpicrasamhselpioaCnineyroitneultefkytnearyeoomeaDnficnsamenoesaeedcctfoeDdtealvonnhccryeiTnynuntioneoetttyfdinhcshotmdsn-ieptojmtnesletoeoegfyhiytwhohddndmafcxumefpumaaehfyotttdeinnthgsricuntnimiTsmuedopghegaektaentepnsCoshhsihtahiscngiiisoc,r,icssttloottaDooeoynyheeeaoncntnahncwsDwneriofyilysnnttstopomaerodnreeotoitdrdsvioTttpeidni.txwmmmthcmesinuhCmh,Itmciil,lencitifysauaopaDttmkrtuoaiefcahhtnnthtateceeaenDnfirhoraaeeionyhtlcsordycgycashtssummttepthrmuemntishebmricoohoennoecpeooeadoeevnorpaaaaniodipsnteuicnecbrdghusditnar-rnalkudoaewtearssdebdahldenercpegorfsrylriddetoessu2ieetoetbemeendiphslop,cfetodnyred3pf.oibxioauninaupf,iaeoaSnt7yicrifielncArcnncoccr:fsarsit,nrotes8ic.edtivolsaieepvkhihdrtmd-aoopeoIosaitereainuynorltneclhnsontevcannhlpcnethyial-noneetorntctyeydteostgahdmseodraefgmorsteftplwededcshsltnsbiiaTaaoaTheuoousnn.eypiitnrtntftsClrenxCsdmmhuaorrdibtesctienuDetmoherdDihceeeduotsspci-oeypDnimyreDbtesevidheuatnttmenliaasf-e.isiawumt,dftbibjfouturytngfeoeterIirshiesesfpaoeocdnlcco.otcintceirdptcsuyumttppetdkiTtzyyrt.ohttcpdhnhoheaoereihtAoenectydyoo-ystasniidneirpessess.,,t.- INTRODUCTION 2,3,7,8-Tetrachiorodibenzo-p-dioxin (TCDD) is a chemical that can be 75 7525 0003691 hmtrtreonifbaciwceiisomftmiia[mnnnnnihouhuMiehddynbtnereenhrctdrsayafepnreeeetoedttmeeapmroencnercmubaommnpcntrmScetrCokpasyyogeiticureeaetaeenicuiniasehcefpneeednsooocdoanstdiedfaswstsnddyaaen,encsnsdesiee-isrltitdtplveeleeinyaacrcaidmirhsynTmosstwepchhaioytitely,hveienndt2oCsmnlisktseyfaadiimelhlstsyouoeemnc,enanphsdmDes4ouemacsecofasavseovuooeesrp,encDn.fhnisaasle5fflrwusdseexesTtaiidretldIrltlostna,aocuapitaloninh-taeheCni,arlsoftrthinmmdtnsnlnMieoaermlctaribnDtuyecirldboi[tshsfAmhcecyticuluepoie1onDhiticnyedtmhemnlalnctptihponyidhe]llimeeh.-v(lkodlsttsrh.,recoiCpiee-psywieneceemmemitmyAamoriricsihnteto,ngydsnrhotreiohoft,ssineosng,ouseuii-spneioniucyinddricncsnueoincttmhsiaasf[milauiyAnhiseniisesdihv4salleffSttpcdtesttalterat.firse)tnou]oeaaaucnfraressabswmageTsnsoanpalytetddtat[aamynerresuxncniatssC1ntt(i[noo,nare,deetyirrTsoddrto51miednDadnf]eostaanmnaoybt-m,.h,ettdeptm1gcDc6tmmhvooe,wfyfoehuTeeuhir3fplogd]ecfotemllfteoceueiiy.ihl,ealaTestatinmfzvetcjsuecrnhner,tyteihTMm)eedhterahnceaaiaoimcaidedstCmitgemaspmntyoctgiy[pfoase.tuiocutm2pDtlwtfnpooorrivsafehieisoitcTsfiroe,ierodnnDgethheygcy3nosmoeeTnnrtdheee.a,ipsteemsro5mnaaiamn.CutreiitrseooIanlgnre]hfhertsuTecxtDrcnnbo,esriohtepeastnscpteCTpacdpiefteDotyifai-shinodaoogtthfodhnshpCDinmltiLnlstrsusipFbioe-yteoyrrtD*iDubseveospuryxseotnaoaTrAnv,dtereo[Denaepedoslirn'ref7reCaetih-npiin4eiaponuhertshnrrm--txascephendugyshU>sehhocs,lapdmxpueopaqD1rosetbtlyaeniaomaeep-i,hnenrtclsu2finsmoahg-issrmeSceoitTecki]iinigocubeaoenusnt.bntsaiiipr-dgeClsitmpdhecumSdddsrtepletoiallrtme,bteceDeryueiuodhirhteunfenmcenalendeoertscsooaDeixdnlamcicsdutndssaiwelntrnupddso-eti]etsebdc,butmmeiioeemunilmnhedyrhnovertaliwelfsbyxretiehtorloeitefittophe(aistsnmhpoe(eduwysadntndsynPuhacuonceoytsvteticegbenwhpkloHahutedtrsmliehstlaertttanthtueaxiidttelhAnhnhuecmbtotvhhyooiuoyroiriyodknodnescgeeyes)eeefefs,-, ertm(tpanoinenaoxsoidrdncunieoenln-o.titsntofpF[eixn1rneitinac7inhstlaier]eflcv,iaolcoyed-dsbrnempeaeirttuhseuioaneanmmxtigttrsiaeoftoozirtccpicrfarnyahaotttcitzyhpooiocohsenoloinaisbunfgoamacelofcynestfmndzepbtirelynprkainaachiyml.dtoliiSiaoaoinndnictncngorsricooxmeowerpiafngpheosaL,eorannsergifssttueattahsehn[ln)esr1ceotiw5otarimr,noeo1emrnele6seeiaoh]ssaisntantulaaunosrntnoddcehcdydiieezentiosoicninomgnlocufeTmtfdndmhCeeeumeidDsfcnioa.ceaDectinhert-rdeonoeexpsrcrppehyehnoodaacsdngunaesoecdndes D0IN0686 134 j\ MATERIALS AND METHODS TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin (purity 98.6%, Lot No. 851:144-11, 76 7526 0003692i aoadkxnriisnaaismcdlolhyllayivslwepdeoixrtihoplinve0tirodr,i1meeadp.e5grnebo,5ntyvst,i,d1Dgte5hroai,wts5dhes0eCtaoohdccreeikfmt1foes0inorc0eleaunltatitCigtoTocnaomCcnwDopcanaeDsncn/edyktnr,iglatuMrbttiaoeoitdddniolysanw.nwidMote,hfiiMgc5aeh0titc.wlhuee.ga,rs/Umet .9lSi.n.pAtFuao.br)rtawsttheoadesf Animate TTtineChmpeDSpylaDpesrre,taeciatcciulfeliTricvaerenspedixoamlvtfehaeorroloige8dsexo0npl0aa-e*nftrrodCiermesw(,eNoanau.tntVetsdrb.wrRaaeedldolretcleSiobcbwnai,ttriuRasrsmmioemtdi.tneiBoacrutedeectadwaumuenm,rsdeTeaethorreeerfnaiNaotrhelnedwe-tshathaeestirrgiltinhalhencetidconosIxe)nnr.iscdatitiitettyiudotnoaesft. Zinc anafyeie mrstiFnhaeigmesofFuttreayeoolrttrucsotwoigmotlnhiibrtoidlhcetyafitadiai1nsobece0entser0deotusryrmtamflpiernltonidiwnoomeafzn-aatistntiishohricdngeesicnllsauooaoasntltfmpewcbdizerpuainanlsrwettcnirrowieaetnihrcatniosaoo5rnsnafd0edssare0pqwudzirjium.eniaPelrct,oeeeouahadcfnksoaidlnslhlcatiotsaeutowtnoiligalmdt-lhchteaeatidercsdtdhf.wwlaoasbeaodmrstleeeuoerrltc.iaipomonAtmnidpofsptnteuwahrrnseaepdmsdreeueicrwxsstutierinhtdlothge. Experiment with thymoein --0s(ttoGritf.ofu2ae1aTna0d5lcvt*hitmmeenoiesdoCuflete/nor.n31ifsnnt0fJ5i.e,5nucTmg,Amgst)temLot,goxup/bopfim.src,tioertieUolhstN,htrpry.npSeoumtatac..qphAestoassieB.sveil)inPltdennyhwdMjuevaiamo0cnitasnt3jboiwedoM9ercdneir0tsar1ii,s,looey0loainsfkplfevsioindx1negroepid,dlTeonuol4nCiybstnT,ieaaDodtCp8csneDttr,Dsteeo/o(rr1kwDv1ii1galiT-ade,imbsCnfesi1dodslaDgt5tdul/eaDimcbynrnaeyedlwnbda)adnayeDtrwdhtidgpr1yaahi.ao8zmsltsse.Aittmqd(uonCiXunscaataokaotd.alattfesclrGv6otomsoi.honnptlGyoulachdrmsemtyrseetnoetrodweteurnrsiiopaianaalnessftl, dsplieothcteoeossrnpodrhefaacttneheid-yvbemtudhfoifdrsediarneil.wdyIensseuakabl,idnctduehiteta(ioPndneoBo,sSue3)ss.pwiIunnepjresetchptdieeoorunfcibsrolsoentdftrwaaonehledilgkitht,trei(grp5rl0roeeudtcpiergsi)evsooepdrfelc2PotBwipvSeu(.l1pyI5sn. Ttptihhegeer) etmhnled. AoLtfiomtxeriuntluhcseoa2ns2st-eadnyaty[1oex8fp]teharenimdthecynomtnatolaspiinneerd(io1dbm,etthgw/emeaelnn)im1so2al.ul5stwiaonenrdew6k2ail.sl5eedns.tgZimeinnacdtecodotnowcxeiitnnh/ cvtroiaanTbtdihiolieinttiyowtnhwasys.ammsCudeesalelstoeusrfrumeshdpianeilnnefdssoieowfrnuitstmhhwe.aenrdueymmebaeedxrecolubfsyimounicsietnegswtaaasngrdleamstshoehvoefidmnaoulgnsedunesirzpeesrnt,esrtiohilnee D0W0686135 7527 0003693 mwainsimadujmusteesdsentotiaal mcoendciuemntr(aMtiEoMn ,oGfib3coXF1140*) bvuiaffbelreednuwciltehatTedrisc(e0ll.0s/2m5lMin) and supplemented with 20% inactivated fetal calf serum, streptomycin (100 jpAgt(dhiiPnhgloaiuWnte/rl(omtyevCiMcsnel(stoes)isyc,nntlplireaGsnere(nAtPcniwi(dbil,eHSflecaikPrpcAottaueh)ei,aotbnarhwcWnreiotacmseinirevvrl(,valliieNliatceLcnylisoausaitmede,(6nar1dd0cUebn0e,0dyop0dLDenpm.an2eIsAbnU,aCgsmmlfNl/.aiat,m/el,amosBrsrrolS)mkewwf.f2c)ia4obksAeacyledeeni)hrnten).dohntwAfctia)i1laulmalfta2lotlestet.r,vri5iravosoKd5aldnmuude0tr1imnsefoyinrldtninue,enl.g,icUgdTro1o,ea0fwJncwtLhso1ampetaeni)rCfmtu,8eluf6t!liytlttlae-iteoMwpfudedofgrtldaueespc[serd6ohWcccn-h.eypu3ecoltHClorloautekuohjncrtpeurueashwelmnvlyaltdaateucmiatleenreiigddninegr- wtnroaipnsl-disctoaimnteeuclianutletaudrPceausc)lktuwarredersel(iqienxupicdroeussncseitnds/tmiallsainttiho(cenpdmciof)fuepnreetrenr3c.eXRbe1es0tuw*ltecseen(lmlss)et.idmiaunlavtaeldueanodf Experiment with endotoxin wesaTTdefAace1inotnhxfnc2ohwaulfjrtpadisII7seeseimdnonole4evo:cclire,tiutaetmtBwdetoni3oslsymhsdsaogx,feoeee8eydweirefero30nixdus,knntt--efTpahmDitstraownwlr4,eC,eieirfrairtwnatafiDdykivtztecwsdmd1hiheDiirotlneode0dnhlseexeeetcait0Lountoodpukn0dpiueatnxoe-cdMdmrs,saobinro1,ileontngleysibniasdom.t,tnsdde5oloTchwDin.emdexseo,eeCat5tnniSxieynnfrtnaDtatteh,iasdirln,r1SmmfwettDa5oofcua3i51oetaetieSi/l--dti0dnkocmnilow,ogsy4t,txaegnhrohM.diyio2wssonnetwb5sEsf0rmifeeo0caeweanm,rltdhdneT1lahefMdae.iikydtci)0Cosgereoc-riaec0rweontDonowTnofkl,ntsioaDiwdm2dCxuenncsiib5aoewtmDtgneEfhodurlpaea0haD.eaneibmtis(tdsscejuolca/sioiadwokan(rtoorptsrlsh0elgitlclleoeoe5avydui.edoip(2nng0e.b0mrsioodfdh.0speia.fmnluf1idtIeetyMckinesrcTnsoelmcdtigeia/alheCfe1tnlrsecfheslpeo0tettcDcn/,deee1htrcehdEirtDrae0ygbahstioii.lml),oevnirlig4et.lyrcceiiot8ddut,odbtdhosAxyheasneooleihn,ieleatnddnaioex.iEnemmnycsrbfpinaSei.enmdnieina.wduomcrcgal0diIlsroesenioolnip.vmteicw1egltwgowileihvteoieh,exwmceteieirnetnhritrarOnano)okdtlegeel..l.. Clearance of Listeria monocytogenes ktpiiholTlnainghoogecfcryLaetnosisisstbietsaernimaacneemdaoosufncreoLelcidi-symttseohrrgidoaeriuntmlteyeosd(n[wo1iicm9tyh]tmi.onMgu2enanidlteeyasS;yiwssn)iaosasnfct-omesmrpicetbechiine(f3iaci--ntito4prnhawvaoeegfenoknocsouynstoo-lidssnpi)soecwcauienflriadce wvtinyeetpnuigeobhua4tst.bley)Fd.wooOuirtrnahdella2yoy.,4rsoaXntwfct1eeor0aJdthawLeyiesseltaekasrtfifatoienrrmtu4othbnwaeoteciioeynkntjoset,gchetweinoieatnhsn, iomt0rhgaoealrsnswp5is0lmeereMseng(siSnTtworCcaeuDirnleDaLt/wekd2eg4igibn2hot/er7dda3y. D0W0686136 i 1 78 000369; ills/m l in 0.025 M) /tin (100 itlotuhreemdagincanavalin H jotkheywmeeid1dr1uectpus lllatacutereder Counting ilavlaedlueanodf ]|1*il imoioicgeictaol ide" Tin *iw. adIeniwgthe^wetO)ko, iSicu0er.1vwivmeinrleg. ninlildgnsleetwhoteirhrradeel ihspmleeennt,. >-ieddnsgspi)osibencwcouateirdnlfraaiydce-e2i4g2h/e7d3, hoofmLoisgteenriiazed[2a0n]d. sTerhiealnduilmutbioernsowf ebraecptelaritaedwtoasdeexteprrmesisneedthaesvli0albogle/scpolueennts. Reduction o f nitro-blue tetraxolium ttKsjcvipfcoaSm(Ltrbm8spnthheGoeuhaei-achgwhf-rdcncaeeerlmttagcLaauliNhhemiiaucsbstmlTsgvg2hreyuerisan.asBeoctigeaepuCormetmbtilisOohsdaeiTcvmfylqpxrr.euyDr,rmpebiue.othu(rftmoSiohcGhDesrecA.yaceGoiepsiiereefnttoegpmIca/ifih-etdkedkneoeosnpmsicadwamNtskuitg1encirahollaanghts.aooeiec3rwBnhsttnneceoidrrturnmbei)a[ss-riedTseelef2uaoooyaisrstot"wsbacnps1msdpuciee5irtuonutemEptt]haaryseei,idl2sthpveitesiUvndouunenes1d;rsaacpaswguwbd,beb)0uretcpl1thsem;calneeoeavemeh0yilethl0etntttdmrnttditea0aieuhpgaeha0ndooosKemecrmrdhetontanmnneirxmeectenXrrudtcbsNogradpe.ouiaotht(tpiopenclesernBnSrcetlaFprri-oalhds/arhragniotebemmTso.trdniiathoeeldkmvhlpeewl2gerpurpbeiwyehdwchr.eetiheere,iwuneewhasainOpardreaaatodttigneehaycaigotseadnowtucefd,si;steayafwdrcpso(hrctsqatul.stad2euirhaiimt.aihetsuntn0ninsFersziisaoaaeh%oruasece[giphoo"tnfci2dunlownssenletuersateieewb1uotdfuwc3btebtdfrifiitaorhs]tme)onloehyi3ypise..tiktfafrtsncna0etaahoehhRuttOdtlc(c;sotfntlirybNeetydromeh2peosfnsmidweatemrvylB.sueihltefit5aeotufaaghewpr4dTosespnTNarslthph3amehatree)nthetoCi7sdBtuotmotodde0tolr*asiniDekncTpwo.ennkegEa)5rad5finrcDC,tjleizednfinaasrunueeamnt3erawmhegeesciccebi--elocanbf-dsnllfiotelosioetaonapaaeec4eewws'vlcpdtyysiascxk1ou-teNqtieetewraeo0oetapnBsbiruadiwvBe%rdsnlntorueiwlcaaceasatsoaeTt,teinmcsatihntsydhsind*pkeaohCtnfpwsetrpoi,edbh-ld-irieaOeaet1otMeedrfieCdba4ratddnl;0?rcmdmiimdtiaht0onuoheumteecaesooyiartdngacerlamdnseioytrnzporatcmin^cluunaitvitereadebsro5adnhashrnMmnoeiarloeyin0aeneeieeare-.lt Statistical analysis Student's sons. t test was used to make one-sided treatment--control compari RESULTS Twt2gsthoar2remoe-Teitdugahehraphteeemysitsn,-pmeeowbconflidfonuetteornstomceegtdtfreiioonscdToifelsgCfnatniPhDsnodiHfedtgaDiciiAavTTl-aylee,tCCnxneCttpDrDhliyoontDyDhnsmeT-ylgodeAmaoxrwbpsopuaieluuonensrppsdIwesisw.ndPewtBjhoiWpegioectuhntMdhthpityo.setcihn.SsasoTeysenmprmdhmourpenometiahtsrsroebiyezenngsmodipentdsenuoicydwgsnscnsawiwiotiinnvfhnaeidecscTingateatahhennhpbstettyspsllremyroaocotIforufixLeontstidhhnmtIwruyesnoamcew3lteigegudeTeglhrsynrbCetcoeshoDretudahaolpDlyllesff., 79 D0W0686 137 1 -7529 0003695 r \* DOW0686138 TABLE I B O D Y A N D T H Y M U S WEIGHTS'A N D S E R U M ZINC CONCEN T R A T I O N S O F 22DAY-OLD TCDD-TREATED MICE T H A T RECEIVED DAILY INJECTIONS WITH THYMOSIN* D o n of T C D D 0*g/kg) Tbyanaia txoataaat Body woigbt (g) ThynaaaaE^t Zlae (g) (e*g/t) 0 0 1 3 .1 * 0 .9 .. Ut 1.1 *0.2 10 0 12.4 * OB* 5 0 1 10* 1.2 * 0.6 10 lowdoao* 11.2 * 1.0** 50* 10* l 10 ki^t doaaa* 11.2 1.2*-* 68* 7* it * Maaa ntoaa * SJ>. (body weight 8-- 10 anhnala, thymaa wight ad sine nialyaaa of 4-- 6 anhnala). Mica w o n u p o a d to 0 or 10 Mg TCDD/kg by maUrnal traatmant pcat- u b O y oa dqi 1,4,8,11,15 aad 18. ___ * Significantly (P < 0.05) d U f m t b o a eoetroi group (ao T C D D aad ao thymosin). * Significantly (P < 0.001) difforaat from eoatrol groap (ao T C D D aad ao thym o aia). * Significantly (P 0.025) difforaat b a a thymoaia oaetroi groap (10 Mg T C D D aad ao thymoaia). * Pbr aehadala aaatext. mtmomofciteT.tatenofPfhlatglor-erefoeeonmrtfsrriepce-psTcaoumoeCtnllomtlpsDsMeobeDonnep-fantseeetixsrovhepfenateohnnmsfiyedinerimmdcsttehtuhomwytsewfmiiitcctoshhueoeesnlTdixttkwCiphrdeoeeyDlirlymgnDigmhoeurtmtvosewenurcanetpaersslsk.umlwSserl.detoiintdlrihycneueecedanetnhedhdsceaiborgnretynoceaiseTxsfudeiiCcnldtatD.hnaDTerteirhrietngrysceiemuvrxaesepotcnamrseseiipennestsniieTntbtdr,aietltbioahhtlntyeee TABLED MITOGENIC RESPONSE O F T H Y M U S CELLS F R O M 22-DAY-OLD TCDD-TREATED MICE T H A T RECEIVED DAILY INJECTION WITH THYMOSIN* D o n of TCDD (Mg/kg) sss A PHA (epn) A Cob A (epn) A PWM (epn) 00 19,001 * 10,774 20,895 * 6,517 6,494 * 2,647 10 0 8,097 * 2,731* 10,779 * 5,345* 2.108 * 1,879* 10 low doaaa* 9,986* 8,248 14,616 * 4,989 3,714 * 1,485 10 high doaaa* 11,878 * 4,677 11,244 * 2,598* 2,542 * 1,135* M oan valoos t 8.D., 4-- 6 aafanaU par group. Mica w o n pond to 0 or 10 g TCDD/kg by mofearnal traatwoat poatoaUliy oa days 1, 4, 8,11,15 aad 18. RooUta an upraid aa tfaodlffaNoca batwoau athnnlatadaad aoo-atimulatodcaltMNS (lacpm/3 X 10* call*). * Significantly (P < 0.06) diftaroat t o n eoatrol groap (ao T C D D aad ao thymoaia). * Far aefaodaloaoo text. p' f tv *' 000 696 TABLEm EFFECT O F 4 W E E K L Y DOSES O F T C D D O N T H E SUSCEFTIBILrTY O F MICE T O ENDOTOXIN Doaa of T C D D 0S/ki)* 0 1.5 5. 15 50 0 1.5 60 Incidanca of mortality1' 10 m C andotoxin 250 m ( andotoxin 0/4 0/5 0/4 1/5 0/4 6/61 1/4 6/6 2/4 ND* 10 m | andotoxin 100 c andotoxin 0/6 0/6 0/6 1/6 2/5 6/6 t 500 m | andotoxin 2/6. 3/6 6/6 * In two axparimants, m ala mica (3-- 4 waaka of ago) w o n intubatad orally,onca a waak for 4 waaka, with T C D D inacatona-arachis oilcarriar. k Intraranoua injaction with andotoxin (E. coil. LPS) tarodaya aftarlaatintubation.Mor talityacorad aftar48 b. * N D " not dona. adt2oni5ed0dencMiadnguostteheondexdioo5nn0t.oeAMxdigfnetTeawrtChatDshiDnelettgihhnreajoelu1cpf.t5oioarMnnagdlolT1fmCi1nDic0DtehMiegngr1ote5hunepMd,go5wTtoahCxneDridneDa,1s52ga-rUMooguucptoT.noCItfnDroj4Delscatgnsiuroiomrnvuaiopvlssf- T A B L E IV EFFECT OF 4 WEEKLY DOSES OF TCDD ON BODY A ND ORGAN WEIGHTS OF MICE* Doaa ofT C D D (vc/kf) Body waight (6) Thymus waifbt (ms) Splaan wai|bt (m|) 0 1.5 50 30.7 s 2.2 28.7 a 2.5 26.0 x 2.3e 66 x 20 65 x 12 34 x 10e 106 x 13 97 x 23 89 x 8b * Mean aaluas t S.D., 6 animals par (roup. Mala mica, 3-- 4 waaka of aca,warn intubatad orally, onea a waak for 4 waaka, with T C D D in acatona-arachia oilcarriar. *P < 0.025. r < 0.005. 81 D0WO686139 7531 '1003697 I TABLEV EFFECT OF A SINGLE O R A L DOSE OF T C D D O N BO D Y A N D O R G A N WEIGHTS A N D O N S E R U M ZINC C O N C E N T R A T I O N O F MICE*. A N D O N T H E SUSCEPTIBI LITY TO ENDOTOXIN D o m ofT C D D (Mg/kg) 0 100 Body weight* (f) 19.3 1.8 18.7 t 2.4 Thymus weight* (mg) 70 i 15 5 1 1 14b Spleen weight* (mg) 120 t 23 97 t 19* Zinc* (mg/1) 1.0 t 0.2 k 1.4 t 0.5 Incidence of mortality' * 20 Mg endotoxin 100 Mg endotoxin 500 Mg endotoxin 0 NO100 5/5 0/6 5/5 6/5 ND in* Mean T ain* t S.D., 7 anim al par croup. Female mica, 3-- < waaks of aa, w a n intubated oaca orally with T C D D aeatona-aradm oil earriarand killad aftar 5 days. b F < 0.06. ' Intraeenoua injaetion with andotoxin (E. coll, U S ) 6 day aftar intubation. Mortality corad aftar48 h. 4 N D - not dona. dTiowlbmTaawaoLeebis1nenniddpte0fifoCiCvseiorljscitcpT.idieemoartthD1DtrgmojerlchIyyehpnes0ihnnaoDrtDgyatohede0itlxttwhwmassrd/htyrtikgekrodeothmeoMeaaedaurgaifroowielnclsofSgls,asetrlsitufoebiuhitsgteeenrgotTnwanlyneepoartmhterngnlcdostCohd,ncyoroideaataoeooefinysa5Donnsfils5trantofj0vtsmleeoddLDees0lewpidri0ttnxna.cnr-mhsde.l/ewfedstieE,ksgareneomMiixipeieooggrevncld:5tpnsgdsedntrhehoejite0oobceenem,utneeta(o0rxtwfceoeTsndikoiacidifntordemmMlniiachydita1mithonmlydybhlgar0oteanape.octhil0lntigLndnaowmeAlanfco2%eentrcogeePutox,n0esVoaaeidhe1fsSititgaosdmrfgnneu)M.e.htnem5.teddhfdeoe.aedoigineAitsrnmneMtTltofwfedi2haLfleftrws5grhlasetryeonetoiPshorluTedgiactsowdtS,o(ei5nstlgehCinrfai2,w,txnea0irtiohvyD5fwyroittToit1nfeeshhi0McmwDhtsu;ntaT0n.asehhogeMtbbot,nCiTlercratnosTleugogtecehe1nuDhvulidtuaCaTfgvy0TesIeiDtatsrbsretcV0Da6lCmyhrroncaeobo)ltDe,eosouDeapohdelwfdsdbn0neptrtneDiyadtumigoat5,rVebtfn5o1rrcwidesitrgneiionIos0oee.allysnwr.ee5aidlurnd0tnctsToiuhrwpytog-a.oituijthsmobhahrmhsnsihAepptwgrneeeaeataiooyaedgsotcntewc5lmeoecssherTsrauuecin0diildfthntenfinaunCmalsgiidMeganTdcutsldwshtDnoailhbtshiCgtepoitsangtieyeteDolceeotTthnDhnddyrohnoroxws-daCcieoruDemxufwtiioganiaxhnomf1Discs5nolnepspevycb..pfaDi50crtadioemsgeTznlrawyeteerpso/ihiohtbMakhMnetneeeuatsltolsnidndylgiegcesgsssgse- D0WO686140 j 1j\! 82 tf *) I j) q q 98 TABLE VI EFFECT O F 4 W E E K L Y DOSES O F T C D D IN MICE O N B O D Y A N D SPLEEN WEIGHTS A N D O N D A Y 1 A N D D A Y 2 SPLEEN C O U N T S O F LISTERIA MONOCYTOGENES* 00WO68614 1 Dose of T C D D (0/kg) Body weight <) Spleen weight (>() Listeriacount* ("logyspleen) Day 1 p.i. 0 50 23.4 i 1.4 21.7 * 0.6 140 i 10 06 t 18d 4.89 0.40 4.58 i 0.24 ,f Day 2 p.i. 0 50 21.7 < 1.5 21.2 t 2.4 178 t 23 124 t 29d 5.13 a 0.31 6.08 t 0.27 * Mean values - S.D., 5 animals par (roup. Mala mica,3-- 4 waalu of age, wart intubatad orally, once a weak for4 waak* with T C D D in acatona-arachia oilcarrier. b Animali were inoculatedintravenoualywith 2.4 x 101 Listeriamonocytogenesorfaniama 4 days after the last intubation; the number of viable Listeriaorganisms par spleen was determined after I and 2 days. de rr << 00..0051.. rwombiteroedegcdinauagyutnchsesitswedsTmaeCiw,ingsDewihgtrDthanesseifdsiotTciihdfgaCennmDnitsfoDiaiecctmfaefganeerlcktotliteiyunlrlopenslt.dohpFtwelhoeieneennrnauemslmlidynafb,carreoyTaormsCpaoshDfhfttareDogperwe-aeenrtirxneieptdoidonocnusauTeecnladtaadilotbinmaolcennooainVmcfwtrINraoIeolp,Brslh.eiTnmaI.sgntiiegwcsnea,o.idfneiSdocxpairptlnieedoterinlndyi, TABLE vu EFFECT O F 4 O R 5 W E E K L Y DOSES O F T C D D IN MICE O N N U M B E R O F PERI T O N E A L M A C R O P H A G E S A N D O N M A C R O P H A G E REDUCTION O F NBI* Dose ofT C D D (eg/kg) No. of peritoneal macrophages (x 10`) Reduction of NBT** Four doses 0 50 1.94 < 0.68 2.23 t 0.35 32.2 * 22.2 37.0 8.9 Five doses SO0 1.81 * 0.54 2.08 t 0.31 23.8 17.1 37.0 14.0 * Mean values - S.D., 4-- 3 animals per group. Male mice, 3-- 4 weeks of age, were intubat ed orally, once a week for 4 or 5 weeks with T C D D in acetone oil carrier. b For detaiiasee text. 83 7533 0003699 DISCUSSION hctapnmtTbtszhlctlttapaermtmrcoafpcpbbmibeyyhhmhhhhiiomiyeoatrdlrcatotfrollvoyoyihnermmaeiyyraipofereialeuetnttserFecrtTtcnvmioeesicoecadmgmsusetmStncstobetrvehrsprappepe.sphtbyesnahcitgeeaenueaocyahsdudnteuuhnhhhrtooheetitdanoyhesal[mrttnhIemceeeosescmynaonotsrytude2moauitfnnTiord-es,fiyoneoernectdcectdidciaee2foorntihuantaystccyscuyleennwnsihoeedeohoonu]s,cdTfasii.etnststaoitduspegmcfiah.mibehgeofoefueleruel,ossTlroClemneltippeosyoisstlnriTtnisssfsTcnaircTlanmnapaaseiehs,ecwDewmcoteasoefcodneta[ihmrrCniyrcdrCtmienislonos2ouDhbumufedlstainvoiputoeueoppetsDshlydrDspymufc05nefsarmineltoscnsoeehticeuycependoopiD,lnkt.cntiiitstx]xDd0ctao3piefonatobmdonhnsuuweo.neioooppyt.otytracl2aiotn-tiib4keeapretlerIrbftTtelmallueiblyylsriidrai,anar8teonsr.ti[hxynMltdttrstClstimhnrtqpsyeee2knenhyihmeopifemehcyc[atkddgeMbradausauD8naeeaodwsdr1hyooa(pTetauminTeultiuira,etet]ptntnumasnofeeDse4engrhct[lniliolCsanonnsectCoeocylhytcsc/tso2ac.bto]fimsi.t.sdiettoodtnetrneDhnnmkepedus.ohmhuehi4Dyioe[IcssdesdotitnTclycaechsVoDeenAbnnumati]ayarDpitteftonleywmtrdreays,hstlupyosyidaoha,rmcnhtdyilteox,i/nooewiakxtysatpnirzmsaefaouhTe[isloopgen,sshheeotsltifseimst2erndhtsasehthtneCcudesiit,lyrodosLelPo-hunsoceervs7dnyih[yyraoi-ddmpwsmDexbsgarVennnHnedwoceeietdoibts]etbenyfmheitpteueerohelricDgdes.ianerruiyphAohoesarnssooaoisTenvtadelodoRateeceusine[eassiesaeoilrcnetrhtccwami2Ctnoonxecnoccdaptnidutetaceyratsofhan,zcent6ptreaitnfdhgaDuot,rim,ttaixsrrriTrssudebnnt]cholrdaessneennypwehlybeeGaiDmoaielnCeodirnwynnnpenmagsfucspooonhnndftradsCaplfupaitsemroDiodtrnuw0ididsamagoeeaciyBipeuhktelnpooooernctnime.estp)cDeehotdl.enrmf0hsnyrsctetntipoyfgidcptfoldSneeabisrsrkeal2TystteemaeteteiydaclmrsieatnddhoatueiAauordooetkdetnnicavvhhted,ttrejiemihliu-anlesenttntirreonemieddouiesyfeenfuaeiosetorgcaroossrtieneoondtrnhtiioftdsoticohepexnet/saotnsztpexohlorenreetmediteevdyesoTifeilifenmdasteMcycsdhooyntmcdpteliydbtpsmphaodencaCueeltdlamxpyfrTchdoaaimhryurocwlowyfcna,acii.eirmaoDnlatab[eseCtetneontraeooamerclihlo2haneumhpiei-smAsfyittDarosttteDvsftndiiesotredse3eneiboiehminwaovehedttatmneeaoahndyc-runDinuhp,a]defstccvdiimynrbitlnrppi,ounoc.peinanddcrathiuteTyteimceTnisontiwthrrofmpdteorNlhireiefoyvumbaliiotirfselfCteoeaCnoeeooasuairnutwroeosmpiilallrsccbfedEscvsrantunniltnroecoDocgtcsiDtsrtye[uiteoaeatueTdegstu-tm.tthoacyceme1dooDcadrieleauddcDhlcieytnnoCspctosaeoofeme3netcninfyheerpea-eftdtnpmsns-narodltfaDoetep]rdisdeftettraluroaothhdrvohdootx.ennohhelTshxkereeoDintidirtascluyarebtpsc.ytnindnattnyyypeEnelycCttoterrtwpomotametLmslowToddmiipmdeoamtiasasncuvtooteomDxtxntosepPxshulehehneoahuteoiranotliennoioiiineruotDudnnistSilntcnnceccvesedasse]hfn]defrlsiy.,tsst-, . 84 7534 0003700 s i atrophy. The endotoxin hypersensitivity, which has to date only been studi mtheynmt uosf tes. The rno/ci oLuPldS :p3tthohinytyemmnthtoiiec- arltosteoexmics r!orr]idt.eiestnEropsvtxoheonincefi human DD at a i i lI * troTterecbeeTtssTghhhpuandeonbtllCChuoidteelnesddoFsfetDDoaecuhnnoaosiiLrcdinmcDneDytettefavehtooic-danleciovSlilxx[tomlrdedho-ieuil3-tmiinyosyknastnnsi2irlsn,isieceowgenlea]o-e,lndsualhS)incobw,shnfs.isiroa-yuseanogtiaioflnepNltrtsWaestectdhenftaroearsrkaBade:neraaihnbritaasTssnIhpea(deaeefntienp1a,imspeimlnndgdsr)hrpwroisaoBgstmauteeerhitoggtanncet,pasaisuuraee[erevteoneogt8drnsimnaeimhednBsdot,oinzms2ywettiecyieyfnlcoa,[9bnyutinshm3yano]tldsnmioipeitlo3leslneoogineheisvkt]isgctcsaincohee.osirasteonnmerondoxeealuldaoyaiilfo[rostcwnylfe1isetehrn-eptnomLi0prehdgvtoaxndhhh,ot.leaui3rrifhueoaycssnattmec1tgseettpTditaecioiet]gseoebtCsutaahxs,horahsa[niflcDieatls1oatattinoithehoeertcrnaD2icTinvarynk.e,dh,dyits3ieCsaRttdntyiifa-ywo0teo(Doclipetni3eedgev]oegroerdxDt),redrianrhotfienner-asejyspsla(siudeeeebtnitn2,strlnenisigeodd)etndoc(oesahwtsnusogueonihfnetetetcothinshnierttenoTevireehhhkrncdxgiCnieeorohnetteimyThpenmDaosfeoemvceuaCpswnTDnpiboftpaiezemDiCnnzrnto,wcipcytoyor,ascDhDaharmdmiaycetredcatDo-cnutthfsroeeneeilooffdscovex,meriepin[espagapoeCc7rms^esbndhitsnoteiiun,iotndiasoo8nitpioeocgonieaniiyaa]nndnnkeer,f.f-.l., iUty- ainVdX thei, feet on iditions f TCDD mlxpaolsseodr I ipTpminnah-adrlaycapigcrmgheaorsatppa.eghhpoSahotchig,cmyeyatpstiti.leecatrsrhhflauyeanpn,desctnthndidoeouontetrodeoxtusofiuenlmttismohayapocprafceoiorrptmeshhdeeabngiseneeintxseidvdpboieteutryofitfmx,eisicenatnnstododntewistbtcoihtouxhetsihfsyLerTdei.ns-ugcmineltlopltosnrhfaooenpcaydelpttreromtreigeavaesticnoirouoeonssf ari atttrwiaoennrs]e REFERENCESli 1 B.A. Sehwetx, JJS. Norris, C.L. Sparacbu, VX. Rowe, t J . Gehring,JX. Emerson and ire n ot C.O. Garble,Environ. Hlth Petspect., 5 (1973) 87. d a ta j, 2 XD. Courtesy and JJL Moon, Toxicol. Appi. Pharmacol., 20(1971)396. hiynmduues- i \ 3 D. Neubert, P. Zona, A. Rothenwmilner and HJ. Marker, Environ. Him Parspeet., 5 (1973) 87. 4 D. Firestone, Environ. Hlth Parspoet., 5 (1973) 89. k>rewdiitnh- 4 1 I 5 K.D. Crow, Tirana. St.John' Dermatol. Soc., 58 (1970) 79. 6 4. Klmmif and KJi. Sehuls, Dermatologies, 115 (1987) 640. 7 NP. Buu-Hoi, PJL Chanh, G. Basque, M.C. Asua-Gelade and G. Saint-Ruf, Natur- otozin wise.,4 (1972) 174. eisbpcseoepsretvdi-- 8 B.N. Gupta, J.G. Voa, JJL Moors. JG. Zinkl and B.V. Bollock, Environ. Hlth Par- pact., 5 (1973) 125. 101.9 M.W. Harris, JLA.Moore, J.G. Voa and RK. Gupta, Environ. Hlth Petspect..6 (1973) n cell- 10 RJ. Kociba, PJL Keeler, CJi. Park and PJ. Gehring, Toxicol. Appl. Pharmacol., 36 mrtdaleintyt (1976) 553. 11 J.G. Voa, J.A.Moore and J.G. Zinkt, Environ. Hlth Petspect., 5 (1973) 149. iym us 12 J.G. Voa, J-A.Moore and J.G. Zinkl, Toxicol. Appl. Pharmacol., 29 (1976) 229. 13 J.G. Vos and J_A. Moore, Lit. Areh. Allergy AppL ImmuaoL, 47 (1974) 777. 85 D0W0686 143 75e T Q xqJ 0003701 DOW0686144 14/J.E. Thigpen, RJL Faith,E.E. McConnell and J.A. Moore, Infect. Immun., 12 (1975) 1319. 15 W. Groth.Zbl. Vat. Mad. A, 23 (1976) 31. 16 B.L. O 'Dell,P.M. Newberne and J.E. Savage,J.Nutr., 66 (1958) 505. 17 8.L. Snyder and R.I. Walker, Infect. Immun., 13 (1976) 998. 18 J.G. Kreeftenberg, H.G. Loctan, JJ). van Ramahorst and E.C. Beovary. Develop. Biot. Stand., 34 (1977) 15. EJ.19 SJ>. Tripathy and G.B. Maekanew, J. Exp. Med., 180 (1969) 1. 20 Rnitenberg and L M . van Noorte Janaen, ZbL Bakt. Hyg., L Abt. Orig. A, 231 (1975) 197. 21 G.G. Krueger, B.E. Ogden and W.L. Weeton, Clin. Exp. Immunol., 23 (1976) 617. 22 L. Adorini, L. Ruco, S. Uecini, G. Soravito de Franceechi, C.D. Baroni and G. Doria, Immunology, 31 (1976) 225. `I 23 A. White, Thymua honnonea: a new field in endocrinology and ita aignificanee for boat immunological competence. In: D.W. van Bekkum (Ed.), Activity of Thymic Honnonea, Kooyker Scientific Publication Rotterdam, 1975, p. 17. 24 P.W. Beatty, KJ. Lembacfa, M.A. Hoiacfaerand R.A. Neal,Toxicol.Appl. Pharmacol., 31 (1975) 309. 25 R.E. Kouri, H. Ratrie, S.A. Atlaa, A. Niwa and D.W. Nebert, Life Sci., 15 (1974) 1586. 26 A. Niwa, K. Kumaki and D.W. Nebert, Mol. Pharmacol-, 11 (1975) 399. 27 J.G. Voa, Crit. Rev. Toxicol., 5 (1977) 67. 28 F.M. Collina and G.B. Mackinaw, J. Immunol.. 101 (1968) 830. 29 ILS. Khera and J.A. Ruddiek, Adv. Chem. Ser., 120 (1973) 540. 30 Q. Jonea and JJ. Oreig. Experientia, 31 (1975) 1315. 31 J1.1G1..Zinkl, J.G. Voa, J.A. Moot* and B.N. Gupta, Environ. Hlth Petapect., 5 (1973) 32 J.A. Cook, N J L DiLuzio and E.O. Hoffman, Crit. Rev. Toxicol., 3 (1975) 201. 33 R. Kiiach, G. Flciachaer, K. Kamiaaka and LM. Anna, J. Clin. Invert., 55 (1975) 1009. SOP 4 `J 86 0003702 i V . ' ? ." 1- - C y ':' >; 'V ?'..> / j : *Y\.'. y: / := ' * 'J ; V^if, .'v-: - .v ^ r r ~ nT.'*v .' O v :\ \ ' ;."*> .;-:;v PS*.- +:t4 ,-u .- :: . ' *.v v r r .; . l i- : <r - > Jr v -,.';-/V-'.\5 - ;.* -s Arch. ToxicoL 40. 161-188 (197B) 6TOSprXingIeCr-'VOertLagOIVG7s Y Review Article iMneSdeicvaelsoP,roItballeyms Raised by the TCDD Contamination G.Reggiani CBnicil Research. F. Koffmana-La Rocha A Co. ' O iamachar StraOc 124, CH-HXJ2 Basal, Switzerland O o rOrNooBi CT> 00 vi ccnn Aurbbsatnraacrte.aT,hraeiSseedvesesovearcaclimdeendticwalhperroebTleCmDsOlinwkeadswpirtohdtuhceednoann-dspreecleifaicseitdy oonf tahne CO citlyinoicfallesfeioantusraensdofstyhmepetfofemcstscfaruosmedth^e*etxhpiosstuorxeinanindtthheeawniidmeavlaerxipeteyriamnednts.evTehre asissseosfsmtheenttoxoifnthinethheeaeltnhvhiraoznamrdesnht 3adndtoonretlhyeodnetfhineitdioetnecotfioannbayrecahceomnitcaaml ainnaat'e.ydmlbkenyseioaotwshnunesrce(tlcoifhnxoliironcra.palrEcsenyvcmealu)cpduatiapnotmpgioesnoaroroefadftthtaelhelemTasCoptsoDstptOruoonlatnlotygixolyiincnrewecfdfhaeuisclcdttisrnheogennfouhanrnultdmyheayprnooesus,xsntpihgboelseputeyrpoperp.ieclvOeae.lnfsTtktihhvineee doefgtrheee acansdeisn.tensity was mild and spontaneous healing occurred in the majority the fTivrearnfsuienncttidonecsrheaasvee obefetnhedelylemctpehdobcyyttehseainmdmpeadsisaitneglyseigstnasbloisfhiemdpmaiormniteonrtinogf tOkeifmdtnh,eetyhh,etehcaaeltrhbbolcohooyndddrritaeitpoenr,ofodauft ctathnivedeppooorprgupalhnaytsir,oitnnh.meNceoetanobtrvoaelilrstmanpdhataphseorblioepgehnyeroraeflgtnihseteerrvleiovduesru,pstyhtsoe -oTwrdceuheCistntephcDmtCCoestDc.molhiotcmasaeoteeplfxdoacpitrofdhoinsnesnitugmrsorohttiehlloaraerrientnvpadaeiccSsatcthleuiwdvoarereeenlosnlyooutasfgeshmthohalcoeebscxnrumaygbrmoeretdteeeiodnnircxmaaiilctnleiotisotnonshrjxeuoistcreefpiirvtteaahyesstr,etcoeoacginnuuethrensUreaisecdnShnAeobtifuynpfalerdtncehttdhgceienomSEanepnupcvrclruueeiotesvdapsoibeeoa,lauentcbhsdctoaoittatdhhcettechhniiineret-- ' ^o r Key words: TCDD release - Delimitation of contamination --Human health protective and preventive measures. 0003663 I Paper prewired at the 3th International Conference on Ocetmational Health In the r*h. I So y National Cancer Institute CARCINOGENESIS Technical Report Series No. 23 1978 PB 276 71 DOW 518027 mrm^ ^ l 0COM,AW BIOASSAY OF PIQLORAM FOR POSSIBLE CARCINOGENICITY CAS No. 1918-02*1 NCI-CG-TR-23 REC'DA.I.C. MAY 1 5 1978 U.S. D E P A R T M E N T OF H EA LTH , E D U C A T IO N , A N D W E L F A R E Public Health Service 0002S06 BIOASSAY OF PICLORAM FOR POSSIBLE CARCINOGENICITY 518028 O O Carcinogen Bloassay and Program Resources Branch Carcinogenesis Program Division of Cancer Cause and Prevencin Nacional Cancer InsCiCuce Nacional Inscicuces of Healch Bechesda, Maryland 20014 U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health EHEW Publication No. (NIH) 78-823 0002907 BIOASSAY OF PICLRAM FOR POSSIBLE CARCINOGENICITY Carcinogenesis Program Division of Cancer Cause and Prevencin Nacional Cancer Inscicuce Nacional InsciCuces of Healch CONTRIBUTORS: This reporc p resanes Che resulcs of Che bioassay of p i d o r a m for possible carcinogenic ity, conducced for Che Carcinogen Bioassay and Program Resources Branch Carcinogenesis Program Division of Cancer Cause and Prevencin Nacional Cancer Inscicuce (NCI) BeChesda, Maryland. The bioassay was conducced by Gulf SouCh Research Inscicuce, New Iberia, Louisiana, lniclally under dlrecc concracc Co NCI and currendy under a subconcracc Co Tracor Jlcco, Inc., prime concraccor for che NCI carcinogenesis bioassay program. O H-*. 00 O ro ^ The experimeneal design was decermined by Drs. J. H. Weisburger*2 and R. R. Bares*2 ; che doses were selecced by Drs. T. . Shellenberger4 2 , J. H. Weisburger, and R. R. BaCes. Animal creacmenc and observaclon were supervised by Drs. T. E. Shellenberger and H. P. Burchfield4 , with che ceehnical assistance of Ms. D. H. Monceaux4 and Mr. D. Broussard4 .. Histopachology was performed by Drs. E. Bernal4 and B. Buracto4^ and the diagnoses included in chls report represent the interpretation of these pathologists. Animal pachology cables and survival cables were complied at EG&G Mason Research Inscicuce^. Statistical analyses were performed by Dr. J. R. Joiner?, using methods selecced for the bioassay program by Dr. J. J. Gare2 .* Chemicals used in chis bioassay were analyzed under the direction of Dr. H. P. Burchfield, and che analytical results were reviewed by Dr. S. S. 01in7. This reporc was prepared ac Tracor Jlcco under che direccin of Dr. Marshall Steinberg7, Director of che Bioassay Program; Drs. J. F. Robens7 and R. tf. Fogleman7, toxicologists; Dr. R. L. 7542 ill 0002908 \ \t20X19 M O O Schueler7 , patholigist; Ms. L. A. Waltz7 and Ms. Y. E. Presley7 , technical writers; and Or. E. W. Gunberg7, technical editor, assisted by Ms. P. J. Graboske7. C The statistical analysis was reviewed by a member or members of the Methemstical Statistics and Applied Mathematics Section of NCI (Or. John J. Cart, Mr. Jun-oo Nan, Or. Hugh M. Pettigrew, and Dr. Robert E. Tarone served as reviewers on an alternating basis). i The following other scientists at the National Cancer Institute were responsible for evaluating the bioassay experiment, interpreting the results, and reporting the findings: Dr. Kenneth C. Chu Or. Cipriano Cueto, Jr. Dr. J. Fielding Douglas Or. Dawn G. Goodman Or. Richard A. Criesemer Mr. Harry A. Milman Or. Thomas W. Orme Or. Robert A. Squire^ Or. Jerrold M. Ward ^Carcinogenesis Program, Dlvlson of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Heslth, Bethesda, Maryland. ^Now with the Naylor Dana Institute for Disease Prevention, American Health Foundation, Hammond House Road, Valhalla, New York. ^Now with the Office of the Commissioner, Food and Drug Administration, Rockville, Maryland. *Gulf South Research Institute, Atchafalaya Basin Laboratories, P. 0. Box 1177-, New Iberia, Louisiana. ^Now with the National Center for Toxicological Research, Jefferson, Arkansas. iv 0002909 ^EG&G Mason Research Institute, 1530 East Jefferson Street Rockville, Maryland. ^Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville, Maryland. ^Mathematical Statistics and Applied Mathematics Section, Biometry Branch, Field Studies and Statistics, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. ^Nov with the Division of Comparative Medicine, Johns Hopkins University, School of Medicine, Traylor Building, Baltimore, Maryland. ^ 0 ^ ^ ^ qq O CJ O rAA t' v 0002910 D W 518031 SUMMARY A bioassay of technical-grade picloram for possible carcinogen icity was conducted by administering the test chemical in feed to Osborne-Mendel rats and B6C3F1 mice. Groups of 50 rats and 50 mice of each sex were administered picloram in the diet at one of the following doses for 80 weeks. Time-weighted average doses for the rats were 7,437 or 1*4,875 ppm; those for the mice were 2,531 or 5,062 ppm. The rats were then observed for 33 weeks, the mice for 10 weeks. Matched controls consisted of groups of 10 untreated rats or 10 untreated mice of each sex; pooled controls, used for statistical evaluation, consisted of the matched-control groups combined with 30 untreated male and 30 untreated female rats or mice from similar bioassays of three other test chemicals. All surviving rats were killed at 113 weeks; all surviving mice were killed at 90 weeks. Survival was adequate for meaningful statistical analyses of the Incidences of tumors in rats and mice of both sexes. Mean body weights of the high-dose rats were lower than those of the matched controls during the first part of the study; however, beginning at approximately 80 weeks, mean weights of controls were lower than those of treated animals. Body weights of the mice were unaffected by the picloram. In rats, a relatively high incidence of follicular hyperplasia, C-cell hyperplasia, and C-cell adenoma of the thyroid occurred in both sexes. However, the statistical tests for adenoma did not show suffleant evidence for association of the tumor with picloram administration. An increased incidence of hepatic neoplastic nodules was observed in treated male and female rats as compared with untreated animals. This lesion is considered to be a benign tumor. In male rats the lesion appeared only in three animals of the vii 000291 51S032 y low-dose treatment group and was not significant when compared with the controls; however, the test for positive dose-related trend in females was significant (pooled controls 0/39, low-dose 5/50, high-dose 7/49, P 0.016) and the incidence in the highdose group was significant (P " 0.014) when compared with that in the pooled-control group. There was also one hepatocellular carcinoma in a low-dose male rat and one in a high-dose female rat. In both males and females, there was a possibly treatment-related lesion of the liver diagnosed as foci of cellular alteration. The incidences of this latter lesion were, female rats: matched controls *1/10, low-dose 8/50, high-dose 18/49; male rats: matched controls 0/10, low-dose 12/49, high-dose 5/49. Thus, there is evidence that p i d o r a m affected the livers of rats of both sexes, but more particularly those of the females. No tumors were found in male or female mice or male rats at incidences that could be significantly associated with treatment, and it is concluded that p i d o r a m was not carcinogenic for B6C3F1 mice or male Osborne-Mendel rats. In female rats, however, the incidence of neoplastic nodules of the liver, benign tumors, was associated with treatment with pidoram. It is concluded that under the conditions of the bioassay, the findings are suggestive of the ability of the compound to induce benign tumors in the livers of female Osborne-Mendel rats. r i vili 0002912 - Strictly Confidential Coeriis_ti_n cf euiden.. ologicni studies on the long W 1--i hazards of chlorinated dibenzodioxin.... Tforking Group of the NISHS and the IA?.C, Lyon, 1C. and 11. Junuar V Mortality Study of persons ex/osid to 3ic::ine aft or an accidc-nt cn ^ o* Novenber 13,- 1953 is Ludwigsfcafen, Gsrnsny. to >i! cz oi Introduction: 00 ro ^ According to the Volume 13 of the IARC-Mcrographs and to cur imp's ledge, several accidents have occurred during the past decades, in which person- cane into contact with trichlorophencl-dioxin, yet there have been no prospective epidemiological studies completed on persons exposed to TCDD. This report describes the findings of a..long-term fellow up study after an accidental chemical reaction during which TCDD had been Q eleased into a building of a factory which was then occupied by workers and also lateron was used for the ongoing production of other chemicals. Event description: The event happei^a on Nov. 17th, 1953 ard was described in a publication about the acute ana sub-acute sequelae of the event as follows (4,2): i During the hydrolization of 1,2,4.,5-tetrachlorooenzol into 2,If,5 - ; trichiorophenol, which was carried out in methanol-sodium hydroxide : by temperature of l0 C and high pressure, an accidental reaction took place. Due to the explosive increase of pressure and tenperatur: within the autoclave an uncontrolled reaction led to "toxic chlorina ted carbohydrates". This occurred in a building four stories high * in which the different stories are connected by several "windows" C and openings for tubes and connections. The autoclaves were located in the second floor, but also reached parts cf the ceiling cf the first floor, from which the feeding tubem went up tc the second .eye; floor and to the autoclaves. The slit opening for these tubes was ` covered by a metal sheet. "7 * -* *T.he reaction warn followed by the development of a ste leav, '.-through a broken security valve which turned into an'indteense vappoorr- ous haze which filled the autoclave room in .short time. This hazzee^ also invaded the other rooms in all four floors and the' staircase connecting all four levels.of the building. After "several minutes: the clouds were dispersed or had been precipitating in the form of - a visible laysr, covering the appliances, walls, window glasees, doors,and other surfaces all over, so that the room was reasonably \ 1 clear to be entered. * 7543 0C06710 rv. DOW 1373183 -2 - Tiie workshop or autoclave room' ras situated in a fire-proof part of the building bich had, throughout the four stories, a special masonry. ' In this special -part of -the building several industrial hygiene measures were taken .subsequently, because of the i-rcediate incidence of cases *-ith chlor-acne in the rorkers exposed to the event. The measures taken 'sere: ; V/hitecash of the hole building ( calls and staircase covered cith line) The corkers cere protected by van-uer-C-risten-Hasks and full protective cloth as a fully gas-proof combinations, a After this ere of sis painters covered all metal by a special paint , called rust-transforner, and lateron cith red iron-onide paint. 'The floor cas covered.cith Acronal, a plastic layer, and silicon-solu-t tion. A second layer of Cpparol foil cas folkxred by several cashings Crith clearing and neutralizing chemicals. After this procedure the total surfaces cere flamed and the autoclaves cere boiled rith benzene. Despite this elaborat e cleaning programme these measures had been ineffective as ras found out lateron. 5? The medical history of a corker cho cas exposed to the contaminated area five years after the event gave evidence of a still remaining hazard at this site. In the interim time no cases of illness had been observed among the employed persons, despite the fact that a full production programme had been going on in the rooms,-making use of the autoclaves, too. The particular hydrolization process described above *.:hich precipitated the adverse chemical reaction had been discontinued since November 1953 and also had the production of trichlorophenol been abandoned. T(_ one incident case of a health damage in 1953 is of particular interest because of its fatal outcome of the illness of the mechanic, cho had been exposed to the substance, j* . .. .The 57-year old man used a .'elding kit auring the repair at one of the autoclave and therefore core all the prot ective cloth and mask. Tnile heating a bearing of the stirring mechanism 'Xj he several times lifted the mask to nips off the ere-at from his forehead. The heat developed by the relding flame caused a vaporization of the lubri cant of the bearing. Four day afser this uorii acute dermatological and *'>*-. neurological symptoms developed. Six months later the person became hespit"lized for pancreatitis and liver enlargement. After nine months a tumour -- n the left upper abdomen and symptoms of an acute inflammatory process" - caused another hospitalization, during rhich the person died. Autoosy revealed pancreatic necrosis and perforation of stomach and bulbus duodeal* liver abscesses and chlor-acne of the trunc. 0006711 In '19.68, tea years after this incidence, sufficient hnorledge available then necessitated the total disrution of the nhole building nhich mas effected in early 1969 according to a detailed plan. This activity renalned so far without any detectable health sequelae or acute symptoms. The experiences rith the acute and subacute clinical reactions have been - | t* presented and published at the KSDICEZK - Congresses in Ludrigshaf en (1972} Haifa (1976),and recently in San Francisco as far as mortality tras determined. The presentation in Haifa considered only the medical histories of 42 resp.55 symptom carriers for evaluation. There trac no rent ion. of any exposure data and measurements being .missing nay be due to the fact that in 1953 the substance ras still unidentified and could only be determined and tested in aninal studies after that event. I ^ v a o , however, suggested that the typical effects seen in the cases would not seen to be caused by a substance or compound rhich ras an intermediate or transient product formed only during the unintended chemical reaction but that the sublirate-like substsnee had to be suspected the agent in question. The possibility can, therefore, not be excluded that individual multiple gj 3exposure occurred to the unsuccessfully removed sublimate in the subse- ouent mericd. Zpidsmiological study: Kethodology: The epidemiological investigation rac initiated as a consequence of the CC <1 cm OC a* discussion of these descriptive, clinical presentations and 7/as further pC-'-pted by the recent event at Seveso. The fatal incidence in 1958 seemed to be indication of long-term persistence of the substance in the originally contaminated area and therefore a cohort could have been composed by all those persons v;ho possibly may have entered the rooms after 1953 until 1969* In order to 4-f have some certainty about exposure and also to establish a sufficient y ^ latency period to detect all possible fatal hazards it mas decided to begipthe. follor-up study mith the core group,"i.e.all those men exposed . . curing the accident and during the immediate cleaning and repair mori-:. This cohort comsxprised 75 persons. The time elapsed since, theevent ran M almost exactly 24 years at the closing c^t^ of the study. The follor-up as successfully carried through for all 75 persons rhich is quite a ^ common sxper .encs given the registration system in Germany. . In Table 1 the number of person years is given together rith the nerson years of a co-carison group (matched reference cohort). Ihe follor-up technique can be demonstrated. ^ " y- As coon as a person has left the factory or the -last residence, the follow .up is extended as many tines as necessary to determine the vital status.From all deceased persons- the`death certificates sere requested iron the public health offices of administrative centers. The total of. 1525 person years reflects an average observation period of almost 20 years per individual. The expected number of deaths has been calculated on the person-year principle, the reference groups for the population sociality. C su^t^u by At*. oj_TM&S o--4 JkBtSTzsLr Froblems ana pitfalls: In this study one rajor pitfall has been avoided vhich is called the ^ sealthy worker effect, because two aspects of this often fallacious elenent of cohort studies did not apply. Hany of the workers were already exposed for soke tine as being employed in the factory and the exposure so dioxin ~as an additional experience ofh| uraticn. For this reason of the observed deaths rith the total population mortality can be done without correction for duration of exposure. Thin has been shown to be influence for results in such studies where long tern exposure necessarily require long-tern survivals in the cohorts which are not to be anticipated in the general .cn 8 T E l MOfl _ly. It is- 'hoped, though, that the comparison nf: with an internal reference group helps to overcone this possible influence of nultipis ,, exposure under the assumption that also in this group there existed the sane possible exposures to other substances as the-study group. The selection of comparison persons at randon was done manually for 'reason of convenience^ such more efficient and proper procedure .would have been the use of a randon digit generator, and the automatic selection of persons matched by age and date of entry into the.factory,^ One pitfall generally encountered in such kind of studies is the problem of validity of the diagnoses on the death certificates. '.The attempt was made to validate the diagnoses, especially rhea cancer vas mentioned. The result of the data collection for validation is ' shown in Table 3* . .... Q006713 .. . - 5- Conclusions: Tcfir.g into account these problens and pitfalls and the fact, that results of epidemiological studies are usually not able to prove a cause! relationship but can be considered as p ointers indicatin"'*"0 possible risk the following conclusions can be drawn: Although based on a comparatively small absolute number a malignant neoplasms have increased consistently above expectation for th possibility of a mere chance event. The effect is, however, shown to' be significant only in one age-group. The statistical test applied to this finding indicates that the observation of an excess of stomach carcinoma in this age cannot be r considered to be due to the small number of cases alone. The mortality of all causes in the dioxin-exposed group did differ slightly from the expected deaths in several co.-parison groups. . This evaluation may be stipulating the n:ed for further studies into the question whether the observed increase of cancer of the . fctonach in one particular age group can be dusly associated with th exposure to dioxin. . ... DW |373186 G o U M * * , P-3-. -- S c L ^ l ^ H 7 2 1 a 1 /n-tf. I ci:x o .Tit> '". J G o C H f t r f V i - P 4 u i 7 -/ fiLut-iste. 1U 0 fl'kajftoT*. cLu-rtL (?( 7 ~ 0- . Ubtfu.. 99^6714 Jb&J~ L r t f e 7 7 3 2 V. J M - ST2. ISA '554 1 Cbw teepher* He. 2, PP 199 " 211 f 1978* Pargaaoa P re ss. P rinted in G rant In t e r n ' S O i t i O N * ! ' ;* X . V 6 L {, M O O poctcgmaanEiTsSn,nacxiansc2Ho-po-rscsgcabsBtxt(poctuzeqsi)psui nas hiss ssracmwnsx lana Sadolf Pacar Swiss Padani Baaaareh Station '** (S - 8820 Wdensvil, Switzerland . - -X- . . 8 Christoff#r lappa Bepartasnt of Organic Chariatry. Unirermity of Qm 8 - 9d 87 Qaan Sweden (leoeired in 0C 10 Pabruary 1978; acoaptad for publication 20 Pabroazy 1978) ' ~~ In tro d u ctio n " Polychlorinated dibenao-p-dioaina (PCSOa, structure aaa Pigun l) nxe n (roup of oonpotmda that bare bean the subject of aueb eonoarn recant year*. Stay are koovn aa highly atable einoT contaminants la a variety of prodneta like chlorinated phecola. phsnczy aeida and baxaehloxobanMna. Sbey wore isrolead la aararal iadaatrial accidents. the aoat recent occurring at Sereso, Italy. Moreover, they hare bean identified in fly aah and flea gases from aoniclpel laainantare and iadaatrial heating facilitla*.1*2 I fi Pigure 1 Structure and mastering (Chemical Abstract Syetaa) of polyehlorlaatad diben-j>--diozia (PCS&a). 1 7555 0002556. DOW 39772 300 le. 3 Za 11,75 pcaitlooal laoaar* rt ior te* PCD* osa to algfat caloria* tona, ad a autor of tb*a* ara Imown aa axtraaaly haaardou* cospouada. Satani woric of HeCoanall *t al. Mani at al.^ and 2radia h a a abovn that poaitioaal iaoaara azy graatly la acute tozicity and blological aetivity. Poeter of 1000*10 000 ara fouad fer oioaaly ralated lama Uka tha 3,3,7,9* aad tha 1,2,3,9-tatra-CDD. Coaaaquactly, it tacca** laparteat to liaatlfy tha dlaczat* iaoaar* foud la raziona predaste. tela Idaatlflcatioa la dlffieult to obtala ualag aorual aaalytical tecfaalqnaa. Vlth tha azeaptloa of aaaa poetimarrj, othar apaetzoaeopie aatboda uaa largar aaooata thaa ara rary oftea arallahla aad aaay tlaaa thara la a aaad fez paro aaaplaa or araa alaci* axystala. Tai7 oftaa, Idaatlflcstleaa bara to soly oa CC rateatloa tlaaa, oapaelally la airiuraa bar* aaay aad dlffaxaat PCSBa ara proaaat. tela la oapaelally trua for blological aad cariaoimatel aat^laa bara tha PCSBa ara praaaat oaly la alante guatiti** togatear ite a aoltltuda of otear eoopouada. Iran tea boat aapazatiea tacbaiquaa aueb aa blgb* raaolntion CC ualag giara oaplllazy colina aay to laanffleiaat to aaparata all poaltioaal 1amara of acaa PCSBa. Za Pigna 3 la a aaaa fragni!tog-aa of a ayathatlc aaapla ahoviag tea olatlea of 10 dlffaraat tatra*CZ[Da, all contalnlng 3 teleria* ateaa la ate of tea oarfeca ring* of tea diorla aolaoula. ilteougb tela oolnaa glraa a raaaaaabla aaparatloa of teoa* lassar*, Ita offlelaasy for aepazatlag all 33 tetra-CT) isooara oold to atlll far tao lev. Tigna 3 Maaa fra^aatograa (a/* 330) abowlag alutioe ef 10 tetea-CSD lassar* ite 2i3 teleria* ateatltatloa patter* praparad by alcroacal* pyrelyaia of dlffaraat trltelexopbeaateai 90 a 07-17 giara oaplllaiy oolnaa, 300C, laotearaali colma offislaaey 140 000 thaoratleal platea. 0002557 ! Io. 2 201 DOW 397724 Jhich internet hae U n deroted te th a ui apeetrocetry of PC3Be. Sha electroa-ispact (Zi) maaa opeetra allov ac eaa? identiflestion of K3Se ai dietinfuichine thea fres ather pclpchloritsted eocpcundj n eh ma dlbenaofarana, diphenyl ethar, biphes?la and naphthalaeee. Thia Identification la baaed oc th intassa Molecular iena (K*) alto tha arpeetad ios eluaterinc due to tha ehlorlaa iaotcpes asd tha eharaetarlatie franasi*tloo idth fossati ef *-C0Cl and K*-2 COCI.6 Za toe high asea racin (/a > 200) nasali? sai far toa 1B identificatioa ef PCBDs, different positienei laeaara ahov ne pronounced diffaxancaa. Hoveras, la prarlona papera 70 Ma bara reportad sosa ebaarvationa h c diffarancaa in toa 'loeer sasc raaga ef diffarant toesero fer tha tetra and hexa-CBDe. Sha prablaea aera relatad te ispuritie in Indaatrial psedaeta (baxa-CTDe) and an anrlrenBantal contaminati at Sereno Italy (tetzadDa). Zb tola papar e rvpart a aora cenami appreaoh toat alleva tha Identification of a eubetitution pattern ef a K S O bjr ohaarration of Ita loveraae lena, la enbstltatlon < pattars ve defina toa saber of chiarine etnea la eaeh earbon rise of a aolaesla (ooo Tic* l}. Vxoa Sabio 1 It la oridant toat too nabar of pooltlonal laeaara of a 7CHD . ean be anbdlrldod Iste realiar crespe Ito tha asse oabatltotlen pattern. Cena#quanti?, toa nabar ef posolbla iaceare cas be redaned leadinc to a acre aiaple idantificatios, hieh ia of iieportanse aloe In th diocnooion of toe poaoibla precursore to toaoa haaardooa eesponda. toa partisi aaaa apactm of 34 dlffezant KSSo ara reportad, toa noe of tola nav teehnlque ia Ulaatmtad la acne applicatiosa mistad ta recant 7CSS problesa -- " * " < 4 anrirrmsantal poliution asd initnatrial produeto. to b lo 1 Z m b a r o f la o sa ra a d ra b rt lt u t lo o n a tie c f PCSP '`" Cospcunda lianmabaecrhoefarebhelnorrliBsaea* ltoa ebso.rro of - : smo-CDDa - dl-CODo - ---- tn-CBDo totm-CBDa ponto-Offi ' homCBT>o hapto-CTOa ete-O O oae Timore 1 1 2 1 3 2 4 3 2 4 3 4 3 4 4 0 .. % 0 " '4 16 02 1 12 01 1e 2 13 12 2 12 24 34 32 41 otoftatolens earbaa r 2. 10 - 14 22 14 10 2 1 a er i t)). 0002558 i i 1*. ' I ;i *i :Ii ! 203 So, 2 Jgsrjejtsestjjl' SifozvBca cea?c'me Che TOO lacas usad la tha praaiat atudy a listad la Sabia 2. Seat of tbaaa lacas vasa obtaiaad aa rafarenca caspaunii fra tha Bouree* listad otben vara preparad by alereseala pyTolyii of diffarent potaaaiua polyehlophaastM ondar canditioaa prrvioualy dasezibad.^ PC3D iacsars preparad by tbia reata vara charoctarirad by JE vitbout actual laclatian. Elista eolotlona o PCSD* la aatbylana cblorida at caneaatrutioca o 9-20 eg/jil vara osad er GC-15 analyaia. CC-HS calva 1 /tonigan 4000 qoadxupole CC-S tostrxunt equippad vitb S anurea and /insigan 6111 data aystan vaa osad. C u lea aeurea eenditicoa vara 70 a7 asd 250C. Saaplaa vara injaetad (1-9 pl aplitiesa) en a 90 a O.JS ua XS 07-17 {lu capiUary eolia conplad to. tba S r U a piattona eapillary totaraca. Iba eolia vaa temperatura programad tren 100 to 160C vltb 20/aia and to 290C vltb 2/eto. & a alution tasparaturaa o tba diarant P C S 1ansiara ara giran la Sabia 2. Masa speeir vara cardad by patitira seanntog (n/a 39-450; 1.4 sac/aoan) oatog tba data ayater a teaulti sed Blgcuaalon Panarti regarte oa gasa aeactroaatrr of PCDDa is prrrleualy nsetianad, PCSDa a adily idaatiied by SI nasa epeetrematry. fhxeugb tba paaaoa c aolacolar loas, tba ion eluaterlsg dsa to tba eblorina isotopa and rea tba typieal ra^estation ana ean diatingniab tbaa Ara otber cblerinatad pollatanta. fia stability o tbo PC33 ayatas la indicatad by intana# (usually basa peales) K*-iona and aleo by airly stroag donbly obarged solacular iena (H2*). Kajor ragsastatlon laadtog to lena to tba biliar nasa zanga la das to tbo expulsin o C0 and O.*vitb fenati o H+-C0C1 and M+- 2 00C1. Mtoor ragaentatioa is by losa o Cl*, Clj and aonattoaa EC1 iros K+ and tbo sajar fragant lona vltb oraation o M^-Cl, h*~CX., M*-C0C1-C1, X^-COCl-Cl-, M -2 C0C1-C1 ote. Zsana o vareos PCSDa giro vory similar apaet in tbia aaaa rango and difeati*tien batvaan lscnara o tba basi o iena to tbia fien la. topoaslble. Xa tba levar saos ranga tha sajar leu prsoant to tba C nasa apaet o PCDDa a C^-, Cj- and Cg-apaciaa, tba lattar orsad by lsaion of tbo carbon-e^rgsa bttads o tbo diorto syatoa and ellovsd by tba leas of Cl' and pnerably Q j ate. Sha solttog loas do oofitain infornatico os tba nabar of eblorina atoas paant in aacb oarbon ring of tba original PCB8 . lassa o a PC3D vltb tba essa eblorina subetitatioa patta giva la tbia gioa vary similar aaaa apaet too, bat eloarly diarant fres tbo o laeaa vltb a diarant ubatitullen patta. Tfcmgb no zaet impuntati aodsa v a adunad fres tbaaa aaaa spactral data, tba psanea or aboanea of charmetartotie loas alleva an idaetlfieatlon o tbo eblorina eubstltutlen pattarsi o PCSDa. oZ^GEMOa * : - i 0002559' DOW 397720 fi J i3 i I S S 9 9 ff B |B1^B1B19B1 1%4 it( JJ4 vi > A 4 A A iA'* M J0 tD\ aKN^KMiMO 9 N N fl 1 I HVoi 'S 'S f* CM 's 's J fi c l N f 's' a'a's'a's's SS 2 i ^ 3 hU i - h U J . p l I p p l i l I I i 11" 1" m i i i U n Iiliiijs* n i fr il 3 1| m B i 3 i $ f l j l S filt f si S . !HOsOa^lQ ^SO' a* SO ese iss sssssssss assa-jl bssseg ss 3 i5 A3 B reS5 ltcsgri l i m w t l c u t t i a 35 B Ir- O Hm O H H O H' fml mH H tmf \mN < OHNflflflflflfl ^MNMNNflflNfl H( <*' I f lf Ml fMl MN NMfMM Mi mM OMi i irt i H m\ N \ mn ia Us! 9B *t*lSO f- 4> Itili $ 4 I I I (I N I rBli w im n AM HlHi B. i:M a 5 j fsdkosr!-^ A^ N M ^ s O (isososo* hw\ 4 h^ H N H * 4 n *\*> * W O J N C * fl fl fl fl I * * rI Jv <4dshS*|(lS\JoA Os u> so r*sor*0t*fli * 'fO' O*'O'O*N 4 4 4* * * NH Hf l HM HN Hfl HM HN MH KNSNN\ M*% i> i . I fablt 2 03. m LO C5 o LO CJ o o o 3 . t. i *. I . V f. t.- 204 e. 2 Xt aboold ba polnted out tbat a earaful obacrratisu of u n apaetra le thia b u s maga la raqolxad and aay ba eeaplleatad b7 tba pracanea of hai luminili panica froa aaoplas or GC olona Bili aajr ba elrcoaractad by oalnf aueh fatturaa m background aubtraetion and ramine apaelfie naaa ebrooatopana ta detaralna tba praaanea or abaanca of a ebanotarlatic lOB in tba apaetra of a PCUD. Biffarmeli la tba levar m runa af rg eoectra ef PCBPa Mono-, di- and tri-CSDa Za Tifon 3 va raport partiti nata apaetra of sono*, di- and tri-CUDa vlth diffarant eblorina aubatitotion pattarne. D W 397727 Tifar* 3 Tortiti naaa apaetra (n/a 40-13) of nono-, di- and trl-SSa. a) 2-Mono-C38> (ltO)| b) 2,3>(2t0) and e) 2,7-dl-CBS(l:l)| d) l,2,4-(30) and a) 2,3.7-trt-C3S> . (2il). Chiarina aobatltatlen pattern In parantbeala. Xn eaaa ef tba nene-CDDt, 2 lsaoara azlat vltb 1 ( 0 cblorina aobatltatlen. She partiti naaa apaatroa ef 2-none-C3B la praaantad in Tifare 3 a. Chlerlna eontalainf lena In tba levar naaa ranca aaas to piar D i n a r rela vltb tba axeaption ef 1?* at e/a 109 and 110. nen- balofeoatad carbon rlaf vltb 4 hydro*n ateaa (4 B) yialda lena at n/a 76 (C^E^*) and K J 8 Z 6 m O(1 2I. 205 52 (C^E^*). Eaeh of thaa ioni la accorpaci! b7 pemcj that rvsult (ree tha Iosa of ona and tvo Stradicala. Stasa ions ora obsarrod froa all PGJSs contatala a oon-halogonatad (4 E) earbon ria. 2hs otbar earbon rise contatala tha Cl-atoa caa also produca tha loa at /a 75 (CgEj+) te. la caca of tha di-CTDa, lacaars of tvo sotips alati a flxst roup ***<-- hoth ehlorin etnea la tha aaaa oarton ria (2 0 ), a sacoad grtnsf eoa ehiorlaa atea la aach of tha ria* (lil abatitation). Marta! dlffaroaeaa la tha aaaa paetra of iaoaar* of thasa tuo gcvtift aria!. 2 ,5 -Ui-CD (Plur 5 b), an iaoaar wlth 2 t0 ehlerlaa nbstitution, pialla lena at / 7 6 (C^I^*), 5 2 (C^S^*) aad thaix aceoapsapla loas fxca tha aoa>halogonat*d ( 4 X) corteo ria. 2ha dlhaloanat*d (2 E) cartaa ria pialla iosa at a/ 109 (C^SgCl*. 'laor), 97 (CjBjCl^) ad 74 (CgEj-^). 2 .7 -Oi-CBD (lil subatitntioa, l u i spaetroa figura 3 c) pialla iosa at a/a 1 1 0 (CgEjCl^, aiacr). 75 (CgHj4-), 51 (C^H^*) atc. froa tha monohaleaat*A (3 E) eartoa ria*, fiwsa iosa vr* prasaat la tha apaetsa. of all FCEBa eoataiala cartaa ria* sabatitutad bp a ainl ehlorin atea. Sri-CHD axiat vith 3*0 ad 2tl chiaria*-abatitatioa. 1,2,4-Si-CID'(Tifar* 3 d, 3*0 abatitatioa) pialla loaa at /a 76, 52 *te. froa tha as&-haloaaat*d (4 E) earbon rie. S u trl-halo*a*t*d (1 E) earbon ria plaid a charmetaritic loa at a/a 108 (C^ECl*). Ehi lon la feoad la tha aaa* paetra of all TC&Bs -- a -&i-halo*natd earbon ria. 2.37-Tri-03B (Tifosa 3 a, 2il ubatitstien) pialla loaa at a/a 75 (CgE^4"), 31 (C^E^*) te. fece tha aano-halogoaatad (3 E) earbon ria. Taaka hieh ppaar at a/ 109 (C^HgCl* minor). 97 (CjHjCl*) ad 74 (CgE^4') ara eharactariatie of a dl-haloanat*d (2 E) cartaa ria. taira tra Tifar* 4 Tartial atta paetra (a/a 45-165) of tatra-CEDa. a) 1.2,3,4-(4i0), b) 1.2.3 .8(jtl) ad c) 2 ,3 ,7 .8 -tatra-an) (2 i2 ). .-v 206 o. 2 3917729 A total ot 2 2 tetra-CSDe tzlit* vith 4 *0 , 3 * 1 and 2 t2 cblorlae ouhatitution. 2 be i u i epeetna of 1,2,3,4-tetre-GID (figure 4 a) 1* fren tha oaly iosar of tba 4<0 gmip. Xana it l/i 74*76 and 5^52 ar Indicativa of a noa-balogeaated (4 E) carbn ring. & a felly balogenatad aazboa ring yielda ehloriaa eontalnlag lona at a/a 142 (CgClj4), 1 3 0 (CjClj*) and 118 (C^Clj*): theee lona ara fomd la tba naaa apactra o all fCUDa ecntaiaing a fully balogenatad carbn ring. r-.` 1 ,2 ,3 ,8-t*tra^3S doaa beloag to a poup o 8 lacaere vith 3 < 1 cblorlae oabatitatloa. Xta naaa apactra (figure 4 b) abova 'lona at n/a 75 (CgE^4), 51 (C^E^4) ate. fren tba nono-halogenaied (3 S) carbn ring. & a tri-halogenated (1 E) carbn ring laida tba abanetariatic ion at n/a 106 (CO.BC14), cenen te all PCBB tbia onbatltatlea. & a largaat gravrp o all fCEDa ara tba tetra-CESa vltb 2*2 ehloriaa eobetltatloa ( 1 3 laceara). Maaa apactra of 7 leonera of thla group vara atediad and abovad only nlner dlffaxansaa. Xn figura 4 c, tba partlal naaa apactra of 2,37,&'*t*tea-CSS la reportad, fin dl-baloganatad (2 E) carbn tinga yleld tba ebarmetarlatlc lona at m/o 109 (Cgl^Cl4), 9 7 (CjSjCl4), 7 4 (CgEj^) and 5 0 (C^Ej4) tba Ion at n/a 7 4 vaa tba noat intanaa Ion In tbia naaa ranga of all leonera of tbia peop. fcata- and haxa-CBSa fanta-CSEa axlat vltb 4*1 (2 lionera) and vltb 3*2 cblorlae anbatltatloa (12 lecaera). Xa flgore 5 a. va preaant a partlal naaa apactra of 1,2,3,4,7-poata-CaSf an laonar vltb .4*1 cblorlae aobatltatlen, Tba nono-halogenated (3 E) carbn ring la ladlcatad by Intanaa lona at a/a 7 3 (CgE^4), J1 (C^Ej4) etc. B u fully balogenatad carbn ring la Indleatad by tba cbaractarlatlc loe at a/a 142 (C^Clg4). --- Sha naaa apactrun of 1 ,2 ,4 ,7 ,8 -pente-Q8> (figura 3 b) abova lona at n/a 10 9 (CgE^Cl4), 97 (CjHjCl4), 7 4 (CgEj4) and 50 (C^E^4) Ana te tba di-balogenatad (2 E) carbn ring. Sha trl-hlogenated (l E) carbn ring la ladlcatad by tba Intanaa Ion at n/a 106 (CgBCl4) and tba ion at n/a 9 6 (CjEQ4). Eaxa-CDDe axlat la tvo groupi 6 laonara vltb 3*3 and 4 leonera vltb 4*2 chlorlne aobatltatlen. Toar leonera of tba flrat group and all 4 X tba aacond groop vera avallabla. laonara of tba aana groop ylald virtually ldantleal naaa apactra. Xa figura 5 e, tba naaa npaotxun of l>2l3.4r6(8*baxn^0D (4*2) la abova. 9ba dl-halogonated (2 E) carbn ring la `ladlcatad by n/a 10 9 (C^E^Cl4), 9 7 (CjE^Cl4) and tba intanaa lea nt n/a 7 4 (C^E^4) and 5 0 (CjEg4)! tba folly balogenatad carbn ring la ladlcatad by tba cbaraetorlatlc Ion at /o 142 (CgClj4). Xa flgora 5 d, tba naaa apaetrua of 1 ,2 ,3.6.7.S-hexa-CDB, aa laonar vltb 3*3 cblorlae aobetltutlon, la Aova. Srl*tealocenated (l B) carbn rlnga ara ladlcatad by lena at a/a 145 (CgZClj4), 131 (CjHClj4) and tha vary latasea Ion at a/a 109 (C^BCl4). laonara of tbia group are eaally dictlagulched fron tboae havlag a 4*2 ehloriaa eubatltutloa paitan. O O < ; -7 5 6 2 0002563 Se. 2 207 Vb'ZLlJ,?. W Q Sgase 5 Partial asas apectxa (o/e 40-150) ot peate- and hsza-CS&s. a.) l,2,3,4,7-(4:l) andb) 1.2.47fl-peat*-CBD (3x2); e) 1,2,3,4,6,3-(4*2) and d) 1,2,3,6,7.8-haxaCSD (33). Santa- and octa-CSDs Hepta-CaDs ( 2 iaoaara wlth 4*3 chloriae aubrtitaticn) and oeta-Cim (4f4) ara auffielaatly eharaetarized by theix hlgfaar aaaa leu. Serertheless, theix lovar ranga nasa apaetra asa laelnded for eoaplatanaaa. Bie featurea oatliaed for ttaa lovar PCSDe are forthar eklldatad by tha nasa apaetra of tha hepta- and oeta^OOa (7igure 6 ). Both hapta-TJI) laoaaza ylald Tlgrnally ideatlcal nasa apaetra vlth tha tsi-halogenatad (1 S) earbon rings Indleated by tha intansa isa at a/a 108 (CgECL*); .tha ully balagenatad carbn ring la indicatad by tha loas at a/a 142 (CgClj*) ate. Xa casa oS tha oeta-CSS, tha loa at n/a 142 (Cg&g*) baeeaaa tha lasgeat pask la tha levar nasa ruge. Its prtsanea and that of leas at a/a 1 3 0 H ate. ls indicativa of fully halogenatad camben riags. 7563 0002564 206 l o . 2 DOW 397730 H*us* 6 Tartial s uo apaetra (/ 45-160) e t o) l2 ,3 t4 .6 7 &-baptn-CHD and b) oeta-CSB. tiT lev e lem of ?ga>8 Zb fobia 3, va ammiri la aoaa of tha ebaraetarlstle Iosa of VCSSa In tha levar osa xa&ca, thst ora lndicatlr# of tba dlffarastlp ehiorinatoi oasbos ring* aad allea a datar minailon of^tha chiorina rubatitatioc pattasi of aa indir!dsol K S D lassar. Cabla 3 Charaetariatie Iona indicativa of th dlfferant chlorinated corion rtars of TCTOa Soabar of ebloriaaa a/a Taleaa and cocpoaitlon of aoaa ebaraetariatio iena a ob earbon ria* -102 *'i -77531<(((CCVV4*E44jp***))) .. ; * seLC(e#**>) no (c^a*) 1095J7(((CCc5^^HHH2jjaCQl`***`))) ---- -- ' sa 4- . , - -- ' 12S84(((CCCg^jHHHCCall^^*))) *"'10s7s ((ccs6aa**)) . 113413((CCgSjiCnl2^) HI 113108 (((Ccc4f5inCn1222***))) * orleu edetiloed *"*,-r ~ - .- -._ - .. -- . -. i 7564 j 00V255 DOW 397731 1. 2 209 Application FCHD In fir ac?i PCSDc aad PCS? ranging froa the tetr- to the octschloro coerponada wove identified la 2 ersral fly ash aszpla f n t a srariclpsl laclarater ad as indatrial heating facility. She aajor PC2CD war identified aa iaoaan dorsad la tba jyrolyaia of the sort season polychldropbaaatea (2,4,6tsl-, 2,3,4,6-tetr- aad panterhlorophenate) Suiaa identifi cation vara baaad ea chlorine enbatltution pattern iron aast epaetral data reported on . bar* aad by eoaparlaan of retaatlon tlaae of refernee PCSBa on high-raeoluticn glaai capillary eoitms.. la Plgsra 7 ia a Base fragnaatepraa of a fly aah aaspla isos aa industrial hasting facility shewing the elution of tatra-, pasta aad baxa-CSBe. 9te two ' najer tetra-CBDa had 2:2 chloAaa eubatitation (1,3,6 ,8- aad 1,3,7,9-tetxa-CIffl fro 2,4,6txicblorspbaaata). A minor tetra-CBD bad also tba 2:2 eubatitution pattern aad tba aasa zotaatioa tlaa as the'oxtxasely toxic 2 ,3 ,7 ,6-t CIP. S u third aajor tetzm-CSB, luting imariltaly after tba 2 ,3 ,7 ,6-iaooar, coaid now ba identified aa 1 ,2 ,},ft-(or 1 ,2 ,3 *7 -) tatxa-CBB. fiaia iacoar with 3 ` 1 cbloria* eubtittloa ia a coadaaaatioa proact ia tba pyrolymia of 2,4-db'aad 2 ,3 ,4 ,6-tatraehlorephenata. figure 7 Mass fragsantagraa (/ 320, 3*4 aad 368} of a fly ash aaspla abcvlag alntioa of tatzm-, pent- aad hexa-CDDa; 30 OT-17 glaaa capillary colon, 200-240C, 2 e/aias sasplo Infection ia tatradeeaaa. 7565 C0025GR 210 It. 2 Sb* three aajor penta-OSe (peak* a, b and c, Tlcusw 7) fomd la fly aab had a 32 ehlorlaa substitution patters. they war* the eoadeaaatiea product* la tha pyrolyaie of '2,4,6-tri- aad 2,3,4,6-tatracbloropbansta. ia additional pnta-C32) had a 4*1 ehlorla* oobotltutlan pattern aad as ldaatlcal retention tla* aa 1,2,3,4,7-Pnt*-CZD. bin leaner la tha eeodcaaatlea product la tha pyrolyei* of 2,4-di- aad paataehlorophaaata. Za eaaa af tha baxa-CIS*, tha Bala laoaar had a 4*2 ehlorlaa aubatltution pattern. Zt aa Identified aa 1,2,3,4,6,8-baxa-CIffi, a eeadaaaatlea product fornad la the pyxolyola of 2,4,8-tri* aad paataehlorophaaata. Tour additional baaa-CTDa la fly aah had tha 3*3 ehlorlaa aubatltution pattern, They era tha dlaarlsatlen product* foaed la tha pyrolyala of 2,3,4,6-tetracblorophesate (1,2,4,6,7,9-, 1 ,2 ,3 ,6 ,8 ,3-, 1,2,3,6,7,8* aad 1,2,3,7,8,9faexa-CD). TCTDe 1 a ?rro eoll fit-, trl- aad tetrs-CBSa or* oteerred la tha aeil at Seraso, Italy after aa accident a at a fthanlral plant prodsclay 2 ,4 ,5 *trlehloropba3 ol. ia reported, the aajor tetra-CSD found had an Identical aaaa apectrua (2 * 2 ehlorlao aubatltution) aad It did eo-chroaato*rapb vlth 2,3>7,8-tetra-CX oa aeroral different laas capillary eolnrms. Bo ldaatlfleatlena of other TCS&e were (Iran la that report. Zt vaa nev shown that tha alaor tetre-CSB preaant at an approrlaata laral of 4)6 ralatlr* to tha 2 ,3 ,7 ,8-iacaer haa a 2 * 2 oblerlaa oahatltutlon pattaxa, Indicating Ita fozaatlon fzoa trlehlorophanatea, proamiably fros 2 ,4 ,3 - aad aa laoaorlc tzlchloxophaaat* inpurity praaaat or fezaad la tha raactor at tha plaat. Zts foraatloa ms confirmed by alezoseal* pyzolyaaa ltb mixtures of 2 ,4 ,3 * aad ethar trlehlorophanatea (2,3,4*, 2,3,3*, 2,3,8- and 2,4,^trl-CF). the Bala ocedaasatlcn product of 2,4,3- nsd 2,3,3- or 2,4,8>tnehloph*aat* (1,3,7,8-tatra-CXO) had aa Idaatleal cotaatlea tlaa (50 a 07-17 at 200C) aa tha aiaor tatrm-CSS food la Saras soil. tha dl- aad tri-CBD* praaent la this aoil had 1 * 1 aad 2 * 1 ehlorlaa aubatltution pattern*, respectively. 1 b* dl*CZB did eo-ahrenatograph vlth 2 ,7 - and 2 ,8-di-CBB, two laoaars not separated oa a 30 a 07-17 eoltan undar tha eoadltloas used, ihe trl-COP did an nhwatnyraph 1 th tha 2,3,7-oehetltuted laoaar. SI- ead trt*CSDe could have been fornad la tha raactor Itoolf, pooclbly froa trl- and tatrachlorebeaaana, or by photolysis af 2,3,7,8-tetre-CSD la tha enrtrofaat. ia aaalysia of tha raactor content left after the accident oould five tha flaal answer. PgBPe fron nhotolvele of octa-C3D Seehlorlaatloo to lover TCSSa vaa ahovn to oceur by tJT-photolyels of oota-CD la arsenic aoiveats. 1 0 , 1 1 The major bepta-CSB fornad vea tha 1,2,3,4,6,7,9-oubatltutad laoaar, ladiestins a preferential leaa af lateral (2,3,7- or B-) ehlorlaa atoaa. 1 1 Tha photolysis produet* are now analyzed to datacnlna tha ehlorlaa substltutloo patterns of the lower TCSSa foxaad. Ihe major hexa-CSD feraed bed e 3*3 pattern aad an identical retention tine aa 397732 oo i i i t ti! 1 a 7586 00025G7 Me. 2 211 -1,2,4,,7,9- (or 1,2,4,,8,9>) htxa-C&D; tuo aiaor isoaers bad 5*3 ed 4<2 chierica subetltstlon pattarne. 1 aajor ad * aiaor psta-QB vere forati both bario# 3 * 2 ehiorine eubatitution pattaras; tt aajor la erpeeted to ba tba 1 ,2 ,4 ,,9- tubatituted lassar. Za casa of tba tetra-CBSa, a aajor and a Binar isoasr vara forasi, botb vitb 2(2 eblarlna wbatltutioa. tba major tetra-C39 did eo-chroaato|paph vitb l,4,,9*tetra-C3D iornad in tba pynlyaia of 2,3,-tritilioropianata. Senlifht photoiyai of oeta-CES did jiald th aaaa iaooars. tba reaotiea aebaaa dedacad i n a tba data abova beva tbat ehlorlae ateas ara m o v e d praferably ixoa a lateral pealtion oa tba carbn ria# vitb tba bichar eblorlaa subatitutioa. Zt inrtber ladieataa tbat.tba ertrenely torio 2,3,7,8-tatra-CIID ia set libai? baia# iozaad ina tba pbotelTSia oi bifbar PCSBa. TCSPa in comarelai ccntechlOTOSheaol A aariaa of cossemiai 7CP and PCP-Sa aasplas vaa aaalysed ior tba pnaenea oi ?Q3s and KSPa. 12 arpa* vitb hlh PCSS contant abovad, ia addition to bapta- aad octa-CSS, tba pnaenea f t 3 bexa-CESa la sasrl? eonstant iaoaarle ratios. tbasa 3 iaoaan bad 3 3 eblorlaa subatitutioa pattarne and vara ldentlfied as 1,2.4,,7,9- (or 1,2,4,,8 ,9-), 1,2,},6 ,6 ,9- (or 1,2,3,,7,9) and l,2,3>78-heza-C3D. Unarpaetedi?, seas 7C?-Ka aasplaa abovad tba pnssaea oi e tetn-CSD at lavala oi 0.1 to 0.3 ppa. Sbia iaoaer bad a 4*0 eblorlaa anbatitatioe pattern aad idaatical ratantlon tinsi on aererai (Issa eapillarj oelnona aa 1.2,J,4-tetre-CE, a vary uajyaastrieal isonar. At pnaaat, va bava no arplnailon ior tba pnaaaca of tbia ltoctz. .xfT'- V;1. ' __ Seferescvi 1. I. Olla, 7 .L. Terneulea and 0 . lutsl&fer, Chanoechere. , 43$ (1977). n.2. 1.1. toar and H.-7. Soaahardt, Ulti. Cab. lebesmlttsluntcn. Irr.. la pnss (1978). 3 - S.X. KoCoaaall, J.A. Ileon. J X Haaaaan aad K.V. Sarria, ter. Ani. Thara.. 146 US7). ... .4. A. Solead, S. Giovar aad A.S. Senda, J, Bici. Chea.. 251. 493 (197). 3. <1. Sandias, 731. Vaabia#toa, CSA. Personal Comunicatloa, 197. I SJL. Sonar, J. Chnaatoar.. OJ, 2 9 3 (1 9 7 5 ). 7. 1.1. toar, J. Cfanaaton.. 9 3 (1975). 8 . -1.1. toar, Anal. Cha.. ^2., 918 (1977). 9. 1J. Sosar, !.?. Sesabardt aad C. lappa, Chamoashvre. ia presa (1978). 10. S.C. Cnaby, A.S. Vea#, JJL Sliasar aad X.A. Voolaoa, Science. 173. 748 (1971). 11. U . toar, J. Chnaatcrr.. 129. 303 (197). 12. 1.1. Soaer and 1.-7. Sesabardt, J. Aa. txff<*- *..1 . Chea., . 362 (197). 397733 o O 7587 0002 5G8 \5 7568 153 DOW 08948 KS b(e&8f- C ( & o ' 'I ., .,, ` *. ..T8c(iaT2wr2c7n1nTayr9ah39*JoCoecylnr40j-rC-texrr7blf4DoU0-eeoiCD88oj4mcail8Dd,lh9cnio2dosDma^ees9yC5elishtT.;Td)*i.tei.I1tynrtrc;rrohhi1*e.ocdemntn4nee9Naorcfrcg47etlroiaioI8Kmahenea8Exrmccefese.MbcieaytftImenldiyeop(e1nnclvz5doatclnShedtiiiyoe4.snntrti.stsmceiyoneniAy-fhhrtnd,.loeeiehonRyrynaccimffoetfbdKrtreehimifpe2yranrsn.eeolaer.le-lgeemos3eesaxnaTvhjihvme.mcyrjsra7ee.dyiecaeIl;llg,ymaia'nhetds8n-ncjso!atWr4-el*oeriedriolf4navTidylecole8blcettlirwaovrh,irnytDilaro.hadueihat^bvsToneslsMvcoysaie,cgCndyhmnnirholonirJ.nlteDcrobo.hidihtEoehseruDaySemveoasPeccPydirirdsitaeds*thvcrbdiiir.roeosleaoheko(1nxdnsrnaoc,IU*hycmyontnr4eeomnlif42thaaxcNbtfo8ca4fssechyi.o-CmorndeonetymroenrLtEth/iaolawcsdtJednrcr2sxdlhrcdatiive17ivriy,dcisaoi,vno47alydrtfxabi(ntmo0ot,rttt5ehsuioeooy9nmn)deaeetrt-,):. , . hacTactlllrmi1uielnahncyh*viCraitdatr4ddrneseidoDtnu5vsrr-imtoi0cmlniTohDhithaexgmyneyaCronrystd)imtdl1Do1ebtorapa*i*lofyonisen4Dr4nxeigogc4A4dncyT88ettclirec,eilneCveCnraaiavwaveenansdndeDtealseChuedenyslesDssiIcmtyesc>iaaednwn.hificisntc(ndrctehaaociluordveeirPtometcnhhynesirmeteiielezaysssvonmrysbc,.hiamostthpmirhyern)tvirTasomedeeaoodthvsrtchfagciroepeeoceenwpircdrsvarrcellosamielcoft.ttblrnithoieynbnacyt(irasotrcAnaThei)tnanrocie.CeudtstnmhuiTaitdhytDvhetsoheinyiemenooDfeitdtsghrnacseiorneeroyjhc(yssyboxtaayorciolisyfsereninclhynhlsltrcchaoduyihlcalysnrflmdueceohnttsrdsolatmyeooiaoecpswdspbcced,hourrywatrocigmicPrsvohvcgtbyp4hicmtoaieto4ethoiaoyrslKrnneee--tt, \ PESTICIDES 00009 ^A 7569 53( a /rsA 7570 Z>C. 4S/3 46/6 47/4 rlin z results of ed subjeeU. 2. i is considered . Th m sxim sl :ted that short -Q be allow ed), rably 8 X l l " in tro d u c tio n , al affiliation, the author to J articles. Au* e sum m ary in be provided ne o r tracine is, includine ts f >ssy ne i e o f same decree typed (also 3 be used in iust accorri* need alpha nate places. Je, volum e, with Chtm e follow inc >f the book, ?es cited in references hnts can be er w ith the 4 of cover) air m ail to MuEt lasteivo inerR/Ne soerathr c-Hh ,o4ll7an(d19B7i7o/m1e9d7i8ca) l1P4r1e--ss160 141 O A REVIEW O F TH E GENETIC T O X I C O L O G Y OF C H L O R I N A T E D oj DIBENZO-p-DIOXINS * ^ o VJl J.S. WASSOM 1, J.E. HUFF 3-3 and N. LOPRIENO 4 1 3E n v iro n m e n ta l M u ta g en In fo rm a tio n C e n te r , B io m e d ic a l S c ie n c e s S e c t io n , In fo rm a tio n 3C e n t e r C o m p le x / In fo r m a tio n D iv is io n , O a k R id g e N a tio n a l L a b o r a to r y , O a k R id g e , T N 3 78 30 (U .S .A .); U nit o f Chem ical Carcinogenesis, International A g en cy for Research on Cancer, 693 72 L y o n C ed ex 2 (France); and * Laboratory o f G enetics, Institute o f A n th ro p o lo g y, Pisa U niversity, 56100 Pisa (Italy) ((RAeccceeipvteedd 2283 FMeabrrcuhar1y917987) 8) o O CO <1 Summary oC*O. Information from both published and unpublished sources considered rele vant to the understanding of the genetic toxicology of chlorinated dibenzo-p- CD CD dioxins is summarized in this review. Interest in writing this paper was stimu lated by the fact that this class of compounds, particularly 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD), has gained notoriety as an extreme environmental and industrial hazard. The potential for human exposure occurs in the work I place when dioxins are formed during the synthesis of a number of comm e r j cially important compounds such as 2,4,5-trichlorophenoxyacetic acid, hexa- I chlorophene, and pentachlorophenol. Environmental contamination m a y result ,, from manufacturing processes and from dioxin contaminants in marketed products. Research on dioxins as potential mutagens was initiated because of their structural similarity to acridines, a class of known intercalating agents. T o date, only 4 dioxin compounds have been evaluated for mutagenicity: the di-, tetra-, and octa-chlorinated derivatives and the unsubstituted dibenzo-p-dioxin. Since only a few of the many possible structural forms of dioxins have been tested, no definite conclusions can be made about their potential mutagenicity. Fur thermore, the positive mutagenicity and cytological effects reported thus far W ork sponsored b y the N ations] Institute o f Environm ental H ealth Sciences under Interagency Agreem ent 40-247-70 end the T oxicology Inform ation Program /National Lib ra ry o f M edicine under Interagency Agreem ent 40-274-71 under U nion Carbide C orporation contract W -740S-eng26 w ith the U .S. Departm ent of Energy. A b b r e v i a t i o n s : B P . benzo[o)pyrene; D C D D , 2.7-diehlorodibenro-p-dioxin; O C D D , octachlorodibenzo-p-dioxin; 2,4.5-T, 2,4.5-trichlorophenoxyacetic acid; T C D D . 2,3,7.B-tet^acU orodibenso-pd io xin ; T C P E , 2.4.5-thrichlorophenoxyethanol. 7571 0002426 142 with the few dioxin isomers examined seems to depend on the position of chlo rine substitution. The most active form of the molecule is the 2,2,7,8-derivative (TCDD). Data available for assessing the mutagenic potential of T C D D are conflicting and scarce. Differences in testing results reported in these studies could be attributed to solubility problems with the test chemical, treatment protocols, 1C. ACIMPME purity of test samples, or toxicity. Because there are conflicting data, addi tional experiments are needed before the mutagenic potential of T C D D and other dioxins can be determined. Studies exploring the promoting effect of dioxins on the mutagenicity of other compounds are also recommended because o 0 A experiments have shown T C D D to be an extremely active liver enzyme inducing agent that enhances the mutagenicity of certain polycyclic hydrocar bons such as 3-methylcholanthrene in vitro. CO The importance of discerning the hazards to human health from dioxin c o m pounds became apparent after an accidental release of T C D D from a chemical <1 CX - . . e .i plant contaminated the Seveso, Italy area in July 1976 *. This accident revealed that insufficient data were available to properly evaluate the long-term O c k c n z o -/- ! health risks posed by dioxin compounds. Several research projects were there O F U - 1- Struct fore initiated after the Seveso incident; it is hoped that m a n y of the questions concerning the mutagenicity of T C D D and possibly of other dioxin congeners stimulatec will be answered as a result of this work. dealing w analytical summariz Introduction rently be mutageni Chlorinated dibenzo-p-dioxins are a group of chemical compounds which are a result c among the most toxic and hazardous pollutants in the environment. These review th compounds, collectively referred to as dioxins, are impurities associated with The o: certain end products resulting from the treatment of chlorinated benzenes at. effect is elevated temperature and pressure under alkaline conditions. The most notable ~ was pro; contaminant of this group is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (See- (see Fig. Fig.,1) which m a y be formed along with other dioxin compounds during the acridine? manufacture of several commercially important products such as the herbicide Acridine 2,4,5-trichlorophenoxacetic acid (2,4,5-T) [25], the fungicide pentachloro- The t phenol [39], and the germicide hexachlorophene [39]. of chen- Suspicions of the possible long-term health hazards of dioxins arose after it dioxin, was found that 2,4,5-T was teratogenic in the rat and mouse [15]. Shortly thereafter it was discovered that the 2,4,5-T sample used in this study con cally pe festatio: tained about 30 p p m T C D D [15]. It was primarily the report implicating T C D D as a contaminant of 2,4,5-T [15] that led to its further evaluation for teratogenicity [16] and its eventual testing for mutagenicity [21]. These and tural co tive has Because other similar reports published during the late 1960's and early 1970's also * In January 1978, a joint International Agency io r Research on Cancer (1 A R C )/U S National In sti tute of Environm ental Health Sciences ad hoc W orking G roup was convened on the C oordination o f Epidem iological Studies on the Long-Term Hazards of Chlorinated D iben zod ioxin s and C h lori nated Dibenzofurans. The participants recom m ended that 1A R C coordinate the follow -up o f the accident-exposed individuals from five countries (1A R C Internationa] Technical Report N o. 78/ 001,1978). S*' Sr' eh- th. Y. At di. the position of chiob ' -3,7,8-derivative C D D are conflicting se studies could be reatment protocols, nflicting data, addintial of T C D D and promoting effect of ommended because ctive liver enzyme polycyclic hydrocar- h from dioxin comD D from a chemical 6 *. This accident iluate the long-term projects were there b y of the questions er dioxin congeners mpounds which are nvironment. These ies associated with in 1 benzenes at .The most notable ioxin (TCDD) (See pounds during the ch as the herbicide icide pentachloro- uxins arose after it >use [15). Shortly in this study conreport implicating her evaluation for v [21). These and early 1970's also ^cetrheRndnCatioho)cnde/ailUtofhSoRxelieNlnoCaptwtooair-onoturndpdN*iC!oonh.faInlt7tosihot8riein/- !i ACRIDINES 143 VS aOCh. AC*iD*n acaroiNE o a a n g e r* IC,h J,n (Ch ,),Chnm 2*^0 aaoflavine 2hci OumacaiNE NToaocHLOaioc DIOXINS y/ Hj*T v N > ^ nN*2-2HC* TIt p a Fl a v i n E ---- D E N IO ~ #-D O X lN 0 2. r-CHCHLCOOSEN20P-D'OXIN Cl V0 2. S. 7, 8 - T C T R A C H L 0 * 0 DiBEN ZQ --a -D iQ x iN F it. 1* Structural com parison of acridines and dioxins. 4.2.3..,7.S.S OCTACh l W O - DIBENZO- # -D ioxin stimulated toxicological studies on other dioxin derivatives as well as studies dealing with issues such as environmental contamination and movement and analytical detection of these compounds. Detailed state-of-the art reviews summarizing work in these areas have been published [35-- 37,39) or are cur rently being prepared for publication [10,20]. Since the question of potential mutagenicity, carcinogenicity, and teratogenicity of dioxins has been raised as a result of the incident in Seveso, Italy * [51,69], we thought itimportant to review the information available on the genetic toxicology of these compounds. The only theory proposed to explain h o w dioxins m a y exert their mutagenic effect isthe suggestion that these compounds intercalate D N A . This mechanism was proposed due to the similarity in structure between dioxins and acridines (see Fig. 1); therefore, information comparing the mutagenicity of dioxins-and acridines has been included (see "Comparative Mutagenicity of Dioxins and Acridines"). The basic dibenzo-p-dioxin nucleus isnearly planar and has 8 possible points of chemical substitution. From the monochloro- to the octachloro-dibenzo-p-' dioxin, a variety of derivatives are possible and some have proven to be chemi cally persistent and biologically active [30]. The lethal dose and toxic mani festations are structure- and species-dependent. Of all the possible dioxin struc tural configurations, T C D D is the most widely known and tested. This deriva tive has been called one of the most potent small molecule toxins known [30]. Because of its extreme toxicity and potential mutagenicity, carcinogenicity **, 1Seveso, Ita ly (population approx. 17,000) is located near the city of M ilan. In July 19 76 , the Seveso area was accidentally exposed to high levels of T C D D as a result of an explosion in a nearby chemical plant. Fo r a com prehensive description of this accident, the reader m ay w ish to consult the book by John G. Fu ller entitled "T h e Poison That Fell from the Sky**, Random House, New York. 1977. 1A t least 24 long-term experim ental carcinogenicity studies are in progress on various chlorinated dioxins (M -J. Chess, H. Baruch, J.E. H uff, and L. Tom atis. 1 A R C Inform ation Bulletin on the Su r vey of Chem icals Being Tested for Carcinogenicity, Num ber 7, Lyo n , January 1978. 46 0 pages). DOW 374401 J44 and teratogenicity [16], laboratory use should be carefully controlled and a rigid safety protocol followed [8]. There are several general articles which refer to the possible mutagenicity of dioxins [4-- 6,68], but the bulk of the material reviewed came from a number of published papers that contain experimental results [7,13,17,27,28,38,40,41, 43,57,64] describing the evaluation of these compounds for mutagenic and/or related cytological effects. Information from unpublished sources is also included [9,49]. Summaries of all these investigations are presented in the fol lowing section. Genetic toxicology testing $ Jackson [40] evaluated highly purified samples of 2,4,5-T * and T C D D for cytological effects in the African blood lily (H a e m a n t h u s k a t h e r in a e Baker). Treatments involving both compounds in varying proportions were studied. In contrast to the no-effect result with a highly purified sample of 2,4,5-T, dra matic inhibition of mitosis was observed in cells exposed either to 10'4 molar 2,4,5-T containing 0.2-- 1.0 pg T C D D per liter of water ** or to a 10'4 molar solution of 2,4,5-T containing an unknown level of T C D D as a contaminant. Similar results were obtained when treatments were limited to T C D D alone (0.2 pg and 1.0 pg T C D D per liter of water **). These treatments also induced formation of dicentric bridges and chromatin fusion with formation of multi nuclei or a single large nucleus. Because these effects were not evident in the pure 2,4,5-T sample, Jackson concluded that the cytological effects produced were due to the T C D D contaminant. Davring and S u m m e r [18] reported results obtained with a commercial sam ple of 2,4,5-T in which dioxin contamination was less than 0.1 ppm. This formulation was evaluated for cytological effects in a wild-type D r o s o p h ila m e la n o g a s t e r population by exposing adult flies, 24 h after eclosion, to 250 p p m 2,4,5-T in food. Results indicated that this 2,4,5-T formulation affected early oogenesis and caused sterility. However, it was not unequivocally stated that the observed sterility was of genetic origin. The formation of multinucleated cells after treatment with T C D D has also " been observed in mammals [12,29,42]. For example, Greig et al. [29] treated female Porton rats with single oral doses (50-- 400 pg/kg) of T C D D dissolved in diraethylsulfoxide or arachis oil. Histological examination of liver cells 60 days after treatment with 100 pg/kg T C D D revealed that parenchymal cell structures were altered and many were multinucleated. N o mitoses were observed in any of these multinucleated cells. The only other abnormality detected was an occasional pyknotic nucleus. These results were interpreted as indicating that T C D D had interfered with the capacity of liver cells to maintain their correct morphology, thus leading to death and/or structural disorganization. Buu-Hoi CD -I a* 4b O > * Further inform ation resarding the cytoloftica) effeeis produced by 2.4.5-T w ill be available soon in a paper entitled "T h e G enotoxic Effects of 2.4 .5 -T " b e in i prepared for publication in M utation Res. b y W .F. Grant. Jackson (4 0 ] reported that the m axim um solu b ility of T C D D in water was 0.2 MS/1 and that the 1 . 0 M i l l treatm ents were therefore probably subjected only to a water-saturated solu tion (0.2 MS/)). et al. Wistar and R rats tr 2,7-dic multin had be Vos carein< liver. I of T C induce studies treatec 25 pg/ ploidy Gret dibenz induce ments doses 5-day 6 h afi was di. peritoi receive ing thi 29 da\ of chr- studies dichlo: tial foj group admini a staticontro in a la mosorr tocol rats re weekly consist treatec tions. an efft for ch increas The tr 757(f0c|2429 Uy controlled and a ib lUtagenicity of ame from a number ,17,27,28,38,40,41, >t mutagenic and/or ed sources is also 3resented in the fol- T * and T C D D for k a t h e r i n a e Baker). :s were studied. In !e of 2,4,5-T, draner to 10*4 molar r to a 10"4 molar is a contaminant. to T C D D alone :ents also induced mation of multiot evident in the effects produced commercial sami 0.1 ppm. This :ype D r o s o p h i l a id n, to 250 ulaun affected uivocally stated T C D D has also al. [29] treated D D dissolved in er cells 60 days cell structures bserved in any tected was an ndicating that i their correct tion. Buu-Hoi *t*iovn*iJi*nblMe luotaotnioinn utl\ and that the t*d so lu tio n (0.2 145 et al. [12] have reported similar observations in liver and myocardial cells of Wistar rats after single intraperitoneal injections of T C D D (10 mg/kg). Khera and Ruddick [41] observed that mitotic figures in myocardial cells were rare in rats treated in utero during days 6-- 15 of gestation with 250-- 2000 pg/kg of 2,7-dichlorodibenzo-p-dioxin per day. Kimbrough et al. [42] have also reported multinucleated cell formation in the livers of N e w Zealand rabbits whose ears had been painted daily for 4 days with 0.2 ml of a 20 pg/ml T C D D solution. Vos, Moore, and Zinkl [67] have suggested that T C D D could be a hepatocarcinogen due to its specific cytological effect on the proliferating cells of the liver. When 58-week-old male C57B1/6 mice were treated with single oral doses of T C D D (100, 150-- 200 pg/kg), an enlargement of liver cell nuclei was induced. The L D S0 in these studies was calculated to be 114 pg/kg. In subacute studies, 100 4-month-old male mice were divided into two groups and either treated for two or six weeks with weekly T C D D doses of 0, 0.2, 1.0, 5.0, or 25 pg/kg. Liver cells of mice treated with six doses of 25 pg/kg showed poly ploidy, vacuolization of nuclei, and an increased mitotic rate. Green and Moreland [27] were the first to test dioxins (TCDD, 2,7-dichlorodibenzo-p-dioxin, and the unsubstituted dibenzo-p-dioxin) for their ability to induce chromosome aberrations in mammalian cells. In one of their experi ments [27] the dioxin compounds were dissolved in dimethylsulfoxide and doses of 10 pg/kg were administered daily by intubation to male rats for a 5-day period. Analyses of bone marrow preparations from animals sacrificed 6 h after the last treatment were negative. In a second experiment [27], T C D D was dissolved in an anisole/corn oil solvent (1.5% v/v) and administered intraperitoneally to rats in doses of 5,10 or 15 pg/kg. Another group of test animals received 20 pg/kg of T C D D orally from the same test solution. Animals receiv ing the higher doses, 15 pg/kg intraperitoneally and 20 pg/kg orally, were killed 29 days after treatment. Again, bone-marrow preparations revealed no evidence of chromosomal aberrations in any of the animals. As a result of these two studies [27], the authors concluded that the test compounds (TCDD, 2,7dichlorodibenzo-p-dioxin and dibenzo-p-dioxin) appeared to possess no poten tial for producing chromosome aberrations in the bone marrow of male rats. A -group of positive controls treated with triethylenemelamine (0.375 mg/kg), administered intraperitoneally and orally, and used as positive controls showed a statistically significant increase in chromosome abnormalities over untreated controls. In contrast with these earlier results, Green, Moreland and Sheu [28], in a later study, found that T C D D significantly increased the number of chro m o s o m e aberrations in rat bone marrow when a different experimental pro tocol was used. In this experiment [28], male and female Osborne-- Mendel rats received T C D D doses of 0.25, 0.5, 1.0, 2.0 or 4.0 pg/kg by gavage twice weekly for 13 weeks. The T C D D samples used were dissolved in a solvent consisting of one part acetone to 9 parts c o m oil. Bone marrow from the treated animals was assayed for mitotic inhibition and chromosome aberra tions. N o change in the mitotic index was observed at any dose, however, an effect was found in cells from both male and female animals when assayed for chromosome breaks. The treated female groups showed a significant increase at the 4 pg/kg level as compared with the 0.25 p g fh g level (P < 0.01). The treated male group showed a significant increase at the 2 /rgand 4 pg/kg r* 146 levels as compared with the 0.25 yg/kg level (P < 0.01). The authors cautioned that even though the results were significant, the observed activity should be regarded as only weakly positive. Czeizel and Kirly [17] cornpared.the frequency of chromosome aberrations in the peripheral lymphocytes of 76 workers employed at a chemical herbicide producing factory in Budapest (Hungary), along with that of 33 control indi viduals. The manufacturing process at this factory favored the formation of dioxins. Of the workers surveyed, 36 had been exposed to 2,4,5-trichlorophenoxyethanol (TCPE) or Klorinol and 26 to Buvinol (a combination her bicide containing T C P E and 2-chloro-6-ethylamino-4-isopropylamino-l,3,5triazine). The remaining 14 workers had never been engaged in the production or use of either of these herbicides. T C D D contamination found in the final herbicide products has been reported to be less than 0.1 mg/kg and generally not more than 0.05 mg/kg [66]. The frequency of chromatid-type and unstable chromosome aberrations found by Czeizel and Kirly [17] was higher ( P < 0.001) in the factory workers than in the controls, regardless of whether or not they had been directly involved in production of the herbicides. However, aberrations were more frequent in workers preparing T C P E and Buvinol than in the other factory workers, but the difference was significant only for the chro matid-type effect. Chromosome aberrations have also been reported in studies of Vietnamese populations exposed to the herbicide 2,4,5-T in which T C D D was present as a contaminant [32]. In these studies, a higher incidence of chro mosomal abnormalities was reported in individuals brought in contact with this herbicide as a result of forest defoliation. These studies, however, have been criticized as being statistically unsound (see Hay [31,32]). Preliminary results of a cytogenetic investigation of TCDD-exposed indi viduals from the Seveso, Italy population have been reported by Tenchini et al. [64]. Examinations of the affected population were confined to w o m e n w h o became pregnant immediately before or after exposure to T C D D and w h o had chosen to abort because of the possible teratogenic effects of this compoundr Chromosome analyses of maternal peripheral blood and abortive fetal tissue were scored for the presence of numerical and structural chromosome varia tions; no significant change was found in the chromosome number of any sam ple analyzed. Also, no significant increase was observed in the frequency of structural chromosome aberrations from maternal blood samples. There was, however, a higher number of structural aberrations in the fetal tissues than in maternal blood samples or fibroblasts from adult tissues, but the frequency of these aberrations did not appear to be greater than that expected to occur spontaneously in cultures of comparable cell types. Tenchini et al. [64] pointed out that these preliminary data do not answer the question of whether the higher frequencies of chromosome aberrations found in fetal tissues were due to chromosome damage caused by T C D D exposure. In another preliminary cytogenetic study related to the Seveso incident [46], no chromosome abnor malities were found in the peripheral blood cells of 90 workers selected from the chemical plant which was the source of the T C D D contamination in Seveso. Likewise, no abnormalities were found in chromosomes from peripheral blood cells of Seveso residents located in the area most severely contaminated with T C D D [46]. Hay [31] has cited results from a study done at another industrial nonmui sonai c< and tht TA53 five res', with thi TA1532 because protoco Salmoni culty in strains 1 Nebert, strains 1 due to : treatroei pound. 7 Come Ames S standarc ing care concern regarding the frequency of chromosome abnormalities in workers pre sumably exposed to T C D D during the production of 2,4,5-trichlorophenol. In this study, 800 workers with chloracns presumably caused from T C D D expo sure were investigated and the frequency'of chromosome abnormalities deter mined. The abnormalities observed among these workers were reported to be not greater than the statistical norm. T C D D was first evaluated for mutagenicity by Hussain et al. [38] because of its structural similarity to acridines (Fig. 1). The assay systems used were (1) E s c h e r ic h ia c o li strain Sd-4 which measures reversion from streptomycin dependence to streptomycin independence, (2) S a lm o n e lla t y p h im u r iu m strains T A 1 5 3 0 and TA1532 which measure reversion from histidine dependence to independence, and (3) prophage induction in E s c h e r ic h ia c o li K39 cells. T C D D was mutagenic in E . c o li Sd-4 and in Salmonella TA1532. T C D D had a weak prophage-inducing effect and did not show any mutagenic activity in Salmo nella TA1530. Parallel studies with acridine orange in E . c o l i Sd-4 and acridine mustard in Salmonella TA2532 were used as positive controls. Corollary results with these control compounds, coupled with the well-documented knowledge that Salmonella strain TA1532 detects frameshift mutagens, seem to indicate that T C D D was mutagenic via intercalation with D N A . Seiler [57] evaluated T C D D and octachlorodibenzo-p-dioxin (OCDD) in S a lm o n e lla t y p h im u r iu m strains G46, TA1530, TA1531, TA1532, and TA1534 and found T C D D to be a strong mutagen in strain TA1532, a doubtful mutagen in TA1531 and TA1534, and a nonmutagen in G 4 6 and TA1530. O C D D was reported as a questionable mutagen in strains TA1532 and TA1534 and as a nonmutagen in strains G46, TA1530 and TA1531 [57]. McCann [49] (per sonal communication) tested T C D D in the Salmonella system using a spot test and the standard plate test with strains TA1532, TA1535, TA1537, and TA1538, both with and without metabolic activation. In all experiments, nega tive results were obtained. The results [49] found with TA1 5 3 2 are in conflict with those obtained by Seiler [57] in this strain. McC a n n 's negative results with -- T A 1 5 3 2 cannot be directly compared with Hussain's et al. positive studies because these authors [38] used a liquid incubation procedure in their test protocol. The negative response which McC a n n [49] observed with the other Salmonella strains (TA1535, TA1537, and TA1538) adds further to the diffi culty in understanding the potential mutagenicity of T C D D , especially since strains TA1537 and TA1-538 are sensitive to the action of frameshift mutagens. Nebert, Thorgeirsson and Felton [53] have also reported negative findings with strains TA1 5 3 5 and TA1538. The differences in results for T A 1 5 3 2 could be due to m a n y factors, such as the solubility of the T C D D sample, its purity, treatment protocols, and the problems involved in handling such a toxic c o m pound. Table 1 summarizes results from these studies. C o m m o n e r [13] tested the unsubstituted dibenzo-p-dioxin in four of the A m e s Salmonella strains (TA1535, TA1537, TA1538, and TA100) by the standard plate test to determine the reliability of the test system in distinguish ing carcinogens from noncarcinogens. Negative results were reported for all strains with the concentrations tested (1.0,10 and 100 pg per plate). Dimethylsulfoxide was used as the solvent in the preparation of all test samples in this study. 148 TABLE 1 M U T A G E N IC IT Y O r D IO X IN C O M P O U N D S IN S a lm o n e lla ly p h l m u r lu m D ioxin Isom er TCDD OCDD Dthenr.o* p -d io x in Stralna detecting baae-palr aubatltutlona > G4S 0 0 0 -- 0 TA1530 0 0 -- -- 0 TA1635 -- " 0 0 0 -- TA100 0 0 0 0 0 _ Strains detecting fram eshlfts b TA1531 0 0 0 7 - 0 TA1B32 _ 0 44- 7 0 TA1B34 0 0 0 7 7 O TA1537* _ 0 0 0 0 TA1538 , --. 0 0 0 1 Sec alio Table 4 for adescription o f (he Salm onella alraina. 150 , not teated; -- , negative results; t, poaltlve reaulta; ?, doubtful mutagen. Reaulta tbow n were obtained ualng different experim ental protocols. Reference No. 49 53 SB 67 57 13 &- 'CO> * 9 M s 5 90frEZ,E M O d a 3 h> c f - HO.w -6o- ' 2 n 2 n- * H52 - ato i* 2 fir y??a?>^> S it s ?. 3 i 149 Further evidence for the possible intercalating reaction of T C D D with D N A has been reported by Kondorosi et al. [43]. These investigators checked T C D D and several electrophiles (methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, and ethylene dibromide) for their effect on the transfection of Q0 E N A . The loss of transfectivity in this system indicates a reaction with single-stranded nucleic acid. Intercalating agents which do not have active sidechains (e.g., alkylating groups) would be expected to give negative results in this test because they react primarily with double-stranded D N A . All the agents studied gave positive results except T C D D . Using T C D D at concentrations reported to be mutagenic in E s c h e r i c h i a c o l i Sd-4 and S a lm o n e lla t y p h im u r iu m T A 1 5 3 2 [38], Kondorosi et al. [43] found no effect on the transfection of Q0 E N A . The authors stated that these results confirm the assumption that T C D D 'forms a physical complex (intercalation) with D N A rather than reacting chemi cally with the nucleic acid. Toxicity and dominant lethal studies with T C D D have been reported by Khera and Euddick [41]. Groups of 20 male Wistar rats were dosed orally with 4, 8 or 12 pg/kg T C D D per day for 7 days before mating (survival results are shown in Table 2). Surviving males were caged with two untreated virgin females for 5 days and this regimen was followed for 7 sequential mating trials. Females were killed 9 days after separation from the males and viable embryos, resorption sites, and corpora lutea were counted. N o evidence was found for the induction of dominant lethal mutations during post-meiotic phases of spermatogenesis although the incidence of pregnancies from all mating trials in each treated group was reduced. Histological examination of surviving males showed normal testes, but the epididymides were inflamed with sperm granu loma formation which is analogous to changes seen in the autoimmune reaction following bacterial infection or the response of tissues to foreign bodies [41]. Other evidence that T C D D produces adverse effects in the testes has been reported by Van Miller and Allen [65]. In this study, male Sprague-- Dawley rats were fed diets containing various levels of T C D D for 65 weeks. Animals receiving 0.05, 0.5 and 1.0 p p m of T C D D in their food died within 4 weeks, and autopsies showed a noticeable decrease in spermatogenesis. Seiler [58] ^reported that a 0.4 mg/kg dose of T C D D administered intraperitoneally to male mice produced an approximate 5 0 % reduction in the rate of testicular D N A synthesis. T C D D and other dioxins have also been implicated in causing adverse effects in the testes of a number of other animals (mice [50], chickens [54], guinea pigs [50], and monkeys [54]). The fact that T C D D apparently reaches the testes [41,50,54,58,65] and the report that T C D D has a weak chromosome-breaking effect in rat bone-marrow cells [28] support, to some degree, oo * uu. TABLE 2 S U R V IV A L IN M A L E W IS T A R R A T S A F T E R T C D D T R E A T M E N T Doee (K (/k() Dead animal S u rv iv o rs Mean survival time (days) 12 20 8 11 42 0 17.7 9 20.1 18 36.5 * N o m ortalities w o n observed in the control (roup. Source: Khera and R u d d ick 141)'. / 0002434 150 TABLE 3 T H R E E -G E N E R A T IO N R E P R O D U C T IO N ST U D Y O F M A L E A N D F E M A L E S P R A G U E -- D A W LE Y R A T S IN G E ST IN G >PCDD D aily dote Treatm ent Observations {M tfk I) * to m atins F0 F1 FJ (day*) 0.1 0.01 0.001 9 0 (F 0 ) Decreaaed fertility: 109-- 116 poor aurviva] in F j ( F i and F 2 )b offspring BO (F0) N o effect on 1 0 9 -1 1 6 fertility ;F j offspring (F , a n d F 2 ) b and litter size appeared norm al B0(F0 ) N o effect on fertility 1 0 9 -1 1 6 (F , and F j)b ec Significant decrease in fertility ; F j offspring had decreased survival rate and reduced growth rate; sm aller litter size N o effect on fertility Significant decrease in fertility; F 3 off* spring bed decreased survival rate and reduced grow th rate; sm aller litter size N o effect on fertility * Anim als w e n m aintained continuously on diets containing sufficient' T C D D to provide the dose levels show n. ^ Offspring com prising the F j and F j generations were potentially exposed to T C D D during gestation via placenta] transfer and during lactation. A nim als from these groups began ingesting T C D D diets at 14-- 21 days o f age and were mated at approxim ately 130 days of age. c Due to adverse effects observed in F j parents, treatment was discountinued. Com piled from M urray et al. 162]. the possibility that T C D D m a y have the capacity to act as a weak dominant lethal agent. Further preliminary results reported by Murray et al. [52], in a three-generation reproduction study of Sprague-- Dawley rats ingesting various levels of T C D D , adds some additional support to this possibility (Table 3). Mul tigeneration studies, however, are not proper indicators of dominant lethality, nor is the production of adverse testicular effects an indicator of mutagenicity. These data [52] and the data cited previously [41,50,54,58,65] suggest a need for further testing. Even with the present evidence and speculation, it is not clear whether the adverse effects produced by T C D D in the testes are the result of direct T C D D activity or the result of some other toxic response trig gered by T C D D . Modifying effects on mutagenesis The importance of metabolism in mutagenesis studies has been considered because m a n y compounds thought to be nonmutagenic are converted to muta gens after metabolic activation. Polycyclic hydrocarbons are a notable example. Inducible enzymes in various body organs, particularly the liver, are involved in the metabolic conversion .of promutagens. Agents frequently used to increase these enzyme levels include Aroclor 1254 (a polychlorinated biphenyl), phno barbital, and 3-methylcholanthrene. T C D D has been reported to be a potent stimulant for hepatic aryl hydro carbon hydroxylase activity in male rats (Sprague-- Dawley), chick embryos (Leghorn), and female mice from several inbred strains (C3H/HeN, C57B1/6J, and BALB/cJ) [55]. When compared with results obtained in the rat with 7580 ' 0002435 ZW S P R A G U E -- D A W LEY Fi c -se in rin g viva) grow th sue ity Significant decrease in fertility; F 3 offspring had decreased survival rate and reduced growth rate; sm aller litter sire N o effect on fertility OD to provide the dose levels to T C D D during gestation via ingestin g T C D D diets at 14-- ed. C om piled from M urray et as a weak dominant rray et al. [52], in a ats ingesting various Dilitv (Table 3). Mul ti nant lethality, tor of mutagenicity. 3,65] suggest a need peculation, it is not i the testes are the toxic response trig as been considered converted to mutaa notable example, iver, are involved in Jy used to increase 1 biphenyl), pheno- epatic aryl hydro?), chick embryos H/HeN, C57B1/6J, ^d in the rat with 151 3-methylcholanthrene, T C D D is 30 0D0 times more potent in inducing aryl hydrocarbon hydroxylase activity [55]. Felton and Nebert [24] used liver frac tions from C57B1/6N and D B A / 2 N mice pretreated with T C D D at doses rang ing from 0.1 to 100 pg/kg to assay the in vitro mutagenicity of 3-methyl cholanthrene and benzo[c]pyrene in the A m e s Salmonella test sytem using strain TA1538. A significant increase in 3-methylcholanthrene mutagenesis was observed in test samples pretreated with liver fractions from TCDD-induced mice. The mutagenicity of 3-methylcholanthrene followed quite closely the hydroxylase activity and cytochrome P!-450 (P 448) formation. The muta genicity of benzo[a]pyrene was not affected when subjected to this same pro tocol. Berry et al. [7] reported that T C D D appears to be an exceptionally potent and broad-spectrum toahsplacental inducing agent for carcinogen-transforming enzymes found ijj/'tfarious tissues. Pregnant Sprague-- Dawley rats were given single intraperitoneal injections ofT C D D (0.2-- 6.0 Mg/kg) dissolved in c o m oil on day 17 of gestation. Animals were killed on day 20 of gestation, and homogenates were made of maternal livers,'lungs, kidneys, adrenals, and placentas and of fetal livers, kidneys, and skin. These homogenates were then treated with [3H ]benzo[a ]pyrene (BP) and were assayed for the effects of T C D D pretreat ment on the tissue-mediated covalent binding of B P to D N A . Tissue samples from the livers, lungs, and placentas of TCDD-pretreated dams, as well as sam ples from livers, lungs, and skin of their fetuses, showed an increased capacity to covalently bind B P to D N A when compared with tissue samples taken from noninduced control animals. Berry et al. [7] stated that "The nature of the T C D D enhancement appeared to be tissue-specific, and moreover, the data from the binding in vitro appeared to correlate well with the metabolizing capa bilities of the tissue and the metabolites formed (BP-7,8-dihydrodiol, BP-4,5dihydrodiol, BP-9 phenol, and BP-3 phenol)". The effect of T C D D on the covalent binding activity of benzo[o]pyrene to D N A and the mutagenicity of 3-methylcholanthrene raises the question of whether T C D D can influence the biological activity of not only other poly cyclic hydrocarbons but other compounds as well. The significance of this possibility will not be fully understood until additional data are available. Comparative mutagenicity of dioxins and acridines The testing of T C D D for mutagenicity and its initial classification as a muta gen was brought about by its structural similarities to acridines (Fig. 1). The following sections summarize, and compare the mutagenicity of acridine and dioxin compounds as measured in identical test systems. These results neither prove nor disprove the possibility that T C D D m a y act as an intercalating agent. The exact m o d e of action, of T C D D and other dioxins will be clarified only after further experimentation. B a cteria Sa lm o n ella ty p h im u riu m Tests with several genetically defined histidine mutants of S a lm o n e lla t y p h i m u r iu m a r e presently used in a variety of applications for the evaluation of 152 chemicals for mutagenicity 11-- 3] (Table 4). Salmonella strains used in the evaluation of TCDD, O C D D , and the unsubstituted dibenzo-p-dioxin are shown in Table 5, along with results for several acridine compounds. E sch e rich ia co li Sd-4 E . c o l i Sd-4 is a mutant derivative of E . c o l i B and is used to detect rever sions from streptomycin-dependence to streptomycin-independence. Documen tation of the molecular mechanism resulting in this mutation was not found but reportedly results from a base-pair substitution mutation [10]. See Table 6 for comparative results of T C D D with acriflavin. B a cterio p h a g es (prophage in d u ctio n ) A search of the Environmental Mutagen Information Center data base revealed no information on the ability of acridines to activate la m b d a phage in E . c o l i K39 cells which were used to test T C D D [38]. Reports noting activa tion of la m b d a phage in another E . c o li strain and negative findings for phage induction in S a lm o n e lla t h o m p s o n were found and are shown in Table 7. In an extensive review on prophage induction in lysogenic bacteria, Heine m a n n [33] listed acridine orange as having a moderate prophage-inducing abil ity. Hussain et al. [38] stated that T C D D had a weak prophage-inducing effect and that such results agreed with data obtained from studies with acridines. The data in Table 7 indicate that acridines, considered as a group, could be classed as weak when tested for prophage activation. Presently, insufficient data are available at this writing for a true comparison. DOW 374410 TABLE 4 C H A R A C T E R IS T IC S O F T H E A M E S S A L M O N E L L A T E S T S T R A IN S U SE D TO E V A L U A T E TH E M U T A G E N IC IT Y O F T C D D A N D O T H ER D IO X IN S Strain M utation (site in histidine operon) O ther characteristics Type o f m utation detectad G46 TA1S30 TA1S31 TA1S32 TA1S34 TA1536 TA1S37 TA1S38 TA100 h it G46 his G 46 h it C207 his C3076 h it D30S2 his G 46 his C 3076 his D 3052 his G 46 N orm al lipopolysaccharide coat; D N A excision-repair present Partial lipopolysaccharide coat present: D N A excisionrcpalr deficient Partial lipopolysaccharide coat present: D N A cxd sioa repair deficient Partial lipopolysaccharide coat present: D N A excisionrepair deficient N orm al lipopolyucchride eoat; D N A excision-repair present Lipopolysaccharide deficient; D N A excision-repair defective Lipopolysaccharide deficient: D N A excision-repair defective Lipopolysaccharide deficient; D N A excision-repair defective Lip o p o ly saccharide deficient; D N A excision-repair defective; R factor plasm id present Base substitution Bass substitution Fram eshift Fram eshift Fram eshift Base substitution Fram eshift Fram eshift Base substitution Com piled tram A m ti et aL 11.2) and Sim m on |60). D02437 T y .V 'ir./fcV'-v . -- * trains used in the -dioxin are shown d to detect reveridence. Documenon was not found [10]. See Table 6 Center data base e la m b d a phage in jrts noting activa'indings for phage in Table 7. : bacteria, Heineage-inducing abile-inducing effect es with acridines, group, could be ntly, insufficient 'O e v a l u a t e t h e L ) o i m utation detected Base substitution Base substitution Fram eshift Fram eshift Fram eshift Base substitution Fram eshift Fram eshift 3ase substitution SI CtC>Z0 e<>r cne nin ru*> nie < H IO I B IB < H ntD < A t- 5 E Nr> < cH V I 4 + 9 ito <H *. lele elei eoe I ,/ O HOo .p? >- \:s VC e H * 154 TABLE 6 Mammals M U T A G E N IC IT Y R E S U L T S W IT H E i c h c r i c h a coli Sd-4 Chem ical tested Results * R e i; Dominant Mailing an TCDD A c rifla v in e 4 38 19 cals reported of their revit * 4 , Positive results. in Table 8. Positive re some rearrar TABLE 7 induces pre< P R O P H A G E IN D U C T IO N Freese 126] Chem ical tested H ost organism Re sult! * Ret. adding or dthe activity TCDD sehcrichio coli K 3 9 4 38 ported by tl A cridine orange P roflivine Ethacridine lactate m onohydrate E s c h e r ic h ia c o l i 16 S a l m o n e l l a t h o m p s o n 19 Soim one/io t h o m p t o n 19 * 4, Positive results; -- . negative results. 4 - 61 the results c a62 test, couplet o62 testis [41,5( can act as a damage to t pounds are and m a m m r TABLE 8 M A M M A L IA N D O M IN A N T L E T H A L S T U D IE S dine compc mammalian Com pound Reported results * Ref. O Mutation re TCDD A cridine orange A c ii flavine 9-Am inoacridine IC R -170 b Q uinacrine hydrochloride T ry p a fia v in e _ -- -- -- 4 -- -- 41 47 22 22 63 22 22 The Sevt effects of 1 an attempt exposure, i and other I * -- , Negative results: +, positive results. * A c v ** both an intercalating and an alkylating agent. domestic a: plant [46] way includ tests in m k TABLE 9 of peripher C H R O M O SO M E A B E R R A T IO N S T U D IE S IN C U L T U R E D M A M M A L IA N C E L L S Com pound Cell type Reported result! * Ref. rabbits), cj crossing-ov Discussion TCDD b R at bone m arrow cell! * (w eak) 28 ! TCDD b 2.7-D ichlorodibcnzo-p-dioxin Rat bone manrow cells Rat bone m arrow cells -- 27 27 Of the i D ib e n z o -p -d io x in Rat bona m arrow cell! -- 27 tested for P ro fla v in e H rL a cells 4 69 [9, 27, 28 A cridine A cridine orange Hum an diploid fibroblasts Hum an diploid fibroblasts 4 4 69 69 as the uns most freqi ; * +, Poiitive result*; -- . negative results. been cone b Results from theft experim ent! [27.28) were obtxined using different experim ental protocol!, especially tested alo; in the dote range texted, d o lin g fchedule. and aolventi used. a&ufc. :is* Ref. 38 61 62 62 Ref. 28 27 27 27 59 59 59 Protocols. eipecieUy DOW 374413 Mammals 155 Dominant lethal studies Mailing and Wassom [47] in their review of the mutagenic action of chemi cals reported that only 6 acridine compounds had been evaluated, at the time of their review, in the dominant lethal test. Results from these tests are shown in Table 8. Positive results in the dominant lethal test are believed to be due to chromo some rearrangements rather than point mutations. Therefore, a mutagen that induces predominantly point mutations would go undetected in this test. Freese [26] stated that acridines produce point mutations via intercalation by adding or deleting bases in O N A. Thus, negative results would be expected if the activity of T C D D resembled the intercalating action of acridines as sup ported by the negative results reported by Khera and Ruddick [41]. However, the results obtained by Green, Moreland and Sheu [28] in the rat bone-marrow test, coupled with the evidence that T C D D does produce adverse effects in the testis [41,50,54,58,65], still leave the unresolved question of whether T C D D can act as a weak dominant lethal-inducing agent or cause some other type of damage to the genetic material of mammalian germ cells. Several acridine c o m pounds are known inducers of chromosome aberrations in plants, Drosophila, and mammalian cells in culture [59], but no reports on the evaluation of acri dine compounds in the rat bone-marrow test were found. Results from other mammalian studies with acridines are shown in Table 9. Mutation research in progress The Seveso incident revealed a lack of information concerning the health effects of T C D D . As a result, a number of research projects were initiated in an attempt to obtain a better perspective of the hazards posed by T C D D exposure. Most of these investigations are taking place in laboratories in Italy and other European countries. Information is also being collected on people-or domestic animals exposed to T C D D as a result of the accident at the chemical plant [46] which caused the contamination of the Seveso area. Projects under way include fertility studies, dominant lethal studies in rabbits, translocation tests in mice, point-mutation tests using hamster cells, cytological examinations ' of peripheral blood from exposed individuals and exposed animals (cattle and rabbits), cytological examinations of fetal tissue from abortuses, and mitotic crossing-over and recombination studies in yeast. Discussion and conclusion Of the m a n y structurally possible dioxin configurations, only 4 have been tested for mutagenicity or other related effects. These are the di- [27], tetra[9, 27, 28, 38, 41, 49, 53, 57], and octa- [57] chlorinated derivatives as well as the unsubstituted dibenzo-p-dioxin [13,27]. Of these, T C D D has been the most frequently tested but the results obtained from these studies have not been conclusive. For example, positive results were reported when T C D D was tested alone for cytological disturbances in the African blood lily [40] or for 000244 -r% :?* 156 mutation induction in S a lm o n e lla t y p h im u r iu m [38,57] and E s c h e r i c h i a c o li [38]. Positive results were also reported, when 2,4,5-T test samples contami nated with T C D D were evaluated for cytological effects in Drosophila [18] and the African blood lily [40]. On the other hand, negative results were reported with T C D D in S a lm o n e lla t y p h im u r iu m conflicting with those studies reporting positive findings (see Table 1). The octachloro derivative ( O C D D ) was a doubt ful mutagen when evaluated in the Salmonella test and the unsubstituted dibenzo-p-dioxin was nonmutagenic [13], Mammalian studies with dioxins are scarce. The only data found showed that T C D D gave negative results when tested for dominant lethal effects in rats [41] and weakly positive results when assayed for the induction of chromo some aberrations in rat bone-marrow cells [28]. These data, coupled with studies showing that T C D D does reach the testes [41,50,54,58,65], indicate the possibility that T C D D could act as a weak dominant-lethal inducing agent or could cause damage to the genetic material which manifests itself in some other way. The dichloro derivative (DCDD) and the unsubstituted dibenzo-pdioxin were both negative in the rat bone marrow test [27]. The cytological effects produced by T C D D in the liver of treated animals ' [12,29,42,67] m a y be attributed to the toxic effects produced by the short term high dose levels administered in these studies and the fact that T C D D tends to concentrate in the liver. Preliminary results obtained from cytogenetic analyses of maternal blood and abortive fetal tissue taken from persons exposed to T C D D during the Seveso, Italy accident are inconclusive [64]. Inconclusive results have also been reported [31] from studies of peripheral blood cells taken from individuals exposed to T C D D within the chemical plant which caused the accidental expo sure at Seveso as well as from individuals residing in heavily contaminated areas. O n the other hand, workers exposed to the herbicides Klorinol and Buvinol, which m a y have varying levels of T C D D contamination, had a higher incidence of chromatid breaks in peripheral lymphocytes than did nonexposed individuals [17]. Increased frequencies in chromosome aberrations were also reported in Vietnamese exposed to TCDD-contaminated herbicide 2,4,5-T, but these studies have been challenged and are of limited value [32]. The information implicating T C D D and other dioxin compounds asmutagens is scarce and conflicting; therefore, more work isrequired before the mutagenic potential of these compounds can be determined. Factors which have con tributed to the difficulty in understanding the experimental results with dioxins, particularly the T C D D derivative, include the extremes in toxicity of these compounds and their varied sensitivities to different treatment protocols. Solubility * seems to be one of the more outstanding problems encountered when working with dioxins. To obtain comparable data, all future studies should be done under similar treatment conditions whenever practical and with * Inform ation o n th * solu b ility of T C D D can be found in Table ] of the paper b y W .B. Crum m ett and R J . S le h l entitled " Determ ination of Chlorinated D ibenxo-p-dioxins and D Jbenxoiuiaru in V a riou s M aterials''. Enviro n. Health Penpecl.. S (1973) 16-- 25. See also the paper by C. B o tri, A . M em oli and P . A lhaique entiUed "T C D D Solubilization and Pholodecom position f n Aqueous Solu tio n s". En viro n . Sci. Tecbnol., 12 (1 B 7S) 335-- 336. tamples the acti< being st strains r only on such as Ames S or testir genetic dioxins also be prediox dichlorc in this : ward it . 2,3,7,8TCDD, vations detect i desirabl of certr promot pounds are pot TCDD, the Sev Ackno' The ronmei F. Gra Labora Joyce '. Interm Health Special Sr tii is A b m m ** ftf m T 7] and E s c h e r i c h i a c o l i ' 4 r ` ' samples contamiin jsophila [18] and /e results were reported those studies reporting g ( O C D D ) was a doubtand the unsubstituted lata found showed that i lethal effects in rats induction of chromose data, coupled with 50,54,58,65], indicate t-lethal inducing agent lanifests itself in some substituted dibenzo-p7]ver of treated animals roduced by the short* the fact that T C D D es of maternal blood to T C D D during the results have also been ?Jten from individuals i the accidental expoleavily contaminated bicides Klorinol and in<* i, had a higher than did nonexposed aberrations were also erbicide 2,4,5-T, but [32]. npounds as mutagens aefore the mutagenic Drs which have conmental results with remes in toxicity of treatment protocols, oblems encountered i, all future studies -Tpractical and with paper by W .B. Crum m elt ms and D jbenxofurans in the paper by C. Botr<, A, p oaiiion in AQ ueous Solu* 157 samples of known purity *. Mutagenicity assay systems that are sensitive to the action of intercalating agents should be considered first when systems are being selected for additional tests. For instance, reexamination in sensitive strains of Salmonella, particularly TA1532, is needed since this strain is the only one which has shown a positive response (Tables 4 and 5). Experiments such as these will help resolve the conflicting results observed thus far in the A m e s Salmonella test. Likewise, retesting for dominant lethality in mammals or testing in other in vivo mammalian systems which measure damage to the genetic material of germ cells should also be given consideration. The testing of dioxins using the multipurpose S a c c h a r o m y c e s c e r e v is ia e strain MP-1 would also be of interest since Fahrig, Nilsson and Rappe [23] have found that the predioxins, 4,5,6-trichloro-2-(2,4-dichlorophenoxy)phenol and 4-chloro-2-(2,4dichlorophenoxy)phenol induce forward mutations and mitotic crossing-over in this yeast strain. Pentachlorophenol (isomer purity 99%) induced some for ward mutations and mitotic gene conversion in this same yeast strain, but 2,3,7,8-tetrachlorodibenzofuran, a compound which is structurally similar to T C D D , did not induce any genetic activity in this organism [23]. These obser vations make further testing of dioxins in this or other yeast strains which detect mitotic crossing-over, gene conversion, or forward and reverse mutations desirable (see Zimmermann [70]) **. Evidence that T C D D is a potent inducer of certain microsomal enzymes points to the need for additional studies on the promoting effect which dioxins m a y have on the mutagenicity of other c o m pounds. Particular attention should be given to formulations in which dioxins are potential contaminants. A better understanding of the mutagenicity of T C D D , and perhaps other dioxins, will be obtained once studies stimulated by the Seveso tragedy have been properly evaluated. Acknowledgments The authors are grateful to Dr. Heinrich V. Mailing, National Institueof Envi ronmental Health Sciences, Dr. Sidney Green, Howard University, Dr. William F. Grant, McGill University, and Dr. Richard F. Kimball, Oak Ridge National Laboratory, for their review and helpful comments. Thanks are also due Dr. Joyce McCann, University of California (Berkeley), Dr. Vincent Simmon, SRI International, and Dr. Georgio Bronzetti, National Institute of Environmental Health Sciences, for providing the authors with unpublished information. Special acknowledgment is given to the Environmental Mutagen Information 374415 * See paper by O . A nilin e entitled "Preparation of Chlorodibenzo-p-dioxint for Toxicological Evalua tio n ", in: EJH. Blair (Ed.), C hlorod ioxin t -- O rigin and Fate. Advance* in Cbemietry Se ri N o. 220, Am erican Chem ical Society, W ashington, D .C ,, 1973, pp. 1 2 6 -- 135. * A fter this m anuscript was written. Bronzetti (personal com m unication) (9 ) sent the authors result* from som e prelim inary experim ents he had done w ith T C D D in the yeast strain D 7 of Saccharo m yces ccreuuioe.The genetic end-points of these experim ents were m itotic crossingover, gene con version, and reverse m utation. Retults from these experim ents, w hich were done w ith and w ithout m etabolic activation, revealed that T C D D induced a high rate o f killing, and no crossing-over or gene conversion. A weak reverse m utation inducing effect was observed as well at the presence of several "petite co lo n ie s" on the plates screened. The T C D D concentrations tested in these experi m ents were 1, 2, 4. 6 and iig/ml. A n acetone-- corn o il solvent was used in the preparation of the T C D D samples. * 0002442 ^^37 156 Center staff for reviewing the manuscript and providing a valuable system for accessing the chemical mutagenesis literature. W e are especially indebted to Wilma Barnard for her patience in typing this manuscript through its many revisions and updates. 27 G rr Ph* 28 C rf I* ' 29 Gre dio- References 3 0 G ri! 31 Ha> 32 Haj 1 A n a . B.N,, E.G. G urney. J.A. M iU cr and H. BarU ch. Carcinotem as fram cshift mutagens: metabolites 33 Hei and derivatives of 2-ecetylam inofluorene and other arom atic amine carcinogens. Proe. Natl. Acad. Scl. (U .S.A.). 66 0 9 7 2 )3 1 2 8 -3 1 3 2 . gen gen 2 Am es, BJf.. F.D. Lee and W .E. D urston. A n im proved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. (U .S.A.), 70 (1973) 782-- 786. 3 Am es, B Ji., and C. Yanofaky. The detection o f chemical mutagens w ith enteric bacteria, in: A . H oilaender (Ed.), Chem ical Mutagens: Principles and M ethods for Their Detection. Plenum, New Y ork. 1971. pp. 267-282. 4 Anonym ous, Another herbicide on the blacklist. Nature (Lond on), 226 (1 97 0) 309--311. 5 Anonym ous. A close loo k at T C D D , A gric. Res.. 20 (4) (1 97 1) 8-- 10. 6 Anonym ous, Herbicides better for birds than bacteria. Fo od Cosmet. Toxiol., 11 (1) (1 97 3) 149-- ISO . 7 Berry. D X .. T J . Slaga. K M . W ilson. P.K. Zachariah. M J . Nam kunc. W Jd. Bracken and M .R . Jucbau, Transplacental induction of m ixed-function oxygenases in extra-hepatic tissues b y 2,3,7,8-tetraehlorodibenzo-p-dioxin, Biochem . Pharm acol., 26 (1 9 7 7 ) 1 3 8 3 --1388. 8 Boer, F J , F.P. van Rem oortere and W.W. M uelder, The preparation and structure o f 2,3,7,8-tetraehloro^p-dioxin and 2,7-dichioro-P-dioxin. J. A m . Chem . Soc-. 94 (3) (1 97 2) 1006-- 1007. 9 Bronzetti, G ,, Unpublished data. 10 Bronzetti. G ,, and E. Zeiger. The toxicity of 2.3.7.8-tetrachlorodibenzo^'dioxin (in preparation). 11 Bnrsick, D J .. and E. Zeiger. A com parison of chem ically induced reversion patterns of S a l m o n t l l a t y p h i m u r i u m and S a c c h o r o m y c e t e m u u i a t m utants using in vitro plate tests. M utation Res., 14 34 Her del- 35 Hu: .- col) 36 Hu: dib- o and O 17 $ 37 Huchi 38 Hu? C O ten- 39 In i Ox the Ox 101 4 0 Jac M of a y 41 K h let) (1972) 2 7 1-275 . Ser 12 Buu-Hol, N J ., Pham -Huu Chanh. G. Sesque, M .C. Azum -G elada and G. Saint-Ruf, Organs as targets of "d io x in " (2.3,7.8-tetrachlorodibenzo-p-dioxin) intoxication, Naturwissenschaften. 59 (1972) 174-- 175. 42 K ii of 43 K r 13 Com m oner. B ,, R e lia b ility of bacterial m utagenesis techniques to distinguish carcinogenic and non- Rh carcinogenic chemicals. Environm ental Protection Agency Report N o. EPA-600/1 -76-022 (1976) 44 Le 1-- 103. agi 14 Cook, TJd.. and CJC. G oldm an, Hyeanthone and its congeners as bacterial mutagens, J. Bacteriol,, Be 122 (1975) 549-- 556. '_ 45 Le 15 Courtney, K B , D.W . G aylor, M X . Hogan. H X . Falk, R .R . Bates and I. M itchell, Teratogenic evalua At tion of 2.4.5-T. Science. 168 (1 97 0) 8 6 4 -8 6 6 . 16 Courtney, K .D , and J.A . M oore. Teratology studies w ith 2,4.5-trichloropheaoxyacetic acid and 2.3,7.8-tetrachlarodibcnxo-sa-dioxin. Toxicol. A p p l. Pharm acol-, 20 (1 9 7 1 ) 39 6 -- 403. PP 46 Lo 47 M. 17 Cxcizcl, E , and J. K ir ily Chrom osom e exam inations in w orkers producing K lo rln o l and Buvinol, in: Ha L . Banki. (Ed.), The Developm ent o f a Pesticide as a Com plex Scientific Task, M edicine, Budapest. 48 Me 1B76.PP. 239-256. m> 18 D fvring, L _ and M . Sum m er, Cytogenetic effecta o f 2,4.5-trichlorophenoxy-eeetic a d d on oogenesis and early embryogeneais in D r o m p h O a m clanogaafcr, Heredltas, 68 (1 97 1) 1 1 5 --122. 19 Demerec, H , G. Bertani and J. Flint. A survey o f com pounds far m utagenic action on . coll. Am . Nat,, 85 (1951) 119-- 136. 20 Dinger. C .Y ,, J X . H u ff and H .B. Gerstner. Treatise on chlorinated dibenzo-p-dloxins and chlorinated dibenzofurans at biological and environm ental problem s (in preparation). 21 Ehrenbcrg. L _ On the m utagenic action o f phenoxy acid preparations, E M S New sL, C (1972) 27. 22 Epstein. S .X . E. A rnold , 1 . Andrea. W. Bass and Y . Bishop, Detection o f chemical mutagen b y the (1 49 M< 50 Mi in 51 M 52 M- ge Ph dom inant lethal assay in the m ouse, T oxicoL A p p l. Phasm acoL. 23 (1 97 2) 28 8 -- 325. 23 Fabric. R - C.-A. N ilsson and C. Rappc, Genetic activity o f chlorophenob and chlorophcnol im purities, 63 Ni ar in : K J l. Rao (Ed.), Pentachloropbenol: Chem istry. Pharm acology and Environm ental Toxicology, A- Plenum . New Y ork, 1978. pp. 325-- 338. 24 Felton. I X and D.W . Ncbert, Mutagenesis of certain activated carcinogens in vitro associated with 54 N cl genetically mediated increase in m onooxygenase activity and cytochrom e P j-4 5 0 , J. B io l. C he m , 250 (1975) 67 69 -67 78 . 55 P< ar 25 F ish bein. L , Mutagens and potential m utagens in the biosphere. M utation Res.. 21 ,(1973) 220. 56 Pr 26 Freese. E ,, M olecular m echanism o f m utations, in: J.H . T a ylo r (Ed.), M olecular Genetics, Academ ic 0 0 C 2443Press. New York. 1969. pp. 2 0 9 -2 6 9 . oi j lir"' a valuable system for ir. pecially indebted to uscript through its many . t r a i n m u t a g e n s : metabolite re carcinogen. Proc. NaU. Acad. test system fo r the detection and _S.A.>, 7 0 (1 9 7 3 ) 782-- 786. w ith enteric bacteria,in: A. H oi* ir D etection, Plenum . Mew York. .26 (1 9 7 0 ) 3 0 9 -- 811. net. T o x io l.. 11 (1 ) (1973) 1 4 9 - W .M . Bracken and M Jl. Juchau, itic tissues by 2.3,7,8-tetrachloro- on and structure o f 2.3.7,8*tetra. (1972) 1006--1007. zo-p-dioxin (in preparation), reversion patterns o f S a l m o n t U e 0 plate tests. M utation R e s- 14 G . Sa in t*R u f. Organs as tercets of vissenschaften. 69 (1972) 174-- jstin gu isb carcinogenic and non* N o. EPA -600/1*76*022 (1976) ra d . m utacens, J. BacterioL, 1 1. M itch ell. Teratogenic evalue- -ichlorophenoxyacetic ad d and 3 7 1 )3 9 6 -4 0 3 . d u d n g K lo rin o l and B uvinoL in: :title T a sk, M edicine. Budapest, ienoxy-eeetic a d d on oogenesis m m 6--122. Jtageaic action on . coll. Am . enxo-p-dioxins and chlorinated ). S M S Newel., 6 (1 9 7 2 ) 27. 1 o f chem ical m utagens b y the 7 2 )2 8 8 -3 2 6 . Is and chlorophenol im purities, rd Environm ental Toxicology, ' ge n s in vitro associated w ith rome P ,-4 6 0 . J. B io l. C b cm - *n Res.. 21 (1 97 3) 220. M olecular Genetics. Academ ic 27 Green. S.. and F 3 . Moreland, Cytogenetic evaluation of several d ioxins in the rat, Toxicol A p p l' Pharm acol.. 33 (1 97 5) 161. gg Green. S - F. M oreland and C. Sheu. Cytogenetic cflects of 2.3.7.8-tetrachlorodlbenzo-p3ioxln on rat bone m arrow cells, F D A By-Lines. No. 6 (1 97 7) 292-- 294. 29 G rsig. J.B., G. Jones, W.H. Butler and 7.M . Barnes, Toxic effects of 2^.7.B*tetrachlorodlbenxo-pd ioxin . Food Cosm et. Toxicol., 11 (1 97 3) 686-- 695. g 0 G ribble. G .W - T C D D : a deadly m olecule. Chem istry. 47 (1 97 4) 16-- 18. g l H ay. A .. Id entifying carcinogens. Nature (Lond on). 269 (1 9 7 7 )4 6 8 -- 470. 22 H ay. A - V ietnam k d ioxin problem . Nature (Lond on). 271 (1 97 8) 697-- 598. 23 Hcinem ann, B., Prophage induction in lysogenic bacteria as a m ethod of detecting potential m uta genic. carcinogenic, carcinostatic. and teratogenic agents, in: A . Hollaender (Ed.), Chem ical M uta gens: Principles and M ethods for Their Detection, Plenum . New Y o rk . 1971. pp. 235--266. 24 Herbold, V .B., W. Buselm aier and G. RBhrbom , C ritical survey on liver m icrosom c test, a system for detecting point m utations. A nn eim . Forsch.. 26 (1 9 7 6 ) 1706-- 1707. 35 H u ff, J.E.. and J.S. Wasaom. Chlorinated dlbenzodioxins and dibenzofurani: an annotated literature collection. Environ. Health Pergpect.. 6 (1 97 3) 283-- 312. 36 H uff, J .E - and J.S. Wassom. Hazardous contam inants: chlorinated dlbenzodioxins and chlorinated dibenzofurans. in: Proceedings of the 2nd A nnual Conference on Environm ental Chem icals: Hum an and A nim al Health, Colorado State University, F o rt Collins. Colorado, July 23--27, 1973, (1974) 176-- 197. 37 H u ff, J .E - and I S . W assom , Health hazards from chem ical im purities: chlorinated dibengodioxins and chlorinated dibenzofurans. Intern. J. Environ. Stud., 6 (1 97 4) 13-- 17. 38 H ussain, S - L . Ehzenberg, G. Ld froth and T. GefvaU, Mutagenic effects of T C D D on bacterial sys tems, A rabio, 1 (1 9 7 2 ) 3 2 -3 3 . 39 International Agency for Research on Cancer, Chlorinated dlbenzodioxins. in: IA R C M onographs on the Evaluation o f the Carcinogenic R isk of Chem icals to M an, V o L 16. L yon . France, 1977. pp. 41-- 102. 4 0 Jackson. W .T - Regulation of m itosis. 3. C ytologies] effects of 2,4.6-trichloropbenoxyacetic acid and o f d ioxin contam inants in 2.4.6-T form ulations, J. Cell ScL. 10 (1 97 2) 16-- 26. 41 Khera, K .5 - and J.A . Ruddick. Polychlorod ib enzo*p 3ioxias: perinatal effects and the dom inant lethal test in W istar rats, in: E J l. B lair (Ed.), C hlorod ioxin s -- O rigin and Fate, Advances in Chem istry Scries N o. 120, Am erican Chem ical Society, W ashington. D .C - 1973. pp. 70--34. 4 2 K im brough. R J )., C .D . Carter, J.A. Liddle. R .E . Cline and P.E. Phillips. Epidem iology and pathology o f a tetrachlorodibenzo-dioxin poisoning episode. A rch. Environ. Health. 32 (1977) 77--36. 48 K ond orogL A - I. Fedorcsak, F. Solym oty, L . Ehzenberg and S. Ostcrm an-Golkar. Inactivation o f Q ff R N A by electrophiles. M utation Res.. 17 (1 97 3) 14 9-- 161. 4 4 Legator, M .S - The host-mediated assay, a practical procedure for evaluating potential mutagenic agents, in: F. V ogel and G. R Shrb orn (Eds.), Chem ical Mutagenesis in Mam m als and M an, Springer. Berlin, 1970. pp. 26 0 -- 270. 4 5 Legator. I U n The use of indirect indicators for m utagenicity testing, in : S.S. Epstein (Ed.), D rugs of A buse: Their Genetic and Other Chronic Nonpsychiatric Hazards, M IT Press, Cam bridge, Mast., 1971. pp. 171-- 176. 46 Loprieno, N ,, Com m unication of w ork in progress. 4 7 M ailing. H .V ,, and J S . Wassom, A ctio n o f m utagenic agents, in: J.G. W Bson and F.C. Fraser (Eds.), H andbook o f Teratology, V o L 1. Planum , New Y o rk , 1977. pp. 99-- 162. 4 8 M cCann, J , E. C hoi, E. Yam asaki and B.N . Am es, Detection o f carcinogens as m utagens In tlie Sal m onella/m icrotom e test: assay o f 3 0 0 chem icals: discussion. Proc. Natl. Acad. Sc L (U .S.A .), 73 (1 9 7 5 )9 6 0 -9 6 4 . 4 9 M cCann, J ,, U npublished data. 60 M cConnell, E.E., and J-A . M oore, The com parative toxicity o f chlorinated dibenzo-p-dtoxin isom ers in m ice and guinaa pigs, Toxicol. A ppl. Pbarm acbL. 37 (1 97 6) 146. 51 M cG in ty. L ,, The graveyard on M ila n 's doorstep. New ScL. 71 (1 0 1 4 ) (1 9 7 6 ) 38 3 --386. 52 M u rray, F J , F .A . Sm ith, K .D . Nitsehke, C.G. H um islon . R J . K ociba and B.A. Schw etz, Threegeneration reproduction study of rats ingesting 2J.7.S*ietrach ioeod lb sn zo*p 3ioxin . T oxicol. A p p L Pbarm acoL. 41 (1 97 7) 2 0 0 -2 0 1 . 63 Nebert, D.W ,, S 3 . Tbotgeirason and J S . Felton, Genetic differences in mutagenesis, carcinogenesis and drug toxicity, in: F J . de Series. J.R . Foutg. J.R . Bend and R Jd . Philpot (Eds.), In vitro M etabolic A ctivation in Mutagenesis Testing, Elsevier/North-H olland. Am sterdam , 1976, pp. 10 5 --124. 64 N orbaek, D .H ,, and J.R. Allen, Biological responses o f the nonhum an prim ate, chicken, and rat to chlorinated dibenzo-p-dioxin ingestion. Environ. Health PerspecL, 6 (1 97 3) 233-- 240. 65 Poland, A ^ and E . Glover, Com parison of 2 .3.7,8-tetrachlorodibenxo-p-dloxia. a potent inducer of aryl hydrocarbon hydroxylase, with 3-m ethylcholanthrene. M ol. Pharm acoL. 10 (1 9 7 4 )3 4 9 -- 359. 66 Propping. P,, G. R Sh rb o m and W. Buselm aier, Com parative investigations on the chem ical induction o f p oin t m utations and dom inant lethal m utation in m ica. M ol. Gen. G e n sL. 117 (1 9 7 2 ) 19 7 -- 209. DOW 374417 s t r t * if--11"*""1"-""'"" 160 67 Seiler. J.P., A survey o d the.mutagenicity o f various pesticides. Experientia. 29 (1973) 6 2 2 --623. 66 Seiler. J.P., Inhibition of testicular D N A synthesis by chemical mulaeens and carcinogens, Preliminary results in the validation of a novel short term test. Mutation Res.. 46 (1977) 3 0 6 --310. 69 Shaw, M.W., Human chromosome damace by chemical agents. Annu. Rev. Med.. 21 (1 9 7 0 )4 0 9 --432. MuEtlasteivoinerR/Nc.-.- 60 Sim m on. V . F ,, Unpublished data. *' 61 Smarda. J.. J. Koudelka and V . Kleinwachter. Induction of bacteriophage and colicin by meant of 62 aScmriidthin.eHo.rWan..geE.ffEexcpteorfiepnhtyias,ic2a0l (1964) 500. and chemical changes on the liberation of phage particles by lysogenic strains o f Salmonella, J. Gen. Microbiol.. 8 (1953) 116-- 134. 6 3 Stout. SJC., W.M. Generoso and S.V>'. Huff. Chemical effects on total reproductive capacity of female mice: a study of genetic and cell killing effects on oocytes. E M S Newal., 6 (1 9 7 2 ) 16. GENETIC 64 Tenchini. M.L., R. Giorgi, C. Crimaudo, G. Simoni. P. Nuzzo and L. de Carli, Approaches to examine* tion o f genetic damage after a major hazard in chemical industry: preliminary cytogenetic findings on T C D D w xposed subjects after Seveso accident, presented at ttie Expert Conference on Genetic Damage Caused by Environmental Factors. Oslo, Norway. M ay 11-- 13,1977. G.W. GRIGG 6 5 V an Miller. J.P.. and J J l. Allen, Chronic toxieity of 2,3,7.8-tetrachlorodibenzO'P<dioxln in rats. Fed. M olecular an Am . Soc. Exp. Biol.. 36 (1977) 396. (A uttralia) 66 Vasarhelyi. E ,, 1. Hegedus and S. Szoke. Production and analytics of K lorinol (T C P E ) and some of its related compounds, in: L. Bank! (Ed.). The Development of a Pesticide as a Com plex Scientific Task, Medicina. Budapest. 1976. PP. 3 9 -- 61. 67 Vos. J.G., J A . Moore and J.G. ZinkL Toxieity of 2.3.7,8.tetrachlorodibenzo*p^lioxin (T C D D ) in (((ARReeccvceiesipvioteenddr2el5ie C 57B1/6 mice. Toxieol. APPl. Pharmacol.. 29 (1974) 2 2 9 -2 4 1 . 66 Waldbott. Effects o f Environmental Pollutants, Mosby, St. Louis, 1973. o 69 Walsh, Seveso: the questions persist where dioxin created a wasteland. Science, 197 (1977) 1064-- O 1067. Contents 3J 1 70 Zim m crm ann, F.K., Procedures used in the induction of mitotic recombination and mutation in the yeast Seccharomyces cercvisiae. Mutation Res.. 31 (1975) 71--66. I ,i r^. OSBICCAInCDAcCAiRcnyahooonNctoncter((l(e((p((e((tuoucUo1(nk((i2fit((iAi1r23er)iHiimilvire1m2g)ci)n)i)ioabn))r)tr))))i)liooerecgaoeiuc)uSEReOoCSnIwfdtIrAnagitsDMICNpnieeintcafelehncienotnilnfvnsaodoNegnohhsecneeerden]vastinursioeuidcesspefAciru<tsiirt:i:l:;: Occurrem Couma rouna odi \5t \ r CHAPTER 6: O COMPARISON OF THE HEALTH EFFECTS or CO' OF AQUATIC HERBICIDES OD tO CO Based on absorption and metabolism/ it is possible to determine a comparative index of likelihood of injury to internal organs due to environmental exposure to aquatic herbicides. In order or increasing risk: 1. Endothall is poorly absorbed through the skin, lungs, or gastrointestinal tract unless the membrane is first damaged. It is minimally metabolized by animals or intestinal bacteria, is not lipid soluble, and has not been reported to alter metabolism. 2. Diquat is poorly absorbed through the skin, lungs, or gastrointestinal tract unless the membrane is first damaged. It is not metabolized by animals, but is metabolized by intestinal bacteria to unknown products of unknown toxicity which are partially absorbed. It is not lipid soluble and does not accumulate in the body. However, it alters metabolism in many, if not all, organs by its ability to be reduced to a free radical at the expense of NADPH and to then' autooxidize, producing hydrogen peroxide or superoxide free radicals. 3. 2,4-dichlorophenoxyacetic acid is lipid soluble and rapidly absorbed through membranes of the skin, lungs, and gastrointestinal tract. It is not metabolized by animals, but its derivatives (salts, esters, etc.) re hydrolyzed to the acid. Although it is degraded by soil bacteria, it apparently is not changed by intestinal bacteria, possibly because it is absorbed too rapidly. Up to 32% of absorbed 2.4- D is found in cell nuclei. It is rapidly distributed to all tissues and is not excreted as soon as the above lipid-insoluble herbicides. 4. Dichlobenil is lipid soluble and rapidly absorbed by all routes of environmental exposure. It is metabolized by animals to a variety of known and unknown products, some of which are highly toxic uncouplers of oxidative phosphorylation. It should be noted that this approach to risk assessment gives exactly the opposite order from an assessment based on the acute oral LD50. LD50 is not relevant to low-dose chronic exposure, but is the basis for the hazard levels assigned to pesticide labels: caution, warning, danger, in order of decreasing lethal dose. Dichlobenil and 2.4- D formulations used for water milfoil are labeled "caution," diquat is labeled,"warning," and endothall is labeled either "warning" or "danger" depending on its concentration. None of the four herbicides has passed all of the currently accepted tests for the determination of safety of low-level exposure to humans. Endothall has been subjected to adequately designed 0005353 7592 DOVi-jo53824 ^testing, but the final results of the mouse carcinogenicity test are -not in yet, and an outside investigator has detected mutagenic poten t i a l in a very sensitive but reliable assay properly performed. Diquat has not been adequately tested for teratogenicity in mamijnals, but inadequate tests suggest possible hazard, and one study in amphibians shows teratogenicity and embryotoxicity at concentrations lower than proposed for Lake Washington (Anderson and Prahlad, 1976). Chronic feeding tests have shown that diquat induces cataracts in rats at 2.5 mg/kg/day and in dogs at 5 mg/kg/day orally. Diquat was posi tive in one short-term carcinogen-screening test, the induction of DNA repair synthesis. It was negative for carcinogenicity in both rats and mice in less than adequate tests, which could give false negative results. 2,4-D was found to be both teratogenic and embryotoxic, with effects noted as low as 0.5 mg/kg/day. This injury was induced at even lower concentrations in the presence of a primary microbi"al breakdown product of 2,4-D, 2,4-dichlorophenol. 2,4-D was shown to cause point mutations in animal cells, to damage DNA in a manner simi lar to ironizing radiation, and to stimulate cell division. Testing of 2,4-D for carcinogenicity is inadequate according to present stan dards, but those inadequate tests demonstrate significant tumor increases, particularly in sarcomas of the lymphoreticular system in both rats and mice. (Inadequate studies can give false negative results, not false positives.) One study for tumor promoting activity was highly positive, using the mouse skin system. The breakdown pro duct, 2 ,4-dichlorophenol, is also a good promoter of skin carcino genesis. Dichlobenil testing for teratogenicity and carginogenicity is so inadequate that no conclusions are justified. An adequate reproduc tion study indicated that dichlobenil inhibits growth of young rats .at 50 ppm and decreases fertility at 100 ppm. It has not been tested for mutagenicity in any animal system, and microbial system are notor iously unreliable for chlorinated aromatic compounds. Present testing standards are certainly minimal for the deter mination of safety to humans. They do not assay for tumor promoting ability or for effects on the functioning of the higher nervous system such as loss of intelligence, loss of ability to concentrate, or induction of emotional instability. Since only the manufacturer of endothall has met these minimal standards of testing for health effects (one test not yet complete, however), but all four herbicides are registered by the Environmental Protection Agency and approved for use in public waters, an attempt was made to determine whether EPA files contained additional relevant unpublished studies which would justify registration. Catalogs of documents in the EPA files on dichlobenil and diquat were obtained from Friends of the Earth, and a request was sent to EPA under the Freedom of Information Act for all of the documents listed under GG0533* -59- 7593 053825 >23820 neurotoxicity, oncogenicity, chronic feeding, reproduction, and tera tology. This totaled one document on dichlobenil (chronic feeding) and eight documents on diquat (neurotoxicity, oncogenicity, chronic feeding, and reproduction). After two months, two documents on chronic feeding of diquat were received, and after six months the EPA closed the request without sending anything more. The two documents received are reproduced in their entirety below: #123373-0007 Chronic - Oral Rat: 500 ppm. w/w. for 16 months did not affect the rate of growth, during the growing period, and no toxic signs have, been observed. #123370-0034 Chronic toxicity Chronic toxicity studies show that 500 ppm in the diet of young rats of both sexes produced no detectable effect. It should be apparent even to the casual reader that these do not contain the minimum essentials of an abstract (species, strain, sex, number of animals, doses, formulations, route of administration, dura tion of study, effects looked for, methods of analysis of data, conclusions) and certainly provide no data appropriate for regulatory decision making. It is possible that the two reports above are from the same study, not repeated studies, reducing even further the total number of relevant studies in the EPA catalogs. Since the primary argument of other government agencies which routinely expose the public to these herbicides is that they are registered with EPA and therefore must be safe, it should be deter mined whether EPA really has no data on the health effects of dichlo benil and diquat, or merely defies the law. The Federal Insecticide, Fungicide and Rodenticiae Act states: Section- 10. (d) (1) All information concerning the objec tives, methodology, results, or significance of any test or experiment performed on or with a registered or previously, registered pesticide or its separate ingredients, impuri ties, or degradation products, and any information con cerning the effects of such pesticide in the environment, including, but not limited to, data on safety to fish and wildlife, humans and other mammals, plants, animals, and soil, and studies on persistence, translocation and fate in the environment, and metabolism, shall be available for disclosure to the public. Much of the information used in this report was obtained from the chemical companies and has not gone through the process of peer review required before publication by reputable scientific journals. This is also true of the unpublished manuscript and publications of government -6C- QijOjJoj 7594 BV i l 053826 agencies. The reader should consider this when using such information -for decision making. ( One final conclusion of this study must be that published reviews ' of this subject are extremely unreliable, quoting other reviews with no original data ^and thus giving the appearance that many more studies have been-done than in fact have. This author welcomes correspondence concerning any additional relevant studies which may become available. V -61- 7595 '3 V / J 053827 BIBLIOGRAPHY Anderson, Ross J. and Prahlad, K. V. (1976) The Deleterious Effects of Fungicides and Herbicides on Xenopus laevis Embryos. Archives of Environmental Contamination and Toxicology A, 312-323. c 0005357 c; 7596 -62- unoset iissssuteil'l :j * j.-..vauucc wlsu.4.u a x j-Cieaornmg trolled pesticide use defended 68*90 l won ,i By Richard C. Klenitz Journal Madison Bureau Madison, Wis. -- The state's pub lic intervener urged the Agriculture Department Thursday to- write stronger protections for ground wa- ter into proposed rules for pesticide ` use. "Pesticides do not belong in our waters," Asst Atty. Gen. Thomas -. Dawson stated. Ax the first of a series of hearings on revision of the state's pesticide regulations (Ag 29), a spokesman for the state's vegetable processing in- : dustry argued that the proposals on overspraying, posting of treated fields and pre-spraying notice were impractical. Alvin Randall, executive secretary of the Wisconsin Canners and Freez ers Association, said itwas not prac tical to try to eliminate allrisk for all people. Adverse effects. *ikewise Russell Weisensel, exece director of the Wisconsin Agri- - Business Council, suggested that as rules become more rigid they ad versely affect more small fanners. H e said his group wondered whether there was much benefit to be gained from the revisions. "People have to take some of the risk themselves," Randall said. George Klacon, of the Green Giant Corp.. said people should be educated to recognize that there are pesticides being used in fields. He said, "I don't believe people can put up enough signs for those passersby who might want to take a few ears of com." Weisensel added that normal trespass laws should suffice. Wants to know Carole Ann Barth, a researcher for Citizens for-a Better Environment (CBE) who said she was asthmatic, also commented on the matter of posting and prior notice. "I don't expect the world to stop around me," she reasoned,- " but I. want to know what is happening' around m e so I can arrange m y own activity." Dawson, who intervenes for the F '<c interest in environmental matt .asked that the rules be written to prohibit any application of pesti cides that would enter the state's waters. H e noted that Wisconsin relied heavily on ground water for domes- Turn to Hearing, Page 7 FromPage 1 tic use. He proposed that whenever the agency received credible evi dence that a pesticide had been iden tified in ground -water,' an order should be issued prohibiting its fur_ther use in the recharge zone of the " ground water basin. Dawson said that aldicarb -- a chemical used to treat a potato pest and which has caused problems in N e w York -- has been detected in ' the Wisconsin Central Sands region. He pointed out that his proposal would ban its use until it could no longer be detected. The proposed Department of Agri- . culture, Trade and Consumer Protec tion rules would require that resi dents of adjacent land be given notice i of aerial spraying if requested. The same provision would apply for beekeeoers when soraviakJs cnnrtiicrpd within three miles of theirhives. Other revisions would restrict day time spraying where crops or weeds visited by bees are in bloom; require a permit for use of chlordane; include municipalities as commercial applica tors. and require permits and en closed storage of pesticides. Dawson 'objected that the rules did not require annual reporting of pesti cide use. He said the information was needed to properly monitor human and environmental effects. Weisensel described Dawson's proposals as unbalanced and said they caused the agency a great many problems in drafting the rules. Dennis Dixon. Elkhorn, president of the Wisconsin Agricultural Avia tion Association, said he "was not surprised that the activist minority is not satisfied." >- --- --,-u the statement in the proposed rules that any use resulting in overspray was negligent. Dixon said some pro* vision should be made for equipment failure, adding that aerial application; Should not be singled out because 'there was no evidence of harm to human health. ; Dixon said notice should be limited1 to toxic materials. . Ray Geymann. of Oconomowoc CanningCo., proposed that beekeep-' ers be required to own or lease for aging rights in order xo qualify for the prior notice protection. Further more, he said all beekeepers should, be licensed and a fund should be de veloped from honey receipts to fi nance a system of coordinating appli cation information and notice. Geymann also said a 1.5 mile radi us for beekeeper notice seemed more > *V C005353 D A N IE L P M E Y ER d ir e c t o r o r p u b l ic a f f a ir s 7597 "T" Group C O N SO LID A TED PAPERS. INC W|71ISS>C4O2N2 S3I3N66RAPIDS WISCONSIN S U ! ISS 7598 IS S CHAPTER 2: HEALTH EFFECTS OF 2,4-D (2,4-dichlorophenoxyacetic acid) AND ITS DERIVATIVES O' CJJ CO A. Introduction CO ^ The herbicide 2,4-D was prepared in 1941 by the interaction of 2,4-dichlorophenol, monochloroacetic acid and sodium hydroxide, and a similar process is used in its commercial production. 2,4-D is a systemic herbicide widely used for site preparation and conifer release in forestry, for control of broadleaf weeds in cereal crops and sugar cane, and on turf, pastures and non-cropland. It is also used to control the ripening of bananas and citrus fruits, to delay preharvest dropping of some fruits, and in some countries as a"fungi cide for the control of Alternaria rots when lemons are to be held for storage. As a component of "Agent Orange," 2,4-D was used to defol iate jungle areas in South Vietnam (IARC, 1977). The EPA has approved the use of 2,4-D, marketed as "Aqua-Kleen" by Amchem Products, Inc., for control of several aquatic plants including water milfoil. Label requirements include a "caution" against accessibility of the product to children; applying to water used for irrigation, sprays, dairy ani mals or domestic use; or contact with skin, eyes, and clothing. B. Metabolism of 2,4-D A total of nine human male "volunteers" in two different studies have been ed a single dose of 5 mg/kg of 2,4-D (Kohli et al., 1974; Sauerhoff et al, 1977) to determine its metabolism. Essentially all of the 2,4-D was absorbed from the gastrointestinal tract. It was distributed widely through the body and excreted in the urine unchanged. No metabolites were^detected. No symptoms of illness or abnormal blood chemistry were detected. 95% of the dose was recovered from urine in six days. v The metabolism of isotope-labeled 2,4-D in rats has been deter mined and is in agreement with the human studies above (Khanna and Fang, 1966). Radioactivity was found in all organs and tissues exam ined, with 9% to 32% in the nuclear fraction. All was unchanged 2,4-D, suggesting that the chemical in the cytosol was not peptidebound as found in plant tissues. 2.4- t D is lipid-soluble and therefore rapidly absorbed from the lung (3urton et al., 1974), when assayed in rats. 2.4- / D amine alt administered orally was readily absorbed by rats, pigs, calves, and chickens, but 2,4-D butyl ester was much more slowly absorbed and the circulating chemical was all in acid form, indicating that the ester was hydrolyzed during absorption (Erne, 1965). Absorbed 2,4-D was distributed rapidly through the body with C&05334 -16- t 7599 highest levels in the liver, kidneys, lungs, and spleen. It is readily passed through the placenta in the pig and into chicken eggs C. Impurities and Breakdown Products 03 PHENOL to Synthesis of 2,4-D Technical 2,4-D available in the United States is about 98% pure (IARC, 1977). The primary contaminants are bis (2,4-dicnlorophenoxy) methane, bis (2 ,6-dichlorophenoxy) methane, and 2,2 ', 4 ,6 '-tetrachlor-^~ odiphenoxymethane (Huston, 1972). No toxicological information was found on these; they are not listed in NIOSH's Registry of Toxic Effects qf Chemical Substances 1977. Incomplete reaction of 2,4-aichlorophenol during synthesis of 2,4-D would leave some of this pre cursor contaminating the product, but no published evidence of this was found. 2,4-dichlorophenol is a major product of the breakdown of 2,4-D by microorganisms (Paris and Lewis, 1973). No. 2,4-dicnlorophenol was detectable in mice which had been injected with 2,4-D acid or its butyl or isooctyl esters (Zielinski and Fishbein, 1967), but this route of administration bypasses the intestinal tract. Intestinal bacteria may be able to hydrolyze 2,4-D to 2,4-dichlorophenol also, but the metabolic studies above in section B would indicate that this does not happen. However, the abstract of a Russian study which is not available in the United States, states "in pregnant rats receiving a single 50 mg/kg oral dose of 2,4-D the metabolite 2,4-dichlorophenol was detected in the placenta, the amniotic fluid, end the embryo and in the mother's milk after delivery" (Antonenko, 1977). 2,4-D during its breakdown in he environment must contain 2,4-dichlorophenol and lead to human ekjfosure through food and water. 20% 2,4-dichlorophenol promoted the appearance of skin tumors in mice following a single initiating dose of dimethylbenzanthracene (43% 7 6 0 0 ^with papillomas and 11% with carcinomas after 15 weeks, vs. 7% and 0%( -17- 0005335 Y / 053783 in initiated controls). After a longer period of application to the skin, it was able to induce tumors without prior application of the initiator (75% with papillomas and 6% with carcinomas after 24 weeks). After 39 weeks of treatment with 2,4-dichlorophenol, 62% of the mice had carcinomas. The test substance was dissolved in benzene in these experiments, and control animals were treated with benzene alone a(Boutwell and Bosch, 1959). This indicates that 2,4-dichlorophenol is a'Skin carcinogen in mice, or at least cocarcinogen with benzene tfhich is not carcinogenic by itself in this system. CO z* . J 2-chloro4-ketoadpc add / * chlorosucdnic add i sucdnic add 3-ketoadipic add V succinic add Fig. 1 SPer1aet9teft6eeha0nrw;lsea.on,y(cn1se01so)a9:fn6dF7m;eiW(m1c(a7r)lleo)kAybeiDlryaauln1xed9bndSuedE7rgy;vrFaaen(a4dstu)asat1tBi9lo51.e,n99l;6lo41af;91n79bd05(u2;7t);(1y(l81T(5))de)esETdBtvijeeeaodrnlcasl'anoecfdaneAed2t ,tleS4ax-.alrDar.l.,in.i,hd1eM19r1u699r9561r;349t;.6er9(9s;(J6a)(rAZeLJ.uoouss SOURCE: Doris F. Paris and David L. Lewis, "Chemical and Microbial Degradation of Ten Selected Pesticides in Aouatic Systems," Residue Reviews, Volume 45, 1973, p. 107. f 2 ,4-dichlorophenol, like other polychlorinated phenols, can form cnlorodibenzo-dioxins when heated (Epstein, 1970), but not the infanous TCDD (2,3,7,8-"t^trachloroaibenz o-p-dioxin) found in 2,4,5-T (Cribble, 1974). The most likely di oxin produced by heating 2,4-di- 1 cnlorophenol would be 2,7-aichloroci benzo-p-dioxin, which is reported to be minimally toxic (Schwetz et al , 1973, Dew Chemical Co.). Mo deaths occurred in four male mice gi ven 2000 mg/kg or in two female rats given 1000 mg/kg. Mo signs of toxicity were observed in these 7601 G00533S animals. This dioxin was negative in the rabbit ear assay for chloracne. Rats treated with 100 mg/kg/day (gavage) on days 6 through' 15 of gestation gained slightly more weight during pregnancy than controls but showed no toxicity. There was no effect on fetal body measurements, or incidence of resorptions, or gross, soft tissue, or skeletal anomalies in seven litters examined at Dow. However, Khera and Ruddick (1973) of the Health Protection r3 Branch, Department of National Health and Welfare, Canada, found significant teratological injury to the heart muscle in fetuses of rats given 2 or 1 mg/kg/day of 2 ,7-dichlorodibenzo-p-dioxin orally on days 5-14 of gestation. Microscopic examination of the heart revealed C2 > edematous separation of myofibrils that had resulted in compression thinning and fragmentation of myofibres. Mitotic figures were rare, indicating that growth of the cardiac tissue was suppressed. Such * lesions were not found in rats given 0.5 or 0.25 mg/kg/day or in the Ocontrols given the solvents anisole and corn oil. cn 03 2/7-dichlorodibenzo-p-dioxin should not be formed during produc*^tion of 2,4-D because the reaction is carried out at a lower tem^perature than is required for dioxin formation. However, it, could be ^ f o r m e d from residual unreacted 2,4-dichlorophenol if heated during storage or transit, and from the microbial breakdown product .during fires in treated brush or slash. One author has reported detecting a trace of hexachlorodibenzo-pdioxin (HCDD) in one of 28 samples of 2,4-D tested for content of higher dioxins (Woolson et al, 1972). This was probably an artifact of laboratory contamination since there is no apparent way for this to form directly from 2,4-D production. i* Other products of microbial or plant degradation of 2,4-D (Paris and Lewis, 1973; Ashton and Crafts, 1973) are not listed in the NIOSH directory (NIOSH, 1977). Microbial products include 6-hydroxy-2,4-D, various cnlorocatechols, and various cnloromuconic acids before finally becoming succinic acid, a normal physiological component of all cells (Paris and Lewis, 1973). Catechol has been shown to be an efficient cocarcinogen for mouse skin in the presence of minute amounts of benzo(a)pyrene, but the chlorocatechols were not tested (Van Duureri and Goldschmidt, 1976). Esters of 2,4-D are split by microbial action to release 2,4-D acid which is degraded as described above' (Aly and Faust, 1964). D. Acute and Subacute Toxic Effects * The acute oral LD50 of 2,4-D for humans is reported to be 30 mg/kg, and for. /ats it is 375 mg/kg (NIOSH, 1977). Five incidents in which agricultural workers were poisoned by 2,4-D when they returned to the fields from one to 14 days after spraying were described by Radionov et al, 1967. Symptoms reported were consistent and included headache, weakness, dizziness, nausea and vomiting, sore 1 7802 -19- 000533? WW / 053785 throat, irritation of the nasal mucosa, substernal pain, and loss of consciousness. Recovery took four to nine days. Workers engaged in the spraying of 2,4-D and its derivatives from aircraft for three to five years and longer complained of rapid fatigue which usually cleared overnight. They periodically had headaches, pain in the liver and epigastric regions, and poor appe tite. They had impaired taste sensitivity to salty, sour, bitter, and sweet test solutions. There was a deterioration in sensitivity to odors also. No changes in blood chemistry were found (Fetisov, 1966) . In addition to the symptoms above, peripheral neuropathy has been .j reported in humans hours or days after exposure to 2,4-D on the skin and probably simultaneously by inhalation (Goldstein et al., 1959; Berkley and Magee, 1963). The symptoms progressed through several weeks until pain, paresthesias, and paralysis were severe. Disability-j .was protracted, and recovery was incomplete even after the lapse of years. Numbness and aching had extended proximally from the fingers and toes, and the patients became unable to walk because of pain and' ;-U weakness. They also suffered moderately severe sensory deficits on tests of touch, pain, and temperature. Autopsy of a human fatality due to ingestion of 2,4-D revealed widespread plaques of acute demyelination in all parts of the brain, j with central petechiae (Dudley and Thapar, 1972). Multiple petechiae j( were seen throughout the white matter of the brain, and the kidneys j were hyperemic. i The nervous system of rats, cats, and dogs was studied after 2.4- D administration parenterally (Desi et al., 1962). A reversible inhibition of cerebral electrical activity was observed in the acute experiments, and in chronic experiments the same was present to a gra dually increasing degree. According to conditioned-reflex experi ments, the higher nervous activity suffered severe damage. The point of attack seemed to be the reticular formation. Lesions in this ! region paralyze the function of the cerebral cortex. v Pretreatment of rats with 250 mg/kg 2,4-D three hours prior to administration of 9 mg/kg ^4C-2,2-D greatly increased the level of ^4C in rat brain and spinal fluid as compared to plasma level (Elo and Ylitalo, 1977). The increase was much more striking in the brain (11-fold) and spinal fluid (39-fold) than in the liver (4.5-fold). The likeliest explanation of this is that 2,4-D impaired function of 1 the blood-brain barrier by causing capillary injuries.* Cattle and sheep which died after 5 to 34 daily oral doses of 2.4- D alkanolamine -salt were necropsied (Palmer and Raaeleff, 1964). Lesions included liver and kidney degeneration, the heart usually con tained hemorrhages, and there was usually an excessive quantity of j pericaridal fluid. C005333 7603 -20- Mice acutely poisoned with 2,4-D had dark and mottled liver, kid neys, and spleen, blood-stained fluid in the upper intestine, and wide 1ilation of*the blood vessels of the lungs, liver, and kidneys Bucher, 1946). In dogs that died two to nine days after a single dose of 2,4-D, ymptoms included ataxia, progressive increase in spasm of the hind egs, and myotonia (Drill and Hiratzka, 1953). Sneezing, eye rubbing, .nd diarrhea were observed, but not vomiting, and some dogs showed ividence of meningeal irritation. Autopsy showed inflammation and iecrosis of the intestinal mucosa, hepatic necrosis, and renal tubular iegeneration. Myotonia in animals exposed to acute and subacute treatment with :,4-D has been described extensively (i.e. Bucher, 1946; Heene, 1975; Janon et al, 1978). Injection of 2,4-D into aarenalectomized and lydrocortisone-treated rats did not cause the myotonic response seen ifter injection into normal rats (Buslovich and Koldovskaya, 1972). Myotonia was present in an elderly man after ingestion of a Lethal dose of 2,4-D (Dudley and Thapar, 1972). ' 10"4M 2,4-D caused uncoupling of oxidation and phosphorylation in isolated rat liver mitochondria (Abo-Khatwa and Hollingsworth, 1974), and 2,4-D- inhibits synthesis of cholesterol and fatty acids in rat liver (Olson et al, 1974). It increases the quantity of RNA and pro tein in rat liver and stimulates RNA synthesis in isolated rat liver nuclei (Chang et al., 1974). S. Tests fpr^Developmental Toxicity 1. Summary of Research Five assays for developmental toxicity of 2,4-D in rats have been published, using widely different doses and different strains of rats. Knera and McKinley of the Food and Drug Directorate of Canada used oral doses of 50 to 150 mg/kg of body weight/day during organogenesis and found skeletal malformations at 100 mg/kg/day and above. Schwetz et al of Dow Chemical Company used oral doses of 12.5 to 87.5 m 9/<<9/day during organogenesis and found skeletal anomalies at 75 m 9/ :<9/day and above. They also found an increase in fetuses with sub cutaneous edema at all doses, and general growth retardation.. Konstantinova et al. of the Scientific Institute pf Rural Hygiene in Saratov, USSR, tested 2,4-D and its breakdown product, 2,4-dichlorophenol, separately and together, and found developmental toxicity in the form of internal pemorrhages after 50 mg/kg/day of 2,4-D and after 1 m9/5cg/day of 2,4-dicnlorophenol and also after a mixture of 0.1 "'S/^S/day of each. Ak'eksashina et al of the Byelorussian Scientific Research Institute of Sanitation and Hygiene in Minsk, USSR, did two different experiments. One used intraperitoneal doses of 0.1 or 0.5 mg/kg/day throughout pregnancy and found growth retardation and -21- 0005333 hemorrhaging into the abdominal cavity at the higher dose and vascular distention at both doses. The other experiment tested single doses of. 1/2 LD50 given intraperitoneally on various days of gestation, and found increased resorption of fetuses with reduced litter size, growth reduction with increased size of brain ventricles, and hemorrhaging into the abdominal cavity. A test for developmental toxicity in hamsters was done by Collins and Williams of the U.S. Food and Drug Administration. They used oral doses of 20 to 100 mg/kg/day during organogenesis and found a dose- dependent increase in fused ribs at 60 mg/kg/day and above which was significant only if repeats were pooled. . Two studies of the teratogenicity of 2,4-D in mice have been done. A large series of tests using different strains of mice, dif ferent doses, and different esters of 2,4-D was completed in 1968 by Bionetics Research Laboratories, and has now been published. -Oral " ..f administration of 100 mg/kg/day of 2,4-D acid during organogenesis caused a significant increase in fetal mortality and percent of abnormal fetuses, with most of these being eye and jaw malformations. 2,4-D acid produced a significant increase in fetal abnormalities in four of six adequately-sized groups of three strains, when given subcutaneously at 100 mg/kg/day. Subcutaneous administration resulted in a significant increase in abnormal fetuses in one strain after 48, 94, 100, or 130 mg/kg/day of the isooctyl, isopropyl, butyl, and isooctyl esters respectively. This was a repeat of a previously nega tive study. The anomalies were mainly of the eye and jaw. All of these subcutaneous administrations were dissolved in dimethylsulfoxide, which' is a teratogen for the nervous system of the hamster (Ferm, 1966). However, the DMSO controls in this mouse study showed no significant increase in developmental toxicity over untreated controls. The other mouse study was done by Courtney of the U.S. Environmental Protection Agency. She used oral doses of 150 or 250 m g / k g / d a y of 2,4-D and four of its esters during organogenesis and found growth retardation at both doses and cleft palate at the higher dose. Other malformations were not assayed. Two developmental studies on pigs have been done by Bjorklund and Erne of the Swedish Royal Veterinary College and National Veterinary Institute. A pig was fed 500 ppm of 2,4-D amine while pregnant plus six weeks after. The fifteen piglets born were underdeveloped, apathetic, and did not want to suck, and ten died the first day. No malformations were observed. In the second study, eight-week-old pics were fed 500 ppm of/2,4-D amine for 12 months. After one month, 2 out of 5 in the experimental group developed malformations in the hooves on the forelegs, making walking difficult. 0003340 7605 -22- 2. Conclusions 3 The four manifestations of developmental toxicity have all been demonstrated in animals treated with 2,4-D or its esters or amines. *. Malformation: skeletal at high dose (60 mg/kg/day or above), Ceripheral circulatory system distended at low dose (0.1 mg/kg/day) CJfcnd after single high dose (1/2 LD50), eye anomalies at 100 mg/kg/day. CO Malfunction: subcutaneous edema at 12.5 mg/kg/day and above, >/Sftemorrhage into soft tissues and body cavities from 50 mg/kg/day to CO .5 mg/kg/day. Growth retardation: prenatal at 0.5 mg/kg/day and above. Post natal after 50 mg/kg/day or when treatment continued during lactation. Lethality: pre-implantation not reported. Post-implantation at 100 mg/kg/day and above, or after a single high dose (1/2 LD50). Postnatal in progeny of rats fed 150 mg/kg/day, or in a pig fed 500 ppm. The above summary includes both oral and intraperitoneal treatment. 2,4-D is rapidly and completely absorbed from the gastro intestinal tract and is not metabolized in animals, so the serum con centrations should be similar by the two routes. Erne, 1966, has demonstrated the ease of placental transfer of 2,4-D in the pig. 2,4-D also rapidly penetrates the placenta in rats; 24 hours after a single dose, 16.8% of that dose remains in the uterus, placentas, fetuses, and amniotic fluid (Fedorova and Belova, 1974). The effect of 2,4-D on the circulatory system was synergistic with that of its microbial breakdown product, 2,4-dichlorophenol. When the customary 100-fold safety factor is applied to the effective dose (Wilson, 1973, p. 156), the acceptable tolerance is equivalent to a 50 kg woman drinking 40 ml of treated water (2.5 ppm) daily during early pregnancy at a time'when 50% of the 2,4-D has been degraded to 2,4-dichlorophenol. The situation is not quite that simple, however, because breakdown continues to other untested compounds, and part of the '2,4-D would be degraded in plants where 2,4-dichlorophenol is not part of that pathway (Ashton and Crafts, 1973). Many of the studies reported here were done in other countries, raising the question of the similarity of synthesis and contaminants. No studies indicated that the synthesis and purity of these products is different elsewhere. 2,4-D caused developmental toxicity in all four species tested, including studies dene in four countries. It is interesting to note that the hemorrhaging found in all of the Russian rat studies using lpw/doses was not found in any of the high dose American studies, ihis may be a strain difference in rats, or a dif ference in the orotocols of observation. CG05341 7606- 2 3 - F. Tests for Effects on Reproduction and Fertility 1. Summary of Research c A study by Schwetz et al., of Dow Chemical Company, claims to show m that 2,4-D has no effect on fertility, but it actually contains no :,'f "* such experiment. 68ii3 The only study of the effect of 2,4-D on reproduction and fer- tility was done by Hansen et al., of the U.S. Food and Drug Administration. They used rats for a three-generation study and found no effect on fertility. 2. Conclusions On the basis of a single study in a single species, there is no indication that 2,4-D might interefere with reproduction and fertility in mammals. G. Tests for Mutagenic Effects and Other Short-Term Tests for Cancer 1. Summary of Research Assays of 2,4-D for both forward and reverse mutation in bacteria and bacterial virus are consistently negative (Fahrig, 1974; Shirasu et al., 1976; Simmon et al., 1977: Andersen et al., 1972). These included tests run in the presence of an activation system prepared from the livers of rats or mice. This is to be expected since 2,4-D is a chlorinated hydrocarbon and these as a class are negative in the Ames test., - 2,4-D caused a significant increase in recessive lethal mutations in Drosophila melanogaster when it was fed to male flies at 1000 ppm for two weeks (Magnusson et al., 1977), but was negative when the flies were treated with a 9mM solution for 3 days (Vogel and Chandler, 1974) and after unspecified treatment (Fahrig, 1974). v 0.001 mM to 1.0 mM 2,4-D with or without rat liver activation induced unscheduled DNA synthesis in SV-40 transformed human fibro blast cells'in culture (Ahmed at al., 1977a). 0.01 mM 2,4-D in the medium of primary human fibroblast cultures caused DNA damage leading to increased removal and reinsertion of bases. The type of repair was that seen following ionizing radiation (Hart et al., 1977). 0.01 mM 2,4-D the culture medium caused a significant increase in forward mutation*to ouabain resistance in the Chinese hamster V79 aneuploid lung cell line (Ahmed et al., 1977b). 0.01 mM or 0.1 mM 2,4-D in the culture medium of bovine fetal muscle cells caused the mitotic cells to exhibit unipolar and tripolar -2 CG05342 7 6 0 7 OGiGSO spindles and a variety of other abnormalities including malorientation of the mitotic apparatus in relation to the axis of the cell. Myo blasts in initial stages of myogenesis were noted to be in mitosis in treated cultures, suggesting that 2,4-D may have a stimulatory effect on myoblasts which normally are in a post-mitotic stage (Basrur et al., 1976). No evidence of nondisjunction or chromosome loss was found in Drosophila after both males and females had been fed 100 ppm 2,4-D for their entire larval period (Magnusson et al., 1977). Chromosome aber rations were not detected in human lymphocyte cultures (Fahrig, 1974) but no methods are given. No chromosome breakage activity was found 24 hours after intraperitoneal injection of 100 mg/kg 2,4-D in mice, as indicated by no detectable increase of micronuclei in erythrocytes of bone marrow (Jenssen and Renberg, 1976). 2. Conclusions 2,4-D causes point mutations in animal cells without liver acti vation, damages DNA in a manner similar to ionizing radiation, and stimulates mitosis. It does not cause chromosome breakage or nondisjunction. H. Tests for Carcinogenic Effects 1. Summary of Research Only three laboratory studies for the detection of carcinogeni city of 2,4-D- or any of its derivatives appear in the literature. All other reports are discussion or reanalysis of the data of these three studies, none of which uses optimum methodology according to the guidelines in Chapter 1. All doses used were lower than the 1500 ppm used in the 3-generation reproduction study in which adults tolerated chronic feeding and even pregnancy without lethality (Hansen et al., 1971). This study indicates that the MTD for rats is at least 1500 ppm. If mice are equally tolerant to the chemical, as suggested by their similar LD50 the MTD for mice should be at least 750 ppm (mice eat twice as much per day in relation to their body weight). The study done at the U.S. Food and Drug Administration in the early 1960's comes closest to being adequately designed, with a maxi mum dose of 1250 ppm, 25 rats/group, and two years duration. However, only a few of the animals had adequate pathological examinations at the end of that time, so small tumors would have been overlooked. There was a high incidence of tumors in controls, which could easily disguise a carcinqg^nic effect in such small samples. Increased inci dence of tumors was statistically significant in total malignant tumors in males, in lymphosarcoma in both males and females, and in breast neoplasms' in females. This study is described and analyzed by Hansen et al., 1971, and Reuber, 1979. 0005343 7608 A mouse carcinogenesis study was sponsored by the National Cancer Institute, also in the 1960's, and the data published years later. It consisted of both oral feeding studies and single dose subcutaneous injection studies. Two strains and both sexes were studied, but only 18 mice were included in each group and all were killed by 18 months. 1 053791 In the original study, strains and sexes were pooled to give a large enough sample size for statistical analysis by the chi-square method, and a significant increase in total tumors and in reticulum cell sar coma was found after subcutaneous administration of 2,4-D isooctyl ester. No other tests were postitive using this approach, which hides strain and sex differences in sensitivity. Analysis of the data by strain and sex, using the chi-square method, indicates a carcinogenic effect of feeding the isooctyl and butyl esters of 2,4-D to females of one strain, with the reservation that the method is not totally reliable with such small samples. Analysis of the data by strain, sex, and organ system using more elegant statistical methods indicated that the induction of reticulum cell sarcoma by injection of the isooctyl ester is most significant in females of the other strain. ro- However, the importance of this observation to human health is limited by the injection route of administration and the knowledge that 2,4-D esters taken orally are probably split into the acid form before absorption (Erne, 1966) so that circulation of the ester might not happen after contamination of humans. This study is described, ana lyzed, and discussed in Bionetics, 1968, Innes et al., 1969, Reuber, 1979, Mrak report, 1969, and Jurek, 1974. Its primary defect is its short duration, but the small group size and less-than-optimal oral dose given the adult animals could also contribute to false negative results. This study also had a rather high background level of tumors in the control animals. 0t A study at the Institute of Nutrition of the USSR Academy of xMedical Sciences included three experiments. The first experiment used adequate numbers of rats and duration of feeding 2,4-D amine, but the dose was not over 1/2 MTD. The background was very low and the tumor increase insignificant. However, the publication (Arkhipov and Koslova, 1974) does not state whether the rats were autopsied and whether histopathology was done, so small tumors might not have been found. A similar experiment with mice (100/group) was also negative, with no tumors in either the control or treated animals. The third experiment involved application of the herbicide to the skin of mice (100/group), with or without prior application of a low dose of an initiator of skin carcinogenesis. Animals treated with either the herbicide alone or the initiator alone did not develop any tumors, but 17.7% of those given the sequential treatment developed skin papillo mas, the premalignant lesion of skin carcinomas, indicating strong tumor-promoting activity of 2,4-D. Again, there is no report of histopathology, so i would be impossible to distinguish conclusively between papillomas'and carcinomas. CGG5344 7S09 -26- 2. Conclusions 274-D bas -not been tested for carcinogenicity according to pre sent standards# but two less-than-adequate experiments give statisti cally significant increases in malignant tumors when analyzed by organ systems. In both mice and rats, the lymphoreticular system was most -- v sensitive to 2#4-D carcinogenesis# but other organs also showed increases. OJ The high incidence of tumors in the controls of both of the ifai American studies is of concern in discriminating between a complete s&: carcinogen and a promoter of existing unintentionally-initiated pre sag neoplastic cells. This situation suggests that# unless the strains ) , used have a genetic predisposition to cancer, something in their -- a environment was mildly carcinogenic. This could be air-borne carcin- ^ ogen from other.tests in the same room, chemically-polluted drinking Cjs water, or contaminated feed. CO -si * It .has recently been shown that small amounts of the strongly tO carcinogenic nitrosamines are present in several varieties of labora^ tory animal feed containing contaminated fish meal, with the highest level in the diet which is recommended by NIH for use in carcinogen icity testing (Edwards, 1979). The 2,4-D testing was done too long ago to have used this particular feed formula, but th diet used also contains fish meal. In this author's laboratory, a single injection of dimethylnitrosamine causes lymphosarcoma in those Wistar rats which did not die earlier of kidney or lung carcinoma, the primary and secondary sites of action of the carcinogen when given intraperitoneally. A very minute amount of initiator is needed when the appropriate^strong promoter is present. The Russian rats and mice were apparently very resistant to spon taneous carcinogenesis or lived in a very clean environment. Although the dose used was only 1/2 MTD, the number of animals used was twice the usual number, making it likely that a carcinogenic effect would be detectable.. This suggests that the carcinogenic effect demonstrated in the American studies may be primarily promotion, an effect clearly demonstrated in the third Russian experiment in the mouse skin system. Cander initiators are tissue-specific, although the most sensitive tissue may differ between species. Promoters may be less specific, affecting more different tissues. Both phenol and 2,4-dichlorophenol, compounds used in the synthe sis of 2,4-D, are also efficient promoters of mouse skin carcinogene sis (Boutweli and Bosch, 1959). 2,4-dichlorophenol is the first-level product of the breakdown of 2,4-D by microorganisms in the environ ment. Second-level products are chlorinated catechols, and their parent compound catedhol has been shown to be a carcinogen on mouse skin (Van Duuren and Goldschmidt, 1576), so these compounds should be highly suspect. 000534a BOW 1 053793 003 The conditions of the American experiments approximate the con ditions of American people: wide exposure to a variety of cancer ini tiators and a 25% spontaneous cancer rate in lifetime studies. Over such a background, the presence of additional nonphysiologic chemicals which can stimulate the rate of development of cancers from premalignant cells must be considered a significant hazard. I . Conclusions About Health Effects of 2,4-D 2/4-D is lipid soluble and is rapidly and completely absorbed through all normal routes of exposure. Its butyl ester, and therefore probably all of its esters, is hydrolized before absorption from the gastrointestinal tract. It is not metabolized in animals, but rapidly penetrates the placenta. 2,4-D as normally manufactured does not contain chlorodibgnzodioxins because the reaction is not heated sufficiently to form them. However, 2,7-DCDD (also known as 3,8-DCDD) could be formed if the reaction mix was overheated or if partially degraded 2,4-D were heated during storage or disposal. This dioxin is far less toxic than the TCDD found in 2,4,5-T, but has been shown to be fetotoxic to the heart muscle in rats. Many cases have been reported of human poisoning by inhalation or absorption through the skin. Damage is primarily to the nervous system, and some such symptoms are not readily repaired. Gastrointes tinal symptoms and irritation of mucous membranes are also seen. Laboratory tests have shown development toxicity of 2,4-D in four species of animals. These include all four classes of developmental toxicity (malformation, malfunction, growth retardation, and lethal ity) but not all four in any one experiment. They include tests of the esters and amine of 2,4-D as well as the parent compound, and both pre- and postnatal adverse effects. A synergistic toxicity of 2,4-D and its primary microbial breakdown product, 2,4-dichlorophenol, on the fetal circulatory system was demonstrated in rats. The abstract of^a Russian study, which is not available in the United States, reports a survey of workers in the production of herbicides of the 2,4-D family and finds that "substantial menstrual and child-bearing functions arise manifested as higher rate of miscarriages, premature births, toxicosis of second half of pregnancy, and the menace of miscarriages during the whole pregnancy period" (Elina, 1974). An adequately designed study indicated that 2,4-b does not inter fere with fertility in rats, although other studies have shown that it causes point mutations in animal cells, damages DNA in a manner simi lar to ionizing radiation, and stimulates cell division. Carcinogenicity testing of 2,4-D has been limited to three studies, none of which meets today's minimum standards. In spite of the inadequate experimental design or assay, two of these studies 0G034G 7S11 -23- demonstrated carcinogenicity to the lymphoreticular system in rats and mice. The third study was negative for complete carcinogenesis, but demonstrated the strong tumor-promoting ability of 2,4-D in the mouse , skin system. In a separate study, 2,4-dichlorophenol, the breakdown product of 2,4-D, proved to be a strong promoter and probably also a weak initiator in the mouse skin system. The positive carcinogenicity studies had high background inci dences of tumors in the unexposed animals, similar to the rate in the human population of the United States, while the negative test had a negligible background. This could mean that 2,4-D is only a promoter of previously initiated, premalignant cells, not a complete carcinogen by itself. If so, it is still a significant hazard because of the similar circumstances of the human population to the high-background experiments. Several human epidemiological studies showing increased cancer rates after 2,4-D exposure have all included persons exposed to CJ! Co other herbicides ticular chemical as well, making was to blame. it impossible to prove that any par si 761000534'? -29- BIBLIOGRAPHY Abo-Khatwa, Nabil and Hollingsworth, Robert M. (1974) Pesticidal Chemicals Affecting Some Energy Linked Functions of Rat.Liver Mitochondira in Vitro. Environ. Contam. & Toxicology 12, 446-454. 55 X 053795 Ahmed, Farid E., Hart, Ronald W., and Lewis, Neil J. (1977a) Pesticide Induced DNA Damage and its Repair in Cultured Human Cells. Mutation Research 2, 161-174. Ahmed, Farid E., Lewis, Neil J., and Hart, Ronald W. (1977b) Pesticide Induced Ouabain Resistant Mutants in Chinese Hamster V79 Cells. Chem. Biol. Interactions 9, 369-374. Aleksashina, 2. A., Buslovich, S. Y., and Kolosovskaya, V. M. (1973) Embryotoxic Action of the Diethylamine Salt of 2,4-D. Gigiena- I r.-, Sanitariya 2, 100-101. Andersen, Kenneth J., Leighty, Edith G., and Takahashi, Mark T. (1972) Evaluation of Herbicides for Possible Mutagenic Properties. J. Agr. Food Chem. 20, 649-656. Antonenko, T. A. (1977) Experimental Data from a Study of the Permeability of the Placental Barrier for the Herbicide 2,4-D (Acid Form) and its Passage with the Mother's Milk During Feeding. Gog. Aspekty Okhr. Zdorov'ya Naseleniya 1977, 177-178. Arkhipov, G. N. and Kozlova, I. N. (1974) Study of the Carcinogenic Proper ties,,of- the Herbicide Amine Salt of 2,4-D. Voprosy Pitaniya 5, 83-84. Ashton; F. H. and Crafts, A. S. (1973) Mode of Action of Herbicides. Wiley & Sons, p. 274. 'Basrur, S. V., Fletcher, R. A., and Basrur, P. K. (1976) n Vitro Effects of 2,4-Dichlorophenoxy Acetic Acid (2,4-D) on Bovine Cells. Canadian J. of Comparative Medicine 4, 410-415. Berkley, Mary C. and Magee, Kenneth R. (1963) Neuropathy Following Exposure to a Dimethylamine Salt of 2,4-D. Arch of Int. Med. Ill, 351-352. Bionetics Research Labs, Inc. (1968) Evaluation of Carcinogenic, Teratogenic, and Mutagenic Activities of Selected Pesticides and Industrial Chemicals. Vol. I, Carcinogenic Study. Vol. II, Teratogenic Study. ^/National Technical Information Service, U.S. Dept, of Commerce. 3jorklund, Nils-Erik and Erne, Kurt (1966) Toxicological Studies of Phenoxyacetic Herbicides in Animals. Acta Vet. Scand. 7, 364-390. 7813CG05343 -30- Boutwell, R. K. and Bosch, Dorothy K. (1959) The Tumor-Promoting Action of Phenol and Related Compounds for Mouse Skin. Cancer Research, 1j), 413-424. Bucher, Nancy L. R. (1946) Effects of 2,4-Dichlorophenoxyacetic Acid on Experimental Animals. Proceed. Soc. Expt. 2. Biol. Med. 63, 204-205. ^Burton, Jerry A., Gardiner, Thomas H., and Schanker, Lewis S. (1974) r^Absorotion of Herbicides from the Rat Lung. Arch. Environ. Health 29, >1-33. 5>- r^Buslovich, S. Y. and Koldobskaya, F. D. (1972) Activity of Hexokinase C?-*in Skeletal Muscles of Albino Rats in Experimental Myotonia. Voprosy Meditsinkoi Khimii 18, 403-406. I Chang, Hsian-chen, Rip, Jack W., and Cherry, Joe H. (1974) Effects of "^Phenoxyacetic Acids on Rat Liver Tissues. J. Agr. Food Chem. 22, 62-65. CO vjCollins, T.F.X. and Williams, C.H. (1971) Teratogenic Studies with CD2,4,5-T and 2,4-D in the Hamster. Environmental Contamination & 05 Toxicology 6_, 559-567. Courtney, K. Diane (1977) Prenatal Effects of Herbicides: Evaluation by the Prenatal Develonment Index. Arch. Environ. Contam. Toxicol. 6, 33-46. t Danon, Jak M., Karpati, George, and Carpenter, Stirling (1978) Subacute Skeletal Myopathy Induced by 2,4-Dichlorophenoxyacetate in Rats and Guinea Pigs. Muscle & Nerve 1, 89-102. Desi, J., Sos, J O l a s z , J., Sule, J., and Makkus, V. (1962) Nervous System Effects of a Chemical Herbicide. Arch, of Env. Health 4, 95-102. Drill, Victor A. and Hiratzka, Tomiharu (1953) Toxicity of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichloropnenoxyacetic Acid. A Report on Their Acute and Chronic Toxicity in Dogs. Industrial Hygiene and Occupational Medicine 1_, 61-67. Dudley, Alden W. Jr. and Thapar, Nirwan T. (1972) Fatal Human Ingestion of 2,4-D, a Common Herbicide. Arch. Path. SM, 270-275. Edwards, G. S., Fox, J. G., Policastro, P., Goff, U.,*Wolf, M. H., and Fine, D. H. (1979) Volatile Nitrosamine Contamination of Laboratory Animal Diets. Cancer Res. 39, 1857-1858. </ -- Elina, V. A. (1974) Effect of Products of Orcanochlorine Herbicide Production .on Specific Functions of the Female Body. Gig, Tr. Sostoyanie Soetsificheskikh Funkts. Tab. Neftekhim. Khim. Prom-sti. 1974, 187-190. C005343 -31- i ^4  j 053797 , Elo, Heikki and Ylitalo, Pauli (1977) Substantial Increase in the Levels of Chlorophenoxyacetic Acids in the CNS of Rats as a Result of Severe Intoxication. Acta Pharmacol, et Toxicol. 4_1, 281-284. Epstein, Samual S. (1970) A Family Likeness. Environment 12, 16-25. Erne, Kurt (1966) Distribution and Elimination of Chlorinated Phenoxyacetic Acids in Animals. Acta Vet. Scand. 7 240-256. Fahrig, R. (1974) Comparative Mutagenicity Studies with Pesticides. IARC Sci Publ. 10, 161-176. Fedorova, L. M. and Belova, R. S. (1974) 2,4-D Detected in the Reproductive Organs and Fetuses of Treated Pregnant Rats; Ways and Dynamics of i-ts Removal. Gig. I Sanit. 39., 105-107. Ferm, Vergil H. (1966) Congenital Malformations Induced by Dimethyl Sulphoxide in the Golden Hamster. J. Embryol. Exp. Morph. 1 6 , 49-54. ^ Fetisov, M. D. (1966) Occupational Hygiene in the Application of Herbicides of the 2,4-D Group. Hygiene and Sanitation 31, 383-386. C- Goldstein, Norman P., Jones, Peter H., and Brown, Joe R. (1959) Peripheral Neuropathy After Exposure to an Ester of Dichlorophenoxyacetic Acid. J. Amer. Med. Assoc. 171, 1306-1309. Gribble, Gordon W. (1974) TCDD, A Deadly Molecule. Chemistry 47, 15-18. Hansen, W.H/, Quaife, M. L., Habermann, R. T., and Fitzhugh, 0. G. (1971) Chronic Toxicity of 2,4-Dicnlorophenoxyacetic Acid in Rats and Dogs. Toxicology and Applied Pharmacology 20, 122-129. Hart, R. W., Hayes, S., Brash, D ., Daniel, F. B., Davis, M. T. , and Lewis, N. J. (1977) In Vitro Assessment and Mechanism of Action of Environmental Pollutants. Annals New York Academy of Sciences 298 , 141-158. Heene, Rainer (1975) Experimental Myopathies and Muscular Dystrophy. Schriftenr. Neurol. 3J3, 1-97. Huston, Beverley L. (1972) Identification of Three Neutral Contaminants in Production Grade 2,4-D. J. Agr. Food Chem. 20, 724-727. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man., (1977) 1_5, 111-138. L 0G053S0 7615 -32- Innes, J. R. M., Ulland, B. M., Valerio, Marion G., Petrucelli, L., Fishbein, L., Hart, E. R., Pallotta, A. J., Bates, R. R., Falk, H. L., g-Sart, J. J., Klein, M., Mitchell, I., and Peters, J. (1969) Bioassay e^nf Pesticides and Industrial Chemicals for Tumorigenicity in Mice: A ^^Preliminary Note. J. Nat. Cancer Inst. 2, 1101-1114. I ss*Jenssen, Dag and Renberg, Lars (1976) Distribution and Cytogenetic Pj-frTest of 2,4-D and 2,4,5-T Phenoxyacetic Acids in Mouse Blood Tissues. qy*Chem. Biol. Interactions 1, 291-299. C3S04 Jurek, 1974 - see Syrowatka and Jurek. Khanna, Suchitra and Fang, S. C. (1966) Metabolism of C^4-Labeled 2,4-Dichlorophenoxyacetic Acid in Rats. J. Agr. Food Chem. 14, 500-503. Khera, K. S. and McKinley, W. P. (1972) Pre- and Postnatal Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4-Dichlorophenoxyacetic Acid and Their Derivatives in Rats. Toxicology and Applied Pharmacology 22, 14-28. Khera, K. S. and Ruddick, J. A. (1973) Polycnlorodibenzo-p-dioxins: Perinatal Effects and the Dominant Lethal Test in Wistar Rats. Advances in Chemistry Ser. 120. Ed. R. F. Gould, 70-84. Kholi, J, D., Khanna, R. N., Gupta, B. N . , Dhar, M. M . , Tandon, J. S., and Sircar, K. P. (1974) Absorption and Excretion of 2,4-Dichloro phenoxyacetic Acid in Man. Xenobiotica , 97-100. Konstantinova, T. K., Ephimenko, L. P., and Antonenko, T. A. (1976) The Embryotrop'ic Effect of the Dissociation Products of Herbicides Based on 2,4-D. Gigiena I Sanitariya 11, 102-105. Magnusson, Jan, Ramel, Claes, and Ericksson, Ann (1977) Mutagenic Effects of Chlorinated Ph.yenoxyacetic Acids in Drosophila melanogaster. Hereditas 8_7, 121-123. Mrak Report (1969) Report of the Secretary's Commission on Pesticides and'Their Relationship to Environmental Health. U.S. DeDt. of HEW, 468-477. Olson, Robert J., Trumble, Thomas E., and Gamble, Wilbert (1974) Alterations in Cholesterol and Fatty Acid Biosynthesis in Rat Liver Homogenates by Aryloxy Acids. Biochem 142, 445-448. i Palmer, J. S. and Radeleff, R. D. (1964) The Toxicologic Effects of Certain Fungicides and Herbicides on Sheep and Cattle. Annals N.Y. Acad. Sci. Ill, 729-731. Paris, Doris F. and Lewis, David L. (1973) "Chemical and Microbial Degradation of Ten Selected Pesticides in Aquatic Systems," Residue Reviews 45, 107. 0003331 Radionov, A. D., Chumachenko, A. N., and Kirilenko, I. D. (1967) The Toxic Properties of the Herbicide 2,4-D. Hygiene and Sanitation 32, 116-118. Reuber, Melvin D. (1979) Carcinogenicity of 2,4-Dichlorophenoxyacetic Acid. Manuscript, personal communication. JOO! Sauerhoff, M. W . , Braun, W. H., Blau, G. E., and Gehring, P. J. (1977) T O The Fate of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Following Oral MI Administration to Man. Toxicology , 3-11. TO TO Schwetz, B. A., Sparschu, G. L., and Gehring, P. J. (1971) The Effect of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Esters of 2,4-D on Rat Embryonal, Foetal and Neonatal Growth and Development. Food & Cosmet. Toxicol. 9, 801-817. Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe, V. K., Gehring, P. J., Emerson, J. L., and Gerbig, C. G. (1973) Toxicology of Chlorinated Dibenzo-p-dioxins. Environmental Health Perspectives, September, 87-99. $ Shirasu, Y., Moriya, M., Kato, Y., Furuhashi, A., and Kada, T. (1976) Mutagenicity Screening of Pesticides in the Microbial-System. .Mutation Research 40, 19-30. Simmon, Vincent F., Kauhanen, Kristine, and Tardiff, Robert G. (1977) Mutagenic Activity of Chemicals Identified in Drinking Water. Progress in Genetic Toxicology. Ed. by D. Scott, B. A. Bridges, and F. H. Sobis, 249-258. Syrowatka, Tadeusz, and Jurek, Andrzej (1974) Carcinogenicity of Pesticides. Roczniki Panstw. Zakl. Hig. 5, 563-576. Van Duuren, B. L. and Goldschmidt, B. M. (1976) Cocarcinogenic and Tumor-Promoting Agents in Tobacco Carcinogenesis. Journal of the National Cancer Institute 56, 1237-1242. Vogel, E. (1974) Mutagenicity Testing of Cyclamate and Some Pesticides in Drosophila melanogaster. Experientia 0, 621-623. Woolson, Edwin A. (1972) Survey of Polychlorodioenzo-p-dioxin Content `in Selected Pesticides. J. Agr. Food Chem. 2, 351-354. Zielinski, Walter L. Jr., and Fishbein, Lawrence (1967) Gas Chromatographic Measurement of Disappearance Rates of 2,4-D and 2,4,5-T Acids and 2,4-D Esters in Mice. J. Agr. Food Chem. 15, 841-844. / CGQ53S2 7618 DOW1079581 MN074379 \ : r* ASSESSMENT OF H U M AN HEALTH RISK ASSOCIATED WITH THE USE OF 2,4-D IN FORESTRY M ANAGEM ENT. m MINNESOTA DEPARTMENT OF HEALTH cn 717 S. E. DELAWARE ST. MINNEAPOLIS 5 5440 CG0G013 7619 DQWJ 079582 ASSESSMENT OF HUMAN HEALTH RISK ASSOCIATED WITH THE USE OF 2,U-D IN FORESTRY MANAGEMENT Minnesota Department of Health Division of Environmental Health Section of Health Risk Assessment 717 S.E. Delaware Street Minneapolis, Minnesota 55iu*0 March 1978 CG0G013 yjjy * 'K (K i DflWJ079583 TABLE OF CONTENTS I II III IV V VI VII VIII IX X XI XII XIII Introduction Physical and Chemical Properties of 2,^-D Persistence in the Environment Residues in the Environment Absorption Acute Toxicity Chronic Toxicity Teratogenicity Mutagenicity Carcinogenicity Comparison of Estimated Maximum Intake of 2,14-- 0 and Acceptable Daily Intake Conclusions and Recommendations References* * CGOoQ^O 7621 DOW]079584 INTRODUCTION Recently, concerns have been expressed by Minnesota residents that the use of 2,4-- D in forestry management may have an impact on human health and on the environment. This report is a quantitative assessment of possible human health effects resulting from the use of 2,4-D in forestry management. It does not contain an evaluation of the possible effects of 2,4-D on the wildlife population, except as a route of human exposure, or of possible effects on human health resulting from the use of 2,4-D in agriculture. The scope of this assessment includes a literature review of the toxicology of 2,4-D and an estimation of 2,4-D exposure by a "maximally exposed individual". From this, conclusions and concerns are drawn on potential health impacts from exposure to 2,4-D and recommendations are made on the use of 2,4-D in forestry management in Minnesota. 0006021 i/ ii (J * DOW1079585 PHYSICAL AND CHEMICAL PROPERTIES OF 2,4-D The herbicide 2 ,*4-dichlorophenoxyacetic acid (2,*+-D) is a member of the major chemical class Chlorinated Phenoxyalkanic Acids (see Table 1 for a partial list of other herbicides in this class). Table 1. Chlorinated Phenoxyalkanic Acids Common Name or Designation Chemical Name 1. 2 ,U-D 2. 2 , u -d B 3. 2,4,5-T M. Dichlcroprop 5. MCPA 5. Silvex (2,U-dichlorophenoxy)acetic acid u-(2,4-dichlorophenoxy)butyric acid (2,4,5-trichlorcphenoxy)acetic acid 2=(2,U-dichlorophenoxy)propionic acid *J-chloro-o-tolyl)oxyj acetic acid 2-(2,4,5-trichlorcphenoxy)propionic acid The chemical formula of 2,U-D is shown below: Chemical and physical properties of pure 2,4-D are given below: a. 'Description: Odorless white crystals b. Boiling point: 160C at 0.^ mm . c. Melting point: 140-1H1C d. Solubility: .Soluble in 95% ethanol and in^acetone, dioxane, and isopropyl alcohol; solubility in water is 5U0 ppm at 20C e. Stability*: Stable up to and including its melting pcint f. Reactivity: Forms salts that are soluble in water Synthesis of 2,4-D ' v The syntheses of 2,4-D and 2,4,5-T are considered together O ' V . *' f t , "* j - i A V, because of their similarities in their production and also because ;of an important difference that has resulted in some confusion over the toxicities of the two herbicides. % The method of synthesis of 2,4-D is as follows (see figure 1), monochloroacetic acid is reacted with 2 ,4-dichlorophenol in the r- presence of an aqueous sodium hydroxide to form 2,4-D (U.S. EPA, 1974). 'S*'* '** The production of 2,4,5-T is similar to that of 2,4-D except that 2,4,5-trichlorophenol is required as a starting material and this is manufactured by the basic hydrolysis of 1,2,4,5-tetrachlorobenzene. Hydrolysis is carried-out under pressure at an elevated temperature that must be carefully controlled to avoid formation of a toxic impurity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is one of the most toxic chemicals known. As an example, the LDgQ value for a single dose of TCDD to guinea pigs is on the average 0.0006 mg/kg CSchwetz, et al., 1973). As a contaminant of 2,4,5-T, TCDD has generated considerable concern over the use of phenoxy herbicides. However, TCDD should not be confused as a contaminant of all phenoxy herbicides. ~ Contaminants of 2,4-D Of 28 samples of 2,4-D tested for content of chlorodibenzo- para-dioxins (this includes TCDD), no TCDD was found in any of t the samples (Woolson, et al., 1972). However, hexachlorodibenzo- <0 O & <2 p-dioxin (HCDD) was'found in one sample at a level of 10 ppm. HCDD ais toxic but much less toxic than TCDD. Schwetz, et al.,(1973), observed tertogenesis in the offspring of rats treated with 100 A mg/kg/day of HCDD. Maternal toxicity was also noted at this dose level. In a concurrent study, chick edema was produced 624 107958H CG0GG24 Figure 1. Diagrammatic representation of the major steps in the production of 2,4-D, 2,*J,5-T. Also shewn is the step where TCDD (2,3,7,8-tetrachlorodibenzo-para-dioxin) arises. No com parable material is forired in the 2,4-D process because of the different, cbnditions for the production of the dichlcro- and the trichloro-phenol. 7825 t' 00 in birds treated with 10 and 100 mg/kg-day of HCDD. 00 i n Another dioxin that may be associated with 2,4-D is ao." 2,7-dichlorodibenzo-p-dioxin (DCDD). Schwetz, et al., (1973) found DCDD to be limited in toxicity. DCDD failed to cause death in female rats given oral doses of lg/kg. DCDD was neither teratogenic or embryotoxic at 100. mg/kg/day. 'r t The herbicide 2,4-D may be contaminated with isomeric 2,6-D (2,6-dichlorophenoxy acetic acid) unless careful control "of the process is maintained. 2,6-D is' more rsistent to biological' degradation than is 2,4-D (U.S.EPA, 1974). Bis(2,4-dichlorophenoxy)methane has been identified as the major contaminant of 2,4-D (Huston, 1972). An analysis of a com mercial sample of 2,4-D indicated that it contained 30 ppm of this contaminant. Smaller quantities of bis(2,6-dichlorophenoxy) methane and 2,2', 4,u'-tetrachlorodiphenoxy methane were also found. The toxicological significance of these three compounds as impurities in production grade 2,4-D is not known. Formulations ' 2,4-D is a strong acid (pka 2.64) that is only slightly soluble iiuwater and petroleum oils. Although the acid is the active form, it is normally converted to water-soluble amines or oil-soluble esters for convenience in handling and application and also to ., * improve the effectiveness of the herbicide. To indicate the nature h of the different formulations, the phenoxy group is designated as R. ?- C&GG-3 Then examples of the various salts and esters are as follows II-u 7626 4 DOW 1079589 Salrs Sodium salt, R-CH^COONa Potassiuin salt, F.-CKjCOCK Ammonium. salt, F-CHjCOONH^ Triethylamine salt, R-CH.-COONHCC-H. ), Triethylolamine salt, R-CH2COONK(C2HuOK)3 Alkyl Esters Methyl ester, R-CK-COCCH- ' 9 " Ph3 Isopropyl ester, R-CH-COOC CH, Butyl ester, R-CKjCOOC^Hg Octyl ester, R-CH2COOCgK17 Heavy or Low Volatile Esters butoxyethanol ester, F.-CHjCOOCjE^-O-C^Hg Fropyleneglycolbutylether ester, R-CHnC00CH2CH(0H)CH?0-C4H g Tetrahydrofurfuryl ester . - H0 R-CH-CCC-C CK, 2 i. A2 These formulations are then mixed with other ingredients, such as solvents-, emulsifiers, thickeners, and wetting agents, to make commercial formulations for specific uses. The salt and amine forms are diluted with water and sprayed on foliage or injected undiluted into trees. Esters are sprayed as oil solutions or as emulsions with water. f The strength of commercial formulations is expressed in terms of the equivalent ontent of the parent acid. However, different pro ducts are not equally effective even on this basis. Esters generally have greater herbicidal activity than the amine forms. CGOGGtaG II-5 / <0 i DOW1 Other studies also suggest that under normal use 2,^-D per- 'ists in soil for about a month. In moist loam soil 2,4-p applied at a rate of 1/2 to 3 lbs./acre persisted for 1 to 4 weeks, under summertime conditions in a temperate climate (Klingman, 1966).' ' .CD Alexander and Aleem (1961) reported that 2,^-D, at normal recom- ' CJT mended dosages, persisted for about 1 month in two types of soil and for about 3 months in a third type. Degradation of 2,^-D has also been studied in forest litter. r" In one study with red alder forest floor material, 9*+% of the 2,U-D was degraded in 35 days (see Figure 3) (Norris, 1971). Compared to . other herbicides studied in this investigation, degradation of 2,4-D was rapid. Figure 3. Recovery of 2,**-D, amitrole, 2 ,*,5-1, and picloram from red alder forest floor material. Amitrole, 2,H-D, and 2,4,5-T applied at 2 -pounds per acre and picloram at 0.5 pounds per acre in water (Norris, 1971) f DOW1 079591 PERSISTENCE IN THE ENVIRONMENT The persistence cf 2,U-D in the soil and water is an important factor in its potential to occur as residues in foods and water or to come in contact with nontarget organisms. Persistence in Soils The persistence of 2,u-D in soils appears to be a function of several factors. These factors include concentration, soil type, formulation, temperature, moisture content, organic matter, and possibly microbial ecology at the site of application. Therefore, it is difficult to assign any finite limits on the time 2,U-D will remain intact. `A mere reasonable approach is to assign a time range in which 75 to 100% dissipation has occurred. The persistence of 2,4-D, when applied at recommended rates, is about 1 month. This is a relatively short time when compared to other herbicides (see fig ure 2)Q(eamey, 1970). MONTHS Figure 2. Persistence of herbicides in soils. Length of bar represents time required for 75 to 100% loss of the compound (Kearney, 1970). CG06G3 7629 Y .D 0 W 1079592 Norris (1966) noted similar results in an earlier study where 14 degradation of triethanol amine salts of carbon -carboxyl labeled 14 2,4-D applied to an alder was measured as liberated C Oj* More than 89% of the applied 2,4-B could be accounted for by the liber ation of radioactive CO2 in 315 hours. In the soil in the presence of moisture, even at relatively low levels, the esters of 2,*-B are readily converted (hydrolyzed) into the acid form (Smith, 1972). The degradation of 2,4-B acid can be shown to be due primarily to the action of soil micro organisms (Allebone, et al., 1975). Persistence in ffater In water, 2,4-B breaks down either photochemically or under the action of aquatic micro-organisms. Light provides the energy for a variety of degradative reactions including oxidation, reduc tion, and hydrolysis (Allebone, et al. , 1975). In surface waters, levels of 1,000 ppb were degraded to 10 ppb within 30 days of application (House, et al., 1971, cited in Allebone, et al., 1975). DeMorco, et al. (1967), investigating the persistence of 2,4-D in natural lake waters, found that in "wanr." aerobic waters the herbicide was degraded within 6 days whereas in "cold" deoxygenated waters it persisted for up to 80 days. Low levels (0.024 ppm) of 2,4-D were found in pond water 1 day after application/of 1.3 3 ppm granular material (Frank and Comes, 1967). The concentration in water increased to a maximum of 0.067 ppm after 18 days and 2,4-D could no longer be recovered after 24 days. Higher concentrations (4.96 ppm) were found in the hydrosoil III-3 CG0GG33 7630 DOW 1079593 after 1 day, and there tended to persist longer. Negligible residues C 0.05 ppm) were detected after 55 days in the soil. Thea increased persistence of 2,M-- D in sediment was also noted bi y. Smith and Ison (1967). They recorded and isolated significant concentrations of 2,4-D (58.8 ppm) in sediment samples removed from a reservoir some 10 months after treatment. Degradation in soil appears to be rapid, occurring in several weeks in most soils and forest litter. Degradation in water is also rapid; .however, 2,^-D appears to persist longer in bottom sediments. The two main pathways of microbial degradation are thought to be via a hydroxyphenoxyacetic acid intermediate or via the corresponding phenol (Allebone, et al., 1275). Apparently degradation goes to completion (CC^) although the exact mechanisms .'e not well understood (Norris, 1966 '. No information is avail able on the toxicity of intermediate metabolites or other possible end products of microbial or photo degradation. CGG5G3Q