Document 10yVm4645jpNGLyyp7YJKdMDd

SSTZT AlOo this experiment. The relationship of the lingular deformations to the ad ministration of 2,4-D is dubious. In a complementary experiment, a pregnant sow was fed 2,4-1) at a dietary level of 500 p.p.111. during the entire pregnancy, and further oil. During a protracted parturition, fifteen live and one stillborn pig let were delivered. The newborn piglets were underdeveloped and list less and showed a high mortality rale, ten out of fifteen dying within 24 hours. No malformations were observed. Autopsy findings in the dead piglets were those of a generalized anaemia. On continued feeding of 2,4-D at the same level to the survivors for another (5--7 months, growth depres sion was evident. On autopsy, no chracterislic gross changes were observed, apart from multiple disc degenerations in the cervical and lumbar spine in two animals. Heamatological, biochemical, and hislopalhological findings were similar to those observed in the preceding experiment, thus confirming the toxic action of the phenoxy acid. The dam developed weakness of the hind quarters and was slaughtered at 0 weeks post partum because of a complete inability to rise. Autopsy revealed excessive degenerative changes in various regions of the spine, which provided a hislopalhological explana tion to the clinical signs. The toxicological significance of these changes, however, as well as of the spinal degeneration in the young pigs, remains obscure. Rats. Ten pregnant albino rats, initially weighing about 350 g, were equally divided between an experimental and a control group. The experimental group received 2,4-D in the drinking water, at a level of 1000 p.p.m., during the gestation period and the following 10 months. The estimated daily dose of 2,4-D was 50-- 100 mg/kg body weight. No unequi vocal clinical or morphological changes were observed during this period. On continued administration of 2,4-D to the second generation rats for up to 2 years, growth depression and a reduced intake of feed and water and also a slightly increased mortality, as compared with controls, were noticed. Clinical-chemical and hislopalhological examination did not reveal any de finite abnormalities. This fairly high tolerance of rats to 2,4-D may be related to the high plasma disappearance rate of the phenoxy acid in this species (sec Distribution, aliove), which most likely is the result of an effective renal excretion. In pigs a lower plasma disappearance rate was accompanied by a lower tolerance to 2,4-1). Chickens. Six while Leghorn chickens of both sexes, originally weighing about 1 kg. were continuously Ted 2,4-1) at a dietary level of 500 p.p.m. Except for one bird dying after 5 months or renal gout, no distinct clinical 33 0001401 5542 . symptoms were seen. The survivors were sacrificed ;il intervals helween 011c and 18 mouths. The autopsy findings were mostly of a noiispeeirie character such as waxy muscular degeneration, provcnlriculnr ulceration, peribronchilis, and generalized anaemia. In another experiment, also extending over 18 months, sixty day-old F iy . H y p ertro p h ic k id n e y . Ik-fll of a .Viuonlhs oh! chirk jjiven 'J.-t-l) aiuinv 111)011 |>. p. in. in tile drinking wider} since 3 days <rhl, as eiiiu|iari*d w ith norm al kill n rv s irijthll of co n tro l chirk of Ilit- same aye. F iy . It. Section u f h y p ertro p h ic kidney. T idm lar dilatation an d h y p ertro p h y . I'A.S. .'1(10 X. F iy . 7. Section o f iiirrinal cluck kidney. Norm al liilmles. I'A.S. .100 : V 0001435 DOW 121538 P-OW 121539 broiler chicks of bolli sexes were evenly divided between one experimental group, receiving 2,4-1) inline (1000 p.p.m.) in the drinking water. and one conlml group. Apart from a reduced egg production in laying liens of the experimental group, no specific clinical signs were observed. Klcvcn treated and twelve control birds died or were killed because of disease during the Fi<j. \S'. SertiiMi of h y p e rtro p h ic kidney. G lom erulus w ith exlreiiudy diluted capsular space. PAS, aim .<. Fiij. 9. Section o f norim il chick kiilncv. N orm al {louirrulus. PAS, atXI 03 0001406 5544 I .4 r experiment. The causes of death in both 'roups won- 111:1inly coccidiosis, leukosis, lymphomatosis, enlcrilis or unknown origin, or inanition of un known origin. Survivors went* sacrificed and aulopsied at intervals between *2and IS nioiiullis. Tlic 2,4-D-lrealed birds, dead as well as killed, displayed a .spectacular kidney enlargement due to epithelial proliferation, a finding which seems not to have been reported previously (Figs. 3--D). The seemingly exclusive development in chickens of this lesion may possibly be related to the fact, that of the species examined this is the only one having a renal portal system which enables inlestinally absorbed substan ces to pass directly to the kidney. Interestingly, the renal hypertrophy deve loped only in those chicks that had been continuously exposed since a few days old to the phenoxy acid. Apparently, this finding provides a parallel! to the observation of Carr (ItkVi), that only very young chicks are susceptible to leukaemia virus-induced renal carcinoma. In both instances the reactivity may lie associated with the abundance of residual embryonic tissue in the young birds. ( 3fi 0001407 ' . 5545 DOW 121540 DOW 121541 Summary The investigations summarized lien- aimed at developing methods for isolating, delecting and determining residues of chlorinated phenoxyaliphatic acids (phenoxy herbicides, "hormone weed killers") in biological mate rials, and at studying the distribution and toxicity of these materials in 'experimental animals. After examining the conditions for isolation of phenoxy acids from diffe rent types of biological materials, an analytical method was adopted in volving solvent extraction of the sample (the technique varying with the type of material), purification of the extract by partitioning between im miscible phases, and separation and identification of the acids by thin-layer chromatography. The eluted acids are determined photometrically. The described method recovers phenoxyacetic acids from various biologi cal materials to the extent of 70--90 per cent, with a standard deviation of 3--5 per cent (at the 4-p. p. m. level). The sensitivity is about 0.1 ,g of phenoxy acid. Applying this method, the distribution of phenoxy acids was studied in calves, pigs, rats, and chickens. After the administration of soluble salts of 2,4-D and of 2,4,5-T as single oral doses of 50--100 mg/kg body weight, the plasma concentrations rose to a peak within 2--7 hours and then declined to insignificant levels over a few days. The plasma half-lives ranged bet ween 3 and 12 hours, depending on species. Oral administration of a 2.4-D ester resulted in considerably lower plasma levels of 2,4-D being attained (no ester was delectable in plasma or tissues). 2,4-D apparently distributed it self rather evenly over the body water, the highest levels being attained in the excretory organs (liver and kidneys). High levels were also found in ovaries, adrenals, lungs and myocardium. The levels found in' brain were relatively low, indicating a slow passage of the blood--brain barrier. Placental transfer in swine apparently was less restricted. The disappearance rate from tissues was fairly high, the average tissue half-lives for 2,4-D ranging between 5 and 30 hours, depending on the species. The highest disappearance' rale was consistently seen in rats and the lowest in pigs. Elimination evidently took place mainly be renal and. In a smaller extent, by biliary excretion of the unchanged phenoxy acid. Repealed administration of suhloxie doses did not lead to excessive accu mulation of phenoxy acid in any of the tissues examined. Except for a 00014 OP moderate increase in (issue levels relative lo plasma, (he distribution pattern did not change on repealed intake. In pigs and chickens, the capacity for excreting phenoxy acids was seen to rise during prolonged exposure. In acute toxicity studies, reversible toxic effects from 2,4-D amine were observed after single oral doses of 200 mg/kg in calves and of 100 mg/kg in pigs, respectively, doses above 300 mg/kg causing severe poisoning in pigs. Hals and chickens seemingly tolerated single doses of 100 and 300 mg/kg, respectively. On repeated oral administration, daily doses of 50 mg/kg could he toxic to pigs, whereas chickens tolerated 300 mg/kg/day for 2 weeks without outward effects. The results suggesl that clinical signs of acute or subacute poisoning are likely to appear, at least in pigs, if the plasma concentration of phenoxy acid exceeds a threshold value of about 200--300 /(g/ml. In long-term studies, signs of toxicity from 2,4-D appeared in pigs at a dietary level of 500 p. p. m. (corresponding to a daily intake of 25--50 mg/kg) and in chickens at 1000 p. p. m. given in the drinking water (equivalent to 50--100 mg/kg/day). In rats administration of 2,4-D in the drinking water at 1000 p. p. m. (50--100 mg/kg/day) over two generations caused a reduced weight gain and an increased mortality in the second generation. No une quivocal morphological changes were observed. In the pig experiments, the main symptoms were growth depression, loco motor}' disturbances, anaemia and albuminuria. On autopsy, degenerative changes were seen in the liver and kidneys. On feeding 2,4-D at the level of 500 p. p. m. to a pregnant sow during the gestation period and for 6 further weeks, no characteristic signs were observed. Autopsy did not reveal any changes attributable lo the intake of 2.4-D. The newborn piglets, how ever, were underdeveloped and exhibited a high mortality rate (66 per cent). Continued administration of 2,4-D lo the survivors resulted in growth depres sion, persistent anaemia and degenerative changes in the liver and kidneys. In the chicken experiments, a spectacular renal hypertrophy was the main finding, together with a reduced egg production. The renal lesion developed only in those birds that had been exposed lo phenoxy acid since a few days old. The chronic oral toxicity of 2,4-D lo the species examined appears to be moderate. Apart from the notable nephrotoxicity demonstrated in chicks, the observed long-term effects seem lo be non-spceific. Attention should be paid lo the high mortality rale in the newborn piglets and the reduced egg production in the chickens, however, which findings, together with the de monstrated aecumulation of phenoxy acid in foetal tissues and in ovaries, might be indicative of a phenoxy acid-induced interference with reproduction. 38 0001409 DOW 121542 References Aamiseii/i, .1.: The occurrence of 2,4-D in weds from cultivated plant* sprayed with chlorinated plicnoxyacclic acids. K. Lantbr llgsk. Anntr ltlfil, 27, 44a-- 431. Anderssun, A. B. Berzins: N igra hcbiciders loxisku verkan p i risk och krfiftor. Svensk l'isk --Tidskr. 1901. 70, 111-- 110. Antlrcnc, IV. .1. S . E. Cooil Studies on 3-indulcacctic acid metabolism. IV. Conjugation with aspartic acid and ammonia as processes in the metabolism of carltoxylic acids. P lant Physiol. 1957, J2, 500--572. Audus, L .J.: The Physiology and Biochemistry of Herbicides. Academic Press, London A New York 1904. Bauer, A'.; Studien ber Nebenwirkungen run Pflanzenschutzmitteln auf Fische und Fischuhrlicrc. Milt. biol. Bund Anst. Ld u. Forstw., Heft 105, Berlin-Dahlem 1901. Berkley, .1/. C. & K .R . .1layer: Neuropathy following exposure to a dimethylaminc salt of 2,4-D. Archs intern. Med. 1903, H i, 351--352. Beuenue, A., G. Zweig Nancy L. Nash: Residue determination of 2,4-dichloroplicnoxycelic acid in dry crops and walnuts. J. Ass. off. agric. Client. 1902, 45, 990--993. Brodie, B .B ., G.J. Cosmides D. P. Ball: Toxicology und the biomedical sciences. Science 1905. U 8, 1547--1554. Brown, C.P. A. R. Mathicson: Dimerization of the chloroacclic a d d s in solution. J. phys. Chera. 1954, 58, 1057-- 1059. Carr, J. G.: Renal adenocarcinoma induced by fowl leukaemia virus. Brit. 3. Cancer 1950, 10, 373--383. Crafts, A. S.: The Chemistry and Mode of Action of Herbicides. Inlerscicncc, New York London 1901. Curry, A .S .: Twenty-one uncommon eases o f poisoning. Bril. mcd. J. 1902, 1, 087--GS9. Dalgaurd-Mikkclsen, S. E. Ponlsen: Toxicology of herbicides. Pharm acol. Rev. 1902. U , 225--250. Davis, J .T . J .S . Hughes: Further observations on the toxicity of commercial herbicides to bhicgitl sunfish. Proc. Southern Weed Couf. 1903, 1ti. 337--340. Prunk, P. .1. R. ii. Grigsby: Effects of hcrbicidal sprays on nitrate accumulation in certain weed species. Weeds 1957, 5, 200--217. Freed, V. II.: Qualitative reaction for 2,4-dichh>ruphcmixyacc|:ic arid. Science 19-18, 107, 08--99. Goldmacher--u. Mallinckrodt, M. L. I.outcnbuclt: Zwei tdliche Vergiftungen (Suicid) mit chlorierten Phcnoxyc.vsigsurcn (2,4-1) und MCP). Arch. lox. 1900, 21. 201--278. Goldstein. N .P ., P .ll. Jones J .R . Brawn: Peripheral neuropathy after rx|>miirc to an ester of dichloruplicnoxyacclic a d d . J. Am. mcd. Ass. 1959, 171, 13(10-- 1209. Herbich, J. (1. Machutu: Vergiftung mil 2.4-l)icldiirphcuoxyessigsfiure (2.4-U). Rcitr. gericht. Med. 1903, 22, 133-- 139. Hill, E .C . II. Carlisle: Toxicity of 2.4-dichkir<iphciH>\yacctie arid for experimental anim als. J. ind. Hyg. Toxiocol. 1917, 20, 85--95. 30 DOW 121543  t'' .4 r ( j Johnson, II. It. M. A 0 . Koumides: A further case M. C. 1*A. poisoning.. lirH. nied. J. 19G5, 2, C29--630. Kldmht, II. I).: WuchsluuiMnduUion nml Wucli'i'loffmHabolivmus im Weizenkulcoptilzylimler 111. Sloffwi-chsvlproduklc der Xaphlhyl-l-csvigs:iurc und -M-Dichlorplienoxyossigsaurc uml dcr Vrrglcich mH jcncu dcr liidol-3-o*igsfuirc und Iienzoesuurc. Pluula 11IGI, 57, 33'J--353. l.r Tournean. U. A X. Krmj: The use of chromolropic a d d for (lie determination of 2.4dichlorophcuoxyucelic acid. Plant Physiol. 1952, 27, 822--827. Mitchell, L. C.: Separation and identification of chlorinated organic pesticides hv paper chrom atography. XI. A study of IN pesticide chemicals; technical grades produced in 1957 and reference standards. J. Ass. off. agric. Client. 1958, i t , 781--80G. Monarca, G. & G. Di Vito: SuM'iulussicazinirc acuta da discrlianlc (acido 2-- t diclorofcnossiacelicd). FoHu nied., Napoli 19G1, 44, 480-- 185. Nielsen, K., II. Kuempe <CJ. Jcnscn-llolm : Fatal poisoning in man by 2.4-dichlorophenoxyacetic acid (2,4-D): determination of the agent in forensic materials. Acta phnrmac. tox. 19G5, 22, 224--234. Po/ihum, R. D. A U. i l . Daoies: A cose of M. C. P. A. poisoning. Bril. nied. J. 19G4, 1, 677--G78. Home, V. K. A T. A. Hymns: 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. Am. J. vet. lies. 1954, la , G22--G29. Schanker, L. S.: Passage of drugs across body membranes. Pharm acol. Rev. 19G2, 14, 501--530. Shillinyer, Yu, I. A L .P . Naumova: Toxicological evaluation of a herbicide--the butyl ester of 2,4-D. Gigiena, Toksikol. i KKn. Xovykh Iuscktofungilxidov, T rudy 1-oi Vsesoyuz. Xauch. Konf., Kiev 1957, 297--305. (Through Chem. Abstr. I960, 54, 25514). Slade, R .E ., IV. G. T em phm an A IV. .4. Sexlon: Plant-growth substances as selective! weed-kVHcrs. Nature (Lond.) 1945, 155, 497-- 198. Sperber, I.: Secretion of organic anions m the formation of urine and bile. Pharm acol. Rev. 1959. 11, 109-- 134. Stahler, L. i l . A E .J . Whitehead: The effect of 2,4-D on polaswium nitrate levels in leaves o f sugar beets. Science 1950, 112, 749-- 751. Smanson, C. It. A IV, C. Shorn: The effect of 2,4-dichlorophenoxyacelic acid on the hydrocyauic acid and m im ic content of sudan grass. Agron. J. 1954, 4<S, 418-- 121. Teresi, J .D . A J . M. Luck: The combination of organic anions with scrum album in. IV. Quantitative studies by equilibrium dialysis. J. biol. Chem. 1948. 174, 653--GG1. Todd, R .L .: A case of 2,4-D intoxication. J. Iowa med. Soc. 19G2, 52, GG3--GG4. BOW 121544 40 0001411 5543 loi 5550 A L B E R T M. K L I G M A N . M. O.. P m. O. M . *;P|TAt. O r U ^'V C H ^IT Y O r ?rN N jri,V A N A 3 6 ' ANO S p Q U r.c ST^CCTS Phii.A3;Ph'A.pa *9iCa J a n u a ry 23, 1968 M r. V. K . Rowe Biochemical R esearch Laboratory 1803 Building The Dow C hem ical Company Midland, M ichigan 48640 CwM O CM H Oo o a D e a r V. K. : This note is a follow -up to m y re p o rt of July 8, 1966. In th a t study, you will recall that 6 groups of healthy adult subjects received sm all and in c re a s in g d o se s of te tra c h lo ro d ib e n z o -p -d io x in . We followed a sp ec ific protocol laid down by you. U nfortunately, not a single subject developed acne n o r was th e re any evidence of toxicity. This e n co u rag ed m e to p ro c e ed m o re vigorously. We then a sse m b le d a new p a n e l of 10 s u b j e c t s a n d a p p lie d 0 .0 5 m l of a 1% s o lu tio n in a lc o h o l chloroform to a one inch square on the back. These applications w ere m ade every other day for one m onth. The treated sites w ere covered with a non occlusive gauze sq u a re . E ach week for 6 weeks, the following lab o rato ry tests w ere done: U rinalysis, CBC, BUN, SGOT, Alkaline phosphatase, and Creatinine clearance. C learly, this exposure was im m ensely g reater than the fo rm er one. 8 of 10 s u b j e c t s s h o w e d a c n e f o r m l e s i o n s u s u a ll y b e g in n in g 3 to" 4 w e e k s . This began as w ith a typical developm ent of com edones. In 3 in stances, the lesions p ro g re sse d to inflam m atory pustules and papules. These lesions la ste d for 4 to 7 m o n th s, since no e ffo rt was m ade to sp eed healing by a c tive treatm en t. Biopsies w ere obtained in 5 instances at various stages. The histologic and clin ical m anifestations w ere in every way com parable to classical chloracne. The lesions were indistinguishable from those obtained by Dow 6X and H alow ax. In no instance was there laboratory or clinical evidence of toxicity. The subjects rem ained well throughout the study. i 551 0000450 9ZO STOO ^ M r. V. K. Rowe Page 2 J a n u a r y 23, 1968 These results im plem ent the conclusions form erly drawn, nam ely: it is m uch m ore difficult to induce acne in the human than in the rabbit e a r. The process begins m ore slowly and the doses required are very much g re a te r. H ow ever, unlike the rab b it e a r, the comedones often becom e in flam m atory. It is a certainty that the rabbit ear is exceptionally sensitive to a c n e ig e n ic c h e m ic a ls and is an e x c e lle n t s y s te m for the d e te c tio n of su ch chem icals. Finally, it m ay be said that chloracne closely m im ics acne vul g a r i s . The only d iffe re n c e is the p au city of a n a e ro b ic o rg a n is m s in the c o m edo. One m ay conclude that b a c te ria a re not very significant in the patho g en esis of ch lo racn e. Very sincerely yours A M K /klf 0 050505425 1 5553 749645) Report To DOW CHEMICAL COMPANY On GROUP (1 Through 6) Submitted By Clover Laboratories, Inc. Philadelphia, Pennsylvania 19101 0004G31 *** f " T, - O D D 4* Code: F 'orehead; B = Back; Meg = MJcrogram GB OU 1 Mcg 3ubJ. A ge 7/eight Race D aily No. btal Site D ose [>086 Doee INITIAL LABDHATOBY Alk C rt. 3GOT Phos BUN 7 * 3 0 fb*. C l. FINAL LABORATORY Alk Hb. C rt. JJG0T Phos 3UH WEC tobi C l. 63 I 22 185 N 0 .2 1 0 .2 K 12 9 .5 11 8743 1515 Cubito 15 10.0 11 9085 l . 4 ! 0.1 2 0.2 F 12 10.4 13 9059 16.5 77 CuAlin 2 29 210 N 0 .2 1 0 .2 F 16 12.8 10 9021 15.2 40 16 11.6 12 9834 15.5 Cu/Min Sub ject 2 Dis Charge 1 3 22 150 N 0 .2 1 0 .2 F 18 12.0 13 7405 15.6 81 15 10. S 10 6573 15 4 Cu/Min 3 0.1 2 0.2 F 64 15 11.5 10 7803 14.1 Ci/MIn 4 23 190 N 0 .2 1 0 .2 F 10.2 11 9981 13.5 47 20 Cu/Min 4 0.1 5 32 185 N 0 .2 1 Sub Ject 5 D is Charges ' 2 0.2 F J1 0.2 F 1 \ 1 19 12.2 12 7705 14.3 74 17 Uu/Min O O O t ^ 9 .4 8 6003 12.6 o.e 11 6390 12.8 54 Ci/M n ia 7 .. 1 4570 13.8 11 """T J 1 1- T 5555 5556 Code: Back; Meg = M icrogram OBOI I 5557 Codt Forehead; Meg = Microgram <3? 0 1 2 Code: P Back; Meg Microgram G P 0 1 __2 Meg DubJ. # A ge 7/etght Race D aily N. Urtai Site ,i P ose Dose Dose 6 23 175 N 0.5 1 0.5 B INITIAL LABORATORY 3GOT Aik Phos BUN 7*30 Great. C l. j 49 18 11.5 9 7573 16LjcVMin FINAL U BORATO?Y 3GOT Aik Hb. C reat. Phos SUIT WBC C' . 14 12.3 8 5009 15.4 6 0.1 5 0.5 B 67 17 12.3 9 7245 14.8Cu/Mlr 72 7 26 130 N 0.5 1 0.5 B 15 9.7 13 6197 13.7 Cu/Min 15 10.2 12 6232 14.5 7 0.1 5 0.5 B 16 10.0 12 7173 15.0 67 Cu/Min 93 8 28 150 . N 0.5 1 0.5 B 20 8.8 12 7475 14.4 Du/Min 15 8.7 16 5680 14.8 8 0.1 5 0.5 B 18 9.1 13 7874 14.3 74 u/Min 84 65 9 22 138 N 0.5 1 0.5 B 12 10.9 13 7121 15.7 Cu/Mln 14 9.4 11 5828 15.4 Cu/Min 9 0.1 5 0.5 B 17 10.( 12 8142 15.5 10 32 163 N ~1 0 1 i 0.5 1 0.5 B 0.1 5 0.5 B 75 20 10.6 11 6819 15.7Cu/Mln 18 14 GG< 1 4 3 3 L-. _______ L 10.2: 14 6927 13.9 -------- - r 1r i 91 11 9731 * 13.7| 96 ___ jCu/Mln; R SS Code Forehead; Meg = Microgram GP O 3 Meg jubj. # A ge Weight Pace D afly N o. Tbtal Cite _____ * Dose Dose Dose INITIAL LAB3RATOPY Alk G reat. 3GOT Phos BUN 7'3C it/. C l. FINAL LABORATORY Alk Hb. C rt. 3GOT Phos 2UH WBC Kdb. C l. 69 1 29 150 N 1.0 1 1.0 F 32 10.4 12 8155 15.5 Cu/Min 17 l a 7 11 9346 15 1 0 .5 2 1.0 F 87 20 10.4 13 7429 14.5 Ci/M in 2 26 148 W 1 .0 1 1.0 F 17 11.1 9 7690 13.2 81 26 10.7 10 6657 12.0 Cu/Mln 2 0 .5 2 1.0 F 16 10.6 9 7405 12.4 72 C i/ Min 3 139 N 1.0 1 1.0 F 19 10.2 11 7726 14.6 97 24 9 .6 15 5481 I5 .O Cu/Mln 3, 0 .5 2 1.0 F 20 4 23 170 N 1.0 1 1.0 F 15 13.1 9 7003 13 99 24 C i/M in 12.S 14 7440 14.3 74 Ci/Min 11.1 8 7757 12.8 555 4 CD 5 5 0 .5 2 1.0 F 28 156 w J l . o J 1 1.0 F 1i 0 .5 -- ------ r ! 2 l.o j F 1i 27 11.9 11 7906 12.2 97 C i/M in -----------r J 67 21 12.7 I 1 6357 12.8 Cu/Min 15 H. c 11 ! 5210 i 12.5 1 1D ii------/ " r ~ r (j J.. L t 19 ll.sj 13 j 8121 12. 73 Jcu/M in; t/ ) n a ss Code: P Back; Meg = M icrogram GROT 3 Code: F = Forehead; Meg = Microgram 0 3 OU 4 Code: P = Back; M icrogram =* Meg GBOf 4 55fi2 Code: Forehead; Meg - Microgram G? Ol 5 Meg jubJ. # A ge 7/,eighkt Race Daily N o. Tbtal Site Doge Dose Dose INITIAL LAB3P.AT0RY 3GOT Alk Phos BUN 7 r3 C Kb*. Qreat. C l. FINAL LA B03A T03Y Alk Hb. C rt. 3GOT Phos 3UH WEC Refe. C l. 83 1 49 210 N 4 .0 1 4 .0 F 18 9 .9 12 7729 129 Du/Min 13 10.7 13 9978 14.0 l 1.0 4 4 .0 F 72 16 11.0 12 9941 14.6 Cu/Mir 62 2 30 210 N 4 .0 1 4 .0 F 25 8 .4 11 8312 15J5 Ct/M in 18 9 .9 10 9947 15.1 2 Sub ject Die charged i 90 3 23 165 N 4 .0 1 4 .0 F 16 7 .7 9 9982 15kl Cu/M ir 12 9 .2 11 8873 14.5 3 1.0 4 4 .0 F 77 14 9 .7 ' 10 7781 14.9 Cu/Mtn 5563 76 l 4 23 170 N 4 .0 1 4 .0 F 14 6 .2 10 9764 1512 CVMin 12 7 .9 11 9367 15.1 1 4 1.0 4 4 .0 F 11 8.1 12 9074 l4.fe C9i/JM in Sub ject 5 Dis chargee 1 __L..,,__1 5 Sub ject 1 Dis chargee CCI_______ m O -- "1-----------r _ o<^ \ k \ I i ! Code: *" - Back; Meg = M icrogram QBOi 5564 <i Code: ' Forehead; Meg = M icrogram QE 0 ' Meg jubJ. # A ge W eight Race D ally NO. Ibtal Site P ose Dose Dose INITIAL LABORATORY Alk C rt. 3GOT Phos BUN W3C Jib. C l. FINAL LABORATORY * Alk Hb# C rt. 3G0T Phos BU1T V/BC X sb. c i . : 93 1 29 166 N 8 .0 1 8 .0 F 15 11.8 il 9537 14.1 Du/Min 16 10.3 12 7391 13.7 1 1 . 0 8 8.0 F i > 15 81 10.5 13 7446 13.9 CuAfin 2 22 164 N 8 .0 1 8 .0 F 12 9 .5 14 9524 16.4 72 16 8 .4 12 5695 15.0 Cu/Mip 2 1 . 0 8 8.0 F 1 Sub ject 3 Dis charge* t 16 8. Oj 11 5521 iI i( 14.8 87 Cu/Min 5565 3 Sub ject Dis charge 1 i 4 38 201 W 8 .0 1 8 .0 F 13 11.1 15 8367 150 99 19 9 .7 10 6591 15*2 C iA ln 4; 1 . 0 8 8 .0 F 72 17 9 .5 18 6761 15.8 C i/ M in f i 33 169 W 8 .0 l 8 .0 F 11 10.2 18 9986 15.2 68 20 9 . a 13 9437 13.8 ... J. Cu/Min i_ i_____ 5 Sub ject Dis chargee r !* _ T ~ ..... p i 1 0 0 0 4 3 4 2 i __ . . 4. . 1 ! .. k t l '\ Code: B - Back; Mcg = Micrograra GROUT 6 ...; * loi Ul A y* O D fe ? - -/ : *;*'. 'vS gT*'- iN ,-^-isr#r, ~"xv:' - V.?. f . - - ^ S $ S J . rv.1`t` . ^ - J r I I - -- r - -. s- N ational Clearinghouse r tk ir . ... iPison C ontrol C enters, A *****' s'W t.i,1-* > ? ... Bulletin /,,y , \ ' v. 'l- Q T ,'.' Vi li.S. DEPARTMENT OF HEALTH. FDCATION ANDWELFARE Public Health Service "K- ;..:i - A w r il L-ioS Washington D.C. 20201 _ V. SE A TK S.FR C M CHLORINATED PHI-lNOXYACETTC ACIDS (2, 4-1); 2, 4 , 5 - Y; M C F A ) S t: w to 00 o Ci in..: r. o i t >a ici'..T'/ ur-uti in-rbl* ici vj-s a r c 2 , 4 - D , 2, 4, 5 - T ; and r e l a t e d c o r n - . . . d a . Vn:l i t e r o : n o t i}u-s<. a r e a c t u a l l y to x ic i s a m a -tr e r oi" c u n t r o - r s y . - R.a*. h u i C.'- r * o u . -.L. ' i'I..- -*; m a y r.ot b e e b n t r o .-e rsy w h e n t h e s e . c h e m i c a l s a r e u s e d a s d i r e c t e d r , . T; :>;.:d ?hc h ou s> ho lei o r in a g r i c u l t u r e , b u t th e M i c h i g a n f a r m e r w ho d e - Th."'1 T'';-. ' n v r l Lati`,u !..t*rc*rt:, iy b o th g i r l s haT a g e n e r a l i z e d e r y t h e m a o f ' - ik.ii., m i n i m a l e d e m a t o u s s w e l l i n g of. th e o r a l a n d v a g i n a l m u c o u s : n .! .r ;i:.e s , a lig h t s w e lli n g o f l i p s a n d e y e l i d s , i n f l a m m a t i o n o f th e r n i u t h , -> .. u-.:jr.u.'i.'i and o l i g u r i a . ^ r e c e i p t .of a f a t a l c a s e h i s t o r y d u e to s u i c i d e in a 4 8 - , e a r - o l d C o n n e c t i c u t ; a re v ie w o f the C le a rin g h o u se s re p o rts w as m a d e. In the p ast th ree"''' i n , th e C l e a r i n g h o u s e lia s r e c e i v e d s e v e r a l r e p o r t s o f p e r i p h e r a l n e u r i t i s -A alt in r a s h .f l o w in g e x p o s u r e to tlie s e p r o d u c t s , in a d d i t i o n to t h o s e . areadv c i t e d : ^ ^ l > ? V - V - - ' . V " ; . .Luw Vj'jTw.*a _ . i - 4Tr.' if *^T* O t h e r syniptbn^p9Ta.t:w'<?r ^ a s s o c i a t e d Wiiii th e ' c h l o r i n a t e d p1ier.oxy a c e t i c .'i;E?TV5^' id derivntiv^i^:.ty.exe; w e a k n e s s o f c h e s t m u s c l e s a s s o c i a t e d w ith p n e u m o n i a Y.:*-: ' =' -.!I*ht<ie.ui:i.aaiunrdi3|urinary fr^ewqiutiei npcoyo;r.i-rmriutsacblielittyo,nel;etfhwaorgbyr;o itnh ce rr se aws eithora tdaexcirae,ase;.-''-:^" ' pf r'*turv*V iwloKt o? th ^ v i c t i m s ..th a t 1 .id* s y m p t o m s ' h a d v o m i t e d . . T h e r e . ^ 4 r-.'j.'urt oi holiw ir.a iio iis n i u c i n h a l a t i o n o f Z, 4 , o - T . A i Kltvli.i.-; oi i ::i D iv j ion o ; D ir--, t H-- .It;;i h> r v h < r.< : oi' H- .dll: rYi:r i :a --j 5568 cccg o e -sI .'U/, Zi . i.. - . . .*% * . >'. w-J,- ,* ,4 - D and its d e riv a tiv e s S erve as a b a se m a te r ia l tro m whirl-, soluble e s te r s and s a lts a r e p ro d u c e d . Tin: LD50 ot th e se p ro d u cts ranjje iro m 300 to 700 m illi g r a m s p e r k ilo g ra m o r a lly . ;*lrf*S.w>; fr ^' . . . . When the chlcrophunoxy h erb icid es a re ingested in sinale la ran d oses -jy .`"V."? they m a y p ro d u c e dc-atn. F o u r e a s e s have been d e s c rib e d in the .lite m ` Jure and a 5th c a se has been re p o rte d to the C learinghouse. A C o n n e-n eu t truck d riv e r in a suicide attem p t ingested a cupful of weed k iller which was a p p r o x i m a t e l y 20 % 2 , 4 - D a n d 40% 2, 4, 5 - T . W ith in an h o u r , 'lie b e c a m e ' . 'Ti-ca n a u s e a t e d a n d b e g a n to v o m i t . A p p r o x i m a t e l y o n e h o u r a f t e r th e in g e s t i o n ^ .' he w a s fo u n d in a c h a i r c o n s c i o u s b u t in a d a z e d c o n d i t i o n . H e w a s a d m i t t e d '*rw - to the h o sp ita l an hour and a half la te r, still conscious, but d iso rie n te d . On his second hospital day he w as on the hospital critic a l list because his g e n e ra l condition w as getting w o rs e . His te m p e ratu re was 104 F , , his blood p re s s u re 70/50, and although his u rin a ry output w as d escrib ed as good, h i s BUN h a d r i s e n f r o m 16 m i l l i g r a m s p e r c e n t to 4 8 . He w a s h y p e r v e n t i l a t i n g and had b asalar ra le s . U. Late in the second day of hospitalization the patient's blood p ressu re was v ery low d e sp ite la rg e d o se s of Aram ine and a trial on Levophed. He had sinus tachycardia (150), a central venous pressure of minus 3, tem perature o f 104 F , h y p erven tilation , blood PH of 7 ,4 3 , and anuria. He a lso h a d t| generalized erythem a. v ' 'V . *. . .I* * .w - . . iC ' : ' ' The patient developed scattered crep ita n t.ra les in the right lung and died o f cardiac sta n d still (no ven tricu lar arrh yth m ias w ere noticed), e a rly in :i the m orning of h is third hospital day, approxim ately 46 hours after the ingestion. . ' This c a se had s e v e r a l s im ila r itie s to the c a s report of poisoning by MCPA (2 m eth y l-4 -c h lo r o -p h e noxyacetic acid). Vomiting occurring shortly after in gestion and un consciousness appearing in a few hours Both h*ad lo w *e7jyr?e dVv'..blood*. p-r e s s-u?r e"*b*u!t in the MCPA c a se it c.ould be\ ma.rtfi*n*."-''*'t**-O'" tained w ith m eta ra m in a l. H owever, ' none of the neurological sym ptom s (facial tw itcK m g^constricted pupils, decreased tendon reflex es, clon ic spasm of u itib s) w ere m entioned in the present c a s e , ' E arly deaths in a n im a ls by la rg e d o s e s of chlorinated ohenoxvacctic acid fVv>-r. ; r!.-*&SkiJi< r i /derivativesjw ere' attributed to'ventricular fibrillation. |n delayed deaths^ tliere w as a^disinclination to m ove, p r o g r essin g to rigidity of sk eleta l m u sc le s (m yotonia) and a taxia. The se v er e c a s e s had p r o g r e ssiv e apathy, " "-/V m uscular w eak n ess o f the hind lim b s, p a ra ly sis, clonic sp asm s and finally - coma. ` i -, C004E07 * *'<j J +*_ i~ An I n g e s t i o n s o f th e .s e h erbicide*-' - n :.. >r a ; / .i c a r c f i i l g a s t r i c l a v a g e and s y m p to m a tic an d s u p p o rtiv e t. :rap^ . P r o d u c t s r e c o m m e n d s Q u in iu . S u 'fa ;. ,n;. ev ery two h o u rs for the f ir s t j . . }' Toxicology of C om m ercial -M).2 g r a m s o r a l l y nyotonia, if p resen t, and to s u p p re s s v e n tric u la r c ti ro , So r e - a s e s m a y r e q u i r e c a r d i a c m o n i t o r i n g w i t h d e f i b r u T- r h -d to th e c h e s t . F e v e r should be tre a te d using phy I* c k s a n d a l c o h o l s p o n g e s ) s i n c e a n t i p y r e t i c s m a y be in .a: .indirr.ted. Parke - Davis PARCODE -' * 0 Crt o X P a r k e -D a v is has r ec en tly initiated a n u m erical product i.!enr ; cion system known as PARCODE. The sp ecial number desigi nt i f ie s each ca p su le, K apseal, tablet and E m plet. The in itials P -L id en tifies the product as a P ark e-D avis preparation and the imprinted number identifies the particular product. Although the com pany's manufactured products w ill henceforth carry a PARCODE identification sym bol, there m ay be considerable tim e required fur utilization of the non-coded products jj' ..-y currently in stock. ' V: .,, ^ Codling o r id en tification , have alread y bn estab lish ed by E li L illy and j Company and the N orw ich P h arm acal Company .' The poison con trol cen ter s^ have been se n t a copy o f the P a r k e -d a y is recogn ition code and w ill be in - form ed if any other pharm aceutical cpmpanieis adopt sim ilar sy stem s. 7*- ., ; ^ ' V* . -, * ACUTE OVERDOSAGE OF DORIDEN '` T'-\" '***V'' " .rti -V' -.. -' ' AOUVi OHI Y ' VII VVVv VTi4VWKv TVV** * -L \bAipbrlfioi rga lrarpohmiems ^i i^n i^r oa tpi nyn <o) fUtphpit&in^no. of!tTeR P k a r m a p p i i f r a iTwn mrirtifilnngv .'to itihmer. t T ' : ; . ...r-:- : ;A : Vr j -, --Vi-.-v V-a -.v:c s s , ' ... 'T i 5570 754506 * -->.`.~ur*r?"V-V5:*U-**.v; /r > '. ! * " * - -. ' - *'' ' ' . _3 . r -:"' .. :.'`"w-Yi v1**-~;r^' , D E F ER O X A M IN E F o r s e v e r a l - y e a r s th e C i b a C o r p o r a t i o n h a s b e e n c o n d u c ti n g r e ta r h on -i..^. th e u s e o f d e f e r o x a m i n e ( D e s f e r a l ) , o l d e r n a m e d e sfc r r i o x a s m m - , . !: the t r e a t m e n t o f i r o n p o i s o n i n g . B e c a u s e t h e r e h a v e b e e n ever-.:; --rib. ie p u b lished in the m e d ic al lite r a tu r e on its effectiveness-, th ere l..i- been a s c rie s of requests for this m edication when 'poisonings have o c cu rred . S in c e th is d ru g w as. u n d e r 'i n v e s t i g a t i o n ,'i t w as only a v a ila b le to the a u th o rise d in v e stig a to rs. The lis t o f in v e stig a to rs, w illing to w p t re - ' . f e rra ls of iro n intoxications, could not be published. -T herefor.-, the N a t i o n a l C l e a r i n g h o u s e i n i t i a t e d a s e r v i c e to tin* P o i s o n C o n t r o l C e n t e r s to p ro v id e the n e a r e s t in v e s tig a to r 's nam e anti telephone n u m b er when an in q u ir y w a s m a d e fo r the im m e d ia te tr e a tm e n t of an in d iv id u al c a s e . We felt th is m ig h t be of ben efit to a s e rio u sly ill p atien t, as w ell as to the in v e stig a to r, by providing him a la rg e r num ber of observations from which to judge the u sefu ln ess of this m ed icatio n . T his drug h as been re le ase d for; g e n e r a l m e d i c a l u s e , a n d u n t i l th e M a n u f a c t u r e r can. c o m p l e t e i t s d i s t r i - b. bution this service w ill be continued. v1 D eferdxbinine'f a chelating agent," com plexes with iron to form fe rrio x am in e. V .w h ich p re v e n ts tbe/ iTon frp m enterifigVihtp^-iurther ch em ic al re a c tio n s . The c h e l a t e i s ''s o l u b l e in w a t e r ; i s e x 'c r e f c d b y th e k i d n e y . : and p r o d u c e . z c h a r a c'^1 f ea .1'* r' i s t i c . r e- d' j dVi* s fUi c o l o r t q .the. " 1- ' ' i u ^*i' n e ^ - ^ h e"rJBpii't * d r ' e' t i c a l la y , d* e ff e r o x a m i n e w i l l bind 8 . 5%'of its w eight of fe rric , xrom th e i n i t i a l r e c o m m e n d e d d o s e ;; o f 1 g r a m w ould, bipid" 8 5 m i l l i g r a m s o f f e r r i c i r o n arid th e m a x i m a l 24 - h o u r, d o sa g e o f '6 g ra m s'w ould bind o y e r one-half, g ra m of iro n , the e q u iv a le n t of'; 2 1 /2 g r a m s of F e S O ^ . 7 1 1 ^ 0 .. ...c.b.. v' .r' - .; . V; ; V. vivi; ; r f -;c` ^ -' ' ' , CG0IS03 5571 loL 5572  DOW 763478 I -> --I - -I - J 7 -r .7 * c :n ".:3 u rrs csPAarcssr pea o? casL l J 'if'iivii 'GL) use c u p l o v e e s o:il t t . .. n z i ' u J T <iu:.;3iift GH-P 551 OATE October 21, 1968 auummIS Marguerite X. Long TITLE Reviow on tho Kotabolion of Phenoxy Compounds In Plant3 -o xj GH-P 551 and Animalo ro oo IM-C^SUTIVE SWCURV UTM t~ ~ u i:iC K i o This gj; tonalvo review w a s undertaken to determine whether phenoxy horblcldoa undergo changes in plants or animals which would result in olgnlficant rooiducs of metabolites which would have to be ac counted for in tho ootabllohmont of tolerances by FDA. The review la dlvldod into sections as outlined in the attached Table of Contonto. 2n Tho metabolic products shown to be formed in plants were short S lived intorcodiates whose nature depended on the chemical structure 30 of tho phonony compound, tho rate and mode of application of the herbicide, tho otago of growth and metabolic condition of the plants and, moat importantly, on the species and varieties of the plants atudiod. In oplto of very extensive work during the past twenty years, no one has been able to identify the products, although hydroxylation of tho ring is believed to occur in some plants. In animals, each compound was excreted unchanged, virtually quantita tively within a short time. Traces of residues were found for only two or throe days in milk from cows maintained in pastures during and following treatment with 2,4-D. It was concluded that there 13 no reasonable expectation that res idues of metabolltos of tho phenoxy herbicides could be present in significant amounts in raw agricultural commodities of plant or animal origin as a result of currently registered uses for 2,4-D, 2,4.3-T, silvox or HCPA. (This review was prepared for the NACA as part of a supplement to pesticide petitions for negligible toler- ancos of those phenoxy compounds in a number of crops. The total compilation Included copies of the 73 references cited herein). EXEC* WEI Pm MKCT KO. CHARGE NO. pachi STAGE psoaLCM no. NSWPHYSICAL PROPERTIES ( * HWrt) DEPARTMENT m e 0E REPORT Summary 56 Q yis CJno *eair ' executivi research * biqprooucti research manaoer * eioprooucts department manage* registration SECTION ( b b t a j *SIOPROOUCTt INPORMATKMCENTER Corporate Res. & Dev. (CWH,JEJ) Product Team Manager (DDD) Ag. Products Dept. Manager (REN) Puerto Rico PS Field Res. *Ag. Products Res. & Dev. Manager (EHB) Davis PS Field Res. & Dev. Vegetation Control Bus. Manager (JHG) Oklahoma City PS Dev. Crop Protection Bus. Manager (AJ.B) San Francisco PS Dev. PS Res. & Dev. Director (KCB) Pesticide Wildlife (EEK) *AgT Products Chemistry Director (RDM) Biochem. Investigations (WRB) Ag.. Products Information Center (RJS) Biomechanisms (GNS) Walnut Creek PS Lab. Winter Park PS Dev. Midland PS Research (FHD) Wayside PS Field Res. & Dev. Field Research Manager (ERL) Product Technical Specialist (JHD) Animal Ag. Prod. Res. & Dev. __ BiocTlem. Res. Lab. Distribution list continued on back 5573 DISTRIBUTION CONTINUED ON REVERSE SIDE IF REQUIRED. EOMIC.n<OPRlTEOINU.tJI. A t-it ^ '4 Distribution continued: E. C. Britton Res. Lab. (GDJ, DUO) Ag. Products Formulations Lab. Formulations life. Department Lako Jackson Lab. Patent Department Pitman-Uoore PS Dov. Manager U.S. Dov. Uanacor Dow Canada Aroa Dov. European Area Dov. Latin Acorlean Aroa Dov. Pacific Aroa Dov. Western Division Research Midland Division Res. & Dev. Analytical Uothcds (MEG) Screening Formulations (CEC) Jonoon, D. J. Williams, C. S. Watson, A, J. Raynor, R. N. Hoaly, U. J. Lynn, G. E. Southwlck, L. i i i CG04347 < i REVIEU ON THE METABOLISM OF FHENOXY COMPOUNDS IN PLAINTS AND ANIMALS DOW763480 Table of Contents Summary Statement Glossary of Terns Review of the Literature I. Metabolism in Plants A. Introduction B. Tabulation of Conditions Used in Studies with Plants C. Metabolism of 2,4-D 1. Hydrolysis of Esters and Salts of the Herbicide 2. Decarboxylation and Formation of the Phenol 3. Conjugation into Water Soluble Detoxication Products 4. Transformation into a Different Compound 5. Beta-oxidation of the Side Chain in 4-(2,4-DB) D. Metabolism of 2,4,5-T and 2,4,5-TB E. Metabolism of MCPA and MCPB F. Metabolism of Silvex (2,4,5-TP) * II. Metabolism in Animals III. IV. Recent Residue Studies by Newer, Methods ! Significance of Metabolites as Potential Residues in Foods and Feed j i V. References ! 5575 i CC04343 5576  PRODUCTION RESEARCH REPORT NO. 106, U.S. DEPT. OF AGRICULTURE 00028C3 r T able 30.-- Sum m ary o f dotage o f various organic herbicides that cause significant weight loss, reduced weight gain, or poisoning in cattle, sheep, and chickens1 B erbicida Dosage Leastnumber ofdosages for-- rate Cattle Shcop Chickens Herbicide Dosage Least number ofdosages for-- rate - Cattle Sheep Chickens 01 O nJ ACbm2222A2l-.,-.foi444(,MdpegappeesaaaiaarA2s,--caectttoccllrrcrc5of,hhhkyerooolcci4ipc-ictpddtcaaeeDptpptcTl,shhoiio5rannrraiyycceryo((imoml-)oo(mill22eneTl.ponceeaaDllDb-aps,,oiaafheninr444ccdttuoacximiineetliiee---tccecndutodhyhDDcrroyhhhai..dnryggheln(cIlll))o(c(lldd1looo,,eMai2yyopoiee-dsirrrae,c2cappmhrt:sto<o4orC,ohoohlrah2c(ipp,mkpplole5paeelnPe-hhatrsinopd-hrhbbnosAolcc)nTuvoyicen.xuuncnppo)en)n(nllmyattooa,h,oelxoyyodta-anxxnfe)eixxllsmno,dyynerty:y.ld))hayia-.nce1ce-ecttlioe 2-Cacldcotarmo-iNdc,N(-CdDiaAllyAl-). 2-Cat(rThciclcCohtraBlomo-CrAio)dfb..eAcn(fCx-dyDilaAlclyAhll)-orainded Mg./kg. 522105000000 .......28..55..1..... ........187..0......._.._.._.._.._11.._00.. 250 3 - 2 4 100 ........................ 10 251500000 ..................4.....................3..4...............1..60.. 250 ............... 5 10 100 10 0 10 521050000 ................8.............. ....40.. .........1..10.. 522055000 ..................3......................1.7..... . . . . .11.00 2152505000 .................1.1........ 1141 10011 1520500 ........2.1...... 621 ..................1....0.. Phenyl urea compounds: 3-(p-ChlorophenyI)-l,1aimethylurea (monuron). 3-(3,4-DlchlorophenyI)-l* methoxy-l-methylurea (linuron). 8-Phenyl-l.l-dlmethylurea (fonuron). 3-(3,4-DtchlorophenyI)-l,ldlmethylurea (diuron). Thlocarbamate compounds: -2.3-Dichloroallyl dilaopropylthlocarbamato (distiate). -2,3,3-Trlchloroallyl diisopropylthlocarbamate (triauate). Ms-Ike- 2252521111550526255200001510000550000050000000 ..... .. ... ... 22211111155052527705205050000550500 ..... .... 1 100 2 10 31 35 42 431 138210 . 110800 111180000 111180000 1 2331 21 . 1117000 11700 n r r - r n o aaoh U Lu d 7$ 1.1 1 L< STATIONS VirnCUlNUMI'.lJK'.IXSKA ANSTAIT / S-104 O'j ST(K-.K!IOI.M r.o Chcnictry Department RECEIVED .dec 2 2 129 r e g is t r a t io n December 1C, 1969. Marguerite 1 . Long, Ph.D. B egistration Section, A gricultural Department, The Dow Chemical Company, P .0 . Box 1706 MIDLAND, Michigan USA Dear Dr. lon g, Thanking you for your le t t e r o f December 11, 1969, I am en closin g some rep rin ts including those you requested. Also included i s a ta b le o i tissu e d istrib u tio n data fo r 2,4-D and 2,4,5-T in chickens, taken from a forthcoming paper (l7.-S. B.iSDaLund and K. Erne, Acta Y et. Scand. v o l. /a k970?T } < , J974- In a p liy sio lo g ica l study under way we have found chlorophenoxy- a c e tic acid s to be excreted in chickens by the proximal con voluted tubules in e sse n tia lly unmodified form (acid -hydrolys- able conjugates making up le s s than 10 fo o f the excreted phenoxy a cid ) and apparently by a transport mechanism in common Y/ith u ric acid and a few other aromatic acid ic compounds (K. Erne and I . Sperbcr. to be published). 2 ,4 ,5-T was found to ex h ib it a higher a ffin ity fo r the carrier than did 2,4-D . I should g rea tly appreciate i f you could le t ' mo have one copy of your finish ed review . Sincerely yours, X. Erne Ph.D. (V et. S c i.) , A ssoc. Prof. 121504 0001371 5579 osss S 9) gable 2 . Phsaoriy acid le v e ls in tiaouoo o f b r o ile r chickens fod 2,4-D or 2 ,4 ,3 -T a t 1,000 p .j .n . in the drinking r/ater fo r 104 days.- Chicken "o. Pkcr.oxy acid Sex 0 21S4/67 2,4-3 . 0 2105/67 2,4-3 C 21S2/67 2,4,5-T C 2133/67 . 2 ,4 ,5 -0 <? 9 o 9 L iv er 4 .0 6 .3 .3 .5 2 .0 Kidney ffieauo le v e ls , y-ug/g, freoh weight Lung Stocacha Snail in testin e 5.0 3.6 20.0 10.0 7 .2 28.0 8 .5 8 .0 85 3 .3 4.X *r . 1 0 .0 1 5 .0 1 8 .0 - 2CG3 (yolk) - 0.4 - 2.0 0 .3 - 2,1 0.7 - 1.6 00013 - CoJrl --i tv 00 h-4, GOSIZT Moa 5552 1 / 3 L ^ ^ n r ' t f - t f -f t o 7 / . 1 Epidemiology, Prevention and Treatment 71-0507-9 r**':v.. 'i- : : .. j. . "iv . j|. An i; /i:ioi. rn were !. A JS.* . .vr'i ; !; .i* ' . Lfx.W.. :r^\ ' i.1.*' tt ;"; \\ :. :hi . L)J" ' ' Jatier. .V.. V vit*** jr ees which wore '.-'laced by aqueous suspensions and cquent replacement of the organoclilorine compounds with -fttnophosphorus or.ci. These efforts included also an .jiicationa! campaign on the veterinary and medical, etc. . peels o f pesticide application. The first reassuring results of J i efforts are now available. The need in 1967 for chemical -eatment of the potato crops, however, precluded achieving - ,e desired results in preventing residue accumulation. In ,-i66. residues were found in 125% o f sugar beet samples, *5~ of potato haulms and 19.4% o f food product samples. In >67, their corresponding values decreased to 6.71 and 6.3 :<ic| In 1967 as compared to 1966, there was a significant :.vp in the number o f mass pesticide poisonings of animals. ..-terinary specialists played no little part in achieving these ii uals. *1-0507. Birbin, S. S.; Alekseeva, A. A.; Bulatov, A. A. jtishchcvo District Veterinary Station o f Saratov Province, ,jtislichcvo, USSR). The poisoning of swine treated with dranosan. Veterinuriyj Av. 8: 60-1; 1968. (Russian) Mass poisoning o f swine with the organomercury .erbicide Granosan was seen on two farms in Saratov Province, tin the "October" Collective Farm in Tatishchevo District, -j of 414 swine of various ages were affected during August 1967], the acute period; 121 died and 145 in the agonal state *.jQ to be slaughtered. By November, another 44 animals had :d. On the " Fcdou.v'' State Farm in Marx District, 211 o f 44 swine were affected. The cause of the toxicosis was the -olonged feeding (up to 30 days) of ground barley that had *.en treated with Granosan and o f granary grain sweepings hich contained a b.:a? percentage o f the preparation. The . rit signs of poisonir.a appeared in suckling pigs and failing .'.is 20 to 25 days after beginning feeding on the treated grain; " sows, the signs appeared in 30 to 40 days. At first, the a-mals refused food -ml water and became restless. There was ~>ie nasal mucous secretion. Then weakness in the hind limbs rpeared, with different types o f movement coordination order. Some animals showed spinal involvement. Signs of rural disorders were quite clear, including muscular tremors, nvulsional jerking of the extremities and tetanic contrac t s of the pelvic musculature: As their conditions worsened, '' - animals lay on their stomachs or sides, developing varying rrccs o f paralysis with loss o f pain sensation, rapid rjthing, etc. The younger swine almost all died within 3 to 6 -.->after the symptoms started; the sows' condition persisted -i-t until death (6 to II days) and 40 to 45% o f the - :m ed animals died. In some instances, the swine suffered : above symptoms, including partial or complete loss of >u. for 2 to 3 months. The autopsy findings were initially ' icme in all the animals, the most constant changes being b-l in the intestines. The intestinal mucous membrane was - '-'red with dryish, dirty yellow or brownish-green deposits 'ciectcd to the undeilying tissue. Liver and kidney findings also described. 'Ihciapy with uuithiol, cardiac agents, :-icilliii .uni glucose were ail ineffective. The fact of a delay 'he appearance o f symptoms following Granosan inloxicamust be taken into account in diagnosing organomercury ' "mugs. The clinical symptoms and pathologicoanatomica! : b<-'s in mass poisonings o f swine with Granosan to a great extent recall the course of infectious dheas.'s such as plague. Aujeszky's disease and paratyphoid, so that mercury poisoning; should be eliminated during a differential diagnosis. 7I-050S. Rusin, K. N.; Tokarev, N. F .; lAroshcnko, N. M_ (Min. of State Farms. Ukrainian SSR, USSR). Cases of swine' poisoning with Granosan. Veterinariya Ao. 9: 61-3; 1969. (Russian) Cases o f severe Granosan poisoning in swine arc reported. In the fall of 1967 on the "V ictory" State Farm ia Nikolaev Province, all the Granosan-treated wheat not used in sowing was stored next to forage grain. The treated, thrashed grain was used as swine feed from November I I , 1967 at 2 .2 kg/day. The contaminated grain was fed up to 26 days, since the initial poisoning symptoms were lacking. At 1 month, th e 2- to 5-inonth-old piglets began to display clinical signs; refuse? of food, depression, muscular tremors, etc. The oral mucosa was hypercmic; the feces were o f a yellow color and in m any eases liquid, covered with a thick, clastic coating, etc. S o m e swine liad temperatures below normal for 5 to 6 days and, as a rule, these animals died (76% o f those affected). The autopsar findings included marked plethora, hypercmic oral m ucosa, enlarged lymphatics, uueven coloring o f die lungs i r o n grey-red to dark red which were o f a doughy consistency, e tc . The diagnosis was based on the pathologicoanatomical findings, the clinical picture and laboratory analysis, all o t which excluded infectious disease. Mercury was found i i samples of- feed and of the parenchymatous organs. T h e treated grain, for example, contained 0.18 to 21.7 mgffcg Granosan. The therapeutic regimen, once the diagnosis waoc established and the feed chang.'J, ir.ciudcd glueTM aa.5 caffeine i.v. and various doses of unithiol s.c. and Lm. But o n ly 34% o f the affected swine were saved. Follow-up over the next 5 months showed that poisoned swine w ere retarded in weight and the mercury salts also had a negative effect on sow productivity (premature, stillborn and aborted young). As a result of this incident, the "Victory" State Farm was noc allowed to supply animal husbandry products to the m arket for 5 months. 71-0509 J Bashirov, A. A. (Azerbaijani Res. Inst, o f Labo* Hygiene and Occupational Diseases, Baku, Azerbaijani SSFL. USSR). The state of health in workers manufacturing th e herbicides, the amine salt and the butyl ester of 2,4-D acicL Vmchcbnoe Dclo No. 10: 92-5:796g^( Russian) A medical examination was done o f 292 persons engaged 4 0in the manufacture o f the title herbicides; 24S were men a n d 44 women. Their ages ranged tram under 20 (nine) to over (19). Professionally, die workers' jobs ranged from laboratory worker to machinery npciator; their tvork exposure ranged from under 5 years (194) to 6 to 10 years (98). The workers^ general complaints included general weakness, rapid fatig ability. frequent liojiiaehes <>f a dull nature mainly localized i a the fromal-p.nictal area which intensified at the end of th e work day (63%), vertigo (33%). etc. Objectively, arterial! hypotension was* found in 59 persons (20.2%). Frequent complaints were noted in 51.7% of those examined w hich were typical of digestive function disorders: dyspepsia, poor 139 GGGG-34 -a ' n 71-0510 Epidemiology, Prevention and Treatment -appetite, radiating pains in the right epigastrium, etc. A slight increase ii< the liver was found in 12 persons. From these findings, SO workers (41 men and 9 women) were selected for' detailed clinical studies o f the stomach and liver as well as of the cardiovascular system. Their case histories showed no particular diseases prior to their present work with the 2,4-0 acid herbicides. A control group of 20 persons with no toxic chemical contact was included. Bradycardia was found in 20% which varied from a, lying to a standing position. Arterial oscillography demonstrated that the mean dynamic pressure while lying was within normal limits but while standing this index showed asymmetry. Fourteen of the SO persons examined had a lowered acid-forming function in the stomach; in eight there was a histamine-resistant achylia; etc. Liver dysfunction was often found: 22 of the 5 0 (versus four of the 20 controls) had disordered antitoxic function expressed as a decreased hippuric acid excretion in the urine. Liver protein forming function was also disordered in 19 of Lite SO: lowered albumin level, etc. Four o f the workers had a lowered prothrombin index. Blood sugar was lower than 70 mg% in 16 (versus two of the controls). The various liver dysfunctions found were significantly more pronounced in workers with longer work exposure to the herbicides. The results o f the survey show that in the manufacture o f the title herbicides, various disorders o f the cardiovascular system and o f the organs of digestion may occur. They point up the need for checking the cardiovascular system and the digestive organs m those entering work and in those already working in the production o f 2,4-0 herbicides. 71-0510. Lobzin. V. S. TSinovpi, P. E. (Dept, of Nervous Diseases, Kirov Military Medicine Acad., Leningrad, USSR). N eurological disorders' in chloroplios poisoning. Zh. NcvmnatuL i i'sr.hiatr. 69(5): 679-83; 1969. (P referen c es). (Russian) -- Neurological disorders in acute chlorophos poisoning described based on 41 case histories, 11 o f which were severe (two patients died), 14 o f median and 16 of he:.: intoxication. The severity and character of the clinical >; , depended on the dose and route of the poison into the Ive-. In most instances, chlorophos had been taken interHalh. ] the qcutc poisoning phase, the predominant symptoms central nervous system lesion and vegetative disorders. L.. frequency of individual symptoms (muscular wejo* disorder of consciousness, headache ar.d vertigo, tiem :> delirium and hallucinations, sleep disorder, etc.) is shown l:;. table. Two individual ease histories arc detailed, illustrative. ; severe chlorophos poisoning. Both patients had respira!.--, disorder requiring intubation and were comatose. In one a 52-year-old man, the poisoning was so severe that in spite. ' various therapeutic measures (atropine i.v.. gastric lavage, et, >. the pafjcnt died of respiratory and cardiac arrest. In i!.. second ease, a 46-year-old man, 3 days-aftcr treatment u i.m. TMB-4, i.v. atropine and'glucose and regaining conseiu.: ness, the patient developed a. delirious syndrome. Aft ; successful therapy for" tliis, he complained of muscular pa::., and difficulty in walking. Objectively, decreased left knee .c Achilles reflexes were noted; clumsiness in .the finger, appeared. A diagnosis o f toxic polyneuritis was made. Im patient developed intense pains in the distal parts of V extremities. After 5 months of therapy, only_ slight impr^c ment occurred. The movement disorders and disorder r sensitivity,in the distal parts of the extremities'proved ver resistant to treatment. O f the 41 chlorophos poisoning caweight developed toxic polyneuritis: the .alter appeal,, unexpectedly and anywhere from 10 days to 1'A months at:, the poisoning. Muscular atrophy was detected usually a'to weeks after the first signs of polyneuritis. A discussion of ti; problems o f therapy is included. / See also 71-0439 71-0444 71-0445 71-0447 71-0451 71-0474 71-0489 71-0520 71-0526 71-0527 71-0535 71-0562 * 71-0569 71-0573 71-0580 71-0583 71-0605 BOW 'J 1 6 7 9 7 4 5584 0C0G23U lo i 5585 Residue Reviews 26, 37-62 (1969) MN069799 Toxicity and hazards to man, domestic animals*, and wildlife from some commonly used auxin herbicides By J. M. Wa y * Contents I. Introduction ................. II. Toxicity nnd hazards to m an............................................................ III. Toxicity and hazards to domestic and ta'.rraiory animals................... a) Acute and chronic toxicity........................................................... b) Indirect toxicity......................................................................... IV. Bees and other insects....................................................................... V. Soil animals-..................................................................................... VI. Fish and aquatic organisms................................................................ VII. Wildlife in general............................................................................ Summary.................................... Itdsumd .................................................................................................. Zusammcnfassung..................................... References............................................................................................... 37 38 41 41 46 48 49 50 53 54 55 56 58 L Introduction This review has been compiled from searches in the literature for references to the toxicity and other hazards that may arise from the use of auxin herbicides, and specifically of formulations of MCPA (4-chloro-2-methylphenoxyacctic acid), S.S. 2,4-D (2,4-dichlorophc-noxyacctic acid) and of their butyric acid analogues MCPB and 2,4-DB, of 2,4,5-T (2,4,5-trichlerophcnoxyacetic acid), mecoprop [ tt-(4-chloro-2-methylphenoxy) propionic acid], and 2,3,6-TBA (2,3,6-trichlorobenzoic acid). Assuming 70 percent of the eight million acres of cereals grown in the United Kingdom to be treated annually with herbicides (W ood- * Monks Wood Experimental Station (The Nature Conservancy), Abbuis Ripton, Huntingdon, England. 37 f 0007424 w * t 38 J.M.Wat ford 1964), it is apparent that the tonnage (circa 2,500 tons) of the compounds mainly used for this purpose is similar to the total tonnage of insecticides and (circa 2,660 tons) (fSutnrgiicckidleasndus1e9d6a6n).nIunal1ly96o2n, Dalaldcdro(p1s96in2)t,hferoUm.Ka. survey of eastern England, found that over 90 percent of cereal Helds treated with herbicides were treated with auxin compounds based on chlorinated phenoxy acid derivatives, notably salts of MCPA and to a lMesCsePrAexhtaesntboefenmegcroapdruoapl.lyAdltehcoluingihngth(eWusoeodoffosrtrdai1g9h6t4fo)rminuflaavtioounrs of of mixtures of compounds, many of these mixtures still include MCPA or other phenoxy compounds and increasingly include 2,3,6-TBA. In addition to its agricultural use on arable and pasture crops, 2,4-D to* gether with 2,4,5-T, is used, or is likely to be used on an increasing scale, for scrub control and control of unwanted broad*leaved tree species in conifer plantations. 2,4-D is also used to a limited extent for control of submerged aquatic weeds. On a world-wide basis 2,4-D is probably used to a greater extent than any other herbicide and this is reflected in the greater number of references to this compound. The extensive use of a single biologically active family of compounds clearly presents a considerable potential hazard in respect to their direct and indirect toxicities to animals including man. It is a measure of the comparative safety of the auxin herbicides that, in spite of their increasing use throughout the U.K. since the mid-1940*s, they continue to be applied much in the same routine way as are inorganic fertilizers, and with as little regard for any possible toxic hazard. During the many years of their use throughout the world there have been notably few authenticated incidents of poisoning of domestic animals or wildlife resulting from the proper application of these com pounds. The fact that fully authenticated incidents of poisoning have not been reported cannot oe taken as an absolute guarantee of safety, particularly with regard to indirect or chronic toxicity, because of the frequent difficulties of attributing the underlying cause of an illness or of death, especially in wild animals. Nevertheless all the direct and circumstantial evidence to hand at the present time seems to indicate that incidents of poisoning have been extremely few and generally under exceptional circumstances. n. Toxicity,and hazards to man The principal routes of toxicity to man are either orally or by in halation; there appears to be little hazard of transport through the skin although individual allergies can develop leading to dermatitis (V a l l e t 1965). Eyes may be directly but are usually only temporarily affected. Hazards to man may occur from the concentrated chemical before dilution, from inhalation of spray or dust during application, or from ingestion of the chemicals in food or in water. Because the great- f 0007425 e :' cn CD a/t CD CD 1 546794 I Toxicity and hazards of auxinherbicides 39 est hazards arc from the concentrated chemical and because man is handling the chemicals in this form at all stages from manufacture to dilution, it follows that he is at greater potential risk than any other organism. However, there are very few reports in the literature of tests or incidents of poisoning of man by these compounds; the majority of these reports refer to accidental poisoning of children. As a result it is now generally accepted that auxin-type herbicides do not present a direct toxicity hazard to man (Bailves 1963) when correctly handled or used for weed control. Kraus in 1945 (in Kepilart 1945) reported that he had taken 0.5 g. of 2,4-D per diem for 21 days with no demonstrable ill-effects. A clini cal study was made in Denmark by Nielsen et al. (1965) on a 23-year old man who had committed suicide by apparently drinking 125 ml. of 50 percent w/v 2,4-D dimethylaminc salt The total weight of 2,4-D in his body was calculated as being not less than six g. (the equivalent of 80 mg.Ag.), about 10 percent of the total weight of active material ingested. The principal damage appeared to be to nerve tissues and the central nervous system. Vallet (1965) gives acutely toxic oral doses of 2,4-D to man of 50 to 500 mg.Ag. body weight, of 2,4,5-T of 500 to 1,000 mg.Ag., and of MCPA of 50 mgykg. No indication is given of the source of these figures which are probably extrapolated from data on laboratory animals. Freireic2 I et at. (1966) have shown that a number of drugs, on a mg.Ag. basis, are more toxic in man than in the mouse, when used as a laboratory test animal, by a factor-of 10 to 15. There are clearly dangers therefore in assuming that a toxic dose to man is the same as the toxic dose to a laboratory animal multiplied by some factor based on the mean differences in weight. In fact these latter authors show for a number of anticancer agents that a very much more accurate assess ment of the ratio of animal to human toxicity can be obtained on a mg./m.* of body surface area, than on a mg.Ag. basis. For this reason Vallet's (1965) figures may be suspect, particularly in respect of the range of 50 to 500 mg.Ag. for 2,4-D, although the lower figure would be of the correct order of magnitude when compared to the residue of 80 mgTkg. found by Nielsen (1965) in his suicide case. Nevertheless, this figure represented the amount actually found and it is likely that a greater proportion of the total 2,4-D ingested was necessary to produce death. In this context it is noteworthy that the acute oral LDgo's to rats of the most common organochlorine insecticides range between 10 and 135 mg.Ag. (Martin 1963J. Edwards and Ritter (1953) have discussed the hazard to operators from inhalation of sprays or aerosols during application of herbicides with particular reference to methods of protection. Monarca and Di Vrro (1961) have described a clinical study of an acute of accidental poisoning of a man in Italy. In this instance a farmer became ill after applying a 40 percent aqueous solution of 2,4-D by habdpump i 00074SF C C 5588 BOW : 1546795 I 40 J.M .W ay against the wind. He was admitted to hospital, suffered a relapse after 18 days, and recovered sufficiently to be discharged after 40 days. Initial symptoms of muscular weakness, vomiting, perspiring freely, and oliguria were noted in the Held whilst a diagnosis of bradycardia, respiratory difficulties, and urinary abnormalities was made after ad mission to hospital. However the authors report that the case was exceptional. F et iso v (1966) has reported similar field symptoms in Russian workers engaged in field applications of 2,4-D. This author concluded that a range of formulations of 2,4-D was "highly toxic to animals in different ways of introduction.'* Whilst reports of minor discomfort following exposure to auxin sprays during field application are rarely reported in scientific literature, there is no doubt that a proportion of workers so exposed do suffer a degree of transitory dis comfort Whether this is of any significance as a long-term toxic hazard has not been determined for man. From the very nature will appear as significant roefstihdeuiersuisnefiotoids ucnrolipkse.lyWthilaltiaaumxsin( herbicides 1964) was unable to detect any residues of auxin herbicides in a number of total danieatlystaicmalplteeschdnoiwqunest.oDtuhcecalinmiatnodf Wseenastituiveirtywa(x0.0(119p67.p).mca.)lcuolfatheids pesticide chemical residues in 'total diet' samples collected on 46 days in 25 American cities during a 699 day period from June 1964 to April 1966. Each sample represented the total amount of food and drink consumed by one person over a two-week period. The total samples represented in all a food and drink supply sufficient for 644 days. Herbicide chemicals were found infrequently and averaged about 0.01 mg./day of which one third was 2,4-D, and half was MCPA and pentachlorophenol (PCF) combined. 2,4-D was found in oils and fats (0.001 mg. in 1964/65) and sugars and sugar products (0.004 mg. in 1964/65,0.002 mg. in 1965/66), whilst MCPA was found in grain and cereals (0.002 mg. in 1964/65), in dairy products (0.003 mg. in 1965/. 66), and in leafy vegetables (0.001 mg. in 1965/66). These amounts are substantially below the limits set for acceptable daily intake by the World Health Organization and United Nations committees. It seems pinrofboaobdlea,rethveerreyforsme, athllatFatouxsitc hazards (1964), from auxin in a survey herbicide residues of water pollution hazards to man from organic pesticides, came to the conclusion that there did not appear to be danger to health at the present time from the background concentration of pesticides believed to be in ground and surface water. However, 2,4-D could persist in lake water and bottom mud for long periods in Russia, quoted by Fuanudsetr, certain environmental conditions. Work suggested that the threshold taste and odour concentrations auxin compounds, especially of phenolic deriva tives such as 2,4-D, that would prove unacceptable to the consumer were very considerably below the threshold concentrations for toxic effects. Aparticular risk might be supposed to lie in contaminated mill- i 0007427 I DOW 1546796 Toxidly nnd hazards of auxin herbicides 'll drawn from cows feeding in treated pasture, but no residues of either 2,4-DB or 2,4-D were found in the milk of cows that had been fed these compounds (Gutenmann ct al. 1963a and 1963b). The beasts were fed 5.0 p.p.m. (based on a daily ration of 50 pounds) for a number of days; the limit of detection for 2,4-DB was 0.2 p.p.m. and for 2,4-D 0.1 p.p.m. Whereas milk might be a source of contamination these results indicate that there is no apparent hazard. ITT. Toxicity and hazards to domestic and laboratory animals a) Acute and chronic toxidly The toxicity of agricultural chemicals to land fauna is normally quoted in terms of the dose that kills 50 percent of a population of test animals (LDS0). Whilst this figure gives a useful indication of the comparative toxidties of different compounds to a given test spetics, the figures obtained in different tests may be influenced by a number of factors. Thus the age and sex of the test animal, method of dosing, and general conditions of the test may have an important bearing on the susceptibility of the animal to the compound being studied. The formulation of the active compound has a considerable influence: for instance 2,4-D acid has an LDfio to rats of 375 mg.Ag. but the sodium EseaasnrtldtenrehHoa(yfs1m95aa67ns60)L1m9dDg5os.4oAn).ogotI.f,tra8esgn0ha5dorudmtlhdtgehb.eAiessogen-.pd,ortitfeohfdpee,ryelhpnoercwsoetpseeyrvaleesornf,bet7ehi0gna0lgtymcBaojpglop./rbrkkeuglct.iuyanl(bRdleeota.hwnIender addition, the LD for different test species may vary quite widely: for example, 2,4-D acid has an LDso of 375 mgAg. for rats, of 100 mchgic.kAsg(.Rfoorwdeoagnsd, 4H69ymmags.A19g5.4f)o. r guinea pigs, and 541 mg.Ag. for Bearing in mind these reservations, the acute oral LDSo to rats of the acid formulations of the compounds under consideration are: 72p0.r4o0.p5m- 9gT3.0A3g0m.0,g2m.A,4g-gD..ABg(W.7, 02o0,o4dm-Dfogr.4Ad0g0a.,nm2d,g3.E,A6v-gTa.B,nsAM1C790P65B5)m.68gT0.Ahmugs.g,,Va2kn,4gd-.D,mMeaCcnoPd-A 2.4.5- T fall in the moderately toxic and the other five compounds in othfetosxliigchittylyoftocxhicemcliacsaslsaqccuoortdedinbgytoFrthaezewrid(1e9ly63a)c.cIenptaedcocmlapssairfiiscoantioonf athnedr2e,l4a,t5i-vTe,-toRxoiwciteieasnodf aHynummasbe(r19o5f 4co)mcomnecrlcuidaeldfotrhmautltahtieon`isneorft*2,c4o-nD stituents of the formulations did not add to the toxicity of the active compound, nor did they clusions were reached by eDxaalcct aaanryd-MpoitxexnetliasteinnganedffePcot.oSisimenila(r19c6o2n) on the basis of this and other work. However, it is possible for sur factants and other additives to enhance- the toxicity of active, com pounds. Whilst there is some evidence with insecticides that mixing f 0007428 5590 ' |546797 42 J. M. Way of compounds leads to synergistic effects on the acute toxicity of the constituent compounds (Keflincer and Deichmann 1967), effects of this sort have not been reported for herbicide mixtures in common use. However, die possibility of dieir occurring should not be overlooked, nor, also, the possibility of antagonism where the effect of one com pound upon another in a mixture is to depress its biological activity. Descriptions of the clinical symptoms of poisoning by MCPA, 2,4-D, and 2,4,5-T of a range of experimental animals have been sum marized by Rowe and Hymas (1954) and more recendy reviewed with comments by Dalcaard-Mikkelsen and Poulsen (1962). Bjorklund and Erne (1966) have subsequently further reviewed and reported on their own results on the effects of feeding 2,4-D to calves, pigs, rats, and chickens. A range of symptoms was produced that was generally similar to those described by the previous authors. Erne ( Iwitia and b) studied the distribution and elimination of 2,4-D and 2,4,5-T in these animals. Amine and alkali salts of both compounds were readily absorbed and completely distributed in the body, but 2,4-D ester was incompletely absorbed and reached only a low level in the plasma and tissues. The highest tissue levels of the two compounds were found in liver, kidney, spleen, and lungs and the levels found in these organs sometimes exceeded the level found in.the plasma. In blood cws some 10 to 20 percent of the plasma level was found. Penetration of 2,4-D into placental tissue of pigs was recorded but there was little or no evidence of penetration into adipose tissue or the central nervous system. Elimination of the compounds was rapid, the plasma half-life being about three hours in rats, eight in calves and chickens, and 12 in pigs. The tissue half-life values ranged between five and 30 hours. No retention of the compounds was noted in the tissues. There was no accumulation after repeated dosing and in pigs there was an increase in the rate of elimination after repeated administration. In all species the main excretory route was via the kidneys. Khanna and Fanc (1966) traced the metabolism of C"-labelled 2,4-D in rats dosed at rates from one to 100 mg./animaL Radioactivity was found in all the organs studied together with some accumulation as early as one hour after dosing. At the one mg. dose rate a concentration peak of radio activity was demonstrated after six to eight hours but decreased there after and was non-detectable by 24 hours. At the SO mg. dose the peak occurred at eight hours and persisted for 17 hours. Extracts of the tissues were shown to contain mainly undianged 2,4-D residues. No radioactivity was found in the expired carbon dioxide, but elimina tion in urine and faeces was dose dependent. At die one to 10 mg. doses 93 to 96 percent of the ingested 2,4-D was excreted unchanged in the urine in the first 24 hours. At the 20 to 100 mg. doses greater amounts of 2,4-D were found in the second 24 hourperiod after dosing, with a linear decrease in percentage recovery with increase iq dose. In experiments with cattle Gutenmann et al. (1963a and b) were un- f 000742.9 5591  < I Toxicity and hazards of auxin herbicides 43 able to detect any residues of 2,4-DB or 2,4-D in milk or faeces of cow's fed five p.p.m. of either compound in a 50 pound daily ration. In these experiments there was no evidence of beta-oxidation of 2,4-DB to 2,4-D. Disappearance of 2,4-D was thought to occur as a result of ScdotilmuJtopiohonsnitiienotnat.hLeIn(r1u9sm6u4be)nse,sqtsuuoedmnieetdaetbxhspeoerrfpiamttieoennotfsoMnBCthPcehAeg, uMettCwPaaLBll,, and by (1964) 2,4,5-T, de and and a number of other herbicides in cattle. All the MCPA fed to a single steer (113.5 mg. single dose based on five p.p.m. of a 50 pound daily ration) was accounted for in its urine over the four days after admin istration. No MCPB was found in the urine of MCPB-dosed cows (total dose of 56.75 mg. and 113.5 mg. based on 2J5 and 5.0 p.pan. of a 50 pound daily ration) on the first day after administration, but subse quent recoveries in their urine showed that 7.2 to 9.2 percent of the MCPB was converted to MCPA. Analysis of MCPB itself, however, was said to be unsatisfactory. It is of interest that a similar conversion omfac2h.4r-oDcBhirtvos)2,(4G-DutceannmabnenpaenrfdorLmiseed1b9y65b).luUergiinllesaunndfismhilk( Lscapmopmleiss were also analysed from cows fed a total of 454 mg. of 2,4,5-T over a period of four days. It was shown that the compound was excreted exclusively in the being recovered. Curlianuexoeveert a period of al. (1964) six days, a total investigated the ofaft4e3o0.f7Cm1g4-. labclled 2,4-D in sheep fed a dose of four mg. of 2,4-D/kg. calculated as the minimum daily dose likely to be ingested from grazing treated pasture. The level of radioactivity in the blood rose to a peak 1.5 hours after dosing but fell to the normal background level in 24 hours. All the material was excreted within 72 hours, urine and only 1.5 percent in the faeces. wBiathlaoyvaekrn9i8s percent in the et aL (1965) dosed a rabbit and six mice with 2,3,6-TBA and showed that a pro portion of the compound was recovered after passing through the animals. These authors discussed the significance of biologically active residues of auxin compounds in animal excreta that might become incorporated in manure or straw. It was shown that 2&6-TBA residues in particular could remain active for a period of months and affect susceptible crops to which the contaminated manure was applied. The same hazard does not normally exist with the phenoxyacetic acid derivatives, where the logically inactive in a compounds are relatively short broken period odfowtimnea.nHdobweecvoemr,eLbiisoe (1966) has pointed out that the excretion of 2,4-D in the urine of cows docs present the admittedly remote possibility of active 2,4-D being tarnadnMsCfietorclrheddsetlfelrion(minanaKdterLpeioaiantercdt(p11a99s46t5u0)r),eDatlolalracepasaourustdcee-dMpttiihkbakleteltchsreeonrpe.ewt aeLre(n1o95a9p), parent ill-effects in cattle, sheep, or horses from grazing pasture sprayed CmcsBY aatnhdcFrAbindwdraxlLor( 1tw95o0)tim(iens Shpcirubnicciedhal1r9a5t7e)s roefp2o,r4t-eDd othraMt CthPeAre. i' 0007430 5592 DOW] 546798 1546799 44 J.M .W ay was no significant difference in the amount of feeding of horses, cows, sheep, ana pigs in untreated plots or plots sprayed with the sodium salt or the isopropyl ester of 2,4-D,-or the isopropyl ester of 2,4,5-T. However, there did appear to be less feeding in plots sprayed with othfecaolwkasnfoeleadminingeosnaltsporfa2y,e4d-Dv.eTgehtearteiowna. sGnooldefsfteecitnoannmdiLlkopnrcod(uI9ct6io0n) found noill-effects on two cattle rromadding 0.25 pints of a 1.5 percent w/v 2,4-D/2,4,5-T mixture to every five gallons of their drinking water for 41 days, Diese authors also reported spraying the skins ora calf, of a cow, of sheep, and of pigs with doses ranging from 0.002 to 0.003 pounds of 2,4-D or 2,4-D/2,4,5-T mixture, with no ill-effects. These dose rates would be of the order of those that might occur in an in stanDceobosfosnpra(1y9d5r4i)ft sprayed MCPA, 2,4-D, or 2,4,5-T on grassed chicken runs daily for 14 days at normal and ten times normal dose rates. 2,4^3-T significantly reduced egg production and the -weight of the birds; MCPA and 2,4-D also affected egg production, mainly in the second week of spraying or during the week after spraying had stopped In all instances there was no effect on the fertility of the eggs and all the progeny reared well, although the dose rates and . 2flFir.kl4eeeq-ltuycDehtnoecfryebdeo(f1ftoo9aup6nhp7del)inciasinnticjpoeonrcuatleicdndtictbehhe.iesEnet'rsxrnicareelegtw(ge1sde9r6wei6nim)thtushhceMhoirwCmeePodgArget,sh.sa2etDv,4es-oroDnem,atechMhaoniCfePatharBnee,d 2.4- DB, mecoprop, and 2,3,6-TBA amongst a range of other herbicides. Dose rates were 10, 100, and 200 p.p.m. equivalent to 0.5, 5, and 10 mgyegg. The percentage hatch was recorded. At the lowest dose there was 100 percent hatch from the MCPA- and MCPB-treated eggs; 90 percent rrom the mecoprop- and TBA-treated eggs, and 80 percent from the 2,4-D-treated eggs. At the highest dose mere was 80 percent hatch from mecoprop, 50 percent from 2,4-D and TBA, and 20 percent from MCPA and MCPB. None of the chicks that hatched was de formed MCPB, 2a,l4th-DouBg,hansodmmeecfeoajther>tbrelaantmcheinntgs.wRaosbenrottsedanfdroRmoctehresM(1C9P57A), reported on various feeding experiments on turkeys with alfalfa sprayed with a low volatile ester of 2,4,5-T at herbicidal rates. No deleterious effects were noted. Calculations were quoted to show that for a one kg. chicken to acquire a lethal dose of 2,4-D from an applica tion rate of one pound/acre, the bird would have to consume in two days feet Sailml tihlaer2c,a4l-cDulaaptiponliesdbtyoRthoeutveeagnetdatHioynmoavser(1a9n5a4r)eafoorfc7a2ttlsequsuarge- tested that for a 770 pound animal to acquire an acutely toxic dose of ,4-D of approximately 1,000 mgAg., it would have to graze com pletely ana ingest all the 2,4-D from an area of 0.17 acre (one pound/ acre es 10.41 mg./ft* *a 93.7 mg./yard2a 112.1 mg./m.*). Accounts are given of direct oral dosing or dermal applications of 0007431 * / M r J ft - 1 0 .Hi srn y 4 * * /o o o su y /A s s o l } o m u m I /A 1 - - . --. - -- u 3 S o t OOO 35, o o o 5593 ~ O . cl*csU /546800 Toxicity and Lizards of auxin herbicides 45 PDa((scuaahoxlleiwmnceea)ph,ra)er,Rardabno-nidMwcdiRediSkaetkadsreneatldlocseehafHnkavyena(mtdr1aia9eBs6itoy4h(()o11o(f99ss55idhe94oew))emipc(e(hzlasatneib(icd1foe9arcr6naas4tit)mot),lreaPy()lpas,ailCgbnmlsyime)a.rKraPkel(asep1lh9eam6atn3erad)rtl (c((c(a111at999ttt646llee345))))),, gave daily oral doses of 2,4-D alkanolamine salt to steers for five days in every seven. He recorded signs of poisoning in animals dosed at 250 mg.Ag. after 15 administrations as opposed to 88 administrations of 100 mg.Ag.; at 50 mgAg. no ill-effects were recorded over a period of 112 administrations. From these results he concluded that although animals could probably ingest enough 2,4-D from concentrated solu tions at any one time to produce illness or death, the chronic toxicity of the compound was sufficiently low to make it unlikely that ananimal willo-euflfdecptsic. kFuurptheenrowugohrkobfyitPoavlemrearpaenridodRaodfetlimefefto(1c9a6u4s)euasninygsesriniogules animals gave the following results: pobk2rgu0fo.0t2ypo,ylm2S414fl....e,eg5ttnSSCShh-AeThhheeageeertgtbeea.ellepyplpueksuttatwsoteonouslrleolu.feberflcreeraucaarmttuketyeedmiildlndlbeefe4rde4to8hsd81mabe1lryttdocdeao3hasi38fltri8yelo3y29rnd,di4odocda-fosoiDetsl2syyse.,em4ssodO-pfooDonasf1f.een0s1i10ta0e0on0sm0fimam1gmf0a.tAggl0eA.rgd/mk.i8gegg8o.d..fAodotafahgffie.ttlethyohareOfed3otcM4rpasirneeCodostPhplaaAoyyimlfllyaeiamnn1mdo0eio0insnsgeaeemllsy.tscgaooo.lrf/tl The toxicity of greater than that of 22,,34,-6D-ToBrA2,4in,5-tTh.eSsetrtarciahlsawndasBosahiodsiteowbicezs(l1ig9h6t4ly) reported that no abnormal behaviour in pigs had been noted following 40 daily doses of 15 to 100 mg.Ag. of 2,4-D, nor from single doses of 2,4-D of 200 to 800 mg.Ag. In short term trials by Bjorklund and Erne (1966), calves and pigs showed definite though reversible symptoms of poisoning after single doses of 2,4-D of 200 and 100 mgAg., respectively. Rats and fowls did not show any sign of distress after single doses of 100 and 300 mg.Ag., respectively, and fowls tolerated daily doses of 300 mg.Ag. daily in their feed for several weeks without visible effects. Repeated daily doses of 50 mg.Ag., however, led to toxic symptoms in some pigs. In longer term studies (Erne 1966a), five young pigs were fed 500 p.pjn. of 2,4-D for up to 12 months but, although various toxic effects were noted and their growth rate was affected, none of the animals died. When 2,4-D was fed to a sow throughout gestation and for a further six weeks, 10 of the 15 underdeveloped and apathetic piglets she produced died within 24 hours and the mother subsequently had to be slaughtered because of abnormalities that developed in her spine. Heavy dosing of pregnant rats, however, with 1000 p.p.m. of f 0007432 I546S01 40 J. M. W ay 2,4-D in their drinking water over 10 months and of their off-spring for up to two years, whilst leading to retarded growth and increasea mortality, did not produce unequivocal signs of toxicity. Continued administration of 500 p.p.m. of 2,4-D in feed or 1,000 p.pjn. in the drinking water of fowls led to reduced egg production and kidney abnormalities. These results led the authors to conclude that the chronic toxicity of 2,4-D to the species studied was moderate. They were, however, concerned about the mortality of new-born piglets, with evidence of movement of 2,4-D through the placental tissues, and the reduced egg production in fowls which they thought might indi cate a possible interference with reproductive processes. In general the findings of other workers support these conclusions on acute and chronic toxicity. In all the work quoted the amounts administered to the test animals for effect, have been well in excess of the amounts they might be expected to pick up from a treated pasture, or in feed derived from crops that had at some time been treated with auxin herbicides at normal dose rates. b) Indirect toxicity Indirect effects of herbicides on grazing animals have been associ ated with increased toxicity of toxic plants, increased palatability of normally non-palatable toxic plants (e.g., ragwort, Senecio jacobaea), acrnedasiensdiuncenditrtaotxeicciotyntiennnt)or(mWaelllylanrodnt1o9x5ic0)p.laHnotws e(vee.gr,.,Fteammpeor(a1r9y5i3n) claimed that, up to 1953 in America, in all cases where poisoning of livestock from herbicides had been reported, the effects noted could be attributed to some other cause. ' Examples have been given by Willard (1950) of cattle eating wild cherry (Prunus serotina), of pigs eating Cocklebur (Xanthium sp.), and of lambs eating thistles after herbiddal treatment with auxins. Instances have been reported of ragwort becoming 'sweeter' from two Lgryanznedanbdy Bocraartttrhleornefseor(d1aa9y5ss2h)oarfitntevpreesratiipogpdal.tiecGdatiiutohcnesbhyayndadrnodbceyBianangliclparc(e1idf9e5r(e2Hn)tCiaaNnlldy) content of the leaves of wild cherry from untreated trees and trees treated with 2,4-D and 2,4,5-T. Their conclusions were that the foliage was no more toxic to cattle after treatment and that there might even bunetrleesasteHd CoNnesi.nBtuhcekleeat vaeis. of the (1961) treated trees than in those fed the alkaloid-containing of the plants Delphinium barbeyi (tall larkspur) and Helenium hoopscii (sneeze weed), after treatment with 2,4-D ester or 2,4,5-T ester, to calves and ewes. o`f the Nheorbiniccirdeeassewdastonxoictietdy.oWf itlhleiapmlasnatsndatCtrribountianbl(e19tGo 3a)papnliacalytsioend D. barbetji, treated with 2,4,5-T amine at various growth stages, and showed lliat the alkaloid content of the plants was increased for several $ 0007433 5595 I fiOiy 1546802 Toxicity and hazards of auxin herbicides 47 weeks after treatment at the vegetative and early bud stages. It was noted, however, that the bitter taste of the alkaloids might make the treaStwedanpslaonntsanedvenShlaewss palatable to animals than (1954) showed that 2,4-D untreated ones. affected the HCN content of Sudan grass ( Sorghum vulgare ssp. stidanense). Initially there was a decrease in the content of IICN but four days after treat* xnent there was an increase over the controls which was maintained fnoirtraatefucrotnhteernt12ofdaleyasv. eSsi.mBiluacrkefeftecatls. were shown to occur with the thought that there might be a relationship between HCN and nitrate metabolism in Sudan grass, an increase in one leading to a decrease in the other. The clinical aspects of nitrate poisoning in stock, conditions under SDwanuhadnivcdilhdisestnotsintaorfalettteh(s1ea9saler6e0p()1ll.ai9knT4etl1shy)eh,taotGvoaexilcibccbeueeermfntfuercleeattpsteoaorifltnendt(hi1tber9ya4lte6eBa)rv,aaerdCselaoecsfyaeucees(tre1tdaa9li5nb7(vp1),9laa4anr0nte)sd,, duction of nitrate to nitrite and the conversipn by nitrite of haemo globin in the blood to methoglobin: the animal dies from asphyxia. Intravenous iniection of methylene blue in doses of two g./500 pounds of body weight gives immediate relief. Nitrate in plants is generally resent in the form of potassium nitrate and increases in nitrate content a(Gveilbbeeernt aestsoacLia1te9d46w, Cithasdero1u9g5h7t).cSounndditioent saalnd(19h6ig0h) soil nitrogen noted a high nitrate content in Urtica spp. and Rubus spp. after heavy rains, followed by preferential grazing of these and other weed species by cattle. A number of abortions in these cattle was correlated with oc currence of high nitrate rather than grazing of the weed species per se. Recent increases in vitamin A deficiency in North American ruminants has been associated with ingestion of nitrates occurring in herbicide- treated plants by PuiLLirs (1964). The accumulation of nitrates in the leaves of sugar beet is well known (e.g., Savace 1949). Increased levels of nitrate in the leaves of this crop as a result of herbicide application have been reported by Willard (1950), Stahler and Whitehead (1950), and Whxiekead et aL (1956). Isolated incidents have been reported of nitrate poisoning of cattle in America as a result of feeding on sugar beet that had previously been sprayed. In one incident in N. Dakota, the nitrate content of sugar beet leaves after spraying was found to vary from 1.81 to 8.77 percent of the dry weight, as against 0.22 percent for un treated plants and a toxic level of 1.5 percent (Stahler and Whtte- head 1950). Studies on forage crops (Berg and McElroy-1953) and on a range of weed species (Frank and Cricsby 1957) have shown which of these may contain high levels of nitrates after auxin application. Cell-free extracts of maize and cucumber from plants that had been previously sprayed with 10 and 100 p.p.m. of 2,4-D were investigated by S ervers and Haceman (1962). Tlic level of nitrate reductase was i 0007434 DOW 1 546803 48 J. M. Wat ia1nn9c5dr2eb,arWseeahdkidtineohwmenaaiodzfeentbiaturlt.art1ee9sd5ui6nc)ephdlaainvnetscsuh(cFouwamnmbteehra.1t9S25t2,u4,d-FDiersceaoibnuesrtehcsemafnoodrremCralatapioridne increases in nitrate content than MCPA, that levels rise to a peak soon after spraying and subsequently decrease with time, and that increases in light intensity hasten decreases in nitrate content It is clear from these reports that nitrate poisoning in stock does occur from time to time and that it is possible for the hazard.to be increased by application of auxin herbicides to nitrate-accumulating plants. IV. Bees and other insects Herbicides kill the plants affect bees (Apis mcllifera) on which the insects feed. In aadndditoiothne, rWinashelcitns if they (1950) has reported that 2,4-D and MCPA were toxic to bees, not only from visiting the trapped on flowers treated bpulatnatsls.oAanstoairneesu(l1t9o6f6d)rrinepkoinrgtecdonthtaamt MinCatPedAwpaartear lysed and killed bees which ingested doses that corresponded to those recommended for weed control. Other workers have reported effects nonTonaonvtbeeasetseoo(sm1tha9eef6tre4eftr)fimeaacepntspdoln(iAHcannatetircooatnaicnrsoeiwfm(hoa1iuv9cxah6i6n1m)9ha6hed2ra,ebvPieictailstduomexgseigcrte-ostJotoepndblaeensethts1s.a9tKi6n2i4n,)4fc.l-oPDw(a1el9mrm6ib0geauhr)t-t has shown that radioactive 2,4-D can be translocated to the nectar of Poinsettia and red clover plants and may be detectable there for two PJtbooaeneltsemhsreher(ae1-vJ9deo6an4bye)sesefano(f1uter9nre6dp0tonr)r,eotaeKetdmfifnebegcnytt(o1GFn9le6ybe0ndebnein)se,gtaJhtonarnditaehlssBaydaornfdbdyaeueCnx(1oind9ni6nhr2eee)lcr.tlblyiPc(aid1dl9uems5set4ert)do-, with 2,4-D or order to enter wthheenhitvhee.yGwlyernenemJaodneetsoacnrdawClotnhnreoullgh(129,45-41)) dust in classed 2,4-D and MCPA as stomach/contact poisons of low toxicity to bees, with LDgo 0.00004 to values of 0.002 mg. 0B.y1r05dym(g1.9c6o2m),poanretdhetootihneserchtiacnidde,sreinpotrhteedratontgael mortality of bees within four days of feeding 30 pg. of 2,4-D and 10 percent feeding m20oprtga.liJtoyhwainthseinn three days rising (1959) reported to 20 percent in five days of that 2,4-D and related com apmouinnedssawletroerntohtetoisxoicprtoopbyeleess,teexr.ceHpatrwahcesnimfoovrma u(l1a9te6d2)asfotuhnedalkthaantola- 30 percent calcium/potassium salt formulation of MCPA was toxic to bees and caused mortality up to 13 days after ingestion. However, part at least of the loss was attributed to cresol impurities, which led to loss of smell amongst the bees with consequent loss of sense of direction. Occasional observations on other insects have been reported. f 0007435 5597 I Toxicity and hazards of auxin herbicides 49 Mellanby ct al. (1959) observed that a spray of mecoprop on oats at ftrhiet)t.imMeaxowf ealhleaanvdy Hfraitrwflyooadtta(1ck96h0a)dtrneoateefdfebcrtooand the fly ( Oscinclla bean (Vicia faba) plants with sublcthal doses of 2,4-D.and recorded a marked increase in the reproduction of them. The longevity of tahdeuplteaapahpidhsidw(aMs uancraoffseipctheudm. Rpoibsii)nsfoened(i1n9g5o9n) ap(Iliss9ou6mr0e))coaarnfdtdeerdAcidnaacgmriensagsaeondndfbeDcrruoaendwdibt(ye1ai9nn6a5pn)laosnthhtoesrwtrpeedeaattehadpathwitdihthe(Aa2cp,4yp-rlDtihc.aoAtsiiodpnahmoonsf 2,4-D amine could enhance aphid infestation in New Brunswick grain fields, probably as a result of depressing the activities of cocdnellid beetles predating on the aphids. In laboratory experiments with coc- dnelh'd larvae treated with 2,4-D amine, there was a fourfold increase in mortality and an increase in time to pupation. There was little fmeworthaoluitrysainmaocntigvsittyt.hIesmadi uanltdbHeextrleasn,ow(1h9ic6h3)uscuonalcllyudreecdotvheartedincarfetearseas in the growth rate of the larvae of the rice stem borer (Chile suppres- salis) feeding on 2,4-D treated rice plants, was a consequence of in creased nitrogen content of the plants rather than a direct effect of the chemical itself. In general, it appears that there is a real hazard to bees--and pos sibly other nectar feeding insects--from applications of auxin herbi cides to plants in flower. Otherwise there would seem to be little hazard to herbicidal insects rates of afrpopmlicadtiiroenc.tHtoowxiedvteyr,otfhethweorckomofpAoudanmdss aantdnDorrmeawl (1965) does suggest that some insects may be more susceptible at particular stages of their life cycle than at others. V. Soil animals Amongst soil arthropods, Davis (1965) was unable to show any differences in poDuluticns of Aeari or Collcmbola sooci^s between un e1trf3feeayctetesadnopn.loDstosailvaainsndi(mp1l9aol6tss5i)nthwoattahshearudenxabpbeeelrenimtsopenrfaitnsydeodfaawnysitihmreiMplaoCrrtPnsAaotufinrsei1.g0Bnioofuilcltaeonnft (1961) concluded that auxin herbicides, based on phenoxyacedc and propionic acid, were the most susceptible to breakdown by micro organisms of the many pesticides applied to the soil. The importance of soil known mfroicmroothrgeawniosrmksoifnAthudeubsre(a1k9d6o4w) nanodf oththeseen.hWerbebicsitdeers is well (1967) has briefly reviewed the literature on the influence of plant growth- regulator auxin herbicides on the host/parasite relationships of nema todes, in which 2.4-D has been shown to increase nematode reproduc tion in plant callus cultures. In addition, plant cell hypertrophy and proliferation, which is a common effect of 2,4-D in many plants, pro vides highly suitable conditions for development of nematodes. In J546804 i 000743*; 5598 S089f-S l MOB 50 J. M, Wat this way susceptibility of a normally nematode-resistant variety of oats could be induced, although there did not appear to be any greater susceptibility of a non-resistant variety. VL Fish and aquatic organisms Under field conditions the toxicity of a pesticide in water is affected by a number of factors in addition to those that affect its performance on land. Thus acidity, hardness of the water, and the sorbent qualities of suspended organic matter in the water may directly effect the toxicity. The trophic nature of the ecosystem, the oxygen status of the water in respect of both producers and demand, and the amount of movement of water both within the system and in terms of flow will affect the concentration of the chemical, its persistence, and its possible toxic side effects. Because of these, and many other interacting factors, the toxicity of a given formulation of a given chemical compound to an individual species will vary under field conditions depending upon the nature of the water body and the immediate environment. For this reason toxicities to fish and aquatic organisms are usually estimated in terms of median tolerance limit for exposure to a given concentration of the pesticide, for a given length of time (TLmx). In addition to direct or indirect toxicity, the effects on aquatic organisms of the removal of the substrate that gives them food and shelter must also be considered. For instance, in one of the Tennessee Valley Authority's reservoirs two applications of 2,4-D controlled con siderable acreages of Eurasian water milfoil ( Mtjriophijllum spicatum). The eradication of the plant eliminated the substrate that might have been colonized by large populations of epiphytic insects such as the larvae of midges, mayflies, and dragonflies (Smith and Isom 1967). Although under some conditions this might have a very serious effect, it has been shown by Way et aL (1968) in an ecological investigation of the use of paraquat in lakes, that although individuals and popu lations of animals may be severely affected in the weeks after applica tion of a herbicide, the species affected may be present again in the season after treatment. It has also to be recognized that very heavy infestations of submerged or floating aquatic plants may interfere with the passage of nutrients and considerably reduce the temperature and dissolved oxygen values of the water (Fisn 1966). Thus, any possible hazards from the use of a herbicide may be outweighed by the advan tages gained from the removal of the vegetation. The danger of significant amounts of 2,4-D appearing in ground water or streams as a result of local applications seems to be slight. Aldiious (1967) measured residues of 2,4-D in drainage channel water after aerial spraying of a Scottish forest at four pounds/acre active ingredient in 12 gallons of water. Up to two p.p.m. of 2,4-D was measured in the seven days after application but none was detectable i 0007437 5599 DOW ]546S06 Toxidty and hazards of auxin herbicides 51 by 2S days. Brown and Nishioka (1967) analysed samples of a water- suspended sediment mixture from 11 streams in the western United States at monthly intervals. Amongst the analyses made were gas chromatograph determinations for 2,4-D and 2,4,5-T. Neither com pound was found in any of the samples. The absence of the compounds was attributed to some extent to their susceptibility to degradation. ecltuadRli.nevg(i1ea9wu6sx2in)o,fhatenordxbiicCciitodypeesh,a(hz1aa9rv6de5sbateonednfism1h9a6od6fe).abCyroaBpneague(e1ro9f6(p16)9es6tn1ico)it,dedBesa,tnhidnatt variations in formulation gave rise to greater differences in toxicity than the differences in toxicity between the basic compounds. Ester lfaotrimonusl.atSioimnsilawreroebsoefrtvenatimonosrewteorxeicmthaadneabmyinLehoorsmteet(a1l9lic59s)altwfhoormrue viewed effects on a number of crustaceans, aquatic insects, and molluscs. Trout ( Salmo tr most sensitive fish utta) are to water npoorlmlutailolyn.reAglaarbdaedstears being amongst the (1958) lias given median tolerance limits for 24 and 48 hour (TLm-i and TLm) ex posures of trout to 2,4-D or 2,4,5-T, or to mixtures of these two compounds, of 9.5 to 250 p.p.m., depending on formulation, compared mtoe1rc,1u5r0ictaoc2e,t0a0t0e.pH.pojlnd.efnor(1s9od6i4u)mdecvhilsoerdataefoorrm0.u0l0a5fpor.pc.mom. fpoarripnhgetnhyel likely toxic hazards to trout from a number of pesticides1 applied at agricultural rates. The following comparative estimates of hazard were given: aldrin - 70, PCP - 7, MCPA-1.5, 2,4-D -1 , 2,4,5-T - 0.5, para quat -1/12, simazine -1/27, diquat - 1/40, dalapon * 1/46, TCA - 1/120, and aminotriazole -1/150. It should be emphasized that these figures refer to agricultural rates of application: hazards from the con centrates might be different. to Perch (Perea fluviatilis) and be affected by MCPA, 2,4-D, orora2c,h4,5(-RTut(iBluasnrdutti1lu9s5)7)araet unlikely rates of application used for aquatic weed control, although ture of 2,4-D and 2,4,5-T was more hazardous. In alactoermmtriearlcsiaBlamnidxt e t al. (1962) found threshold values for toxicity to perch and roach of MCPA of 200 to 215 mg./litre, of 2,4-D of 75 mg./Iitre, of 2,4,5-T of 55 to 60 mg./litre, and of 2,4-D +2,4,5-T mixtures of 5 to 12 mg./litre. These pared rtoesuMltCs PshAowthraanthwerergeresahteorwtnoxinicitHieoslodfen2',s4-D(19a6n4d) 2,4,5-T com calculations (see above). The much enhanced toxicity of the 2,4-D +2,4,5-T mix tures may have been an effect of the particular formulations used, or 1Aldrin l t2^3,4,10,10-hexaclilorx>.l,4,4a^>,8,8a-hexahydro-o-l,`i-<m<o-5,8dimcthanonaphthalene; PCP pentachlorophenol; paraquat l,l'-dimethyl-4,4'- bipyridylium dichloride; rimnzlne 2-chloro-418-biscthylamino-l,3,5-triazine; di quat 9,10-diliydro-8a,10a>diazonaiaplienanthrene dibromidc; dalapon sodium 2,2-dichloropropionatc; TCA tricldoroacctic add; and aminotriazole 3-amino1,2,4-triazole. f 0007438 5600 3 0 8 9 P S / MOO 52 J. M. Wat Dcoauvldis daenmdonHstarbadtceaastslyener(1g9is5ti9c) effect of one established compound on the median tolerance other. limits over a 24 hour period (TLm24) for bluegill sunfish (Lepomis macro- chirus) to a number of herbicides.. Values obtained when the com pounds were added to relatively pure water were 2,4-D - 39 p.p.m., CMoCfPeA(1-92606)pn.po.mted., 2,4-DB delays in 20 p.p.m., and 2,3,6-TBA -1,800 spawning of bluegill sunsh of up p.p.m. to two weeks after treatment of water with the propylene glycol butyl ether ester of 2,4-D at five and 10 p.p.m. However, no other effects were noted on reproduction or on survival of fry. In pond experiments, death of some fish as a result of 2,4-D treatment led.to increased size in the staounrdtvhiDveoairnvsi,dsipv(ri1do9ub6aa3bl)lfyiashna.ds IDan arfvueisrsuthlatenrodftHrtiahulecshgwersietah(t1eb9rl6uf3oe)goidrlel pssuourpntpfeidlsyho,anHvaeuiflcfaehbceltess of different formulations of 2,4-D and other auxins. Their tests showed 2,4-D and 2,4,5-T esters to have TLmMranging from 1.8 to 10 p.p.m. depending on the ester used. Dimethylamine salts of 2,4-D and 2,4,5-T had TLm54 of 162 to 542 p.p.m. and 144 p.p.m., respectively, com pared to the alkyl acid of 8.0 p.p.m. amine salt This work (owfhMicChPiAs roeffe1rr6e3d.5tpo.pin.mC. oapned1o9f662,,4s-eDe above) shows the wide differences in toxicity that can occur in differ ent formulations and the care which must therefore be taken in assess ing the toxicity of an individual product before recommending it for use as In an aquatic herbicide. addition to work on fish, Walker (1962) has reported effects on a variety of bottom-feeding fish food tion of 2,4-D to plastic enclosures at 1.0 otorg4a.n0ispm.ps.mfo.llLowhoinstgea(p1p9l5ic9a) has reported that ester formulations of 2,4-D or mixtures of 2,4-D and 2,4,5-T affected crustaceans, aquatic insects, and molluscs in the range of 0.1 to 3.3 p.p.m. In their investigations into the effect of the use Aofut2h,4o-rDityartesreartveosirfsr.omSm4it0htoan1d00Ispoomun(d1s9/6a7cr)cfoinunTdennnoesmseeeasVuraalblelye toxic effect on benthic fauna or significant changes in mean numbers of burrowing mayflies (Hexagenia) before or after treatment There were some deaths of caged fish but no dead or distressed free fish were found. Observations of free living fish indicated that they ap peared to move out of the treated area, whilst analysis of a number of fish for 2,4-D residues gave no measurable results at the limit (0.14 p.p.m.) of detection. However, analysis for residues in mussels (mainly Elliptio crassidens) indicated that they concentrated the chemitkl from the surrounding water although no mortality was recorded. Analysis of mud samples showed that significant amounts of 2,4-D (up to 58.8 p'.p.m. butoxycthanol ester) were present in isolated sediment samples up to 10 months after treatment. These fairly high levels in the en vironment of a residues found isnedthenestaeraynaimniamlsa.lRsauwchlsas(1t9h6e5m) uhsasselremviigewhteedxlpitlearinatuthree f 0007439 5601 n,,J 46808 Toxicity and hazards of auxin herbicides 53 on the toxicity of herbicides to estuarine fauna, principally Crustacea and mollusca, and described experiments from his own laboratory. A variety of formulations of 2,4-D affected several species of animals at concentrations of one to 10 p.p.m. Larvae of oysters ( Crassostrea virginica) were killed over a period of time by one p.p.m. of butoxyethanol ester and dimethyl salt of 2,4-D, and though oyster eggs were less susceptible, their development could be affected by 2,4-D butoxy-' ethanol ester at 10 p.p.m. These effects might give some cause for alarm when rates of application of 20 to 30 pounds/acre arc required for weed control in estuaries. Doses of this sort would give 7.34 to 11.02 p.p.m. if evenly distributed throughout one acre of water one foot deep. Because of tidal duxes and uneven distribution only very local concentrations at toxic or near toxic amounts may occur in practice. Theresults of these various investigations suggest that at hcrbicidal rates of application of auxins the hazards from acute or chronic toxidties to aquatic organisms are low. Nevertheless in some instances the dose rates required for effective herbicidal action, for example in estuaries or where the chemical is likely to be rapidly dispersed, may give rise to local and perhaps short term concentrations not far re moved from those required for toxic effects on some organisms at sus ceptible stages of their life history. VIL Wildlife in general Hazards to wildlife from auxin herbicides have been reviewed by Rudd and Genelly (1956), Sfbincer (1957), and Mellanby (1967). Whilst with any material having biological activity a risk of acute or chronic toxicity is always present, authenticated incidents of widcscale poisoning of wild animals by these herbicides have not been reported. However, there is the furtner hazard to all forms of wildlife from herbicides of altering the habitat. Whilst herbicidal applications of the compounds to agricultural land undoubtedly modify tne habitat there for wildlife, it is unlikely that individual instances of spray drift of the compounds at sublethal doses, although undesirable, have any really significant effect on habitat of adjoining non-cultivatcd land. This is not to say that some food plants for animals are not regularly killed in hedgerows and other field margins from spray drift, nor that in isolated instances of gross mishandling more severe effects do not occur over larger areas. It is probably true, although there is little or no scientific evidence, that tne continued use of herbicides in the U.K. is gradually leading to the impoverishment of the flora`of the field margins, and consequently to the gradual impoverishment of agri cultural land as a whole as a habitat for wildlife. H e principal problem from the use of auxin herbicides in relation f 000-/440 5 602 546809 I 54 J.M .W at to wildlife, is not toxicological but ecological in terms of scale and intensity of use, especially on non-agricultural and industrial land. Thus the total destruction of a small area of vegetation on one occa sion might be less serious for the wildlife of a region than the selective destruction of vegetation over a wide area at regular intervals. The hazard from the use of herbicides lies in the fact that they can now be used to affect vegetation over very wide areas in a short period of time, thus eliminating 'reservoirs' of wildlife and wildlife habitat that would have otherwise survived. Summary Toxicities and hazards to man, domestic and laboratory animals, wildlife, bees, and other insects, soil animals and fish from MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, mecoprop, and 2,3,6-TBA are discussed. In man only one authenticated instance of death by poisoning has been noted in West European and North American literature. Authenticated case histories of sublethal effects are also very rare. However, com plaints of transient dizziness, sickness, and other symptoms are made rrom time to time by workers engaged in field applications, especially under conditions-where the spray is inhaled excessively. The possibility of man acquiring toxic doses of these compounds in food, milk, or water appears to be very low. Toxicity tests on a range of laboratory and domestic animals with a wide range of formulations of the compounds tend to show greater differences Detween formulations than between compounds. Amongst the domestic animals studied dogs appear to be more susceptible than the others. Acutely toxic doses of the compounds to farm animals and fowls are shown to be greater than the animals could normally ingest from treated vegetation or water following an agricultural application. Hazards to stock from the concentrated materials are emphasized, but the offensive and unpalatable nature of the undiluted chemicals would probably preclude any normal animal from ingesting a toxic dose. The danger of chronic toxicity is also shown to be very low and continuous high dosing over periods of months rather than weeks are required to produce severe symptoms of poisoning. In many animals a high proportion of the active chemical is voided in the urine within 24 to 48 hours of ingestion. Secondary hazards to animals as a result of changes in the chemical constituents of food plants may occur, for w instance toxicity has been associated with temporary increases in the nitrate contents of some plants after spraying. Current recommenda tions for stock to be excluded from treated pasture for a period after treatment should be observed. Hazards to wildlife exist from the use of herbicides but are also associated with other changes in agricultural practices. The effects of herbicides on habitat ore serious for wildlife and should not bo { 0007441 5603 DOW 1 546810 Toxicity and luzards of auxin herbicide 55 underestimated. Deaths of bees have occurred following spraying of nectar-producing plants when in flower. It is possible that similar hazards exist for other nectar-feeding insects. Where other insects have been studied, increases in population have been noted more often than decreases. With some aphids this has been associated with reduction in numbers of predators. No effects have been noted in studies of populations of soil arthropods. Increased activity by nematodes has been associated with the favourable conditions for invasion of the host plant created by auxin induced teratomas and other effects. Toxicity to fish and other aquatic organisms has been shown to depend on the formulation of the compound used. In general, toxicity to fish is low and hcrbicidal applications to water do not present a severe direct hazard. The dangers of secondary effects must be assessed in relation to other methods of control and the environmental char acteristics of the water body to be treated. Some aquatic organisms other than fish may be directly affected at dose rates not very much in excess of those recommended for hcrbicidal use in water. Rsum * Sont discuts les toxicits et les dangers, pour l'homme, les animaux domestiques et de laboratoire, la vie sauvage, les abeilles et autres insectes, la faune du sol et les poissons, des composs suivants: MCPA; MCPB; 2.4.D.; 2.4.DB; 2.4.5.T; mecoprop et 2.3.6.TBA. Chez l'homme, un seul cas authentifi de mort par intoxication a t relev dans la bibliographie de l'Europe occidentale et de l'Amrique du Nord. De mme, les cas authentiques d'effets sublthaux sont trs rares. Nan moins, des sujets affects l'pandage de ces produits se plaignent, de temp autre, particulirement lorsque les conditions ae dispersion exposent une inhalation excessive, ae vertiges passagers et 'autres symptmes et se sentent malades. L'ventualit d'absorption par liomme de doses toxiques avec les aliments, le lait et l'eau semble trs limite. Les preuves de toxicit sur une grande varit d'animaux de la boratoire et d'animaux domestiques tendent montrer que les diff rences sont beaucoup plus grandes en ce qui concerne les formulations qu'en ce qui concerne les matires actives. Parmi les animaux domes tiques, le chien parat tre le plus sensible. Les doses susceptibles de {irovoquer des effets de toxicit aige chez les animaux de ferme et es volailles sont suprieures celles qu ces animaux pourraient ingrer partir des plantes traites ou de l'eau ventuellement pollue la suite des applications en Agriculture. L'accent est mis sur les risques pour le btail des formulations concentres, mais la nature agressive et le mauvais got de ces formulations rendent trs impro- * Traduit par R. Taciuur. 0007442 i ObU4 MW I 546811 I 50 J. M. Watt ' bable l'ingestion d'une dose toxique par un animal normal. Les risques do toxicit chronique sont galement trs faibles, car des doses leves doivent tre rptes, non seulement pendant des semaines mais Sendant des mois, pour pouvoir provoquer des symptmes graves 'empoisonnement. Chez beaucoup 'animaux, une forte proportion des matires actives est excrte dans l'urine dans les 24 48 heures qui suivent l'ingestion. Des risques secondaires pour les animaux peuvent rsulter de modifications dans les constituants chimiques des vgtaux consomms; par exemple, des effets toxiques ont t associs des augmentations temporaires des taux de nitrates de certaines plantes conscutives aux traitements. Il convient de respecter les recommanda tions relatives l'exclusion du btail des pturages traits pendant une certaine priode aprs le traitement. Des dangers pour la vie sauvage peuvent rsulter de l'usage des herbicides, mais ils peuvent galement tre causs par d'autres varia tions dans les pratiques agricoles. Les effets des herbicides sur l'habitat sont graves pour la vie sauvage et ne doivent pas tre sous-estims. Des morts se sont produites chez les abeilles la suite de traitements de plantes mellifres l'poque de la floraison. Il est possible que des risques analogues existent pour les insectes qui se nourrissent du nectar des fleurs. Les tudes relatives d'autres insectes ont rvl des aug mentations de populations plus frquemment que des diminutions. Dans le cas de quelques especes de pucerons, cet accroissement tait associ une rduction du nombre des prdateurs. Aucun effet n'a t observ dans les tudes sur les populations d'arthropodes du sol L'accroissement d'activit des nmatodes tait li aux conditions favo rables pour l'invasion des plantes htes cres par les tratomes et autres effets ds aux auxines. La toxicit pour les poissons et les autres organismes aquatiques s'est rvle dpendre du type de formulation utilise pour l'pandage de la matire active. En gnral, la toxicit pour les poissons est basse et les applications d'herbicides l'eau ne prsentent pas de risques directs graves. Les risques d'effets se condaires doivent tre valus en liaison avec d'autres mthodes de lutte et les caractres de l'environnement des eaux traiter. Quelques organismes aquatiques autres que les poissons peuvent tre directement affects des doses ne dpassant pas beaucoup celles recommandes pour l'emploi des herbicides dans les eaux. Zusammenfassung * Toxizitt und Gefhrdung bei Mensch* Haus- und Laboratoriums tieren, Wild, Bienen und anderen Insekten, Bodcnticrcn und Fischen durch MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, Mecoprop und 2,3,6-TBA * bersetzt von F. Baa. 0007443 5605 DOW 1 546812 Toticity and hazardsofauxmhcrbicidcs 57 werden diskutiert. Beim Menschen wurde nur ein verbrgtes Beispiel eines Todesfalles infolge Vergiftung in der westeuropischen und nordamcrikanischcn Literatur berichtet Verbrgte Flle von sublcthalcn Effekten sind auch sehr selten. Jedoch werden von Zeit zu Zeit Klagen ber vorbergehendes Schwindclgcfhl, belkeit und andere Symptome von Arbeitern vorgebracht, die in der Anwendung auf dem Felde ttig sind, besonders unter Bedingungen bermssiger Spray-Inhalation. Die Mglichkeit fr den Menschen, toxische Dosen dieser Verbindungen in Lebensmitteln, Milch oder Wasser zu er reichen, scheint sehr gering zu sein. Toxizittsteste bei einer Reihe von Laboratoriums- und Haustieren mit einem weiten Bereich von Formulierungen der Verbindungen neigen dazu, grssere Differenzen innerhalb der Formulierungen als zwischen den Verbindungen zu zeigen. Von den untersuchten Haus tieren scheinen Hunde empfindlicher zu sein als andere. Die akut toxischen Dosen der Verbindungen fr Vieh und Geflgel waren hher als sie die Tiere normalerweise aus behandelten Pflanzen oder aus Wasser nach Anwendung in der Landwirtschaft aufnehmen knnen. Die Gefhrdung des Viehbestandes durch konzentriertes Material wird hervorgehoben, aber die agressive und widerwrtige Natur der un verdnnten Chemikalien wrde wahrscheinlich jedes normale Tier von der Aufnahme einer toxischen Dosis abhalten. Die Gefahr der chro nischen Toxizitt erwies sich auch als sehr gering und eine kontinuier liche hohe Dosierung eher ber Monate als ber Wochen ist erforder lich, um schwere Vergiftungssymptome zu erzeugen. Bei vielen Tieren wird ein hoher Anteil der aktiven chemischen Stoffe im Ham 24-48 Stunden nach der Aufnahme entleert. Sekundre Gefhrdung bei Tieren als Ergebnis von Vernderungen der chemischen Bestandteile von Nahrungspflanzen kann Vorkommen; zum Beispiel war die Toxi zitt begleitet von einem vorbergehenden Anstieg des Nitratgchaltes gewisser Pflanzen nach dem Besprhen. Laufende Empfehlungen fr die Fernhaltung des Viehbestandes von der behandelten Weide fr einige Zeit nach der Behandlung sollten befolgt werden. Eine Gefhrdung fr Wild besteht durch die Anwendung von Herbiziden; sie ist jedoch ebenso verbur ' ~i mit anderen nderungen in der Landwirtschaftspraxis. Die Effe...e der Herbizide auf dem Standort beim Wild sind ernst und sollten nicht unterschtzt werden. Tod von Bienen erfolgte nach dem Besprhen von Nektar-erzcugenden Pflanzen in der Blte. Es ist mglich, dass eine hnliche Gefiirdung fr Nektar-fresscnde Insekten besteht Beim Studium anderer Insekten wurde ein Populationsanstieg hufiger als eine -abnahme festgestellt Bei Blattlusen war dies verbunden mit der Herabsetzung der Anzahl der Raubinsekten. Keine Effekte wurden fcstgestcllt in Untersuchungen von Populationen von Bodenarthropoden. Eine gesteigerte Aktivitt durch Nematoden war verbunden mit gnstigen Bedingungen fr den Befall der Wirtspflanze, erzeugt durch Auxin-induzierte Teratome und 0007444 5606 flOIV I 546813 i . 58 J.M.Way andere Effekte. Die Toxizitt fr Fische und andere Wasserorganismen zeigte eine Abhngigkeit von der Formulierung der angewandten Verbindung. Im allgemeinen ist die Toxizitt fr bische gering und die Herbizidanwendung zum Wasser stellt keine schwere direkte Gefhr dung dar. Die Gefahren sekundrer Effekte sind in Beziehung zu setzen zu anderen Kontrollmethoden und zu den Umwelteigenschaften der zu behandelnden Wasserflche. Ausser den Fischen knnen gewisse Wasserorganismen direkt beeinflusst werden bei Dosisbetrgen, die nicht sehr weit ber den zur Herbizi-Anwendung.im Wasser empfoh lenen liegen. References BB--BBBABAABBBABA--orejaaaaendaluu-orael-ualnrtc.adh-rgvdleadnobhmuafiAirRuDlxaipfo(p,i4oc31,e,eelrnyiayenoneisneo1nnuslns2dfltonr8eDesnuaa,,sga,rsa,shRm,d9rel1ukdvihaydehsa,nh,,tLnctusendts.CJHK42h,n.u.nge,,hldeeh,tBsloJon..eeW(N0aF8dIiisrir.aeyrr.MW..tTatnn1Orim:nmbeJJmrbB)t,Mil5h,sAefi.ai.sJ.-MW9.sic.,.ii,t:.Nntea.S.c,kF4cJtchcn.,.r:I^:.5ahBlRr:ig(.-hoB1iBtrriHPn.iTdsl.eda7dWncu1s:oegEE-e0pnrpD..iLSe..:.onhecUEe:edd)bk9sys:h,5sfil.dC.Asxeuh.lrduen.iafia6c:.C,heGboIbs2toes,eRiDerebnIhnaLrcJr0,cr(Rbreiinh5,lcnT.dneasttnLe.E.ot,nt4co.,)rradahteisaettpdanomip.dsFl-eeYaeHn.dLnmeld1eso(CltDdMx.dhrnlrctae.eV1ido.wo5egibe.efWiaiwiic,eoocnsadrTcp9rrecEf,i..Xsecena;eHaennkbroetMrLieaa6arttsl6cst.oesf.b,o,ttSMemei.eClsryGag..r1ap.hso9svpx.baahNEtE.doDMiuiSciJr)snoyPrsatoXinaisdSflala7.n.crneoateSc(its.naatn.vicfucnybftotIgw1b,inAganCmcmkaidiaEnXeGCdhiwvenle9wnesPboge2in,bionsauailribnldt.rat6e.ufs:3tdiamgeatennieddknlnroew.n.ttchr19hspineSWaTsPfdwuurLn:sioat..2Je.7dn)hiidtm,tihoa,snnarn.eyr.a,ceEe(CzkJ,aiy(x4u)klawgB.ddp1borhae:e.Oti1aD.cfsiva-tu3tlnnunsti.d9:fciTnehuDa9lLAnnla6.vnoEaelesosdsi6dnteeSo6i8ge.c24oLgoiAtcere.glfpnsdar,7yx4oasrcte5h.nf.JensrrysRe.Wn,eei)mh.pgo,...n()d3cAsNa.oscdDIf1..oiiPaa(.HSZto7aSnLaciMnotyf1indlf9evhneaennao,1cniLlgeteo49nwwrmol6aeiae2lIooi2d-3isriimos.a..d-5.nzNnrc6b^tLltf,5iusa(swoa.4et4ari8:3Ai3RBi)2th.iWupedklnsnzu-r7t,24Pi,)3t-BTwAxi.:PprDd6epnuig.dm,,c3ufd,acsl-s4agrpLnheCLlira.9nto,oeaeAYdcni-rb3aglre7aain3f.sinlDnccsitoaXpdve5(eonirngcwh8.dz:upnemWCiope4drohdrdyXt(9aenlSrkinsm.lssloi1feclSei-nuie5to:ytydaoIB-af(cmEnkos9aspUtfI4(r4su1imlrauuAtic.cIgo1pl6ih-ixN,onb9tiond.khFqbcutse9pcciId4lgnlhl5m4(oiueuphu.eisnanol.6Fo)epi1ste5l3nrtnrtetldlc.ogugi55scYozozoiaa9csz:)ieSdxxy5hAm)eiryrdon,.rn6o(omsyv.ood)dt,enoEine1tiiarop2ainarn.apwecotekist92ln1.nih)rcetveginshrrssZea.46d2miii.cgBtoahpqcindPrlto9.2,ttuaininAedunCtlriug1irbnrsna)nscFeeaoepdlit0ra.c(neHiblaasosrtcxbois1.nhaL7deAottssuycomeyepii9.Necuh,ponfnirgmbug(6S(ritsJohbetonotnsr11bulB..r.5Fecamee.iaugw.ot9i9zoneP)ttZotirrAderSawa2siii.66dhrtytrfmhsoodgdotc-blno46ieitlt1ionaerchecs2heroonena6c1))ilyny.sh)ess--f-...t..,,, t 000744S 546814 Toxicity and hazards of auxin herbicides SO ?-D--FEC-DDFFDDEFDBBCCDDFB--ixar--erdylraoauaoaauacs--rauanrwnoslv--dvphevcbn-t--rendsseewc.siosiic,aaas4TmrlTAst2P3sSxaCWdSN3N1BCh,H((stsd,koyk,oa,,hac,aotnaCrv1c14c,,28n,ecdohd2Th,Gasoosio,Ftae4eEvsA,netOaeiiW,noeder9i09JnasCox,naoeeeleKBccnm,mo,wnr-o,bSP,..roeS.n.4,(.nis(5ii6.roknnbDndsSefxa.tEd.nc1eiDo.,1dcNd.eMmi.ad.Tr.(tR3AWL.9n6i,Rc:teBRrf.idoa,Z.o,n1N9.fs:Veal-9cJ.ttaAiveC.B)),an.sniCDMllaoh.Wg...l.im.dpeoJN.96:15ht.eon.:eD:R(:..i.77dsc.ceU*.rraa.lear:.SaE136t7Cg,.eEe13imoio,e.,oCaAboSeKoS:nTli.-ne.cFS:nn0nJdi9n1.)5aas)2l.Wopu,gW.1vtlonel33oouhd.ex.:,ca.,aond.roB'ttun7.n6)H-2niosp.P:pf6Lm9re,mmrJeestgte.CcmmTJf,cvd3d4P.i3fa.eatid..roo4Wsa7mYaTa.1ei,lubuaaog.o.e2eesho2ne)v1ressectlilnBg4Csn.EiuncnuiVB.ohnelsnCJip.3dpn(,edeW0tnnl((.uergdc4,aodgty.ic1hdtbAlie.srrt1..e1e0Bs,oinatesnsSg-rhonaPalst2o9lectsi(fe9LwJ69aWdpo.ms.-lo4YIentibu,ai.opypfiuuf1Wp2fons.64euLcmn6in5elNcm3.eoslnsndeelNH.no:cpl95Snd-nnS2a(ck.da6tgpdI99d,lreCudikhm.gnDh1a.t5sfn5caCtn).FiaeairWtd)srrinlsolsctsReLEoo.(e9aatbz7,snpMsot.ooEreuio(uoIVrehm1fnghsinnfyhef6n)W1de.ol)soodr.r.drrs6fui.e9tfft.,nd.sfoepn.solto7ietcS9ffictFtr7-JceMfiiioshe6aood.speho,ReneRsf)PPrvki6aaaea7no2nkac2lmtcebtc.2fh.a7bseanenelrr.ip5ahmson.reli1ie.tcrcehincyp)oo,pVsetifcttcotd.m)xf(i:euatS5y.rtnrLwreatfd*:cwchnohefk.l2l.o1yaleepMcI,1i,on.ese...irp.cgbPvde41aedmE9ncsttanaae33,chx6drnE.eoesaRe:ith1W0rkc1Ye64itttaatf1oeyeeefos,estna:uiese-sSf..46wBetl0ro3toi(rlr,atnorwtxerisntrltedn(osoiv1tWadPo6r)tnciau5.D:Pcascn1hyB:2edeortmeBnu.aan9etnra.ntoei0yd9aaxoJ8cleErtgeoAtsdcit(d5lArwnocxi.nrcfSoer.s5Mo6i:ru1t-cieofcock2ovgeiholeriht(.tps,:A6r(if.Lssl9icnoh.e5Drr.tuilfi)rbyef1en1ylhfaiWcd3r7sesr6n.Oa.g,eWncsicea,ri9g9omCeitak5hevl0stcn7ttbrteii5cgio)2milBn6he6hevL6necoiit3fioe)eth.i4oecsn0hpmdm6aese6ao,arn:dw.e.nftedtlwW,Nuif6sb..eed)roao)f(3ldzttedees.laMTygif.roiisJ1h1ga.JreEbtt5nneaesndbtioh.ui(Co..q5ki,u9eeoCl7ycytnP..n1diimerxusn2rAn4lNtoAdltnaerrosraW9siH.toiri(bga,at(on1ipvewbdotdch:4penl1oma5fae1tytPo(etoecu)iexetai-tpeekr9hodrcL7hmse9.sNscr.efDleedsntoideslep6eoirotttee)dr6osoip.dosklitdee6it-rre..gl2rtdpwcrtht5:.nocPaienbi,ortbdyrVygCi)VhncwCkheysd)*Cfaedfri.iaRaJivc..da.cosdateoodEoeAiooe.nnnheaeptpeBtdndPfaJ'dfsnencncftihdopgoesraeA.fn.secsre)Lortrpbuucrnp.exrosirihsM,siAbLhIogJloeka,mtnolytMcitCtpena.iieiinmntroinpsalfmpa.ecctdretsa.c:3roliWrsnoi.Kicbdai.osbCml,1stddomeogrdLnsctFri.nPt3i.83rtt4en(icof.eaed"ieiecfbahc.oois3Aedtt41iri.MPntrneickohdsdcufnoease7VdccA8dM5fsdlgehrek.Ptecdp'ihec.esciaiShea,casaeicornCelapsLt(((1scondlCa.it.oRodeSitcmng111.kaC1LcPdfsatimcoW.hxdoudoncP999",duMeAihh).fJCeipnhlsctg1655hiVsp,md4cioe.araatp6ai3evltt3a4c3mrbt63einoihfhnulLoideStuyoietr1nl))s)tdni93nhgniee-f:>-f...t.l,....., 000744G 5608 546815 60 J. M.Way --LI---HKCJFGCCHKKXHSFFCt-hoHt-iroeeh.oluririnuan---oh.uuiepp--aay-lltTckmgas-chiJuezddpenannrWaahTv1,dSgd9IaPchp2ctb1,pAcBCtlQd,snosaieEnnqecehrunn-eo0aneynieei9ecuoeo,t2csaeri.aSCuDs-hortdcn4anseuksufuceerfdoccmb5nsa1t,csamnges8sr-re.fh7ltnef,tntten-.,aetn,t-iagbtrcne,,le.ieu.7eerie,,nJDiaaml3if-P.a.2udpndc,Ast.dJ,J,((ciiyCnnihorcBA-.n.iDari,SnucdtSfl,11C,lt.dN,e:onoAiCLt-iqb(rLS6e,4ga.,naiald(.Ccs.tentdbn-99Co1e:Stvap,,.nir.euEtoro1tn,o..psLadd.aehwHCntt,isye5,pn66H9hoMoVae..oebl..ehot9pHTtWd1.,l.SedL:sEzh-asgd036s2ni.n,a'siF.feenl.2WU4.5CT:sTbv.adrce.pN.aJnCee,,ogi:,3tir,nBua7t.fer45Enn.thAeli(laonraindegdLWr:Br,raoeuiIE.nnnac.7diGa(Te--sHeA)dbecn)ox.od)D,knagennl-1DeshgE,c..nHaerMnp.p.o.aPhxgci-:Se).eHidlrrm,..mdJhB(tl6tidci.C-ero.tcan.oa,.stWeayeeieucp1g.i..-eoyshoR6rDrinF.ncuaectonmnn2tGdio,bA-or2CioE9ETcHdsdeMGdLaet0lhnaignlpdc.Re.snLui,.ul.mca4fRRtpie...nlexst4.e,sad)nueoke..in.fbowteesidei6roEa..JItbvm,ynDGA..eWJop1nsdeD4otsfvaow5nsC,aa.r4n)toF.asiseiNryid1.np1Wtltoiei:.Fle.iputin.-.toipaii,eAtfosodriaaps,ChrlefTbT,Fcnfe.hsBpsadno.abaSsBv.yctroYCus3Gerc)nlpD.gsgmdio25.nel.rsnnaihe2ote.ilffe6Hr1oaiac.e.nrldcasnce7r0cd.BedJBre.n,La.1na,0hdnsfploAaC:.ha4letd:odA1odsFe.n,itaor,o.5ddpellwvForitJ.-mswfsnieVLeCor(liaun1ntcDDM(ffRU.ee:.s,Pdkb1nos(WMDardhlltne1.D(idal.iads5sh1heh3:nod9').rca-a.1caS.e'atrsLeen9iCtJAte4ci29isaa..hidteezoni6:i9ontmopotEEC.P.t6aso.inur,ooCrwal(,sss5neattGdxc36m4dfatme3Btnf.anehCO4Dtb:anf1ChpxrriNa4oifoa-h0a)sp,twlno.bad5moc.)B0efeolwDtaao:Db.p)gah1Tcntndoomo..znunidiPaclCenn.cm,isiobateaceBu7ieienon.ie.hes(ttrnyrrrosecealCrfun3misahln3pta)1fyrrswodfeoedmdi1rtdeJyLhJifh)snnf.lslo5ga.v9anLcrloe..r1.e.eaedeho2eSd,einn6b.Tacbd6blDa3fdpss3neNL1re,fC1lto1dvettdE:l42lLine:22sb,apidoosi02C4a.ni2fned(eehoc.oee.-ltnd7cixs)lxs.f3,,c,1arA,id'ecrSyheJso4y.n^fUh.ediops4Ck2nCfH95itsi5.cH.meciat-,sf-e1ic(:cdnhit7A2nWhtulIodi850achrt.N1m1Reu0iha.tLosCehce,sotgaedonic6hF4m0eyi9n98itrAlcsh(neycb.Sntri-aiufneomNov1t)a1ha.cl5beolsdaieitrocdndiprCir.ox(2aas(cgtan(oees9tbl2agPrfaiaomiptc1ce1ltebio1rf6cl:4rabreldsnloo5iir)aprsaeLoOby9a9arc7rr9doginim,.iostlxwo2trhoDycce4plnllol6sW6ofter6d.utucs2ileiepfr)uhn6liyr-g(fa(ecm7dy6i5a.seeus.5nc(ah1Aalesc,tkPdieauebtko)g)l,2t)mRhh2taRol31getoe9Urue.dt.yd,se..sihr,pinrxeeis35wsQWu.4(6rd2eeaemenaneap(ponBhyattnA7idnn,o-4ppP.z1ishnhrn4ixleetatiDpusvee.fcCimas)orqtrt9deea-cyuJnise.ici.(oSahypdJxpBuipern.5ocna5s1edhc1rca.sla.tiAiHteliecnf90s,a)occe97.inDfanuPissaWeDCmmaectonhg,)iLmms.t6nKctr2nttre:ig.ahhnertlshoy2oilae3teolEaodins.becirmaumuln1TcietNltrio)dNrednpi.trracoyEiiatrt9r.oonahoyfosdgozirrdCbnfublfS.cupaxwtxboolesncBraeporliSa(kitSso.hpoiCtClro,tn4addwiihhl1ceerhueaiownnEdtiWsdtr-Cdoiofee,Sefc.9tyrenoentce(fu.aen.hlnnenptre-i62oL-dohrrosSilapsubo.tretrk4voa4eIn6rx,ugrsroat.ni.x4rpnottxceeo6n6taaaJo)fh)yti)toonoeyez.x-r:...l....l,t,,f-., 4f C + r U S sictAj*). t J*CC*uZi~cl 9 3 - ^ 6 ^ 'e ^ 7^# >** /Sa/xa-mx , 0007447 5609 Toxicity and hazards of auxinherbicides 81 * -----PNPMLMMMRRSSSLPRRRSSSS-aaapth-tuiyiaoouuaoaaoe-ernllavinwnu-d--bxbwmlxurnzlmu-ma-besidnl,dBdcwZcPn,mVwbd2C8tal,ElPtcsaarpCtlogcn1s(lc.,reerheea,h,iiEerDaxcisri,ds1nr,oaurkrfel3nesoeece,hiasnR4tvacrrroeeanseo,RJ(Getnellnaucfo9dlest,hlrv),Vlslttes.,,deo12,c..a-t,fowtGpldrlao5,etld,la.s6Jn.,l,ueislh,elk9AaJlc,ed9iCetm.o,aJynrrn-As4oSi2aJM,srdt.M7Kc,crEL,.niweR.nWiofTL6.Rorh:rsy:.a.,edpK-a,ntaGode.Pi)dAiboa,I(vs..4fR.D6n-Pe..t.n.le,eb.K.gT,,,L.L1eNHep,eo(et.erpS.i.sKF.,lS.)aadr,.uFrJaD1Eei,eM5nlsaad9r.,an.A.hC.sBo.lMoodnEkir.:.r:GFonga.,dr:Ea9eonnM-nahC4te.Sia:c.Anowa.eddT.y.h`re,uwdnan..AnAsdd6.snePxP,.n.9.cI.TalRmcoC:eoe:nvsalc,Kddeaino7pi3HJieFSdig)sa.taiJnnfdhneet1aneKctta.Rtphen.e.MsssxaN)6ta.trar:aiEy.nMhl:odn0qso7.uGe.Thooet.dne.reicd.yn.B:p,weipkPdtwpyofoturt.aWcc.arlaet,AceMia.Po.CRdChEhlftt*Emd.se7flo-.nittnattiaihBSiecl6f,siehiaD.bEiABepdtafPec0.biti.dCmecncpcecee1NhhoN,Rdfnsic.aedenpshupsA3orCt.ittetBoeocti.ioC4fmr.leedadseoiPuslJ.dcsrlEe.1.ccha,an)mCcI.t.na3Vattasbw.sHegrwefurI5.ae(uo.ta.FonCo:ir,t8uro2oJraaCbsa1iaatnb0tbnofsRtnxacriyrn.tWf..WFs8sthaoun,flmwt3eTeirtcee9o,rlaegfhiliddemhidoonionocEcnduores9sm3bRHenn5(chlobci.eh2addiiemgbditu(f.:tsl8nan1ia3p1rwont.altre7e,fii:2r.,renn1eaA'heoenJymc.4o9ia9wts,sAStaoeyJ)h,rditdoBr9isa.rfTa(tedI::4(l-lhocm.6tu.bhmo4:ouoeakM?Enn2lnmw1Dsdf5soMi(u-oJfzlueTn5C8iextSAhWfnnJit:nav.Te1dD,Pn9f7edexllFe:yot.sos4r.e5i)geslcubnler.eloei9nsSgnc6Tic)edbi:...euadl.ooyNa-eii2scamwedendo.oSoi5tBslnNs(cd3eriDr(rg.themon-dNsJA,aoaH.td1dltfgmeeiiti9o14bsemahr)aaod.il..egnom:ndcfWa)w9Y:dinooan.d.te-e)n9ttoydrg(soas:dNeeVhneDa.ll6.ranoee-IdNeRnTC6.cdats2iiVtnnebrHrnbecZnsn.i3ifeen5e7rnC20DeyAet9hhtooaecaifceeosiwads)eeqwioatcE,depse):tapon1adnnereaveoe.1cc:ioarle.ou(ifcaezlnenn.nil,t2dckettydayax1fl1NRfafNlrftaeniaeeTaomaediftt2aldndnoae2u54oir9tnloisfs.unfedcndiofbncoa4it,hn.lrWs,naafl42:en)tagm5osattiandihtfeostoorpiresedfydpe,77pdSegybutCvx6capic.eFomls(ioafi4l2..anJ4cha32.ioariitdrta)oo.r1f.kolrdlactre4.(a,ta(9m.aei3P(29eefc.enaw4.ct9oon1oCutdLJsHdl,PAtcayos3,umpaefih,lc.kSffli6s9te,snI5(aoo1o(ontlcr4s.o,CsgAe1ievsot51ia.5sa1ond2Aln-rnthgle81arcnfy5r1opciTnat)N9oNl9d9(.,tp.a.eiej05dcrd9.pl4.ofaccon1,df5uwm)ots6nae.rR(AJr,zuoda)6ipai-rhbn.A9dat1tctfa0nsd4.noDl.nl7(0e3aeelii6flre5ta)oh9tdcilc1m))otpCefisr-su12gi5t2eeeifwrson.o.t2naoo06iihe9sndu90gsoiodd2sheto2slinl)adn4c8piMfee6shee.nnrlnx(tamil.0ortf.iaoso.r)ed1-wr1(gegz(pcLildEw,ootro.cbswassy1dp1lp9nea)hh5miIeccioitafpntgacced.69.tnrior9ttfoe6iu3oa-unanyrihnsheanedea7d6p6tn:cMu5t,a0loaLtsddtawnt.gtose,et2et4rytose4iie)fvmsiokap1trliPonesa3a.oa)Nfse)3naiiwican9ttndo.nsn(ge3ai.a(inlo.raekp2-9usrlna.ta1.ngsnibg9ie.eGnoepo,m(lcuopJt9oVe4rdllbodSCialufO.(inm(Jltihsbasid6ri-doau1ovn1nn.s.uctattDiencyxdAh4soriirjied9i92(arphoocotge1ikpmfee)soNoHst6m,ta6opzne2nnn2o.t,ies4larraSlgoao0car4erbLsi,away,on-etd4n1r4ldgcronniw)tsD)itiirsrsndy)coi::n8-k.nea-o.-.sf.t..., 0007448 Cl C3 00 C3 DOW 1 546817 f 62 J .M .W ay WWWWWWVWW--SWwaahioaaeiilllaubloyiillitnin3pnwsxcti8eC,tdx(,(^(auirshest11of15reiateafootaterfothJehenemsnr99o,9rmelmlnn.,dpnrela,,t56c5r4f1daAheCs,Cas,a,t-tSMd40507tCesn,ry,,DdooiC.Cc,aJS)6)),r:dt.S.pnd.Mi..,..tco.,..e:rE8io.fe.RWLoEofo:(nHA.1.JMN-f.1,nc9ent..Bi.5P.:it.an:9ytt,sCp4.fKh.Ieaeit:ifa5n4TE..(nee(ncsliIWe.,,n1nn6l1:antorvts1JSiiPt9t()9dxutalcc1la..oWoo1P.ea6uo6iiins0xnWKmfdrcdr9obeM8ac4dkeisoeel6lechcs)e()scl.ecilou.aoir41u.copEtre(ddCtofgaste)e9ghuEri.i.ettorfsae.io5rocaipfdcfiean.reonSHeio0dnfaalsnms,neWchnsbu)ldr..c,tsA..)taepeJaosetr:ea.sqCsennPabfow(enftuor1idarlWliFedotd:antafc9raocls.feinaTetrnt6enDnmitst2st1heNhodv5efi,.de1neoPoa4ahet)eaJnm.r,rtcrf-:aewelbeaadctot3rhnfuhboitElcbdnm1fchdCeo.slnoeiift7hetdearLdeefctobaVlbreenttndonrso(ccicsatderf1,os1edlrto.o.xaso6nf9,donhpaptmopP,:62sefptarnlhsfkra,r2sp3Tn.de.4osoyncl3)dsn^uhloedeP1c.tiboddssMelnnd7ovr(..oixats71ocefes.oyJntPnhtf(x9.hc.akhofr1wlO6eceooC,gAt9a7ie7ecwrcrNso4nf6osttaet).eiihnsetdspd..2scorhSoschnh,s)f.Co.cB.2csaee2uSeniA,:DddrOn4n,rtnoi4so.gv,daSttxi5-crkrfisOoDyMro-aikWmoohTmcanlhxtencea.saofeeAWdemrlpoolelJtb.mArrgnied.eedaic8rlcndceay:.4a7oaatCddk10.lCl,doBdeekoo4,.Pnh4ssaCdlg,n.ar0el4Sitotooccmr314ohorIkncioa3i58nnnde.-l.l 0007449 5 Gla 5612 in water by micro..,.3. J. Amer. Water analy-, . .r in s e c t ic i d e :> i 10C7). .it nreanophosphate x C-19019 to Scto Amer. Chem. Soc.# i_^.'.--C : < s C "in _ G l C \*\ L[ DOW 436190 Toxicity and hazards to man, domestic animals, and wildlife from some commonly used auxin herbicides o on sri . <* By J. M. Way fcssgsssgBsass Contents VSHVIuIVIVImIIII....... mSWFabITTBnao)i)ooestriiexxryhllAodIsiiccndlaaca.iiidnun.ttfun.yyieicd.tdrm.ete.iaai.anacoo.nnaq.ltnt.dnsdgu.h.tde.aeo.hh..tn.r..x.iaac...ec..izz.hi..n.rc.a.a.ro...sroir...r..dt!de.n..gy...s.sc..i..a...c..t.....ntt.s....o.o....t.i......os...........mdm....x......o......i...s...m..ca.............i.n...e...t.........y..s............t.........i.........c.............................a................n..................d................................l..........a................b..................o..................r............a................t.........o..................r.............y...............................a...............n..................i..........m..........................a.................l.........s............... RZRueessfauemmreminccn.e.f.sa...s...s..u....n....g...................................................................... L Introduction This review has been compiled from searches in the literature for references to the toxicity and other hazards that may arise from the use of auxin herbicides, and specifically of formulations of MCPA (4-chloro-2-methylphcnoxyacetic acid), S.S. 2,4-D (2,4-dichlorophen- oxyacetic acid) and of their butyric acid analogues MCPB and 2,4-DB, of 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), mecoprop -(4-chIoro-2-methylphenoxy) propionic acid], and 2,3,6-TBA (2,3,6-trichlorobenzoic acid). Assuming 70 percent the United Kingdom to of be the eight million acres treated annually with ohfercbeirceiadlessgr(Wowonoidn- Ripto*nM, HonuknstinWgdooond, EEnxgplearnimd.ental Station (The Nature Conservancy), Abbots 37 5613 C f" k... i , > O 38 J. M. Way ford 1964), it is apparent that the tonnage (circa 2,500 tons) of the compounds mainly used for this purpose is similar to the total tonnage of insecticides and fungicides used annually on all crops in the U.K. (circa 2,660 tons) (S trickland 1966). In 1962, D add (1962), from a survey of eastern England, found that over 90 percent of cereal fields treated with herbicides were treated with auxin compounds based on chlorinated phenoxy acid derivatives, notably salts of MCPA and to a lesser extent of mecoprop. Although the use of straight formulations of MCPA has been gradually declining (W oodford 1964) in favour of mixtures of compounds, many of these mixtures still include MCPA or other phenoxy compounds and increasingly include 2,3,6-TBA. In addition to its agricultural use on arable and pasture crops, 2,4-D to gether with 2,4,5-T, is used, or is likely to be used on an increasing scale, for scrab control and control of unwanted broad-leaved tree species in conifer plantations. 2,4-D is also used to a limited extent for control of submerged aquatic weeds. On a world-wide basis 2,4-D is probably used to a greater extent than any other herbicide and this is reflected in the greater number of references to this compound. The extensive use or a single biologically active family of compounds clearly presents a considerable potential hazard in respect to their direct and indirect toxicities to animals including man. It is a measure of the comparative safety of the auxin herbicides that, in spite of their increasing use throughout the uJC. iltttig the mld-1940's, they~_ eonLiuue to be applied much in Tli<Sama juuUne way us an* lUCTganic fertilizersTand WitVm lilllt regard for nnv possible tu.iii. luzant Liunngthe many yeuis uf their use tlnuughuut the wona mere have been notably lew authenticated incidents ot poisoning of domestic animals orwildlife resulting from the proper application of these com pounds. Tiie tact that fully authenticated incidents of poisoning have noFbeen reported cannot be taken as an absolute guarantee of safety, particularly with regard to indirect or chronic toxicity, because ot the frequent dilticulties oi attributing the Underlying cause ot an illness or of death, especially in wild animals. Nevertheless all the direct and circumstantial evidence to hand at the prfefiHt tlmfe seems to indicate that incidents ot poisoning naVfe bfeen 'extremely few and generally under exceptional circumstances. ~ " "' IL Toxicity and hazards to man The principal routes of toxicity to man are either orally or by in- ' halation; there appears to be little hazard of transport tlirough the skin although individual allergies can develop leading to detmatitis (Vallet*1SG5). Eyes may be directly but are usually only temporarily affected. Hazards to man may occur from the concentrated chemical before dilution, from inhalation of spray or dust during application, or from ingestion of the chemicals in food or in water. Because the great- CO a ril liar.'.. handlin'.: dilution. ergannn tests or majority ii re su lt i present hanKdlmeadi of 2,4-D cal study old man of 50 pi 2.4-D in cquivale: active in nerve tiv Va i.i.: 50 to 5(i and of N these litv animals, on a mg. as a Iahe dangers t the toxic on the m< a ntimhi-i ment of ( mg./m.5 < Valle r's range of lie of the 80 ing./k'. this figiiu a greater produce I.l)iu,S to between 1 Lll'VAI from iuli:* with part Di Vno accidental ill after nj 0002640 ^ ^ ft i Ions) of the total tonnage is in the U.K. 1962), from a >f cereal fields jnds based on CPA and to a irmulations of ) in favour of ldude MCPA 2,3,6-TBA. In ops, 2,4-D toan increasing d-leaved tree ted extent for basis 2,4-D is de and this is mpound. The f compounds pcct to their is a measure , in spjte of !-l , they are inorganic nzard. Id there have of domestic >fthese comisoning have :ee of safety, cause of the of an illness e direct and s to indicate id generally ly or by inihrough the > dermatitis temporarily d chemical olication, or e the great- Toxicity and hazards of auxin herbicides Hfc./v--4'1"!" Qojb ^ ^ 39 ' DOW 43C192 est hazards are from the concentrated chemical and because man is handling lire chemicals in this form at all stages from manufacture to dilution, it follows that he is at greater potential risk jthan any other organism. However, there are very few reports in the literature of tests or incidents of poisoning of man by these->mmpounds; the majority of these reports refer to arridcntal poisoning of children. As a*result it is now generally accepted that auxin-type herbicides do not presenTa direct toxicity hazard to man ( Barnes 19oo) when correctly handlpd nr used-for weed C o n tro r~ ~ Kraus in 1945 (in Kehiart 1945) reported that he had taken 0.5 g . of 2,4-D per diem for 21 days with no demonstrable ill-effects. A clini'cal study was iriade 111 Denmark by N ielsen et al. (1965) on a 23-year old man who had committed suicide by apparently drinking 125 ml. of 50 percent w/v 2,4-D dimethylamine salt. The total weight of 2,4-D in his body was calculated as being not less than six g. (the equivalent of 80 mgVkg.), about 10 percent of the total weight of active material ingested. The principal damage appeared to be to nerve tissues and the central nervous system. Vallet (1965) gives acutely toxic oral doses of 2,4-D to man of 50 t(T5^ mg./kg. body weight, of 2,4,5-T of 500 to 1,00Q mg./kg., arid of MCPA of 50 mg./kg. No indication is given of tbe source of these figures which are probably extrapolated trom data on laboratory gnimnls KnruwirH pt at. M966) have shown that a number ot drugs, on a mg./kg. basis, are more toxic in man than in themouse, when used as a laboratory test animal, by a factM Of 10 1615. There"are clearly dangers therefore in assuming that a toxic dose to rasa is the same as the toxic dose to a laboratory' animal multiplied by some factor based on the mean differences in weight. In fact these latter authors show for a number of anticancer agents that a very much more accurate assess ment of the ratio of animal to human toxicity can be obtained on a mg./m.1of body surface area, than on a mg./kg. basis. For this reason V allet's (1965) figures may be suspect, particularly m respect of the range of 50 to 500 mg./kg. for 2,4-D, although the lower figure would be of the correct order o f magnitude when compared to the residue of 80 mg./kg. found by N ielsen (1965) in his suicide case. Nevertheless, this figure represented the amount actually found and it is likely that a greater proportion of the total 2,4-D ingested was necessary to produce death. In this context it is noteworthy that the acute oral LDsa's to rats of the most common organochlorine insecticides range between 10and 135mg.Ag. (M artin 1963). Edwards and Ripper (1953) have discussed the hazard to operators from inhalation of sprays or aerosols during application of herbicides with particular reference to methods of protection. Monarca , and Di V ito (1961) have described a clinical study of an acute case of accidental poisoning of a man in Italy. In this instancea farmer became ill after applying a 40 percent aqueous solution of 2,4-D by handpump 5615 0002641 o 40 J.M .W ay against the wind. He was admitted to hospital, suffered a relapse after IS days, and recovered sufficiently to be discharged after 40 days. Initial symptoms of muscular weakness, vomiting, perspiring freely, and oliguria were noted in the field whilst a diagnosis of bradycardia, respiratory difficulties, and urinary abnormalities was made after ad mission to hospital. However the authors report that the case was exceptional. Fetisov (1966) has reported similar field symptoms in Russian workers engaged in field applications of 2,4-D. This author concluded that a range of formulations of 2,4-D was "highly toxic to animals in different ways of introduction." Whilst reports of minor discomfort following exposure to auxin sprays during field application are rarely reported in scientific literature, there is no doubt that a proportion of workers so exposed do suffer a degree of transitory dis comfort. Whether this is of any significance as a long-term toxic hazard has not been determined for man. Fromthevery nature of their use it is unlikely that auxinherbicides will appear as significant residues in food crops. W illiams (1964) was unable to detect any residues of auxin herbicides in a number of total diet samples down to the limit of sensitivity (0.01 p.p.m.) of his analytical techniques. D uggan and W eathebwax (1967) calculated pesticide chemical residues in `total diet' samples collected on 46 days in 25 American cities during a 699 day period from June 1964 to April 1966. Each sample represented the total amount of food and drink consumed by one person over a two-week period. The total samples represented in all a food and drink supply sufficient for 644 days. Herbicide chemicals were found infrequently and averaged about 0.01 mg./day of which one third was 2.4-D, and halt was MCi'A and Dentacniorophenoi (fL-i1') combined. 2.4-D was found in oils and fats (O.Otil mg. m lMt>4/tio) and sugars and sugar products (0.004 mg~in 1964/65, 0.002 mg. in 1965/66), whilst MCPA was found in grain and ^cereals (0.UU2mg. in 19t>4/6o ). in dairy products (0.003 mg in 1965/ fed), and m leafy vegetables ( 0.001 mg. in 1965/661. T^iese am ounts are substantially below the limits set for acceptable dailyIntake by the World Health Organization and United Nations committees, it seems probable, therefore, that tuxiC hazards irom auXln herbicide-1residues iirtood are very small, raust (lid-l), in a survey ot water pollution hazards to' man from organic pesticides, came to the conclusion that there did not appear to be danger to health at the present time from the backgroundconcentration of pesticides believed to be in ground and surface water. However, 2,4-D could persist in lake water and bottom mud for long periods under certain environmental conditions. Work in Russia, quoted bv F aust, suggested diat the threshold taste and odour concentrations auxin compounds, especially ot pncnOllC dCHTg^ tives such as 2,4-L>, that would prove unacceptable to the consumer were yen' considerably below the threshold concentrations for toxic effects. A particular risk might be supposed to lie in contaminated milk c: ^ drawn from 2,4-DB or 2 these compr were fed 5. number of c for 2,4-D 0.1 these results HI. To.\ The toxi quoted in ti test animals comparative the figures c of factors. 1 and general the susccpli formulation O.1 <*| . salt has ail ester of 570 and H ymas E rne (1966 addition, tin example, 2, mg./kg. for chicks (Rov Bearing the acid fi2.4.5- T 300 700 mg./kg. prop 930 n 2.4.5-T fall the slightly of toxicity c the relative and 2,4,5-T. stituents of compound, elusions wo on the basi factants an; pounds. W1 5 6 ^ 0 2 S 4 2 rrt-d a relapse after after 40 days, pirspiring freely, .isi.s of"bradycardia, v.ts made after ad<h.if die case was iield symptoms in This author ,-.:s "highly toxic'to t reports of minor u* iield application '."no doubt tliat a of transitory' dis^-terni toxic ha2T.rd at auxin herbicides . l ia m s (1964) was a number of total 03 p.p.m.) of his (1857) calculated !!acted on 46 days .cm June 1964 to our' nf food and pe - The total sufficient for 644 :daveraged about i was MCPA and .d in oils and fats cts (0.004 mg. in vend in grain and 003 mg. in 1963/ 'i. These amounts r.ilyintake by the miltees. If seems erbicide residues ` water pollution conclusion that resent time from he in ground and atcr and bottom onditions. Work shold taste and phenolic derivao the consumer -ations for toxic itaminated milk T o x ic ity a n d h a z -ird s o f a u x in h e rh fc k U -s 41 drawn from cows feeding in treated pasture, but no residues of either 2,4-DB or 2,4-D were found in the milk of cows that had been fed these compounds ( G u t e n m a n x c t a l. 1963a and 1063b). The beasts were fed 3.0 p.p.m. (based on a daily ration of 50 pounds) for a number of days; the limit of detection for 2,4-DB was 0.2 p.p.m. and for 2,4-D 0.1 p.p.m. Whereas milk might be a somce of contamination these results indicate- that there is no apparent hazard. III. Toxicity and hazards to domestic and laboratory animals a) Acute and chronic toxicity The toxicity of agricultural chemicals to land fauna is normally quoted in terms of the dose that kills 50 percent of a population of test animals (LDW). Whilst this figure gives a useful indication of the comparative toxicities of different compounds to a given test species, the figures obtained in different tests may be influenced by a number of factors. Thus the age and sex of the test animal, method of dosing, and general conditions of the test may have an important bearing on the susceptibility of the animal to the compound Being studied. The formulation of the active compound has a considerable influence; for instance 2,4-D acid has an LDMto rats of 373 mg.Ag. but the sodium EesaasnrtldtenrehHoa(yfs1m95aa67ns60)L1m9dDg5os.4n/nk).ogotI.f,trea8sgnh0a5dorudmtldhtgehb./eiekssoegn-.p,dortitfeohfdpee,ryelhpnoercwsoetpseevyraleseornf,bet7ehi0gna0lgtymcBaopjglop./rbrkbeugclt.iuyaxl(bRdleeot.ahwnIedner addition, the LDjj for different test species may vary quite widely: for example, 2,4-D acid has an LDWof 375 mg.Ag. for rats, of 100 mchgic./kksg.(Rfoorwdeoagns,d4H69ymmags.A19g5.4)fo. r guinea pigs, and 541 mg.Ag. for Bearing in mind these reservations, the acute oral LDjo to rats of the acid formulations of the compounds under consideration are: 72p.0r4o0.5pm- 9gT3.0A3g0m.0,g2m./,k4g-gI..A>Bg(W.,702o0,o4dm-Dfogr.4Ad0g0a.,nm2dg,3.E,A6v-gTa.,BnsAM1C790P65B3)m.6SgT0.Ahmugs.g,,./a2kn,g4d.-,DmMeaCcnoPd-A 2.4.5- T fall in the moderately toxic and the other five compounds in othfetosxliigchittylyoftocxhicemcliacasslsaqcucoortdedinbgytoFrthaezewrid(1e9ly63a)c.cIenptaedcocmlapsasirfiiscoantioonf tahnedr2e,l4a,t5iv-Te,toRxoicwiteieasnodf Ha nyummasbe(r1o9f54c)omcomnecrlcuidaeldfotrhmautlathtieon`isneorft'2,c4o-nD stituents of the formulations did not add to the toxicity of the active compound, nor did they clusions were reached by eDxaalcctaaaneyd-MpoitkeknetilasteinnganedffePcot.ulSsijmenila(r19c6o2n) on the basis of this and other work. However, it is possible for sur factants and other additives to enhance the toxicity of active com pounds. Whilst there is some evidence with insecticides that mixing 5617 VB" 0002S43 OOV/436194 o 42 J .M .W ay CD CZJHl of compounds leads to synergistic eiFects on the acute toxicity of the constituent compounds (Keplincer and D eichm ann 1967), effects of this sorthave not been reported for herbicide mixtures in common use. However, the possibility of their occurring should not be overlooked, nor, also, the possibility of antagonism where the effect of one com- pound upon another in a mixture is to depress its biological activity. Descriptions of the clinical symptoms of poisoning by M C P A , 2,4-D, and 2,4,5-T of a range of experimental animals have been sum- marized by R o w e and H y m a s (1954) and more recently reviewed with comments by D a l g a a r d - M ikkelsen and Po u l s e n (1962). B j o r x l u n d and E r s e (1966) have subsequently further reviewed and reported on their own results on the effects of feeding 2,4-D to calves, pigs, rats, and chickens. A range of symptoms was produced that was generally similar to those described by the previous authors. E r n e (lso6a and b) studied the distribution and elimination of 2,4-D and 2,4,5-T in these animals. Amine and alkali salts of both compounds were readily absorbed and completely distributed in the body, but 2,4-D ester was incompletely absorbed and reached only a low level in the plasma and tissues. The highest tissue levels of the two compounds were found in liver, kidney, spleen, and lungs and the levels found in these organs sometimes exceeded the level found in the plasma. In blood cells some 1.0 to 20 percent cf the plasma level was found. Penetration of 2.4-D into placental tissue of pigs was recorded but there was little or no evidence of penetration into adipose tissue or the central nervous system. Elimination of the compounds was rapid, the plasma half-life being about three hours in rats, eight in calves and chickens, and 12 in pigs. The tissue half-life values ranged between five and 30 hours. N o retention of the compounds was noted in the tissues. There was no accumulation after repeated dosing and in pigs there was an increase K Fin the rate of elimination after the main excretory route was repeated via the kaiddmnienyiss.trathioann.nIan aalnldspecainecs (1966) traced the metabolism of C M -labelled 2,4-D in rats dosed at rates from one to 100 mg./animal. Radioactivity was found in all the organs studied together with some accumulation as early as one hour after dosing. At the one mg. dose rate a concentration peak of radio- activity was demonstrated after six to eight hours but decreased there- after and was non-detectable by 24 hours. At the 80 mg. dose the peak occurred at eight hours and persisted for 17 hours. Extracts of the tissues were shown to contain mainly unchanged 2,4-D residues. N o radioactivity was found in the expired carbon dioxide, but elimina- tion in urine and faeces was dose dependent. At the one to 10 mg. doses 93 to 96 percent of the ingested 2,4-D was excreted unchanged in the urine in the first 24 hours. At the 20 to 100 mg. doses greater amounts of 2,4-D were found in the second 24 hour period after dosing, with a linear decrease in percentage recovery with increase in dose. In experiments with cattle G u t e n m a n n et d . (1963a and b) were un- ajjjc . cous jn th. j0 2 , 1 (liluii> coin;: < St Jo . a nu:; stccr ration strilli dose 1 pounc' quent MCPi was s. of 2.-1 machz were : period ox-Hun ' being label!' as the pastur after < the mi urine . dosed portioi animal rcsidi:< incorp in par' suserp same 1. deriva: logical (1966) docs p transit Mi and C- parent sprayp Cnicsi. 5618 000264/J 'of the Feels of ion use. luoked, ic comlctivity. MCPA, :n sumed with RKLUNB sported is, rats, nerally '6a and ,5-T in readily :er was plasma s were i these blood s 1> ervous alf-life i 12 in rs. N o /as no crease pecies Fang ;ed at >11 the hour radioiheree the :ts of dues, nina- 1mg. nged eater sing, dose, iun- Toxicity and hazards of auxin herbicides 43 able to detect any residues of 2,4-DB or 2,4-D in milk or faeces of cows fed five p.p.m. of either compound in a 50 pound daily ration. In these experiments there was no evidence of beta-oxidation of 2,4-DB to 2,4-D. Disappearance of 2,4-D was thought to occur as a result of dilution in the rumen, some absorption on the gut wall, and by de composition. In subsequent experiments B a c h e et al. (1964) and St Jo h n et al. (1961) studied the fate of M C P A , M C P B , 2,4,5-T, and a number of other herbicides in cattle. All the M C P A fed to a single steer (113.5 mg. single dose based on five p.p.m. of a 50 pound daily ration) was accounted for in its urine over the four days after admin istration. N o M C P B was found in the urine of MCPB-dosed cows (total dose of 56.75 mg. and 113.5 mg. based on 2.5 and 5.0 p.p.m. of a 50 pound daily ration) on the first day after administration, but subse quent recoveries in their urine showed that 7.2 to 9.2 percent of the M C P B was converted to M C P A . Analysis of M C P B itself, however, was said to be unsatisfactory. It is of interest that a similar conversion of 2.4-DB to 2,4-D machrochirus) (Gu t can enm be ann paenrfdoLrmisekd by bluegill 1965). Urine sunfish ( Lepom is and milk samples were also analysed from cows fed a total of 454 mg. of 2,4,5-T over a period of four days. It was shown that the compound was excreted exclusively in the urine over a period of six days, a total of 430.7 mg. being recovered. C larke et a t (1964) investigated the fate of C 14- labelled 2,4-D in sheep fed a dose of four mg. of 2,4-D/kg. calculated as the minimum daily dose likely to be ingested from grazing treated pasture. The level of radioactivity in the blood rose to a peak 1.5 hours after dosing but fell to the normal background level in 24 hours. All the material was excreted within 72 hours, with over 98 percent in the urine and only 1.5 percent in the faeces. B a l a y a n n i s et aL (1965) dosed a rabbit and six mice with 2,3,6-TBA and showed that a pro portion of the compound was recovered after passing through the animals. These authors discussed the significance of biologically active residues of auxin compounds in animal excreta that might become incorporated in manure or straw. It was shown that 2,3,6-TBA residues in particular could remain active for a period of months and affect susceptible crops to which the contaminated manure was applied. The same hazard does not normally exist with the phenoxyacetic acid derivatives, where the compounds are broken down and become bio logically inactive in a relatively short period of time. However, L isk (1966) has pointed out that the excretion of 2,4-D in the urine of cows does present the admittedly remote possibility of active 2,4-D being transferred from a treated M itchell (in K ephart pasture to a susceptible crop. 1945), D alcaard-M ikkelsen e t al. (1939), and Goldstein and L ong (1960) all reported that there were no ap- ' parent ill-effects in cattle, sheep, or horses from grazing pasture sprayed at herbicidal or two times herbicidal rates of 2,4-D or M C P A . Giucsby and F arw ell (1950) (in Springer 1957) reported that there 5619 0002645 96T9EAAOQ 44 J. M. Way was no significant difference in the amount of feeding of horses, cows, sheep, and pigs in untreated plots or plots sprayed with the sodium salt or the isopropyl ester of Z,4-D, or the isopropyl ester ot 2,4,o-T. However, there did appear to be less feeding in plots sprayed with the alkanolamine salt of 2,4-D. There was no effect on milk production of cows feeding on sprayed vegetation. Goldstein and Lose (I960) found no ill-effectson two cattle irom adding 0.25 pints of a 1.5 percent w/v 2,4-D/2,4,5-T mixture to every five gallons of their drinking water for 41 days. These authors also reported spraying the skins of a calf, of a cow, of sheep, and of pigs with doses ranging from 0.002 to 0.008 pounds of 2,4-D or 2,4-D/2,4,5-T mixture, with no ill-effects. These dose rates would be of the order of those that might occur in an in stance of spray drift. D obson (1954) sprayed M C P A , 2,4-D, or 2.4,5-T pr chicken runs daily for 14 days at normal and ten times normal dose rates.-2,4,5-T-signifieantIy redneed egg preductiuu and'~tlie wtdgliljof the~birdsr MCPATffld 2,4-D also affected egg production, mainly"in the second week of spraying or during the week alter spraying had stoppedr~In~,Jil Imlaiices llicie m s uu effeet on ihn fyililii/ uf~lhe eggrund all the progeny reared well, allliuugli the duse wies^Juid frequency cf application ere severe than"arc likely to-be found in pun.1liefe. E r n e ^1500) sliuwed Lhai some ru rhs 2.4- D fed to hens~culd be excreted in their eggs. D unachie an d FLETCHEH~t 1907) iu jm fcd liffll's eggs with MCl'A, 2,4-D, M C P B , 2.4- DB, mecoprop, and 2,3,6-TBA amongst a range of nt-W hAphieirU-c Dose rates wefe~10, 100, and 200 p.p.iri. equivalent to 0.5, 5, and 10 mg./egg.-The-percentngc hatch w as recoidid. At the luwtisrdflse there- was 1UU percent hatch from the M C P A - and MCPB-treated eggs; 90 percent from the mecoprop- and TBA-treatcd eggs, and SO percent from the 2,4-D-treated eggs. At the highest dose mere was SO percent hatch from mecoprop, 50 percent from 2,4-D and TBA, and 20 percent from M C P A and M C P B . None of the chicks that hatched was de formed although some feather blanching was noted from the M C P A , M C PB , 2,4-DB, and mecoprop treatments. Roberts and Rogers (1957) reported on various feeding experiments on turkeys with alfalfa sprayed with a low volatile ester of 2,4,5-T at herbicidal rates. N o deleterious effects were noted. Calculations were quoted to show that for a one kg. chicken to acquire a lethal dose of 2,4-D from an applica tion rate of one pound/acrc, the bird would have to consume in two days all the 2,4-D applied to the vegetation over an area of 72 square feet; Similar calculations by Rowe and H ymas (1954) for cattle sug gested that for a 770 pound animal to acquire an acutely toxic dose of 2.4- D of approximately 1,000 mg./kg., it would have to graze co m pletely and ingest all the 2,4-D from an area of 0.17 acre (one pound/ acres 10.41 mg./ft.5as93.7 mg./yard-*= 112.1 mg./m.a). Accounts are given of direct oral dosing or dermal applications of oo u -c n CO auxin b (cow), D a l c a .Pa l m e : (sheep gave d in ever 250 m u of 100": of 112 animaltions a* of the c would ill-effc<. animal 1. : propy2l.c: nksn.no_f_ t Or*. of 2,4,5 butyl c! 4. Th< greater report; 40 dai of 2.4- In: showe* doses did nt mg./k: daily i daily i piss. 1 500 p. effects anima for a piglethad ti spine. 5620 0002S46 dow 436198 Toxicity and hazards of auxin herbicides 45 auxin herbicides to a variety of domestic animals by Kefhart (1945) (cow), Rowe and H ymas (1954) (laboratory animals and cattle), D alcaard-M ikxelsen cf al. (1959) (heifers), Palmer (1963) (cattle), Palmer and Radeleff ( 1964) (sheep and cattle), C larke ct al. ( 1964) (sheep), and Strach and Bohosiewicz (1964) (pigs). Palmer (1963) gave daily oral doses of 2,4-D alkanolamine salt to steers for five days in every seven. He recorded signs of poisoning in animals dosed at 250 mg./kg. after 15 administrations as opposed to 86 administrations of 100 mg./kg.; at 50 mg./kg. no ill-effects were recorded over a period of 112 administrations. From these results he concluded that altnough animals could probably ingest enough 2,4-D from concentrated solu tions at any one time to produce illness or death, the chronic toxicity of the compound was sufficiently low to make it unlikely that an animal would pick up enough of it over a period of time to cause any serious ill-effects. Further work by Palmer and Radeleff (1964) using single animals gave the following results: bk2pogu0rfo.0t2ypo,ylm23441fl....e,eg5ttn.SSSChh-/eTkhheehagreeetgtbeeae.lleypplpuekscttatowsteonouslrelo.ulfeberflcreearucaarmtktuetyeedimildlndlbeeef4r4edtoShsd8m1abe1lryttdocdeao3hsai3f8ltri6yelo3y2r9nd,di4odocdaf-osioDtels2ysyse.,em4ssodO-pfooDonasf1.fene0s1i1t0a0e0on0sm0fimamg1mfa0.tgAle0g.r./gd/km.k8igegg8o.d..f/odkotafohgffie.ttlethyoharefledk3otMa4rpsnireeCoodstlPphaaoAyyimlfllyaeian1mnmde0eio0insnsgeaeemllsy.tsgcaoouVlrftl The greater toxicity of than that of 22,,34,-6D-ToBrA2,4in,5-tTh.esSetrtarciahlsawndasBosahiodsiteowibcezs(l1ig9h6t4ly) reported that no abnormal behaviour in pigs had been noted following 40 daily doses of 15 to 100 mg./kg. of 2,4-D, nor from single doses of 2,4-D of In short t2e0r0mttori8a0ls0bmy gB.jAogr.xluxd and Erne (1966), calves and pigs showed definite though reversible symptoms of poisoning after single doses of 2,4-D of 200 and 100 mg.Ag., respectively. Rats and fowls did not show any sign of distress after single doses of 100 and 300 mg.Ag., respectively, and fowls tolerated daily doses of 300 mg.Ag. daily in their feed for several weeks without visible effects. Repeated daily doses of pigs. Jn longer 5te0rmmgs.tAudgi.e,sh(oEwrenveer,19le6d6at)o, toxic symptoms in some five voung pigs were fed 500 p.pjn. of 2,4-D for up to 12 months but, although various toxic effects were noted and their growth rate was affected, none of the animals died. When 2,4-D was fed to a sow throughout gestation and for a further six weeks, 10 of the 15 underdeveloped and apathetic piglets she produced died within 24 hours and the mother subsequently had to be slaughtered because of abnormalities that developed in her spine. Heavy dosing of pregnant rats, however, with 1000 p.pjn. of 40 J. M. Way 2,4-D in their drinking water over 10 months and of their off-spring for up to two years, whilst leading to retarded growth and increased mortality, did not produce unequivocal signs of toxicity. Continued administration of 500 p.p.m. of 2,4-D in feed or 1,000 p.p.m. in the drinking water of fowls led to reduced egg production and kidney abnormalities. These results led the authors tr> rnnrlnd that Ta chronic toxicity ot '2a -u to tiie species studied was moderate. They Were, hoWVW, concchied about the mortality of new-bom piglets, with evidence ot movement ot 2,4-D through trie placental tissues, and the"Teduced"5gg pTUUtion in towis which they thought might indi- cate~ possible lMterferaiiCe with reproductive processes. ' In-general the fanclings of other workers support these conclusions on 'Scute and chronic toxicity. In all the work quoted the amounts adminisfe'red"t'the test Ullimals foi' effttil, luvt uuuti well in lfuiss of-the amoumsthey might b >Tpecld tu pick up ir6m a treated pasture^or-in -fced-dcrivcd h em cious tint had at smile hmtT 'been treated ulth aaxil"herbicides at normal dose rates. b) Indirect toxicity Indirect effects of herbicides on grazing animals have been associ ated with increased toxicity of toxic plants, increased palatabiliiy of normally non-palatab!e toxic plants (e.g., ragwort, Senecio jacobaea ), and induced toxicity in normally nontoxic plants (e.g., temporary in creases in nitrate content) (W illard 1930). However, F ehtic (1953) claimed that, up to 1953 in America, in all cases where poisoning of livestock from herbicides had been reported, the effects noted could be attributed to some other cause. Examples have been given by W illard (1950) of cattle eating wild cherry (Prtmvs serotina), of pigs eating Cocklebur (Xanthium sp.), and of lambs eating thistles after herbicidal treatment with auxins. Instances have been reported of ragwort becoming 'sweeter' from two or three days after application and being preferentially grazed by cattle for a short period. G rigsby and B all (1952) and L ynn and Barrons (1952) investigated the hydrocyanic acid ( H C N ) content of the leaves of wild cherry' from untreated trees and trees treated with 2,4-D and 2,4,5-T. Their conclusions were that the foliage was no more toxic to cattle after treatment and that there might even be less H C N in the leaves of the treated trees than in those of the untreated ones. B uck et al. (1961 ) fed the alkaloid-containing plants Delphinium barbeyi (tall larkspur) and Hclenium hoopscii (sneeze weed L after treatment with 2,4-D ester or 2,4,5-T ester, to calves and ewes. N o increased toxicity of the plants attributable to application of the herbicides was noted. W illiams and C ronin (1963) analysed D. barbeyi, treated with 2,4,5-T amine at various growth stages, and showed that the alkaloid content of the plants was increased for several O O tU CJ CD CD weeks aft noted, hotreated p! S w ans content o ' there was ment ther for a furl nitrate co relations!)! increase h The cl which nit: and lists c SD a v id s o n und ct c duction cl globin in Intravenot of body v. present in `have been ( G ilbert nitrate co followed 1 cattle. A r currence o Recent inc has been ; treated pi The ac known (r. of this pro WlLLAHD ( et al. (195( of cattle i previously content of LSI to S.7 treated pi. h e a d 1950 a range of of these n Cell-free c previously by B eeviu; ir off-spring id increased . Continued p.m. in the and kidney le that the crate. They om piglets, tissues, and might indi conclusions ne amounts 11in excess 'l a treated time been eer 'sociital y of jacobaea), iporary intic (1953) lisoning of d could be ttle eating 'X a n th iu m nent with l `sweeter' ferentially 1952) and d (HCN) and trees he foliage light even >se of the ng plants i (sneeze alves and .^plication analysed ages, and or several Toxicity and hazards of auxin herbicides 47 weeks after treatment at the vegetative and early bud stages. It was noted, however, that the bitter taste of the alkaloids might make the treated plants even less palatable to animals than untreated ones. Swanson and Shaw (1954) showed that 2,4-D affected the HCN content of Sudan grass (Sorghum vulgare ssp. sudancitse). Initially there was a decrease in the content of HCN but four days after treat ment there was an increase over the controls which was maintained for a further 12 days. Similar effects were shown to occur with the nitrate content of leaves. Buck et al. thought that there might be a relationship between HCN and nitrate metabolism in Sudan grass, an increase in one leading to a decrease in the other. The clinical aspects of nitrate poisoning in stock, conditions under which nitrates are likely to accumulate in the leaves of certain plants, and lists of these plants have been reported by Bradley et al. (1940), DSuanvdidesoi nal.et(1a9l.60()1.9T41h)e, Gilbert et toxic effects al. of (1946), Case (1957), and nitrate are caused by a re duction of nitrate to nitrite and the conversion by nitrite of haemo globin in the blood to methoglobin: the animal dies from asphyxia. Intravenous iniection of methvlene blue in doses of two g./oOOpounds of body weight gives immediate relief. Nitrate in plants is generally present in the formof potassiumnitrate and increases in nitrate content have been associated with drought conditions and high soil nitrogen (G ilbert et al. 1946, Case 1957). Sund et al. (1960) noted a high nitrate content in Urtica spp. and Rubus spp. after heavy rains, followed by preferential grazing of these and other weed species by cattle. A number of abortions in these cattle was correlated with oc currence of high nitrate rather than grazing of the weed species per se. Recent increases in vitamin A deficiency in North American ruminants has been associated with ingestion of nitrates occurring in herbicide- treated plants by Phillips (1964). The accumulation of nitrates in the leaves of sugar beet is well known (c.g., Savage 1949). Increased levels of nitrate in the leaves of this crop as a result of herbicide application have been reported by W illard (1950), Stahler and W hitehead (1950), and W hitehead et al. (1956). Isolated incidents have been reported of nitrate poisoning of cattle in America as a result of feeding on sugar beet that had previously been sprayed. In one incident in N. Dakota, the nitrate content of sugar beet leaves after spraying was found to vary from 1.81 to 8.77 percent of the dry weight, as against 0.22 percent for un treated plants and a toxic level of 1.5 percent (S tahler and W hite head 1950). Studies on forage crops (B krc and McE lroy 1953) and on a range of weed species ( F rank and Gricsby 1957) have shown which of these may contain high levels of nitrates after auxin application. Cell-free extracts of maize and cucumber from plants that had been previously sprayed with 10 and 100 p.p.m. of 2.4-D were investigated by Beevers and H acem an (1962). The level of nitrate reductase was 5623 'OOOSS^g DOW436200 oo 48 J. M. W at increased in maize but reduced in cucumber. Studies on the formation and breakdown of nitrates in plants ( F eiitig 1952, F reiberg and C lark 1952, W hitehead et al. 1956) have shown that 2,4-D causes more rapid increases in nitrate content than MCPA, that levels rise to a peak soon after spraying and subsequently decrease with time, and that increases in light intensity hasten decreases in nitrate content. It is clear from these reports that nitrate poisoning in stock does occur from time to time and that it is possible for the hazard to be increased by application of auxin herbicides to nitrate-accumulating plants. IV. Bees and other insects Herbicides affect bees (Apts meUifera) and other insects if they kill the plants on which the insects feed. In addition, W aulin' (1950) has reported that 2,4-D and MCPA were toxic to bees, not only from visiting the flowers but also as a result of drinking contaminated water trapped on treated plants. Antoine (1966) reported that MCPA para lysed and killed bees which ingested doses that corresponded to those recommended for weed control. Other workers have reported effects on bees after application of auxin herbicides to plants in flower but not at other times ( H aracsimova 1962, Palmer-Jones 1964). P almerTones (1964) and Antoine (1966) have suggested that 2,4-D might have some effect on nectar which made it toxic to bees. King (1960a) has shown that radioactive 2,4-D can be translocated to the nectar of PomsettUi and red clover plants and may be detectable there for two to three days after treatment. Feeding trials of auxin herbicides to bees have been reported by G lynxe Jones and C onnell (1954), P almer-Jones (1960), King (1960b), and B vrdy (1962). PalmerJones (1964) found no effect on bees that had been directly dusted with 2,4-D or when they were made to crawl through 2,4-D dust in order to enter the hive. Glynns Jones and C onnell (1954) classed 2,4-D and MCPA as stomach/contact poisons of low toxicity to bees, with LDjovalues of 0.105 mg. compared to insecticides in the range 0.00004 to 0.002 mg. Byrdy (1962), on the other hand, reported total mortality of bees within four days of feeding 30 jig. of 2,4-D and 10 percent mortality within three days rising to 20 percent in five days of feeding 20 /g. Johansen (1959) reported that 2,4-D and related com pounds were not toxic to bees, except when formulated as the alkanolaminc salt or the isopropyl ester. H aracsimova (1962) found that a 30 percent calcium/potassium salt formulation of MCPA was toxic to bees and caused mortality up to 13 days after ingestion. However, part at least of the loss was attributed to crcsol impurities, which led to loss of smell amongst the bees with consequent loss of sense of direction. Occasional observations on other insects have been reported. li l- pii t! a.' V ( 2, ii< b. ci in m fe in so cr< Ui sil cid ha: he; (P pa. dit! troa 13 ' die. (1C proj org: of s kno' has n>iMi lode tin proli 0002550 DOW 436202 Toxicity and hazards of auxin herbicides 49 M ellanby et al. (1959) observed that a spray of mecoprop on oats at the time of a heavy frit fly attack had no effect on the flv ( Oseinr'lla 4 frit). M axwell and H arwood (1960) treated broad bean (Vida faba) plants with sublethal doses of 2,4-D and recorded a marked increase in the reproduction of the pea aphid (Macrosiphum pisi) feeding on them. Tire longevity of adult aphids was unaffected. Robinson (1959) also recorded increased fecundity in another pea aphid (Acyrthosiphon pisum) after caging on broad bean plants treated with 2,4-D. Adams (I960) and Adams and D rew (1965) showed that the application of 2,4-D amine could enhance aphid infestation in New Brunswick grain fields, probably as a result of depressing the activities of coccinellid beetles predating on tire aphids. In laboratory experiments with coc cinellid larvae treated with 2,4-D amine, there was a fourfold increase in mortality and an increase in time to pupation. There was little mortality amongst the adult beetles, which usually recovered after a few hours inactivity. Isnn and H e u n o (1963) concluded that increases in the growth rate of the larvae of the rice stem borer ( Chilo suppress salis) feeding on 2,4-D treated rice plants, was a consequence of in creased nitrogen content of the plants rather than a direct effect of the chemical itself. In general, it appears that there is a real hazard to bees--and pos sibly other nectar feeding insects--from applications of auxin herbi cides to plants in flower. Otherwise there would seem to be little hazard to insects from direct toxicity ot the compounds at normal lieibloldftl iales of application. However, the work ot Adams and D rew (1965) decs'suggest .Inti, some insects may be fflOtft susceptible~at particular"stages of their life cycle than at others.V. V. Soil animals Amongst soil arthropods, D avis (1965) was unable to show any differences in pooulaticns of Acari or Collembola sn<*n<*s between un treated plots and plots that had been sprayed with MCPA in 10 out of 13 years. D avis (1965) was unable to find any reports of significant effects on soil animals in other experiments of a similar nature. Bollen (1961) concluded that auxin herbicides, based on phenoxyacetic and propionic acid, were the most susceptible to breakdown by micro organisms of the many pesticides applied to the soil. The importance of soil microorganisms in the breakdown of these herbicides is well known from the work of Atjdus (1964) and others. W ebster (1967) has briefly reviewed the literature on the influence of plant growth- . regulator-auxin herbicides on the host/parasitc relationships of nema todes, in which 2,4-D has been shown to increase nematode reproduc tion in plant callus cultures. In addition, plant cell hypertrophy and proliferation, which is a common effect of 2,4-D in many plants, pro vides highly suitable conditions for development of nematodes. In 5625 50 J.M .W at this way susceptibility of a normally nematode-resistant variety of oats could be induced, although there did not appear to be any greater susceptibility of a non-resistant variety. VI. Fish and aquatic organisms Underfield conditions the toxicity of a pesticide in water is affected by a number of factors in addition to those that affect its performance on land. Thus acidity, hardness of the water, and the sorbent qualities of suspended organic matter in the water may directly effect the toxicity. The trophic nature of the ecosystem, the oxygen status of the water in respect of both producers and demand, and the amount of movement of water both within the system and in terms of flow will affect the concentration of the chemical, its persistence, and its possible toxic side effects. Because of these, and many other interacting factors, the toxicity of a given formulation of a given chemical compound to an individual species will vary under field conditions depending upon the nature of the water body and the immediate environment. For this reason toxicities to fish and aquatic organisms are usually estimated in terms ofmedian tolerance limit for exposure to a given concentration of the pesticide, for a given length of time (TLmx). In addition to direct or indirect toxicity, the effects on aquatic organisms of the removal of the substrate that gives them food and shelter must also be considered. For instance, in one of the Tennessee Valley Authority's reservoirs two applications of 2,4-D controlled con siderable acreages of Eurasian water milfoil (Mtfrioplujllum svicatum). The eradication of the plant eliminated the substrate that might have been colonized by large populations of epiphytic insects such as the larvae of midges, mayflies, and dragonflies (S mith and Isom 1967). Although under some conditions this might have a very serious effect, it has been shown by W ay et al. ( 1968) in an ecoloeical investigation of the use of paraquat in lakes, that although individuals and popu lations of animals mav be severelv affected in the weeks after applica tion of a herbicide, the species affected may be present again in the season after treatment. It has also to be recognized that very heavy infestations of submerged or floating aquatic plants may interfere with the passage of nutrients and considcrablv reduce the temperature and dissolved oxygen values of the water ( F ish 1966). Thus, any possible' hazards from the use of a herbicide may be outweighed by the advan tages gained from the removal of the vegetation. The'danger of significant amounts of 2,4-D appearing in ground water or streams as a result of local applications seems to be slicht. Aldiious (1967) measured residues of 2.4-D in drainage channel water after aerial spraving of a Scottish forest at four pounds/acre active ingredient in 12 gallons of water. Up to two p.p.m. of 2.4-D was measured in the seven days after application but none was detectable O O CO CD N> C CO by 23 day? suspended States at chromator. pound wa was attrib Review eluding ai et al. (IS variations than the c formulatie lations. Si. viewed e! molluscs. Trout most senv median to posurcs oi .cc:r.pct:::d to 1,150 tv mercuric a likely toxii agricultur. given: aid quat - 1/1 1/120, am figures ref centrates ; Perch to be alfe> applicatio: turc of 2.-' ct al. (19u MCPA of 55 to GOir These rest pared to (see ahov hires ni.iv i Aldrii dlmetiuHi'n bipyriiUliv tju.il i 9.11 2,2-<l;, lili'ti 1.2,-MriJ.v! C00ZS52 DOW 43l>204 Toxicity and hazards of auxin herbicides 51 by 28 days. Brown and Nisiiioka (1967} analysed samples of a watersuspended sediment mixture from 11 streams in the western United States at monthly intervals. Amongst the analyses made were gas cliromatograph determinations for 2,4-D and 2,4,5-T. Neither com pound was found in any of the samples. The absence of the compounds was attributed to some extent to their susceptibility to degradation. Reviews of toxicity hazards to fish of a range of pesticides, in cluding auxin herbicides, have been made by Bauer (1961), Bandt et al. (1962), and Cope (1965 and 1966). Cope (1966) noted that variations in formulation gave rise to greater differences in toxicity than the differences in toxicity between the basic compounds. Ester formulations were often more toxic than amine or metallic salt formu lations. Similar observations were made by Lhoste (1959) who re viewed effects on a number of crustaceans, aquatic insects, and molluscs. Trout (Salmo trutta) are normally regarded as being amongst the most sensitive fish to water pollution. Alabaster (1958) has given median tolerance limits for 24 and 48 hour (TLmn and TLniig) ex posures of trout to 2,4-D or 2,4,5-T, or to mixtures of these two compounds, of 9.5 to 250 p.p.m., depending on formulation, compared to 1,150 to 2,000 p.p.m. for sodium chlorate or 0.005 p.p.m. for phenyl mercuric acetate. H olden (1964) devised a formula for comparing the likely toxic hazards to trout from a number of pesticides1 applied at agricultural rates. The following comparative estimates of hazard were given: aldrin - 70, PCP - 7, MCPA - 1.5, 2,4-D -1, 2,4,5-T-0.5, para quat - 1/12, simazine - 1/27, diquat - 1/40. dalapon -1/46. TCA1/120, and aminotriazole 1/150. It should be emphasized that these figures refer to agricultural rates of application: hazards fromthe con centrates might be different. Perch ( Perea fluviaiilis) and roach ( Rutilus m tilus) are unlikely to be affected by MCPA, 2,4-D, or 2,4,5-T (B andt 1957) at rates of application used for aquatic weed control, although a commercial mix ture of 2,4-D and 2,4,5-T was more hazardous. In later trials Bandt et al. (1962) found threshold values for toxicity to perch and roach of MCPA of 200 to 215 mg./litre, of 2,4-D of 75 mg./litre, of 2,4,5-T of 55 to 60 mg./litre, and of 2,4-D+2,4,5-T mixtures of 5 to 12 mg./litre. These results show rather greater toxicities of 2,4-D and 2,4-T com pared to MCPA than were shown in H oldens (1964) calculations (see above). The much enhanced toxicity of the 2,4-D +2,4i3-T mix tures may have been an effect of the particular formulations used, or* dbq21ii,u,2pm2a-y,4tcdrt-*iihtc-drAahyianI9lldozio,uo1rrnmiol0nacp-p.drhod-ithpihcyirho1d.lln.rco2oanr,t-i3ce8d,;;a4e,;,TP110CC0sa,iAP1m-d0ai---zahizetnporxeienacnach-tihlaao]cp2orhho-rlcoeaohnc-rleloa,ot4npir,tcho4lci-ara4n,cc5,on6i,lde8-;b;,Sidpsaaiaenb-rthdrhaoeqyxamulmaaaihmdtiyneid-n;orotord-li-l,aae,ll3z'xa-o,dop5l-oie-latn,Ke4>t-i-hz3myi-sdalocom-d;4-i5i,nu4d,o8m'i--- ' ,,s' / 52 J. M. Way could demonstrate a synergistic effect of one compound on the other. D avis and H audcastle (1959) established median tolerance limits over a 24 hour period (TLm=<) for bluegill sunfish ( Lepomis macro- chirvs) to a number of herbicides. Values obtained when the com- pounds were added to relatively pure water were 2,4-D 39 p.p.m., MCPA - 20 p.p.m., 2,4-DB - 20 p.p.m., and 2,3,6-TBA -1,800 p.p.m. Cope (1966) noted delays in spawning of bluegill sunfish of up to two weeks after treatment oi water with the propylene glycol butyl ether ester of 2,4-D at five and 10 p.p.m. However, no other effects were noted on reproduction or on survival of fry. In pond experiments, death of some fish as a result of 2,4-D treatment led to increased size in the * survivors, probably as a result of the greater food supply available to the individual fish. In further trials with bluegill sunfish, H ughes and D avis (1963) and D avis and H uches (1963j"reported on effects of different formulations of 2,4-D and other auxins. Their tests showed 2,4-D and 2,4,5-T esters to have TLm54 ranging from 1.8 to 10 p.p.m. depending on the ester used. Dimethylamine salts of 2,4-D and 2,4,5-T had TLm4 of 162 to 542 p.p.in. and 144 p.p.m., respectively, com pared to the alkyl amine salt of MCPA of 163.5 p.p.m. and of 2,4-D acid of 8.0 p.p.m. This work (which is referred to in Cope 1966, see above) shows the wide differences in toxicity that can occur In differ ent formulations and the care which must therefore be taken in assess ing the toxicity of an individual product before recommending it for use as an aquatic herbicide. j In addition to work on fish, W alxeh (1962) has reported effects, on a variety of bottom-feeding fish food organisms following applica-j tion of 2,4-D to plastic enclosures at 1.0 to 4.0 p.p.m. Lhoste (1959)! has reported that ester formulations of 2,4-D or mixtures of 2,4-D and 2.4.5-T affected crustaceans, aquatic insects, and molluscs in the range of 0.1 to 3.3 p.p.m. In their investigations into the effect of the use of 2.4-D at rates from 40 to 100 pounds/acre in Tennessee Valley Authority reservoirs, Smith and Isom (1967) found no measurable toxic effect on benthic fauna or significant changes in mean numbers of burrowing mayflies (Hexagenia) before or after treatment There were some deaths of caged fish but no dead or distressed free fish were found. Observations of free living fish indicated that they ap peared to move out of the treated area, whilst analysis of a number of fish for 2.4-D residues gave no measurable results at the limit (0.14 p.n.m.) of detection. However, analysis for residues in mussels (mainly EUiptio crassidcns) indicated that they concentrated the chemical from the surrounding water although no mortality was recorded. Analysis of mud samples showed that significant amounts of 2,4-D (up to 58.8 p.p.m. butoxycthanol ester) were present in isolated sediment samples up to 10 months after treatment. These fairly high levels in the en vironment of a sedentary animal such as the mussel might explain the residues found in these animals. Rawls (1965) has reviewed literature 562S Q O rf* o C5 rc o cn on the and ir.. variety ccnci-r. virgini. rth.ui.-l less sir. ethanol alarm \ for wii 11.02 P fool ilc local t practice n it rates of icitics ( the dos. cstuaricgive rivmover! ! ccptible Ilaz.: Runo an Whilst v chronic t roisonin lowevfi hcrbiciili compotu; for wild!, compoun signiiicai: not to s.i in hedge isolated i over laig scientific gradually margins, cultural 1 The p 0002 554 -Hgg.1 1 - - -V lpound on the other. Jian tolerance limits ish ( Lepornis macroined when the cornre 2.4-D 39 p.p.m., 3-TBA- 1,8C0 p.Pjm. ;sunfish of up to two ie glycol hutyl ether 0 other effects were d experiments, death increased size in the >od supplv available gill sunfish, H u g h e s 1reported on effects Their tests showed rom 1.8 to 10 n.p.m. of 2,4-D and 2,4.5-T , respectively, comp.p.m. and of 2,4-D 0 in C o p e 1966, see can occur in differ^ fc ken in assess2 C0 i.-iiending it for has reported effects ; following apolicanm. L h o s t e (1939)! xtures of 2,4-D and olluscs in the rangel e effect of the use! 1Tennessee Valley md no measurable >in mean numbers r treatment. There distressed free fish ated that they ap sis of a number of at the limit (0.14 inmussels (mainly the chemical from recorded. Analysis 2,4-D (up to 5S.8 sediment samples i levels in the enmight explain the eviewed literature Toxicity and hazards of auxin herbicides 53 on the toxicity1of herbicides to estuarine fauna, principally Crustacea and mollusca, and described experiments from his own laboratory. A variety of formulations of 2.4-D affected several species of animals at concentrations of one to 10 p.p.m. Larvae of oysters ( Crassostrea virginica) were killed over a period of time by one p.p.m. of butoxyctlianol ester and dimethyl salt of 2,4-D, and though oyster eggs were less susceptible, their development could be affected by 2.4-D butoxycthanol ester at 10 p.p.m. These effects might give some cause for alarm when rates of application of 20 to 30 pounas/acre are required for weed control in estuaries. Doses of this sort would give 7.34 to 11.02 p.p.m. if evenly distributed throughout one acre of water one foot deep. Because of tidal fluxes and uneven distribution only very local concentrations at toxic or near toxic amounts may occur in practice. The results of these various investigations suggest that at bcrbicidal rates of application of auxins the hazards from acute or chronic tox icides to aquatic organisms are low. Nevertheless in some instances the dose rates required for effective herbicidal action, for example in estuaries or where the chemical is likely to be rapidly dispersed, may give rise to local and perhaps short term concentrations not far re moved from those required for toxic effects on some organisms at sus ceptible stages of their life history. o R udHdaaznadrdGs etonewllilydl(if1e95fr6o)m, Saputxjxincehrer(b1i9ci5d7e)s, have been reviewed by and M e ix a n b y (1967). Whilst with any material having biological activity a risk of acute or chronic toxicity is always present, authenticated incidents of widescale poisoning of wild animals by these herbicides have not been reported. However, there is the further hazard to all forms of wildlife from herbicides of altering the habitat. Whilst herbicida] applications of the compounds to agricultural land undoubtedly modify the habitat there for wildlife, it is unlikely that individual instances of spray drift of the compounds at sublethai doses, although undesirable, nave any really significant effect on habitat of adjoining non-cultivatcd land. This is not to say that some food plants for animals are not regularly killed in hedgerows and other field margins from spray drift, nor that in isolatedinstances of gross mishandling more severe effects do not occur over larger areas. It is probably true, although there is little or no scientific evidence, that the continued use of herbicides in the U.K. is gradually leading to the impoverishment of the flora of the field margins, and consequently to the gradual impoverishment of agri cultural land as a whole as a habitat for wildlife. The principal problem from the use of auxin herbicides in relation 436206 54 J. M . W a t to wildlife, is not toxicological but ecological in terms of scale and intensity of use, especially on non-agricultural and industrial land. Thus the total destruction of a small area of vegetation on one occa sion might be less serious for the wildlife of a region than the selective destruction of vegetation over a wide area at regular intervals. The hazard trom the use of herbicides lies in the tact that tney can now be used tb affect vegetation over very wide areas m a short period or tim erthurelim inating 'rtscrvoirs' or wiidine and wndkie habitat tli'af would have gtlrerwlssTarvlved:-------- " Summary Toxicities and hazards to man, domestic and laboratory animals, wildlife, bees, and other insects, soil animals and fish from MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, mecoprop, and 2,3,6-TBA are discussed. In man only one authenticated instance of death by poisoning has been noted in West European and North American literature. Authenticated case histories ot sublethal effects are also very rare. However, com plaints ot transient dizziness, sickness, and other symptoms are made rom tima to tllB by workers engaged in Held applications, especially under coriditlCSi ivhcrc the spray is inhaled XCesslVly. Tll possibility of"ihH jL`L|uliing uitIi' dnxK bt these compounds in food milk, or water appears to be very low. Toxicity tests on a range of laboratory and domestic animals with a wide range of formulations of the compounds tend to show greater differences between formulations than between compounds. AmomgsT tne domestic animals studied dogs appear to be more suscepuble than the others? Acutely imilti 0s"5f~th compounds to farm animals and fowls are shown to be greater than the ammais could normally ingest from treated vegetation or water tohowing an agricultural application.' Hazards to stock trom tne concentrated materials are empnasized, but the ottensive and unpalatable nature ot the undiluted chemicals would probably preclude any normal animal trom ingesting a toxic dose. TKe dancer or chronic toxicity is lS6 Shown TC'be very 10\Vand continuous high dosing over periods ot months rather than weeks are required to-Tiroduce severe symptoms ot poisoning, in many animals a high proportion of the active chemical is voided in the urine within 24 to 48 hours ot ingestion, secondary hazards to ammais as a result of" changes in the chemical constituents ot rood plants may occur, for insTgneS~t5xicitv~hgS"ljLieii associated with temporary increases ill thtr nitrate contents of some plants after spraying. Current recommenda- tionrfgTstock to be excluded trom treated pasture for penod~after treatment should be observed. 4 7,1 Hazards to wildlile exist from the use of herbicides but are also associated with other changes in agricultural practices, ihc effects of hcibiddes miiiabUUl are serious lor wiidlite and should~not~bo . J1WW1 O kU CO en roo -a underesli neclar-pi hazards r been stud decrease in muni t populatic been asx< host plan Toxicity t on the foi is low an direct ha: relation l- a c te ris tirs Other that, in excess < Sont di doniesthj!: insectes, la MCT1J; 2.: un seul ce. bibliograp: mme, les moins, des temps a; exposent symptmes l'homme dtrs limit'* Les pr boratoirc i ' rences sont qu'en ce rp tiques, le t Itrovoqucr es volaille ingrer p. la suite risques po'. agressive : * Trailui: 5630 J 3 0 0 2 S 5 'c 8 0 Z 9 e t 'M O a Toxicity and hazards cf auxin herbicides 55 underestimated. Deaths of bees have occurred following spraying of ctar-producing plants wlicn in flower. It is possible tiiat simitar Hazards cxisrro'r~oth'ei,'iieLtai-ij.ediiiU Imdcls. Vv'Here othtf lim;tU have been studied, increases mpopulation have been noted more often than decreases. With some~5phftt5 this has been associated with rilducllun in numbers of predators~No effects have been noted in studies of populations ot soil artnropods. increased activity by nematbtlcs has been associated'~with the favourable conditions for invasion of ihe host plant created by auxin induced teratomlS and uQjliT efieels. Toxicity~td~fish rind~ofher aquatic organisms has been shown to depend on the formulation of the compound used. In general, toxicity to fish is low and herbicidal applications to water no not present a severe direct hazard. The dangers of secondary effects must be assessed m relation to other methods of control and the environmental Chat? actenstics-of..the water body to be trpatpd <tnmP agnafjc organisms other thanfish mav be directly affected at dose rates not very much in excess of those recommended for herbicidal use in water. ' /t i i Rsum * it Sont discuts les toxicits et les dangers, pour l'homme, les animaux domestiques et de laboratoire, la vie sauvage, les abeilles et autres 1l insectes, la faune du sol et les poissons, des composs suivants: MCPA; MCPB; 2.4.D.; 2.4.DB; 2.4.5.T; mecoprop et 2.3.6.TBA. Chez l'homme, un seul cas authentifi de mort par intoxication a t relev dan; la bibliographie de l'Europe occidentale et de l'Amrique du Nord. De meme, les cas authentiques d'eifets sublthaux sont trs rares. Nan i moins, des sujets affects l'cpandage de ces produits se plaignent, de ? i >*ii i temps autre, particulirement lorsque les conditions de dispersion exposent une inhalation excessive, de vertiges passagers et d'autres symptmes et se sentent malades. L'ventualit d'absorption par l'homme de doses toxiques avec les aliments, le lait et l'eau semble trs limite. Les preuves de toxicit sur une grande varit d'animaux de la 1 boratoire et d'animaux domestiques tendent montrer que les diff rences sont beaucoup plus grandes en ce qui concerne les formulations ll qu'en ce qui concerne les matires actives. Parmi les animaux domes tiques, le chien parait tre le plus sensible. Les doses susceptibles de irovoquer des effets de toxicit aige chez les animaux de ferme et ft es volailles sont suprieures celles que ces animaux pourraient ingrer partir des plantes traites ou de l'eau ventuellement pollue la suite des applications en Agriculture. L'accent est mis sur les t risques pour le btail des formulations concentres, mais la nature agressive et le mauvais got de ces formulations rendent trs impro- f * Traduit .par R. Tauhaut. ii Tf 00 * 0 2 6 5 7 / i.i ,,W4 'A 56 J .M .W at bable l'ingestion d'une dose toxique par un animal normal. Les risques de toxicit chronique sont galement trs faibles, car des doses leves doivent tre rptes, non seulement pendant des semaines mais pendant des mois, pour pouvoir provoquer des symptmes graves d'empoisonnement. Chez beaucoup d'animaux, une forte proportion des matires actives est excrte dans l'urine dans les 24 48 heures qui suivent l'ingestion. Des risques secondaires pour les animaux peuvent rsulter de modifications dans les constituants chimiques des vgtaux consomms; par exemple, des effets toxiques ont t associs des augmentations temporaires des taux de nitrates de certaines plantes conscutives aux traitements. Il convient de respecter les recommanda tions relatives l'exclusion du btail des pturages traits pendant une certaine priode aprs le traitement. Des dangers pour la vie sauvage peuvent rsulter de l'usage des herbicides, mais ils peuvent galement tre causs par d'autres varia tions dans les pratiques agricoles. Les effets des herbicides sur l'habitat sont graves pour la vie sauvage et ne doivent pas tre sous-estims. Des morts se sont produites chez les abeilles la suite de traitements de plantes mellifres l'poque de la floraison. Il est possible que des risques analogues existent pour les insectes qui se nourrissent du nectar des fleurs. Les tudes relatives d'autres insectes ont rvl des aug mentations de populations plus frquemment que des diminutions. Dans le cas de quelques especes de pucerons, cet accroissement tait associ une rduction du nombre des prdateurs. Aucun effet n'a t observ dans les tudes sur les populations d'arthropudes du sol. L'accroissement d'activit des nmatodes tait li aux conditions favo rables pour l'invasion des plantes htes cres par les tratomes et autres effets ds aux auxines. La toxicit pour les poissons et les autres organismes aquatiques s'est rvle dpendre du type de formulation utilise pour l'pandage de la matire active. En gnral, la toxicit pour les poissons est basse et les applications d'herbicides l'eau ne prsentent pas de risques directs graves. Les risques d'effets se condaires doivent tre valus en liaison avec d'autres mthodes de lutte et les caractres de l'environnement des eaux traiter. Quelques organismes aquatiques autres que les poissons peuvent tre directement affects des doses ne dpassant pas beaucoup celles recommandes pour l'emploi des herbicides dans les eaux. Zusammenfassung * Toxizitt und Gefhrdung bei Mensch, Haus- und Laboratoriums tieren, Wild, Bienen und anderen Insekten. Bodentieren und Fischen durch MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, Mecoprop und 2,3,6-TBA * bersetzt von F. Bau. o O CD c: roo CD werde: eines nordar. letlulc Zeit K andere aSupfradye-:: dieser reiche:: To.\ mit ci: neigen zwischi deren . toxisch, als sic Wasser Die Ci hervort verdm der Au. nischcn liehe 1k lieh, un wird ei; Stundet Tieren . von Na zitt bc gewisse, die Fcr einige ? Eine Ilerbizit in der Standor Tod vor. Bilanzen fr Nek! wurde i Bei Blat der Bau! von Pop durch N Befall d. 00026ar n 5632 . - fcv * Les risTit-s oses clcvecs mines mais imcs graves aporlion des lieures qui m x peuvent les vgtaux ocis des ines plantes commanda>cndant une l'usage des mtres variasur l'habitat ous-estimes. traitements blc que des it du nectar l des aug3im!" 'tions. cm tait jn effet n'a les du sol. '.'tiens favoratornes et ?t les autres formulation ,la toxicit ; l'eau ne d'effets se* thodes de Quelques directement Dmmandes jratoriumsnl P eben 2,3,6-TBA - 4 < . t..;ALi<& DOW436210 Toxicity and haznrds of auxin hcrbicidcs 57 werden diskutiert. Beim Menschen wurde nur ein verbrgtes Beispiel eines Todesfalles infolge Vergiftung in der westeuropischen und nordamerikanischen Literatur berichtet Verbrgte Flle von sublethalen Effekten sind auch sehr selten. Jedoch werden von Zeit zu Zeit Klagen ber vorbergehendes Schwindelgcfhl, belkeit und andere Symptome von Arbeitern vorgebracht, die in der Anwendung auf dem Felde ttig sind, besonders unter Bedingungen bermssiger Spray-Inhalation. Die Mglichkeit fr den Menschen, toxische Dosen dieser Verbindungen in Lebensmitteln, Milch oder Wasser zu er reichen, scheint sehr gering zu sein. Torizittstestc bei einer Heilte von Laboratoriums- und Haustieren mit einem weiten Bereich von Formulierungen der Verbindungen neigen dazu, grssere Differenzen innerhalb der Formulierungen als zwischen den Verbindungen zu zeigen. Von den untersuchten Haus tieren scheinen Hunde empfindlicher zu sein als andere. Die akuttoxischen Dosen der Verbindungenfr Vieh und Geflgel warenhher als sie die Tiere normalerweise aus behandelten Pflanzen oder aus Wasser nach Anwendung in der Landwirtschaft aufnehmen knnen. Die Gefhrdung des Viehbestandes durch konzentriertes Material wird hervorgehoben, aber die agressive und widerwrtige Natur der un verdnnten Chemikalien wrde wahrscheinlich jedes normale Tier von der Aufnahme einer toxischen Dosis abhalten. Die Gefahr der chro nischen Toxizitt erwies sich auch als sehr gering und eine kontinuier liche hohe Dosierung eher ber Monate als ber Wochen ist erforder lich, umschwere Vergiftungssymptome zu erzeugen. Bei vielen Tieren wird ein hoher Anteil der aktiven chemischen Steile im Hrn 24-43 Stunden nach der Aufnahme entleert Sekundre Gefhrdung bei Tieren als Ergebnis von Vernderungen der chemischen Bestandteile von Nahrungspflanzen kann Vorkommen; zum Beispiel war die Toxi zitt begleitet von einem vorbergehenden Anstieg des Nitratgehaltes gewisser Pflanzen nach dem Besprhen. Laufende Empfehlungen fr die Eamhaltung des Viehbestandes von der behandelten Weide fr einige Zeit nach der Behandlung sollten befolgt werden. Eine Gefhrdung fr Wild besteht durch die Anwendung von Herbiziden; sie ist jedoch ebenso verbunden mit anderen nderungen in der Landwirtschaftspraxis. Die Effekte der Herbizide auf dem Standort beim Wild sind ernst und sollten nicht unterschtzt werden. Todvon Bienen erfolgte nach demBesprhen von Nektar-erzengcnden Pflanzen in der Blte. Es ist mglich, dass eine hnliche Gefhrdung fr Ncktar-frcsscnde Insekten bestellt. Beim Studium anderer Insekten wurde ein Populationsanstieg hufiger als eine -abnahme festgestellt. Bei Blattlusen war(dics verbunden mit der Herabsetzung der Anzahl der Haubinsekten. Keine Effekte wurden festgestellt in Untersuchungen von Populationen von Bodenarthropoden. Eine gesteigerte Aktivitt durch Nematoden war verbunden mit gnstigen Bedingungen fr den Befall der Wirtspflanze, erzeugt durch Auxin-induzierte Teratome und I irlt r 1 fi i rf. i l J { ;/ i 5 ,o ry ? ^ 't --- ---------------- . 0 0 0 2 6 5 9 J * 4 I ( J 58 J. M. Way andere Effekte. Die Toxizitt fr Fische und andere Wasscrorganismcn zeigte me Abhngigkeit von der Formulierung der angewandten Vcrbindang. Imallgemeinen ist die Toxizitt fr bische gering und die Herbizifanwendung zum Wasser stellt keine schwere direkte Gefhr dung dar. Die Gefahren sekundrer Effekte sind in Beziehung zu setzen zd anderen Kontrollmethoden und zu den Umwelteigenschaftcn der zubehandelnden Wasserflche. Ausser den Fischen knnen gewisse Wasserarganismen direkt beeinflusst werden bei Dosisbetrgen, die nicht sehr weit ber den zur Herbizidamvendung im Wasser empfoh lenen liegen. References AABBBB-ABB-BBBBAA--endaaajorlaa--elvo--rdaealrulatkcs--rvcahdlonbecmhdff3irk41ciiAuDpmaiplRca,,ee,yolrsaninenniooeets8uf2lnotnndlsrjDe,esaaasu,segsar,,hr,Rmo1ilc,dkdsevydhachh,ntLnaetstkusJdC.hn,FBuh2.,g.shee,nl.satdBJl.n.oeLeoNyW(saLiSrFdraierati..bMWOTnJrJni1reBtm:bnmmitf5M,,siAh.ie.i-.Wai.Jtsis9Mc,,,.t:.,.cni4.JNSFeca.h.tn:,.:ch.cr.5IRhai:(.,-Bgilr1HohBtelPr.iTBrdinddWs.cIan7uEe:esEog0-.LDepnSr.i9.ph:eeo.U:.nescdE)dsnybfh5d:,si.ie.sllCe6x.uru.Afdhin:iaia,CoceC.ba2sbtheeeHoirbRD,0nhaerIJnRrr(bcc,e,ciirTdnh.5lesnto4.nELa)r,en.c,.ottt.ertaraheetpaoie.asstpnm-idelaineFLY.HdnesemedCtDoxi1ll(dMhdnretelracod.Vi.w1eiog5ibife.iceoW,waiccadsncorpTfre.9rc,iE.sKnenaceera:eborHiLketMnaeaa6sctorlettstsob,.6.mSe,epMyrsltGe.irp.Chg.as.1xsaaav9bohtp.o.DEdNEirJuMSyci)sitrosiindoalPaXlaSs.7nafct.antinSraa.ecoennvuf(syt.tftlccno,iIiACgcw.bgam1niaamnndienaiXGwdeEhvCweo2nPisnenl9sobegnu,iblriaaitb.l.nu3lddsgrtfatene6e:eimadidtanki.kcerswor9.nniehshtfpWSa1nTudnP.u2srw.aolJ:td.ienhim,7,)iit.dasthona,n.nerkC(caye.zraEe4iuJ,)w(cyxklpg1.doBaahbtrie.e:D.a-1caOvflilsiu3.tn9suntiD:ldfnaTlcnnuLenai9lvlAiae6n.eEsds6otoie8e.dgsnSoie6oiro2ceetc4glAsn7gl.fr4apya,esdxr.Jct.hrno5n.sefyRr,s)n.me..eWiheop,.gdn).A(a.c3Nsfods.PaI(ci.aBH1o.iZoSainolt7ManS1cflyntnd.aeif9nhnevoghelna1L,ect94onedrlwwiICdoaoe-2ain2lir3oios.a.5m.nsc-Ntd.zblrn,tai,t5wsoft(6us.i3a844.:rae.Ri2uABidmei.nWt3)kp,lzti7-r4s)nuP-62xwc.tTi.3BnArPDdup.pi:m3,gcdsud,-cfag,hsnprlLC4aea,tlriLon9.eoYderaieelgbrnaAa-3a7c3.sninificatlonDs(5npeocdXevwhrinc8g.Cepu:mz-nWhoirddpeiy1odr9(traXhkeinnSmelsl.sci1lsefoS-uon:y5ieaa-tdBtmyfoIk(sc9Eno(a4rtsfsul4pIl1mritA.uau1iol*cIgci6pbl-ino,hiNxon9tqdF.scbh9kductp4eIliglelpp4hhuioe5mo.e(uui6alns.oF)niesp1r5lelnltreucntdt3t.5g5gsaoYcoiaozzios9zi:xce)dy)r5hySArxdmer,iooonr6m.(v).ososd,dienyeEot1nirapina2tioaprn.csekeiatowt2ln91eihgc.)rnhtvicssrsnr2Ze.4da.6mitiicoagqhpeBdioPrln,9t.i2ntCuantiuAndlteibnrrgrius1ann)oesFceeapadtirc(.0nealosHirxsabbtncoia1lsh7hdotetAsscyueoy.pmiei9.ecphouNnb,ifnrugSm((Jgrh6iteosnbout1ns1lbrt.ocBr.e5maFeu.iewagto9i9o.zrPZtr)oencttrieSwiaAeit66yihas.2rriofotstmdghiocdoli4bie6tt4onlnisecthrrahc2ceano*)oe)6l1hnsiyn)..y--s.e.s.t-,f,,. (KMO). 'wg y y aijgi ,iw i CO 05 ro /ijvw'n, y.. > ii b'.A1. u. :: lytu.y, i: N: . T i.ii o A. A JJ Thr I. i:. i i i., O . fi ------- C m .!.. t<l ifi -KA. iu m .. \ C i,iif 1) \l CAAIIIl 1 llilln l. --. I1'. I! ' M ill*!' ( n ', r I ) \ \ I I 1C,V, . L i *ss .* s u it a i: l h \ n , J. T WVrd". ----------, a n d | dilfj 1 H a v iv , N. IH u a m , It S\lini-imr..* f a i r of Khwamh. t am id.,.' I- NC. K : snima!' -- T e n ii * '" rS.i anSd. . I. I l.jru: f i*Sn:, S. NE. W ------- l l c t l i 44 (10 I H lW IV , S i 2.4-1) . 1 m i. C . H Ww Z J H I M . J*. la lln li i 5634 *- 0*0 2 G C * ** - - j . anismen wandten ; m id dii' Geilir- hung zu !schaflen :gewisse gen, die empfoh- * 1. J. Zoul. jpulationx out. Proc. i Scottish Tp(o1u9r06le)v. ch er';' ,ry 1yiobcnut\r,y. ic2)-). ii9cug6hrc5wor)xe.hvlet'ua6-t, reirtbsgizeimdeenigirn. Sagorci.-, if BFeisrclihnemetabontent of nbicidcs as. Ann. N'ooa.t2h4a1y, Mut DOW 436212 Toxicity and hazards of auxin herbicides 59 ------DEEFFFCCDDDDDFFDDBBBC----erdaei----r--oryluaaoaaauuas----ktawuanrocpvsdbhlv--xcn----itndeeseukcssdiwc,taiaals4PTSATtS2c3sNsC,NHmWra1l3CBC,(s(ist,,yak,ooha,eisae,oatncirv141,c2,2hcc,n8oTachG,ostoiood,seEe4AFe,avnsateOiidn,Woiiaee90h9a,sCJnxr,leKBoecmmwnronence,o-,,..PhSS.obnnesi..,r((5b6idki.DS4nsonxefttaEd.n.mce.o,11DcddeN...itdMaira(T.RiA.W9R:63.n,RlrcBfic.daoZ,.,ocnfN1.sl99V-m..J:cttaACa.e)Bv,).i.niasDlMClg..oiW.hl....pNJ,eo9:d65pt1..hoc::Dec(d..:R7.7ici.s.e.U.rEraal:aES1r.ac6t,3.CE7,ngo3e1eaiC.oe,oo,mAbK:oeilSnS.TnkS:cnF-..9n0diJa1nl5)l)saa.2uogp.1W,Wvl3tchno.3doou.e:.c,do.ea,,.tt,'o.u6Bn7trn)H-nip2oPsp.:Li6,rfem9mermteJCsegc.TmmJfc,od4dvi3Pf3aa..d.etomo4r.7WYsaaaiT.auebl,u1g.an.oeeohe2)snve2r1ssieecltnlgsDnuC4.incEs.VBihuoinle.nCJppn3ede(Wd,0tnn.l(g(ecurdoa,t4dgyho.i1cbedltAlrst.r..11e0eeisB,onansStsgnr-aaltsoPhfrl29fi(ctste6w]W.99Lalo.poms-dH4Yen.,ibiuiuofyap.pW1npu.2iso46eunnem65leceNitm3l.oslnenedsHN.l:onp95.Schnd-annS2(lc.gkcd6ap99Rdre,Cdui.k.mhDn1a.gns5ttfcea5Ft.a)iiaGneWr)rdsroil.ssRcktolsEzL.ae9o(bat7n,ps.isoMotEureo(uoo1riVehf1hfcfnnsifychn6neu1doW).)rldsoo.dr.rrt6fsuir.net9ds..f,fs..e.pocttl7ieo9oSifeftc,7FteJgis.oMihsie6a.odohepos,seRf)RvrP6nPkaaacan7oe2amnhtl2ebc.tcf2.bhah7eaenhenr5p.rlaohni.smr1.eierictceniy)woeposp,Vfcicot.tedt).xime(;fyauS.t5rttrLrnadecpnowc:hhe.sfhl12.loyaaelecpM,iI.,1ne.so..egrbpi.Pvede4m1c9teatnEdstan3aec3h,GdxerE.nseoeaRek:Wih0cr1Y164etitatatf1oerefyes.onelsua:ei.es-.fSw6Bstle40ortir(lo,otanrr3wentxisrtdltneoo(vstePi1Waod)6au.tinrcD:acPnh5:c1yBds2eetmornc.Baunat9rne.aoetniyxe0aJd9ao8oolEtrgseAtdic(dt5lwrnAoxi.fSrgcne.or5s6i:Mscu1r-iotefcoc2vkgioehorlyt(.tihep,.:i6rAf(L9sclnshi.eo5Dr1lriti.u)febefs1rynlafh7cdris3Ws6er..naOeg,tW9rcniceao,9gimtakiheee5Clvts0b7ctntrieici65goh)2Blnmih6emvne6oLietif3iio.)eteh.4eacsoh60pdmcaesra6e,ona.:ewn.fetltNwb,udWifeis.6e.)draf)oolz3(betddtes.elirMTygfa.ioahgsi1.EJr1eJttc5nbaeneysnt.iouih.kqo(C.iu5e,9ioy7elcC.tni.Pn1idmresxucn24rtNlAnAoltpanreWrios9siratoiH.ir(abg,ac(1iteovtonecwbdh:olne4pm1a5fae1tyitPoeo(scxt)uieeeiatrte-pkh9dor7mhLc9s.Ntsc.refeliedeDsnetodsileoperc6oti)eer6toscpio.sdodklitt6eeri.g.re-lr2tdwpirth5t.:nbcoieaiPrn,obdtdVyrygCnh)cVewC)hkCsyidafefire.Ra}ai.vce.cc.oataoesEodsooAoin.enineheaitpepBdnftPa.f]dennndccftioghdpoesrean.Afscsrerfrot)eIcpuubrpnxe.sorrii.ihsM,nbsAlohgJ,olekmnttaotylMci.t6ep.aii.:imirenitnaroslpfamc.pcectrste1.eau3orioWisnci.bKiadoi.CssPmlmb,IteoddogrdncLstir.tFnS.i3re(o3cftin4teei.at"eieeecf.aiohboscAistt3inm4M1Pi.tnecdinohdfkutsinoeeeVMA7cdL85isddgieh.srPeeekpcdcheseiSh,eeacaasieiCarnoalpetLs1(((nsoCdld..taicocSRoetcng1m11akL.1CPsfeaticicoxWh.muoonPd9cd99c",sAMu).ihrifCepJnlhhtgcs6,155hcVpm4ieo.aertpaaa6iitivl3tea334erbm6ieo3nnthihnllfuoeutoSdeityr1lns))).i9td3nhgin-eeff:.tl..,....,.., re Y f : ) ,5 > 0002661 O O C C5 TCT CO 60 J. M .W ay 4 FF--FGGG--HILCGKHJHKKKSsohrbhitloruiohieeaulkiehaeo.--yiltplpaurbccicawzsdhneudbJanh,psptdTvBaAaQhrShescdn1I2WgP9cCcah,1ep,tkhnoseieannebn-Eeuncoicoeoyarniq0i,e3eeteru2rac.SDenutf-oC4drseccnumaseoeyrimsmnbsadsnc15scueftrUfg8ae,-,nceetah-7tcetntftbie,,ennc,.-e,t.tre,ia7azu.Deim,ieikn..suJhAlaPc-,3d2pr.iJnfcyiJn,(cd,tABoC(riDctnnc.ecatClir,S,Nuoi1Aof,i,nSil.e.1bSC:6,--(iadLtrLqdi4n,gtlan.(tan..btcCse3rv1.pCEk.oSder.a9ot:otepiaoC1ea,utn,sH.,d.itLdhowyh,Hn6e9na5.eepVM.6oR.tohs.cboSHlp,te9TdWlgn.sFi:lde23szsE6-.,h.sa0a2inn:ileeWfv5..J.bC4,NoTe.aid,sa,ecr.pi,u,n:3geCt7rtB.TeE4eanlt(n.,ang5rhAfB,rriil.ndndeaaL--iWur:Ea7AGd.ro(HLnnce-aTioones.de),bc)edonagen)EnaD1gDe,.lhe.ncHs.e.xgMnrco.pmaPh:.i.Hpxm)lei(deS,Brdl9rJhd.t.ctrioaec.a,nCt1i.o.Wteeagyueeispc..y.eieD6uFsionnhrmionRActr2,.ctCa9ndi2,oobHrCodEEtodTel0dlsccMn.haedCipaane.l,lgsm4lutcR.pie.Rl,.nfueinx.4..R.ek)saei.stbeneeoduno.t.8awEvi..sGtdIerop,fmDne.ilJJydv4ohEAsoD1eCWnfor5waoasn)t,a4sl..ayntpFlrNidsie1lese1ont.i.FWn-..ti:u.iftittspiiioi,apiolpeoCsrATd,eheauca,fsrFb.ofdnsasT.aBpBsCbYcSannvpy.omrr3ud)Ge.Dsclon.ann2g.gno5lrhiel.see2feaHtrial6fe.icnn1e7o.cdBsrJakcd0r,c.cenraesL,nBapa1l.aAi0hot1:.fl.edFo4CdohentA:,sdaodorp,cidlovlri.m5iJwreLF-fntlwoei.aVsrnCtc(eDifn.Uu1e.fs:(DenMWaRbed(,ll1PrnoMisDteDhda.d41.shaiSd51(n:dslh.)ec.atS9o-.crhanaL'a1tCers9'eiJs.9et.e4ai2eeAhiot:nEidnPaoei6.iozoGEt9tp.t(wsmi6soCalo.no5,i,rxsenGartssamdBdf1taf43.c:6Ca3nOteetf4ntmnaDrhib4foaahNCepripoa,-9na)cs0ht.lb5Beamofew.d)wottb)TDolco:n.Dgpoanzadi.h6d1ncuo.aieonm.nPt,lasemBncaicuio.ehbinnteecyci7rnr3o(is.reetlsCrrsifoueapnenfaareum3wrhdy)et1do1fotd)rsimJfhJTiydlhfsnlao.sg.av5n.rol1eLnc9.e.,o.e.eeahbeeidn2Sd6abcdfDTl3.n3an6ebdfsrCpsle1Lv1Ne,dttte1Loll:,ina2Ebds:242s2eiooipna.0ni2eC4heoeft(ice7dcido.s.3.e-,nxl)acfeilsxr,1,cS,4eyrohJn'Adh.oy.s2sfUfnkiIdeHC4itpm9.C-te5fsc.,Heiiaic5s4ni1(d2htcWuA:doc7tnlchi5Imrt8h1i-0.s0sNL1eeRuohacteaohdiCtcn,oahge6e9yr94F30icsn(ArhtnyteSiniliamne.fboar-me1hNv.ho1cs)oe5tadiecorndtlibasnesop.rcCaesae2((9laogf(ribt2Preamihgcacaloitoi11dflepretbr6c:ob13t4air)iaesrenlaroOamrypslobd9a9rso7ie.owir9il2no,rt.tpxnDg4oyreoclhocllfl.dt66usicW6p)stiuler2iylgaden6f-uyh(esrf(.ica.sc75eu6ahA(kem5tcPaesck,n1lbtedil,eua2h))gomRte1)gaUreotRtlh3so,e.d9u..2tee.ysh,neurepis.3iwsireax4W(e65edrin2laermnpoeBhapt(pnnliaFyAtn-d7.nh,.i4oprntxzethei4D1tslvepasiers.uaomdf.)iCeyratqcJoe-u9.iactpneySi.c(ixBJppdhar.5unicecsno15dlesht.AcH1ia.ircliaa0.teetc)fnn9,9coaehDni7ePaWfiDensosgi,maCm.cthmn2)mst6tctnt:rEtfrentag.ri.y2hhhelEatloenooss3mlcd.eeoiianbra1mNetiuTclnrldte)i.otpcrnrrNiEiaortao9yicah.rtgondoysooiifrbznSrfcbuulCfdtBweor.xpaxsaonbeeirlip(kosoSlCtcSta.dihaponr4ltCw,1oieriehchlwheiuinEcateosiioW-n,tpr9codeCeiyfeSuoetLternc(enn.pnhtaeflne6ner2dprihSiLooros,raetsuIblkoor.at64e4,roruagvxtnsnr.r4.naicexxnoota66)tpn)tttJa)oiheyofot:ro-,ez.ey.l...,.,.lrf.,- Lux, D. : Lynn, C. E , l2e.a4vJ5e-sTs.- Martin, J. ' Rl.ickwt Maxwell, 1' broad I. Mki.lanrv, : ----- .Coitl.linAs. ' MonaErnctoam, Cc' Nieldseicnlo, rKof.-. diclilori Palmnfiait,crJi..d Vct. Mi ------, and R. eidos or. P almoh-Jo n ! Zealand ------ Effect i hi* \ y Rawlihx,c i icf C. l Chcsapt RonrnTs, R. Poultry Robinson, A ris), ..a; growth*. Rowi:, V. K. and 2,-1 associati Reno, R. l.,, Savaw1g3iel,,d9Alif:e(..*: Smith, C . K o(1f 926,47-)D.- Springer, P. Pesticidi Stahlkii. L. levels ii, Sihacii, S , preparai Sinirixi.ANn. culture 3tli(e i?ninvo: 5636 0002662 rit. In1uhpoant tmai: i'1tt.e9GSMa3icxt)-,.. Aac-toirokner. n-oenUcxo-l lEyhdrr.o)-. ide to -(i.24.40-, berbi- i. V ichutz L.). logic rc. J. cnoxy cr to Vash, jilvex lonf., ns of ioxye.s Ptontral tune Toxicity and hazards of auxin herbicides ex .--NPPSMMMRRRSSSSSMPRRSLL----aapttamtu--houyiaooi-aaeosr-e-rarvnl-ldwibnwb-i-lkrx-nljalimtbaomdecninlP3dwB,Ptcb2lC,tZmopdEsEtralVaa,cgwellCtn(1chlc,hre,ieekaheic,aei1Di,snuros,fririer3dalhenosfeeisRvn,hae4cetraepc(JosRrolnaeeteG,nfl9dlusonc,a,tl)eVr,vtthe2Is,.eltosd.l,tec-.aGparwlo,bt.5l,,le.l6oad9nu.s,liJe,sl,A9hle,cekcilCJatd.m,nasnyrJ-o4SAMo2ird.,a7sJrtrL,Mc6n,Ki..tEewnRLTiWf.roRhs:d.oy:rea.,-pK.,andPa)ioeGrv.EA(a0dbo,.o4s.RD.Pf-.tnn.e.,,,.bele,.gK1T,,.eNeLHeit..(perne.p)SsK,F..Sla.r,adru.aPieeMJE51,d9,aDslan..Aar..oCBhclsoM.inokErn..d:oGF:dg.,harMEa-e:nn9n4aoCth.acicS:n..oAewad.T'er,d,,wnuy.ando.AdPdn.A6sPs.n9xe.n.Iamc.lTco;RnCav:leends,KcidaienH7Fpeo3Jd)iSitags.aeJnah]fnnideteKRct.teate.Mneph.sssN6)xtat.ar.Ea7:ilynrhatqM:.ousnoOGyoT.ohitene..erdi.b.c:,BynpkditpwepFuftaorow.W.c-crlatatA,ceoMh.i.PaeCdfl*CREttEh.dbse7ml.ofatnitnaitSthieBifc,6lse.EaiihDBadpeeAic0Pft.tie.iGbrnecdccNepeohcN,d1hisR.fnecanedupehps3s.AoCtrtitctiioBt.eeiGoom4l.rsfaudegssdesPJdoi.l.erl.E1accim,nI)aGcna..t3Vabfat.sHwgirewIehuf5e.(uat.ooa.onCFiu8otb,oro:2arJasC1aaatn0bnttsrtfRbnrfaxyci.nWsfs.8.lWilfFuotwan,remr3rceTtee9ogl,aehicriliddemioh2oinnnsEoocodse9cmtubR3eHn5ni(lbcohc.ihe,mdiakeg:td(uidt.flla1na8sniprw14n3oa.l7riefet,2ir.:iaer1nenAtoehenJmcy9a.9ottas-sw,ASoye)h,Jrdir9a.idotrsBfaTDIdt(e4:h:l.(oclm6tbmoh.u4:ooau?nknEMne2lwmss5df1oiMo(-Jufez5nul.Tei8CWtxfStnAn.ihJv:naeDd1,lP7f9ndeexylels:Fortso)54'ei.ecNhsbnrg.lueleoe9Sisbnngi)c6ecd:T..e.d.olaeyuoia-c2sewaeddmin.ootSso5ii(lnNBtes3recDr.e(htor-dsngmcJANaa,Hi.ottd1flgmiwei9tb1o4sia)edmhiral..o.ienangmoc:fdWac9:Y)..inonoad)ttee-ont9dy(grosad:sNeVeinehaD.ll6eZ.raonNe-Ienc.C2R6datdTiisVtnnrrebnHbescnnf.i3eieneen7Sr2eCy0DtAaoehoethia9fciceseowdatwa)qicsctaoc,eceEp):spaannon1.ereaoev.e1ocic:liruli.ociefa(eznleinalntc,nnd2.kxtytdaya1ffN1ltRarfNaenfaieeTandmotieftd2aldinneoau2o54rinlts9sof.drucicebdiofcnialoc4th,n.l,Wnafnsa2me:4)tagmiit5notaseoifhstoorpietrfysddegp7e,7pedJvCSubtxcya6cpe.oiFmslia(oif.4il.a2.4hoeni2arcoi3ttr.ora.)1cofoklrltacera4(t,i9am(eaie.AeP(293f.caniewn49co.tuoo1LdtCsJldP,cyHatso3m,uapeihffk.,gic6lSisf9t,ne.sI5(ao1(ooo.snclt4rso,eesIirACv,o5ti1aa5s1dnto-nl2Ahgr.lnSeSynr1fcc1iopcnaT)t999oNl.pdNti,cea(c50eryRd9..pl4aocncod1,5wfim)6fun.oteazsAr(J,ro6iaapd)r-ih.bnuedA9attnIa.0ncfd4stoDl.l0(7e3aiile6sfe9rln)t5otoacldhci)1)mptfeiC-1r2eusie52etefiws.tr6g.snnoao26ooiihd9an9uosd0ogd22hctoselsin4il0)Mnripsheef6xnn.(enlietdr0ma.lci.otafoo)se1r.wdi-1r(ggEe(zwhdL,lpo.cootcrwsassb1pyp19lniae)5Ihmeciaiocnipttfgeccac6,.9o7ni6tr9trtfueie3ouaneahirynihnnaseed6at,6:cnpt0M5daaol,lusdtdawtn.ogtts2e.ttr4e3yete)4ioevfissmkoiar1ptilPnoe.aaos3)Nasfa)neii3iantwc9sdne.hnnoliaa(.g(nio9r.epa-klu2t9rn.1sa.iagbgnin.eeoCuenpoltm(,oJp9(deroVdbi4llomlSuCdl1O(.fasctnhbaJis6idiraoou-Ivn.nsnc.it9tctuaieynxs4A0bf9oidriiredia(2h6rogcooetppfmk1i)eoNesH6otsm,taozen4on22.ninta,lesr4praSl0oogaerarebsw),,aiaynoet-dlr441lngcn.ndowri)tsDiittrrs)sdnyco:i:k-6.--noe.aos.ft..., / $ii o <i *i 1 ti j Ii 5637 i i 0 0 0 2 6 6 436214 62 J.M .W ay VSWWWWWWWWW--wa-aaaehiiio-lalllu-byotllnllllsdp8nn3(e(t(ci,widC,eakisirs11e1tfeortieiaa5tfooertfaJhetesoesc888ansoer,m,mB.lnndpes1,,56a5asctr4AhCdeC,,,aasstttMt0450dSC7yseBno,r,,Cio.oC))6)JS,ad,:rdt.d.S....pn.,M.c,ot..:er8EeioR.Lf.WoEoRfon(:A-.1JNM.f.1,cne.Bin9t...5P:tnia.y,s:.9ttp4ChfK.Iieatefaa:i5nT.4(E.eein.(eIclW,s.nn1,na6l1n:rostvt1SiJdt9Pi(t)x9duclao.1a.doeoPW1.a6ouisni6x0WndcmKdrfor9ae8cMbd4ikoeseclche6ds)le(l.)sclceia.uoruoo14.dpcEetr(dCtgtofsgheea9)iuEri.ot..efenaistoo5raccpifdnfiear.SsnroeHn0daofalsnsm,ehcnWlsdbu)nr..c,tA..spa)aJeoetsetra:e.qCnsasePnwbfo(nefturiodal1rlWFeitod:danatefr9calo.sefniaTetrtneDn6mnitstt2shh1Nevdoef5i,.deo1ePohena4ta)eanJmrt,crf-e.ar:weebladcatotr3hfnuhoitciEbbnmdf1dhcCe.soenloitif7hredteadecleoftbabV.Irentedtnnorsoi(cccstadceof1,e1rslrot..xoisaf6on,9odntaphpm,opP2:sf6etprnlahkfsr,^a2srp3T.nd4oe.syocn3l)d-sunlhoedec1tP.dibdoMsesnnld7ior(v..oxatcso71efes.notyJnhPc.tfx9h(.akohlfrw1.Oo6eceCgo,tA9air7e7cewrcNsoo4fn6sattet).eiinsphetsds.2d.croohSschhn,s)f.coC.e.B2sa.ee2uSnAiD:,eddrnnO4r,otn4isog.avd,Sxftt5-crklsiryMoDOoi-kramoWcamThonhlx.tecean.asofeeedWArlpmoollJtbe.mArinrg.ediceaedSlcrccyean4:.7ioaaakCdd1t6,lclCdoBek4oe,oi.P4nshdagsC,ln.64raleSittoooc3c1o4mrhoIdncaiokn835nned.l.l- O 0 1CO o; to C71 . i`Ji*- III.. IaDn)r!srC.r. b) S. c) I) J) n 111. 0|X'i. !V. \. ab)) AI*;- c ) S! de)) CK! f) D; VI. C rac: VII. Discu VIII. Eluti' IX. T j t o Summary .. ZlRU'en'fs.eiuirmnemnccen.sf...i. aiimitriTi>vh"aie`r!iiopir; Kuhiimml. t 5638 ** Ur*- Mitlillrpoft, 0002664 U 5639 r /. /). i'jo t / n fU K u * \{-/H 7//' r:*u s. / / ' " ,/f / y sh -? * * / / 'a f f A / / y - t - ' j / C /* ,. 1 1 4. , i `i - c : <A y~ -W v.\ f/y /jo 70-0152. Zink, P. (Institul fucr Gcrichtlichc Medi/in und ; Kriminalislik, Univcrsitact lirh-agfii-Nucrnbcrg, Univmitacts- ofi ttrassc 22, 8520 lirlangcn, Germany). Idcntidcation of various hcrbicidal chlorinated phcno^y-carboxylic acids by means mas* spectrography. Arch. Toxiknl 25(1): 1-4; IW J. (.5 I references) (German) O Cn cr. CD O i Two cases o f poisoning described in the literature I (Gcldmachcr-vnn Mallinckrodt and l.aulcnhach. this journal. ; 21: 261; l% 6 ) served as the stimulus to assess the possibilities ! o f identifying chlorinated phcr.oxycaibuxylie acids by mass . spccuography since the presence o f very small quantities o f the substances made analysis difficult. Comparative \pcvin*- grams were prepared, using the Cll 4 (AtlasAYcrke MA I J n uv. spcetiom ctci, for MCI*A (2-mcjjiy M-clilunt>licnux\ jcctrc -- "" ^ rv^' - a c i d ) , M il'll (yaHi/rM^4-ehimiiO nieihylplivini\y) bulviie _ iCt-u' * ncjd,^liCP (n^hq-(^Quti-2d]LCiliyhieuoxy^:pippionjc^ 8cid)f 2,4T)/(2,4-dicltloruphcnoxyacciic_acid), 2,4-DP (nipha-. <1 1 (;M-dT^TTo!riipIicnoxy)propioj]ic acid) and 2,4,5-T (2,4,5tridildrophcnoxyacclic.acid). The highest peak values arc ^ +t r taGulaTclThi mass units and on the basis o f 1007i. The number o f carbon atoms in each compound was another differentiating characteristic and this could he calculated for all the com pounds from the molecular peak groups, which were high for th e mass spectra o f all the substances. In the two cases o f intoxication cited above, liver and urine were (he specimens analyzed. A crystalline substance was obtained from the urine extract (acidic medium with ether and subsequent vacuum sublimation in tem perature gradients after Schmidt's method) w hich precipitated four insignificantly separated fractions in the sublimation lube on the sublimation layer. The fractions analyzed into a mixture of 2,4-D, MCI'A and M-'PU, a little 2,4 ,5-T and traces c f 2,4-DP; each fraction showed the same m ixture relationships. The liver extract (after Valov) yielded a greasy residue. Mass spcclrograpiiically this proved to be contam inated with a chlorine-free compound. Small am ounts o f 2,4-li were identified by the molecular peak group at 220 w ith the typical 2-diluro distribution as well as peak groups o f . the main fractional part at 162; other chlorinated carbonic ccids were not found. Tims, the analysis showed the com pound to be pure 2,4,1). 5640 0c04S53  *I *I * V. . . I . V s i *> f : *. f * I f I * ^ w 4 i ! ( 44 t I L t I vl . s ' It* 'iv . *- \ ' *. *1**h ^ !, (*ni\ Ai if t \. r ...I .. ,C I !% !( I r ip 4 ' W V t V> I I I I I .' 4 4 > , l i i i t . % 1 4 4 4 I v; to preserve ms r-ecs tfu.v. .criiu- Oi .OoCi'.ILlv^CS, The only salvation for the bceheep in.; industry i:; for each and everv beeireencr to been hammering home, especially to nor city fathers, die in> iiOriar.ce of live honeybee and the deva- iiCvvn *0 . \ Z. C** >v 4t I v V >1 I I . , 1 4I t *' * . . t I SI' i ,Cvr *4* V*Vf % v^ * vh.'.id OCe.S O f demie ol encephalitis and. i ashed : ni air.. v'iji.it ci^i.'iinnlcs .in api*. !e and u*s | fMiiiii^ ICiiiiA r, ^ .. s v*ne ea i thought to myseif as 1 watched plane ioavc the round that possi many of these expensive protjr. have been sold on fear, and icar c rather than on their actual need. .j V ~ ., >. k * V 4 4 * 7 ' >v k*`wi. -- * 4 ?: 3 44 ^ ' t-. Vs n fi i g J 'g ' U g A wg 'J . .T;ihv.Aiwvi s:j;: *VJ VM l\5 OD O W COOPi-KATlVlS studios with ihe * A r i z o n a Ag4*ricultural Ex *ner invert: Nation, Tucson, p l a n t physiologist iKovaru I.. scn4 .*'i O .J , >l..l.l.l..l 1I, i, :n:<:ic:ty of Mo 'ton and C\ilor.'ioioSists O .'jMoill*1 * \ * * O a' J** > . i Robert4 4 S 4 /vis. y tested l'iT. die 'i\ **ides 4 U . V / * .. \ 'l<b e e s iinicr cor.tr 44 V Crv conditions. They fed herbicides at concent rations v' (), id. 100 and 1,000 parts per rr.il- .i.r:i (ppm) in a 60-per cent solution of rue rose#<i*s1IyVru>wp to newly emerged levtls are much honeyhigher .mm normally 1CSS ir. crons. UA.NlCwC-%i 1I*C'li*i*.U'.' I ;Vi<.ojO lila O were: weed hexafluarate, cacodylic acid, and cr thali. Moderately toxic at 10 ppm: P ciuat, MA A, MS MA, OS ivi A, and i 4. I*iUvil. UIt Vi Arn[nc__sa]ts c i _2.4 - 0 and 2,4,: cster of_2,4,5-T, ami potassintTL-sili ieioram apnlicd in water did not creasc bee mortai iiy. li ut an esjer 2.4,S - T .and an oster of sjjvcx anp in a diesel oil carrier causcd"hih n tality on the first day after treairm Ah becs sprayed with paraeuar, ca dylic acid, and Ivi SM A in water c. within 12 uays. i; w**N. ioOJrl.W-^t**wvarxhIcr , at 2 , all 4-D c . oncentrati r/cioram. ons. Si;2.4.5:X >1..* A . , '2-cn Io roothanei mospnor.ie acid, . J ' 7 "1'ii//a rl "iI W' .v; toxic at 100 ano i , 0 0 0 i.i. P ar. cpiii., .iS.>ii'v, OSV / v, While the ili L L ilw ^'vd u ^I 41Akii ;y4. 4I 4* W M4. V proved toxic to honeybees ini. i l l e s e periments are hazardous at rates cuired tor weed control GLAOirVGS IN ot:e C U L T I CG04ao3 5841 DOW 758648 5642 \o<\ y # `DOW 979350 r \ r' l.uol 5'Z'f *!; T/r- !,' ^ i f r' r- un> x~ The Analysis of 2,4-D Acid for 2,3,7,8-Tetrachlorodibenzo-p-dioxin Content Dow Lot No. 091500 091530 091630 091640 PPM Dioxin Run 1 - - ........ Run 2 Run 3 ----------------------------------------------------------------- *- ro ^ W <1 only enough sample for one run <1 <1 <1 <1 <1 ` <1 <1 <1 Recovery studies indicate about 70% at the 2 and 4 part per million level. The above results are based on the difference in response between a spiked and unspiked sample at the 1 ppm level. Peak area was used in the calculations. x W.B.C. 4/13/70 r 0G05736 * 5643 DOW 979351 1 r DIOXINS ASSAY IN 2,4-D and SILVEX The analytical method for 2,3,7,8-tetrachlorodibenzo-pdioxins does not work with 2,4-D or silvex. However, we have bioassayed these compounds for chloracne response in the rabbit ear test and found them negative. (< 1 p p m o f T C D B D ) S e e a t t a c h e d l e t t e r . J C00573? DOW 979352 3 CROP RIDER AMINE 2,4,5-T Diamond Alkali Company Contains 4 lb 2,4,5-T/gallon T r i e t h y l a m i n e s a l t o f 2 , 4 , 5 - T ............ 57% (:7.-.. /.-/ ;i'U*-"- \ 5645 CG0S733 /- 1 THE D O W CHMICAL C O M P A N Y MIDLAND Ap ri l 13, 1970 DOW 979353 G. E. L y n n Ag. Products 9008 Building RESULTS OF CHLORACNE STUDIES CONDUCTED ON ASSORTED 2,4-D, 2,4,5-T, SILVEX SAMPLES A N D THEIR ESTERS, ORANGE A N D DIAMOND'S CROP RIDER AMINE 4T-2. A s p e r o u r t e l e p h o n e c o n v e r s a t i o n o n A p r i l 1, 1 9 7 0 h e r e is a summary of results of all chloracne studies recently com pl et ed on assorted 2,4-D, 2, 4,5-T, Silvex Samples and their Esters, Orange and Diamond's Crop Rider Amine 4T-2. Chloracne T-Number 2578 2586 2587 2588 2589 2590 26t)l 2609 Name 2,4,5-T 2,4,5-T 2,4,5-T . 2,4,5-T 2 , 4 , 5-T 2,4,5-T 2,4,5-T 2,4,5-T 2611 2,4,5-T 2612 2,4,5-T 2602 2603 2604 2605 2606 2613 2669 2670 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2.4- D 2.4- d 2,4-D Reference Number 120449 120349 120369 120419 120459 120479 120070 Butyl Ester 60/40 PPI-93-3 Iso Octyl Ester PPI-93-5 D o w a n o l E s t e r (PiB) PPI-93-6 Lot # 130667 '. Lot # 90530 Lot # 90540 Lot # 90670 Lot # 90970 Lot # 94847 ` Butyl Ester 60/40 ASI Iso Octyl Ester AS-7 2/5/70 Iso Octyl Ester DE 20 A 2/6/70 Chloracne Response Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative 5646 C05733 5847 .oil (-S e e .. SO) 'i)2Vi5. C h a n g e a in e h e sn . c o m p a . o i m o la s d e p e n d ia g b 'c SjcI oil y e a ? In v/ada o n g a r b ee s w e r e .procs: oO ^ 75SSS9 V>j) C 004E62 H8 -- P IL A M ? \ MAT, 9Q143G T o s k k y a a d h a z a r d G to m a n , d o m o s ik a n im a ls , & w ildlife from Gome com m only u sed aunin h erb icid es. W c John M ichael (P lonks W ood E xp. S ta ., 'H untingdon, E ngl R e sid u e R e i\ 1969, 2 6 , 3 7 -6 2 (E n g). A uxin -type herbicides are review ed, w ith som e possibly new interpretations. R epo of toxicity of th e I.in m an m ain ly refer to accidental poisoning children. It seem s probable th at toxic hazards from residues 2 in foods are v ery sm a ll. A cu te and chronic to x icities report in an im als h a v e been based on a m ts, o f I w ell in excess of t l ats. likely to be eaten as pasturage or other food. Howevc it is possible th a t application of 1 to nitrate-accum ulating plat increases th e hazard of n itra te poisoning in herbivorous anim a A p p lication s of l to p lan ts in flow er can cau se a real hazard to be (A pis- m e ilife ra ) and possibly other nectar-feeding insects. F s oil anim als and Ashes th e hazards of I are low , b u t vary w i the form ulation ap p lied . O nly 1 au th en ticated fa ta lity in m from th e action of X has so far been reported.' 102 references. 9Q144t HefBteSfiGS u n 3 m i l b io lo g y . G o rzcla k , Andrz* S yh va n 1969, 113(3), 4 3 -8 (P o l). A review indicates th a t a n of herbicides used in w eed con trol, includ in g triazin es, cldorinat fatty acids and phenoxyacetic acids, and chlorates, cause d turbances in soil m icroorganism s, esp . in forests w hich arc leo disturbed b y hum an a ctiv ities. A lthough herbicides w ere al i. d b ito r y t o so il o rg a n ism s u n d e r c o n d itio n s o f c u lt iv a t io n , it w concluded th a t th e effects w ere negligible and com p letely d appeared during th e year follow ing a p p lic a tio n .^ ^ j t K lenha 90145u B inding of sa lts and esters of 2 ,4 -D , 4-chlorom eth vD h en oxvacetic acid an d 2 .4 .5 -T in soil and natural sorb en t J s 1 ; j O . <6 levels administered, did not result in signs of cholinesterase inhibition. Caroaryl administered daily at 300 rcg/kg to the guinea pig from day 11 to 20 produced a maternal mortality;of 3<# and fetal mortality of 17.5-2, but no terata were observe-.:. After the fetuses were cleared and stained, examination of the skeleton disclosed ;. bone defects appearing mostly in the cervical vertebrae. When carbary1 adminis tration was repeated as a single treatment on selected days between day 11 and 20, terata were found only in seme of the litters treated on days 12 and 16 of gestat ion. The teratogenicity of DM20 in the hamster at 1 mg/100 g body weight-' was con firmed. Thiram was teratogenic when suspended'' in CMC at 250 mg/kg; v;hen it was dissolved in DMS0. the teratogenicity was additive or possibly more than additive.1:.' When DM30 was used as a solvent for disulfiram, the-high levels of disulfiram were more teratogenic than v/as DMS0.alone. Disulfiram as a CMC. suspension vxao not teratogenic. Ref: Health .Aspects of .Pesticides, Vol. `3, Ho. 5, Kay, 1970. 2. A.5-T CAKCELUTIOMS - U.S.A v;^vfi|drv/Tv;r':-'': 'In'Jfiid-April the Pesticide Regulation Division of U2DA suspended the use & all formulations of 2,A,5-T for uses in lakes, ponds or on ditch banks and ' suspended liquid foiralations for use .around the home, recreation areas, and' -y .similar sites. ; '.-V ' On Kay 1 cancellation of all granular .formulations for use around the home, recreation areas, and similar sites, and all uses of the chemical on food crops intended for human consumption v/as announced. Cancellation or suspension v/as .based on new research information which show 2,A,5-1 to'cause abnormal rtovolopr.er.t inunborn mice. The Curgeon General of the Department of Health, Education and 'Welfare says that exposure to this herbicide may present an imminent hazard to women of child bearing age. These actions do not eliminate registered use ,f 2,A,5-T for control of weeds and brush on range, pasture and forests or on rightof-ways ana other non-agrlcultural land. ' Ho cancellation of uses have bec-nr nr.-junced for 2,A-D or 2,A ,5-TP(silvex) which are used in many lawn formulation: . Kef: Chemicals Pesticides Program, Vol. C, Ho. 2, Va y 16, 1970. .1 ddt ma y i?rrB-.y?J5 with p a ii; m edicatioh. acco?'.diho to hCrALE, 3 HEALTH bULLrTlII. The discovery was made oy scientists of the Karolinsk Institute of Gv/ejon who found that the action of antipyrine,- a fever-pain medication, v/as interfered with by pesticides. According to the researchers, the amount of antipyrines In the blood decreased by half in about seven hours in these who were- exposed to : DiTT and other pesticides, v/hile the normal half-life of the anti-pain men Leal .on v/as about thirteen hours. It is believed that insecticides stimulate product:-..a : o-f enzymes in the liver which metasclize the drug more quickly. He:': mnvironmena Jan./reb. 1970. <> e. 1.*. ' ,v t'*-r*,~`'-*''V* "' V.' ' .*,,'.VI ;__nJT.*`' *1--:,r1 C.V'-j* *:' - ..... r > . - , . `'5649 . \ 0 6 t 0 4 8, '6 3 -af a - , " , . \ . ***. pt 'O-1 K'- </&? - M i< n lipidi'iirioIngy. Prcvciitiiin and Treatment J hy i>rgjno|>hiisplitirus insecticides and in reverse if acetylcholinesterase. Atropine protects the tin against aeetylcliolme. but u d im not reverse on o f acet> Icltohnestcrasc. Animals injected with dotes o f organnphosplionis insecticides liave T tieatment with PAM in combination with mba ami lliraki 1 Wilfotd <1*170) and an c i ,VrH j,Y: 1.17; 1*1711) support the use of injunction with atropine as an antidote against tale insecticide poisoning. u J. S.: Lee. II. II.: Kim, II. Y .:Il.n g . W. P.: Lee. of internal Medicine. College of Medicitic. Pusan Pinusa5n2. Kcoasreeas.). Oinical Tavluni obsenraiions A'antnw H a on parathion k k n C h a p a 'V70. 117 rcfcrenccsMKorean) ohservatnins were made in 52 cases of acute osicalKHt studied in the Department of Internal san University Hospital from Januaiy I in H ie totlnwing icsults were obtained. 1lie causes tng were: I ) suicidal attempt IMF.': 1. 2| mtiixica- jving 15**7 )and .1) ime case of intoxication due ig and one due to ingestion of contaminated ales were affected three times nsire often than ersus 25*7). Tlie peak incidence of poisoning in patients was between the ages of 211and 5*>. In onai prevalence, intoxication was mosi common r.especially in July and August. Dosesf (in 21 e were required for full atiopini/aiioii ( I 'i to x Jusesof 10 to 5K_5mg lor clinical tccovcry (X lo pine and 2-PA.M were applied Minulianeinisly in except one recovered clinically. Therapeutically, ry fiom parathion intoxicaiiiHi was achieved in >1pml 1.1 cases (.W 7) died. lAuilmr abstract j. I-..: Mass. H. (Klunk lilt 1leigcnurlslullc uud inkhcitcn. Frciv Umvcrsiiaci. Koetngswcg (5. I rmaiiy). E 605 putsuning in a herd of Berlin rztl. I 'm * hull 2S (J ). I2S-.1) . I<l7tl. (Hi .*rman) lent o f poisiHiiiig foiluwmg (lie use of !-. '>05 a skin spray lo control mange in a group of l`> J two hulls is dcscidicd. The toxicant as a 0.25'* spiavcd in Mich a way lhal the heads of the mangels ol foodiacks were `xposed. The dosage 4 ing paialhHin/kg body weight. Of ihv 21 were affccied. in tlie two hulls, symptoms .n the day of expoMire. One hull alter 10 days of vered: the other died after 4 days o f treatment, at was last 1 develop symptoms did so on the vposure day. The rest nt the cattle displayed otsninng vaiioiisly on pnslcxpoMiic djvs I. 2. 5. le symptoms ohscived were- massive sahvaiiou. tea. muscle tremois. gioaiung. noisy out- aakness ol the hindqiiarleis and paitial hJ retusal of feed, Body lempcutuie and pulse om u l taiigv. 01 the 17 allcctcd animals, live detl. licaliiH'nl consisto l of i.v atropine (50 lo 200 mg/anuiul) ami loxogoniii-Mcick (1500 to 17*0 mg/aiiiinall. This was jceoinpaiih'd hy symptomatic treatinenl. lo eouiiteraet great lluiu loss, iiiilusuin ol glucose and electrolytes were given to a total <>l 54 l/aninul. To cnunieiaxt diatrliea. tiK'dicalnm hy nasal intubation was applied. Other drugs were given to sustain tlie blood system and die liver Willi this treatment 12 animals ( I I cows and one hulli survived. Milk pioductNm drastically declined. Analysis of the hiam of smite die dead annuals sitowed choliiicslctase activny to he at die lowei Itiiui of detectability. In the urine ol' two animals. /Mittrophetiid. a tnetahohte ol I*. (i05. was ohscived. Tlie detailed symptoms of tlie illness, duration ol tieatment and ouieome in each of the 17 altcctcd aniinals i* tabulated. four annuals leeovered wuhoiii iremittent \ general discussant ol the iiatuieot orgarntplHtsphoriis uiioxiva lm and of the telalhiu between the antieliolincsieiase aetivilv of tlicse compounds and.'or then breakdown piodocts and die pallmgemc potccssand its symptoms is ptescnied. K / i. A' w i"t. / 714)709. Pinsent. I* J. N'.. I niK. J. (. (Dept, of Veterinary Medicine. U. ol Hristul. Ij i .: ford llmise. I.anglord. ttiislol. l-ngland). A case of possible 2,4-1) and 2.4JS-T poisoning in the burse. IV/. R er> m i 17(S ): 247. 117ll. TIk* |Mtssihiluy that die ingcslMin of nettles and herbage tlui liad been tteated or contaminated with a mixture of 2.4-1) and 2.4,5-T was irsponsihlc tor tlie illness and death ol several horses is considered. A mixture coni amine tlw above lierbi- cnles was used to destroy nvtlles around tlw cdgcol a pasture ctmtaiiutig six liorscs. Tlie wind earned die quay mlo die gru/.ing arc contaminating tlw water trough and a eousideiable quantity ol tlw licihagc. |-tmi of tlie Inn sc-. weie le-t-wed from the pasture alici 24 hr. The two mhci Imises icniamcd foi 12 days. They were both brouglil in when one ot them became ill. l-.arlv symptoms included depressmn. disiiitcre-i m drinking water. salivatHm. smacking of lips anti lautidice ol the mucous memhianes. Alutieiilaiy stasis devebnted with some abdominal i IisI c iish u i and pain. Ixtcpt loi small aiimuuls ol daik leees adherme to die innciuis mcintsiane die tectum was empty. Ntt leees weie passed flic symptoms also mcliiilcd m uscular tremors, patchy sweating, teguigitaiing foul-smelling stomach eoitteuls ami tlie ma-nuiy to swallow. Water which was taken in altet die jiimul developed a dmran hack out of the mouth, liodv temperature was normal but the pulse increased to m i mm I lie aiiiuul was destroyed alici 10 days of illness. Kcd ami wlute cells ws>re within the miimal range. SCOT values lud increased lo I '? mu-ml. while blood urea increased to 500 mg' l<M ml. lliere were 100 mg of proiviii/|00 ml ol urine. 1lie second iHtise developed smul.u symptoms and died wiihm 2-1 lu alter rer.iov-al tiom die pasture. Autopsy lindiugs included extensive lesions m die esophagus and stomach with dcgvnctaiive changes in tlie liver and kidnevs. Histological examination revealed a proliferation ol squamous epillieliuni o| the esophagus and stomasli wuh some inlcispciscd uieeiaied aicav I here was w e ie laity change in the livei and sonic evnleiiev* of gloineiular ami renal lubuljr damage. Ivvo udiet horses beeau.. ill hut survived. At this wilting, the exact cause ol die liluess and deaths is iimleteriuiued 196 C O O le S i . 5650 ',7-344. Gaiston, A. 17. (Dept. of Biology, Yale U., New o Mavcn, Conn.. 05520). PLbato, jpeospHe aiiid p d ife s. BmSdence 20(7): 405-10; 1970. (17 references) ro oo o Contemporary man is confronted with many problems concerning social unrest, pollution and overpopulation. Some o f the chemicals that man depends so much on to produce f high levels o f agricultural products are .having deleterious *7C O C O / , A h r \r v effects on man and his environment. Chlorinated hydro carbons like DDT, dieldrin and aldrin, once considered very useful, have now become menaces because of their persistence in the biosphere and their potential poisoning o f various kinds o f creatures. Present and future pesticides must be more thoroughly tested and then chosen and used with greater selectivity. Some of the more recently introduced herbicides H ve been shown to be potential herbicida! analogs o f DDT. ne o f the suspected chemicals is 4-amino-3,5,6-trichloropicolinic acid (picloram or Torfon). Under optimum k ^^ conditions in some soils, 20 t o . 50% o f applied picloram T disappeared after 467 days (Youngson et qL , 1967). On other soils low in moisture and poor in inorganic matter and air, only 3.3% o f the applied picloram disappeared in a similar period; It takes between 10,000 and 100,000 parts of exogenous carbon to oxidize one part o f Tordon (Tschirley, 1969). The dangers o f further use o f this herbicide are evident. Another chemical, 2,4-D, can also become a menace under certain-conditions by causing a massive increase in the nitrate r content o f pasture plants as to sicken animals eating these phnts (Stabler and Whitehead, 1950) and must be used very 5651 carefully. Pesticides fabricated around heavy metals, such as !ead and mercury, or other elements such as arsenic (trivalent) Jo not become completely detoxified once applied. The cresence o f rising Q uantities o f lead nnH m^rpnrv n tkn 7 M D 2 4 S --9 0G0486J. G et V orckon i environment lias caused growing concern in public health. S c ie n c e m u st not be misapplied. Thoughtless and . indiscriminate use of- chemicals such as 2,4-D, picloram, (8 ^jKH^and arsenical cacodylic acid by industry, the military . "and other groups and organizations has and may continue to ^ * cause ecological havoc. T he legal structure o f our government - ' as well as - th e :influence o f international committees and . commissions offer many ways to promote rational use and limited control o f our environment and must be employed before it is too late. 7 M 3 4 5 . Anonymous. Agriculture will continue to approve 2,4,5-T regnstetnon. Chem. Eng. News 48(16): 21; 1970. Despite a first round o f Senate hearings, Agriculture will continue to register uses for 2,4,5-T, with restrictions, if any, depending on the results o f tests and research currently under way. Ned Bayley, Director o f Science and Education for Agriculture, holds that there is insufficient evidence to suspend or cancel registration. In reference to the study by Bionetics Research Laboratories showing that 2,4,5-T induced birth defects in laboratory animals, it was stated that "preliminary" government studies indicated that the defects were due to contaminant dioxins rather`than to 2,4,5-T itself. . -, -- -- ------------------------------------- 5653 O W 256993 71-1433-9 Toxicology and Pharmacology \/ eymtBrtiCaheenMlytdhslefyaohdlnarmadBtlhoCabigtoMeipoeo.hnnfTaBihfnnoeCatrtoMmefi.entahdrdnee.isndSpmgeistmochritiienilovadpeprilhcoyeaat.xtenepnadettreitfmhumanetganiyttcshiedcweofismut,hneBgtChiftMrioooxpmaihcniadttnyhtaehotieerf sTSeldalatb(ls1pemsid5DRspoti7iittNtiahramnm1rhhhyiupvfvdels9i0oon1threoeDttegfgose3eeasericooitme7e2tse-pspeaennutfgteiotei1Tkrktrap.1teaAitrrlesoarorsaiMnahadnnelt4leeepcmoa.DmnlyortnrlldstgededdneT3lpenTtuSeiic(sniisDiUuicaisabtdd3n8mslnscihnneafhnaaolitonslrdesey..rT.oidtnregt2akscpeeaao,rneoftrssihatpenBrB2tddcsnettofqheltioaooiedtoccv2dfpirtoEivdacwiauehfoinifdeoonhanifapxe/odnrvnihrasee1rbnnlaeenetdiottipessibtidsereannyrscyrrrsntu6th.osleq,rtneowhgtvypeivcrapaiuinvlhwenuTmaicwawbeeweymrnoovlSahnaleLteeyeae.ieaa.efldnrecctoanisraloclayvnsrafveetdnCittiBeetsntrfdto)oeerretscnthlfaairos(cppeioncyovyroroo,.ensmmynns0atmsie;krto-errtemcoecmrirntnrgenprfai.ternBrreuetiivad0oioaiaowm.mloohapcdnlngovpottdelralwr1neolulhalt.noaTl,itgbeaerrlfeeatho,ytlsflefniotstasatbhdevB(nhil,nrpeMaiotcSayl0evnteawysiehRcsDeeniDhoctloauaseihm.dnopiudnafeeledaa1hcpc.xeupDcrlaDtococotddttossmaeaoatsrel,hCcsenrtlhiorlieTiniolwgTueumyennl.rvlcroeo0eu.cauaDtawrnauospsen-sabitaimtanb(4ftpdi2tlbraubctaitierDaeyndInsgywtlhemtel8eoaept.rumserne,srlyiidewaimranvTldneinad8saprnnstmPoiuni.t0cardsale(fdngeit2sradegoiaucvenhi,1hrdtto.refidoseTriil3nimroee4htseeytra3sitmodenbnniienhdod)ndhtmwdsfap,rvnnfnviepfh.)naregiemCeiirilet,leegnroeteo0nnlisesnWaropetrrzAii)oshrvLerenmfid.ytefi)pvioteeCts5eiahrnue.itatiheaseiciesscdrtcnmmhn,fgAnerdtrtoecdhtetlofaadedhaveiadoiibdroeueooepIomrfstoibloeetecoowteixurfr3DretrhfnrthntyodetrReeeetfhpnsphldmia8heebolsedondnnrczfweooaeioer(ceieetoosirntebaactaiedsn2ndon(usofilledrenardapenfb.nyrGayic)dagvo,rnTwttetbpsdrou:adthdalehg'arewMrtdvihmDsolsetwinbaanasiarirnea2etenaelnnkepfic2ipyrssdaeeOilcodn7dnctctgtsgelsoaatllhtDreehonowacg9atarhTywretlncnfwDisPdraotveiryli-isz,vrttsvaeno1gf-toe8ntoothiitDetim8iotoortuhhe0tnhoahenau4otoeouirmeaaae)ry)nnTln0xegeenenngegr;tssfrtlff... Sm7CdwaaApodDenne1hafeafnervddie-azs.lil11eaemya)Des4lstu.aosegpaaa3Txntrgptnr2nu4ie,hdaCsmto7dd.,yeuo1ii(2BeeLengPsn(Fndd.er2ucthP1ateerfut5a/cw.oyfzfhb)aesufo-la:iianecOlNptna.tt2hctdaenesTeot4rdutg.aohtet1fsogrhderef8if2sftee4dccoa-,sut3og242rgtrwlenhd,-1,e,c3S4Ddeea;efY(t-cv6titrP1tditeiehs.e3cye9ei;ddoelcu-nr7oLAlarhsdsco0uepby)pluerugf.tmio,sisritcgbxroaz(,rwiseiwoFeydn,epnidpCwereerHnet-ehedwr,oglerne.,eemedr2ohcgn(edri,PpwfLhgpo4ciluae)xlhxai-gacdtityD)bcyahrhx.w-eemoa,edepddNcaroeaepaseo-rn.eaSitutDtnaonminrcrCcpitiioediadhrnbpoemergecratnlmtuiywueceitdefepiroi,bdeifa(dnmtcsaiAt.eqFiaBetbalcd(utrRroeir2lda.oeycafe,tnnl4wotnoeTrceo-dntggadiesDhnhceigtit.)ceeeesess;s). Taeetcsnvddmabdcppimmbmoobeeeeearrohueovvnomrvcmsneabbstaeneketeuyrlc.rrrpkpnlllilo,tylyaoeodoloetueMleorop.fpgpsu2xnutosismemenorn4thTcs,auehlsdgwieineeyehgtotbradndgndeeienneispogtatstreom.oaesencyprocEefrdotflsseibahhaeffogscfrnflniteaaugwtyoyechtcdiccssthkcovzfeesehhrlntseeseeoyir2resefot.deril0fsdiaynirwac.u1Oeim,tceeefodnrTshdegufmvfdedngaierehgobigpecirltvcbeseahbfieteitihrthdgtyneerescessyaeivremtdgooomonlewoi3ppxlnncoseeb2farutedcat.mnhirrrsa0laehoetotyoetp2lhnatriferfvolcpeio,ledf4dikecshrdteteae-hgirsmohhesrleDafitmaelatfeiresneintwfoistblbcdneedhuta2dmiwrehhrgrtcsne0ayyreiglralceteetnuitoentssdehmh,h1ogede,straieg9ei.tufbav3antaagitosstset9ttnthigrsupi.2rhhrnfto9do,yeaeh,Feedl34pn,alcoterpel2a4-hhtityrty1tDaeery23odeyep4estddhnxorh.8aaocruyosdaiseeretaocafrTlitetgrfadlasicmuisdsdgnniha-ehtlnoaltsogldehoedeoblycafwegwyfiusafhgr1tntttetebtegdl4hhheahooeoerrasesddeeeg8eeertrft.-.rXXOr"O'osj1i 7Wad1ie-s1n. 4o3s3i35n7.e0Bt6rr)iap.thkIoosswoplshakatiit,oansTe.acAnod.m(pTplheroxepfUerro.timoesfraWotfibsacaoinnDs.iDDn,Tis-Msseearntdsaiitstiioovnne, AMbisctrro.CnIionmptesier,nso.rd3oe1fr(9Nt)ho:e.57104th-92eBs2i-,s5603aB7r.;e19a7v1a.ilable from University 97th71e3-134g13u)6.p.EpyfRf,eocePtlsooefoscf,ildiRaie. ldrDer.itnic(uOolnaretagthoenPieSnttetraristne.sicDUir.s,astCeerotoarvftiaionllncisr.eAaObsersetorg..f. IMntiecrrnof.Cil3om1ps(ie,9so):rd5oe3fr1N9tBhoe-.2701tBh-e6;s71i8s957.1a.re available from University 7Umm1reo-bt1dha4noe3axl7,y.cehK11cl1aoo.prsoy6aosn1tred8,m0Im.1.)Pe.t.Dh(iCiUosocs.mehrolptofaartrIialiolntininvmoeiaAsmbmasmtetrtaUa.lbsr,obiIalninnsstamee-rcCntsh.oafamn3pdD1a(iDing9Tn)a:,, 5M4i0c8roBfC-i9lomBp;sie,1so9r7do1ef.r Nthoe. 71th-5es1i4s5.are available from University 07p11h-0a1t0e423)8p. .eMsKtiiccurihodnbe,i.alPD.iinsJst.eerr(atUactt.iiooonnf wMiathssadcihazuisneottns,, Abstr. Intern. aAnmohregrasnt,opMhaosss-. 31(9): 551 IB; M19i7c1ro.fCilompsie,sordoefr Nthoe. 71th-6es3i8s8.are available from University S7r1tee-ar1ce4toi3-v9a.atonBidsenozb,fioFd-.ciehWteh.my(ilTcphahleoseUpv.haoolurfyaItliaoocwneaty.olIcfohwaoalisCiiecirtsiyete,srlaaos.cf5.2oD2xi4ism0se)e.r tMatiicornofCAilobmpssite,rs.orIdnoetfernNtt.hoe3. 17(1t9h-)3e:s7i51s522.a6rBe-7aBva;i1la9b7l1e. from University 392 G0C 1G37 etQ rr CHDfliCAL BIOLOGY RESEARCH oow c h e m ic a l u s.A SUBMI TTCO OY G. E. Lynn charge 199-0000909 DATS 177-9007000 8/24/71 ----------u-u-- 3-1 - A NB T 2 3 .1 4 -1 1 -1 4 , NB T 2 5 .1 4 -97-5 K NUMICR 2372, 20054. 7797 THE EF FE CT OF 2,4-DICHLOROPHENOXYACETIC AC ID (2,4-D) A N D ESTERS O F 2,4-D .ON R A T EMBRYONAL,, P E T A L A N D N E O N A T A L G R O W T H A N D D E V E L O P M E N T m P . j. Gehring 0 2 ^ r e p o r t e d BY B .A .S c h w e t z , G . L . S p a r s c h u c h e c k e d b y : p . j . G e h r i n g _______________ IN F O R M A T IV E SUWKMKV WITH C O N C L U S I O N ! B A S E D ON TH E S A M P L E R E C E I V E D . A D D I T IO N A L I NF ORMA TIO N IN C LU D I N G TH E E F F E C T r e p e a t e d e x p o s u r e MAY r e R E Q U I R E D AS S P E C I F I C u s e s a n d f o r m u l a t i o n s a r c d e v e l o p e d o r i f p r o c e s s c h a n c e s o c c u r This study evaluated the effects of 2,4-D, the propylene glycol butyl A e t h e r e s t e r o f 2 , 4 - D (PGBE) a n d t h e i s o o c t y l e s t e r o f 2 , 4 - D (10) on fetal d e v e l o p m e n t and ne o n a t a l g r o w t h and surv iv al . D o s e leve ls of 2 , 4 - D p: up to a maximum tolerated dose of 87.5 mg/kg/day or molar equivalents C of PGBE or 10 were administered to pregnant Sprague-Dawley rats on day t h r o u g h 15 o f g e s t a t i o n . F e t u s e s w e r e d e l i v e r e d b y C e s a r e a n s e c t i o n on day 20 of gestation and were examined grossly, measured and weighed. to Following routine preparations, the soft tissues and skeletons were examined. Signs of embryotoxicity and fetotoxicity, such as decreased fetal body weight, subcutaneous edema, delayed ossification of bone, 'SJ_ * lumbar ribs and wavy ribs were observed at high dose levels; considered overall, the responses were dose related. Teratogenic responses, however, were not seen at any dose level. 2,4-D did not affect fertility, gestation, viability or lactation. PGBE and 10 had no effect on fertility and gestation indices, but highest dose levels decreased viability and lactation indices. Neonatal growth and development were not altered by treatment during pregnancy. DISTRIBUTION J. E. Johnton* H. H. Mclatyro C. E. Kiwwnol L. K. Frovol 0. D. McCollistor ^2) F. 0. Amo B. E. Bwytft H. L. Gordon, MD* E. H. Bioir * A. J . Schwort, MD * S . M. MocCuichoen 0 . Kilion, M0 * B. Horvath H. Edwards R. J . Sbavar V. Bo Robinson* B. Hotdor, M0 * L Silvarstoin L. Pitchforth C. A. Coring * cri (5) * G. E. Lynn* C. S. "W illiams* (4) Complete re p o rt QFSTR1CTFD FOR USE OF DOW EMPLOYEES ONLY 5655 0001838 5656 J Japa.-.-ose J c a r n a l o f Ir.o-j= t r i a l Ha a 1th V o l . 12, :;urvhar 3 i O c t c b s r , 137C) jr, sgctiv.:;! i-.2 ^ THE FATE CF 2. -I, 5-TEICHLCHCPHZNOXYACETiC ACID IN MAX A:<:>.i;a M.ATSUWfL'P.A* 2,^,5-M '; = = 7 s ;^ f-'.v r; 15 C 1 T h e effectiveness c{ eh lsrir.ated phensxy acetic acids ss pU n: growth : : ; u h '. 5 : i h is a e -s recognised for i s u r iic r if In e a rlie r re p o rts'. - I ) , 2. d ieh lo rap h en ssy iettic acid (2. 4 -0 } '.vis re tire d to h r r.cn-toxic :o snim nls ir.d mar.. 2 u : la te r. th e tonicity of 2, 4 -0 and 2. 4. o-trichlorouhrr.oxyiretie acid (2. 4. 3-T ) for experim ental anim als hr b ten rev ealed by various p a p e rs)--*). A lthough trc.T.rr.dcus a s o s r.ts ci 2. 4-D . 2, i. 5- T and th e related com pounds h av e brer. --anufaettir- rd m d used, clinical reports or poisoning 3 r: rare. Niels-*:;*) reported a ease of suicide w ith ingestion of d icth y larr.in i sait of 2, 4 -D . He observ ed o.O g o f 2. 4-D in the corpse a: the victim , corresponding to 30 mg/}:g. S ta b u ry i) re p o rte d an o th e r case. H e adm inistered to a patient, suffering from dissem inated ceccidicid n y resis. 3.5 Z of socium sals of 2. 4 -0 through intravenous infusion. T he patient was troubled w ith twiehir.gs of the musoies anc fell into stupor, but recovered. A couple cf years ago, the author found such sym ptom s and signs as nausea, vom iting, -veigh: loss, headarhc, fatigue and skin rash among the workers of the test o p eratio n m ixing an am m onium - sulfam ate derivative, 2. 4, 3-T and two other kinds of chemicals. Though the epidem iological study revealed 2, 4, 5-T to be noxious, the author failed to support the nncir.g because the fate of 2 ,4 , 5 -T in the hum an body w as obscure in those days. L'nsub stitu tcd o r p-ehlorophenoxyacetic acids are excreted in urine by m a n , dog and ra b b i: in an intact form because -of th e eth er bondageS -ti). Clark'-'-), using C'-M abellcd 2 , 4 -D . ctm c.-.atrated th at S 5 ;i of orally adm inistered 2. 4-D was excreted unchanged by sheep w ith in 72 ho u rs. Leigh'-)) cc al. reported the fate of 2. 4. 3 -T in dairy cows. C ut of 4;4 mg fed, 420.7 mg of the 2. 4. 3 -T was t EjjMitn? o: Hf-illS, Hj'salsa o! Mriiria, Sapors. "! \ r t ; T e i far aMl cat ; sn A u j . 5, 13TD. IV, J SA 2 0 1 33:z u z i a m x i : recovered from the urine. These facts suggest that analogically in th e Case of m a n , o rally a d m in istered 2. 4, 5 -T will be excreted in the urine. T h e au th o r examined w hether or not ingested 2. 4, 5-T was excreted in the u rin e unchanged and in v e stig a te d the fate of it from the pharm acodynam ic point of Subjects A volunteer (2$-year-old male} w as ingested w ith 130 m ; (2 .2 m g ;k g ) of 2 .' 4, 3 - T a fte r b re a k fa st. Blood sam ples were collected.il l-to 2-hour in terv als during the first 6 hours and less frequently th e re after. Tw o volunteers (27- ar.d 23-year-old males} w ere given ICO mg of 2 ; 4 , 5 -T o ra lly . U rin e sa m p les w ere collected a : 2- to 5-ho u r in te rv a ls for 72 hours. Collections of 24-haur urine were obtained from 21 w orkers of a chem ical m anure factory. M ethods 1) Determ ination of 2. 4, o-T D eterm ination of 2. 4, 5-T was carried out by X ielsett's m ethod w ith m odifications as follow s. T w enty m illiliters of diluted urine (tw o -o r th ree fold) or diluted blood plasm a (three- or fourfold) w as taken into a 50 m l-esntrifuging tube w ith a glass stopper. A fter adding 1 g of crystalline ta rta ric ac id , 4 g of anhydrous X a jS O t, 2 -ml cf chloroform ar.d 10 sr.f of eth y l e th e r, th e tu be w as vigorously shaken for 5 osis tires and c e n trifu g ed fo r 3 m inutes at 1.200 G. T h e organic phase w as decanted and extraction of the residue was perform ed t-vicc more w ith 3 n l of ether. T h e com bined o rg an ic phase w as ex tracted tw ice w ith 15 m f and 3 mf of 1/20 M phosphate buffer ( p il 7 .4 ). A fte r being washed with naif the volume of chloroform , the combined phosphate buffer extras: w as rendered stro n g ly acid wish 1.5 mf cf SN -HeSO s to bo e x tracted twice with chloroform . T he chloroform solution w as extracted w ith 20 m l of b o rate buffer . (pH 10.5). T he aqueous solution was extracted 585( 2 0 ) `0003537 >*7 vith-2 2.3 .-)/ of c h lu m io rn a fte r i: had been made strongly acid w ith 1.5 si/ of SM -llsSC t. T o r t - r .i v : the turbidity, the o.-cante extract was f.ltrntrd through anhydrous N ataO i. O.'.c m illilite r or tv. a (ur --o re. :n the case of low concent.-atinns) si :kv chic.-a form sclutian was poured into a sm all flask to make it evaporate cautiously just to cryr.css. Tw o m illiliters c: cr.rom otropie a rid in cor.-. 1I;3C< solution was poured into the ftask, which was p rrp a rtd by dissolving 0.10 g of the sodium salt of chro n o tro p ic acid (1.3d ih y d rex y n ap h th airn e-fl.aa.su l.'o r.la arid') :r. ICO mf of concentrated sulfuric arid. Ten m illiliters (or more according to the csr.rer.trations) of Sr.Ci: solu tion p rep ared hv dissolving 25 g at atanr.ous ch lo rid e. SnCIa-2!IaO. in 500 mf of 2N -H :S O i w as added to the flask afte r it had been headed a: 143ab2*C for ten m in u tes. C oncentration v.-is e stim ated a t 573 zap. 2) Identification of extracted 2, 4. 3 -T 2. 4, S -T in the urine was identified by the color reaction w ith ^chronotropic acid ar.d by the apectrophotom etric m easurem ent of chloroform extracts. G as-ehrojnalograohie identification was accomplished after m ethyl ester of 2 .4 . S-T with boron trifluoride being m adc'.S. Experim ental conditions of gaschrom atographic analysis w ere shown in th e feet note of F ig . 2. 3) E stim a tio n s of 2. 4. S -T in th e surroundings of a chem ical m anure factory 2, 4, 5 -T in the surroundings was sam pled w ith a high volum e a ir sam pler (G eim an . T ype A ). 2. 4, S-T a t the breathing point of the workers was sam pled w ith a personal air sam pler w ith a glass ib e r n iter. A fte r sam pling. .'Iter papers w ere rem oved into a continuous extractor, containing a p p ro x im ately ICO m ( of chloroform . 2. 4. 5 -T , e x tracted La chloroform w as estim a te d w ith th e method m entioned above. F .esuK s 1) D e te rm in a tio n o f 2. 4. 5 -T in th e u rin e F o r a p re lim in a ry ex p erim en t. SCO and 00 ug of 2, 4. S -T h ad been added to 130 m / o f th e urin e of an' adult ar.d the urine was analysed to obtain the recovery ra te s as h ig h as 95.6. 92.1?- resp ectiv ely . 2) Identification of extracted 2. 4. 5-T A peculiar w ine-purple color developed when the extract w as heated w ith ehram otroplc acid in the presence of concentrated sulfuric acid. T hu absorp tion sp ectru m o f th e color is p resen ted in F ig. 1, show ing th e m axim um absorption ra te a t 57S zap. Spcetrophotom ctric m easurem ent of chloroform '.solution, one step before the lest of the ptoccuurc, 3S iso 7in lmu if: Fig. I. A bsorption spectrum of 2. 4. 5 -T ex tracted from the urine when reacted w ith chrom o tropic acid at 143 to 14S*C for 10 m in u tes. et>l a ln lill Fig. 2. Gas-chrom atogram cf 2. 4, S-T obtained from the urine. yielded the characteristic absorption curve o f]2. .1. 3 -T . T h e resu it of g as-chrom atographie a n a ly sis is re p resen ted in r i g . 2. on w hich the sa m p le coincided w ith p u re chem icals its reten tio n tim e of a b o u t 3 m inutes. It is clear from these observations th a t 2. 4, S -T was excreted in th e urine in an intact form. 3) D istribution ar.d elim ination of 2, 4, 5 -T As w ill "be seen in r i g . 3 . the p lis m a eoncer.tra- C 21 ) 5658 0003538 j* I lue*/ Ou) 443 line w ith a g rad ien t of --0.505, w hich indicate th a t the absorption is also o f the rst o rder. T h e excretion oi 2. 4, 5 -T in th e urine afte r a single oral a d m in istratio n w as rep resen ted in F ig. 4 and F ig . 5. A fairly large am eunt of 2. 4. 5 -T was recovered from the initial sam ple but only about 45? of orig in al dose w as found in th e urir.e col lected during the first 24 hours a fte r a d m in istratio n . Thirty-six hours after treatm ent, 60Ji had been ?** S^Jiet U ail.>sif list M'<(* :>S3( i 4.-T 4jui)stt;f4 F ig . 3. C o n centrations o f 2. 4, 5 -T in th e blood plasm a. o o r ip. tion of 2. 4. 5 -T rose rapidly during the first 3 fcours and th e peak p lasm a level (21.1 og/m f) w as attained at 4th hour after adm inistration. T he sem i-log arith m ic ear.centratior.-tim e curve -.vas lin e a r a fte r 4 h o u rs' post t r e a t m c a i . j n i i ^ t h i i ' 'n rs i o rd er e lim in a tio n -ra le w ith a g ra d ie n t of --0.C53 o btained w ith the m ethod of least sq u ares.. Zergstime plasm a level (2S.0 fg/.T./) was obtained by extrapolating the linear part of the plot. * T h e differences betw een the observed concentra tions (C) and the respective ones obtained from extrapolation (C Q ) w ere able to be plotted on a 29*Trt*el4 ;#. ia ir S9W* JC G i< i f 4 J ;iA ;a if;i { U ? . F ig . 4. Excretion of 2, 4, 5 -T in the urine. Tla tMus) S S J ctt* i7-T r.old ;!. X ir * l(* t S 3 t;. 100c ( * f 1,4.4-T i t e U i i u r f i F ig. 5. Excretion of 2, 4, 5-T in the urine. recovered and at the end of 72 hours more than 20* was recovered. 4) E stim ations of 2, 4, 5 -T in th e #u rin e as a practical trial T able 1 dem onstrates that a relatively sm all am ount of 2, 4. 5-T was found in the urine of workers. In some of them , it w as im possible to ; detect it w ith this method. T he concentrations of j 2, 4, 5 -T in the working area w ere listed in T ab le " 2 and T able 3. None but the value obtained in the to* * weighing box of 2, 4, 5-T exceeded the rerom j m ended value by A .C .C .I.H ., w hich is 10 m g/m -. to * D isc u s sio n T w o m ajor bands of absorption by the pher.oxyacetie acids a re found in u ltra v io le t region!?). T h e one band occurring in the region of 223-230 m u is probably due to the carbonyl group of the side ch ain ,, w h ile th e o th e r occurring around 2S0 m/f is due to the ben ten ring. Nielsen?) analyzed the concentration of 2, 4-13 w ithin th e ra n g e of 230-33C n /t. In the m ethod, which relics upon the abserp- ( 22 ) 56 5 9 1c 0 0 0 3 5 3 9 449 T able 1. 2 .1 .3 T in the urine of w orkers of a chem ical m anure fue :ory. D ate Name Age Sex U rine v olum e (m l/day) 2.4. 5-T (m g/day) Apr. 9 A . Mo* Y. I. 23 M ale :o M ale so M ale T . Ai. 40 F em ale S . H . 25 F em ale T . T . 29 M ale Apr. lo A . 23 M ale s. s. 0 Male Y . I . 60 M ale ' S . H . 25 Fem ale T . N . 33 F em ale s.x . 21 M ale A pr. 15 T . X i. 40 F em ale Y . 1 . 60 M ale S . S . 0 M ale 3 . N . 41 M ale T . X o. 53 F em ale A pr. 22 Y . O . 33 Fem ale s. s. 50 M ale T . K i. 40 F em ale Y . 1 . SO M ale 1 m illigram s per liter. 2. C50 1.4C0 1.370 1. C20 1. c:o 370 2. C00 2.220 2.400 1.250 1,750 1, 400 750 1, 570 3. 020 1.470 450 250 S30 ISO 1.130 1.5 -- -- 1.3 1.2 0.5 3 .5 -- -- -- 2.7 3.6 1.0* -- -- 2.2 3.5* -- -- l.S * -- T able 2. Concentrations of 2. 4. 5-T in the surroundings of a chemical manure factory. Sam pling pointa Concentrations* W eighing box Inlet of m ixer O utlet of m ixer C enter of v.-ork-rsom 15.4 1.37 0.82 0.52 * m illigram s p er cubic m eter. T able 3. Concentrations of 2, 4, 5-T a t the breathing location of workers. W orkmen C o ncentrations* A B CD s m illigram s per eubie m eter. 0.21 0.31 0.67 0.33 tion 'in the ultraviolet region, it is rath er unsatis factory to m easure the concentrations lower than ] mg/lOO rr.l because of the low abso rp tio n ra te . Using the color reaction with chronotropic acid, I can m easure concentrations lower than that ac curately. , On the other hand, industrially m anufactured 2. 4. S -T seem s to be inevitably contam inated w ith 2. 4. S-trichlorophcnoi, the precursor of 2. 4, 5 -T . or other chlorophenol derivatives. As long as the m easu rem en t is carried out in the u ltra v io le t region, a t 223 m t h e contam inants possessing the ber.cen ring can not be excluded. C hronotropic acid en hances not only sensitivity but also specificity. T he developm ent of the color depends upon a reaction betw een 2, 4, 5 -T ' and ehrom otropic aeid in the concentrated sulfuric acid. I t is said th at th e h e a tin g of 2. 4, 5 -T in the presence of concen tra te d su lfu ric acid resu lts in the fo rm atio n ' of for m aldehyde which then reaets with the ehrom otropic aeidlO. T he reaction, highly sensitive to a sm all am ount of phcr.oxyacetic acid, is relatively non specific. A num ber of other compounds such as acetic acid, fats and waxes m ay react and give a sim ilar color. But in this study^ sam ples free from 2. 4, 5-T did not develop the characteristic purple color. Som etim es when the sam ples w ere heated above 153*C, they developed am ber color, which w as bleached by a stannous chloride solution. Since the w ine-purple color of ehrom otropic acid had the m axim um absorption at 5^5 m p, th e concentration w as estim ated in this region. In prelim inary experim ents, the recovery of 2. 4, 5 -T , added to the urine of an adult, w as satisfactory and th at of 2. 4. 5 -T , added to the hom ogenate of the organs of rats, was shown clsewherclS). T h e id e n tity o( 2, 4, 5 -T , e x tra c te d from th e urine, w as judged by the following c rite ria : a characteristic color reaction w ith ehrom otropic acid re p re se n tin g the absorption m ax im u m a t 575 m^r, coincidence of the absorption m axim um w ith pure 2 . 4, 5 -T in th e u ltrav io let ra n g e a t 233 m ^ , and coincidence of a retention tim e in gas-chrom ato graphic analysis. The acetic acid of the side chain was dem onstrated by the positive color reaction w ith ehrom otropic acid. T he benrene ring w as investigated spcctrophotom ctrically in the ultraviolet region. N ot only the intactr.css of ether bondage but also the intactncss of the chlorine of the side chain were confirmed by gas-chrom atogram s. It can be concluded from these observations th a t ingested 2, 4, 5 -T w as ex ertted in the urine unchanged. Orally adm inistered 2, 4. 5-T was readily absorbed as indicated by the observation th at the peak plasm a C- CO ' 71 CJ (23 ) 5660 0003540 so concentration was attained w ithin a few hours after a single adm inistration. Linearity of the semilogarithm ic eo n rrm ra tio n -tim e curve in its te rm in a l course indicates that 2. 4. 5-T was elim inated from the blood plasm a a: the firs: order elim ination rate. On the other hand, non-linear part of the plot re presents the absorption ot 2. 4, S-T from the digestive trie t. T he absorption rate could be estim ated in d irret'y by plotting the differences b et ween the observed concentrations and respective ones obtained from c.Mrapoiation on scm i-lcgarithm ie scale against tim e, which stands for the absorption Tate'*). D istribution and elim in atio n of 2, 4, 5 -T have rarely been in v estig ated . Only Kurt-E.*ne'>3) re p o rted that the plasma concentration time curves as w ell as the plasm a half-life values of 2. 4, 5 -T w ere sim ilar to those obtained w ith 2. 4-D in 'ex p eri m ental anim als. Accordingly, it is of interest to com pare the fate of 2. 4, 5 -T in m an w ith th a t of 2, 4-D in experim ental anim als. D istribution volum e can be estim ated from zero-time plasm a level and the dosage given at 7.9? of the body w eight, ap proxim ately as large as the total blood volume. It i s said th a t 2, 4-D could regularly be detected in blood cells in concentrations ra n g in g 10 to 207# of the corresponding plasm as. Eased on these findings, tw o thirds of the orally adm inistered 2. 4. 5-T -would be d istrib u ted in the blood in e a rlie r sta g e s of elimination. ClarklD described in his p ap er th a t the radio activity of labelled 2, 4-D rose rapidly in the blood plasm a of sheep during the first half-hour, reached th e peal: a fte r l 1/ hours and d im inished rap id ly thereaftcr. About 15f of the original dose was excreted in the u rin e in the first 1*/ hours. E ig h t and a h alf hours afte r tre a tm e n t. 50? had been recovered in the urine and by the end of 23 hours, over SO; of th e dose h ad been ex creted . In co n trast to these observations of Clark, the peak plasma lev el of 2, 4, 5 -T w as a tta in e d 4 hours a fte r a d m inistration and the plasm a level declined gradually, in d icatin g the p la sm a h alf-life as 11 hours. W ith in 24 hours, only 45* of the o riginal dose w as found in the urine of volunteers. Low elim ination rate, th erefo re, w ould m ake 2. 4. 5 -T m ore toxic in m an than in experim ental anim als. It is m ighty favorable for prevention and tre a t m ent of 2, 4. 5-T intoxication th a t the agent is excreted in the urine unchanged. T h e in tak e of 2, 4, 5 -T could be estim ated by urinalysis. As a practical trial, the urinalysis of the workers was perform ed to find out th at a com paratively sm all amount of 2, 4, 5-T was recovered. In some of them , the ic e n t could not be detected w ith the method mentioned above. It is to be accounted for by the low en v ironm ental 2. 4, 5 -T concentra tions, which were due to several kinds of im prove m ent of operations. Furtherm ore, workers had bent provided w ith such protectors as gloves, masks, hoods and protective suits and they took a bath at the end of duty. In the case of a well-controlled, operation, the urinary exereations w ere occasionally low er than 1 m g/day. Thus, it would be inadequate to detect 2, 4, 5-T with the m ethod m entioned, if the chronic toxicity of 2, 4, 5 -T could not be ruled out in such levels. , Summary T o clarity the fate of 2, 4, 5 -T in th e h u m an body, v olunteers took 100 to 150 mg of 2, 4 . 5 -T orally. Blood and urine sam ples w ere collected and analyzed to find out th a t 1) orally adm inistered 2, 4. 5 -T was readily ab sorbed and elim inated gradually from the blood plasm a, showing a first order elim ination rate. 2) m ore th an 807 of orally a d m in iste re d 2, 4. 5 -T w as excreted in the urine in a n intact form w ithin 72 hours, and 3) elim ination from the blood plasm a and exeretion in the urine by m an w ere slow , as com pared w ith those in experim ental anim als. A ccording to th e m ethod e la b o ra te d , 2. 4 , 5 - T in the urine of workers of a chemical manure factory w as analyzed. * Acknowledgement T h e author w ishes his appreciation to D r. U eda and M r. T arum i for the advice and encouragem ent they have made during the course of this study. References P. C.1) M arth, and Mitchell. J. W .: 2. 4-Diehioro* phenoxyacetie Acid as a Differential Herbicide. S o t. Gaz., ICS: 224--232, 1944. 2) H ildebrand. E. M u W ar on W eeds. Science. 1C3: 55--453, 1945. 3) Bucher. N . L. U.: ESects o f 2. 4-D:ehloropher.o:cr- acetic Acid on Experimental Animals, Pzoc. Soe. E sper. Biol. &. M ed.. 63: 2C4-2C5. 1945. 4) H ill, E. V . and Carlisle. H.: Toxicity of 2. 4 - Dichlorophenoxyacetic Acid for Experim ental A nim als, J. ln dnsl. H ys- & Toxicol.. 29: S3--93. 1917. 5) Drill; V . A. and Hiretcka, T .: Toxicity of 2. 4- DiehloropHer.oxysceiic Acid and 2. 4, 5-T riehlorophe- poxyscetic Acid. Arch. Indusr. Hyg. Occupational Med.. 7: Cl--57, 1933. 5) Oiorklund. N . E. ami X urt-Erne: Toxieologicat Studies of Phenoxyacetie Herbicides in A nim als. A cts vet. stan d .. 7; 391-393. 13o. O' o c~ Co - o' 5661 0003541 7) Nicl>en. K., Ka:mpe. 3 . and Jrnsen-llnlm . J.t rata l Poironinx in Man by 2. 4-[>iehlorop'nenoxyaee:ie Acid (3. 4-D): Determination of the A srn t in Forensic Material.. A e n pharmacol. et toxicol.. 22: 324--221. _ 1SG3. 3) Scabury. J. H.: Toxicity of 2. 4-Dieh'orophenozy- jeetie Acid for Men and D o:. Arch. Environ. Health, 7: 203-2C3. 1953. 3) Levey, S. and Lewis. H. 3 .: T he Metabolism of Phenoxyacetie Acid, ita H nm oio;ues. and some Mono* chiorophcnoxyaccte Acids. New Example* of -Oxida tion. J. Biol. Chsm., ISa: 213-221. 1947. 10) W illiams. R. T .t Detoxication Mechanisms, 2nd - Ed.. 220. Chapman t Kail. London. 1933. 11) Clark. D. E ., Y our.:. J. E .. Y ounser, R. 1--. Kur.t. L. M. and McLaran. J. K.t T he Fate of 2. 4-Diehlo- rophenoxyacetic Acid in Sheep, J. Asric. rood Chest., 12: 4 3 -4 3 . IE . 12) Leizh. E .. V fa jn e r. D. G. and Lisk. D. J.t ra te of Atrazine, Kuron, Siivcx and 2. 4. 5 -T in the Dairy- Cow, J . Dairy Science. 47: 1257--1270. 1554. 13) Banduraki, R. S.t Spectrophotometrie Method for Determination of 2 ,4-Dichlorophenoxyace::e Acid. Bot. Caa.. 1C3: <45--449, 1946-47. 14) LeTournesu, D. and K roz. PL: T h e Use of Chro- stotropic Acid for the quantitative Determination of 2. 4-DlchIorophenoxyacetic Acid. Plant Physiol., 27: 622-623. 1932. 15) Mataumura, A . and A be. S.t Studies on the T oxi city of 2. 4. S-Trieh!orophenoxyaeetie Acid. Report I. On the Determination of 2. 4. 5-T in Tissue Hopenates, Jap. J . Indust. Health, 11: 103. 1559. * 36) Metcalfe. L. D. and Schmitz. A . A .: T h e Rapid Preparation of Patty Acid Esters for Cza Chroma to- . jrmphie Analysis. Anal. C hest.. 23: 353--354. 1931. ,17) N'aka;aki. M.t Dr.-; Transfer in 3io(o;iaal Systems. 1st E d , 153. NANKODO. T okyo. 1 9 3 . 13) X url-Erne: Distribution and Elimination of C h l^ rlnated Phenoxyacetie Acids in Animals. Acta vet. scan d , 7: 240--355. 1356. 2.4.5 - M ; a s 7 s ) (2.4.5-T) on W i f j i r t i i a - l ' D . cJvfcEai-j fc.2-5rL. K'j'tifajaiifitaLfc. 451 '.t'.V 'J W 'i'V 2. 4 .5 -T O fiJK 'L'V tlCSi N'iciscn hr> Zi'Ji& ZL'i L . F i z m f 5 2 . 4 . 5 -T i ? = -i- 1- iiAFJrli 150mg O 2.4.5-7 -f*. a n s i > 4 s?sq-c i \ s a 21.1 /m f ic;iL . 12;A ri:iiT L i:. i i ^ i - o O ' Siti;- R + i 9 f t!iS L i:r;rilfy :0 S '3 0 3 fis '? ia iiL i:tS i!i. 2 .4 .5 - T t i t h o a r t . - i i l - -C f/V iii . 1 - yO E502fc1f 3 < C. 1) SSaT&*i*-C* o * h --^ SiE oU V n i : - / K - a 2 .ic s2 .Li:. 3) iA SiijO 7P3 < o2Sii5ri-.*.lt 233 tn/r 1C*, 9 R iJo u O -riii--S L * *-A?fl -rh/57i -*CK2i LilSSiTc. Ri*2?WiiV 8 ft-C* 9 C S ijiO -= :h .i-aL i:. SA J5?2iSiC 100mg o 2,4,5-T S rE c a -^ L . T'^nnicdp/zoTI?.!?.L7 -^ -5 )2 S iii:2 .4, 5-T crJS LLi:- S24i!2;:-ir.n-C-G-?-iVoii45;i, -usitVj'CJSre 723y,`ul-cso;.'LlL-V-ir,!ci 1 i ; - f)ieJJ'iul o i 1'X.P3.it.F,<Op2S.;ci;-ov''Ci25'lS-j'C2.4. s - T o ^ r t f i A 2 L * t : T o tm><o-Zizi L*C. * % fiS tJ2 rrZ fE3i?f0243-rrj]l?..-o i 'C S `7=2.4,5 -T 4 rS lS L i:. f? 3i%S5ij*:pO 2.4.5-T O S (S li 0.62 mg/m1-->15.4 mg/ mJ -C5,9. S0^ LfcI521dS*jSS:a!!t 3. Smg'day -C> -o/z- if:fi^aO :T ;*-C -tiD 07jL S lci -,-C2.4.5-T CrV*,"''' O' cn C7 <25 ) 0003542 5663 120330 Che.-n.'caf Abstracts Vol. 7 2 . 1 9 7 0 r---- ------- N | i 240 us-l i t 4 kg Ti.i prtrr:< -ri'.n:e, did not d c rt-i.: N O )', NH, u rea. Evidence of binding was found in both shoots and r<its and l*;Oj IcvN i:i the 1. I t s tin .u la if 1 th e activ ity uf the soil of corn. Xo evidence was found for the metabolism of rhlor- mi- ii.H.im an^l inerra-.-d the co n ten t of soil nuti---:. A bromurop. in cucum ber; how ever, some binding occum--! in b->th 30 ``'."r incr. i,: in N content a s n->t-.-l in the pr----. ..f the htrhi-iile. On T ro ia n -tre a te d castar b*-ins the 1 <>f >! was lH% hig h er th an with the c o n tr ol. 1 he con- nt of N' v d P in plants cu ltiv ated in the presence of T rcllan a s hi<l>-r than in the control. A. Rad*-cki 120331s Use of herbicides oo turnip plantings. Puzin.n'te, I. (USSR). K him . Set. Khaz. 1970, 3(2). 131-2 (R u -0 - Dacrhal 4 -8 , diphenam iil 3 -6 , trifluralin 2 -6 , and .un-hen 2 -3 kg. "ha w-re to t e d in 3-year field expis. against weed i in turnips planted in snd-podzols (pH 321-6.3). T he effect of th e -: fcerb-'cides on sh-jots and routs. _ RCVCZ 1293J7y Effect of the ratio of soil to water on the if ., n of linuroc and itriz in e . G r ver, Rajba.as; H ance, R . J . (C m . -Agr. R es. S tn ., R egina, Sa-k.). S o il Sci. 1970, 109(2), 136-3 (Eng). Begbroke soil was prepd. by air-drying and pn-.ing through a 10 meah sieve. T h e soil contained 1.93% C, 15.6% clay, 18.4% silt, and 66.0% sand. Its water holding capacity was about 24% . The pH of a 1:1 (soil :water) suspension was 7.1. Five herbicide concns. were used in the range of 5-25 pp:n lin-arm and 0.5-5 ppm atrazir.e for the 1:10 fsnihw ater) turnip yield wre compared w ith a double hand wc>d'ng as the ra tio ; 5-25 ppm linuron for the 1:1 ratio; and 15- 3 ppm control. T h e yield was always less th a n in the control. The Iinuron and 5-20 ppm atrazin e for th e 4:1 ratio . The linuron m ax. yield decrease (30 and 29 % , re -p .) was with 4 k j dacrhal soins, were m ade in 0.1 S f C aC l, an d th e atrazine soins, contained a n d -5 kg a m ib rn /h a . W ith 3 kg dipht-nutiid and 4 k-g triflu ralin / no CaClj. A dsorption isotherm s were tinear in each ca.-c and ha a min. yield decrease (10 ami 11% , resp.) was obtained. comparison was made using th e Freundlich relation with values Diphenamid was also most effective, even at 3 kg/ha, against of k reported. There was a 5-fold increase in the adsorption of Raphanus raphar.iUrum or S te lti'u i media, killing 73.3% when linuron a t the 1:10 ratio (ft 12J ) compared to the 4:1 ratio before thinning and 32% before h a rv e st. D acthal in d trifluralin <k 2.7); adsorption in th e 1:1 m ix t. showed k 4.4. For atrazine were less effective, and low levels of am iben had no effect; a t 3 th e difference between slu rry conditions (1:10) and the 4:1 kg/ha amiben was toe'e to turnips. J . Maleic ratio was approx. 3-fold, th e k values being 0.8 and 2.1, resp. 12033' t Preharvestir.* chem ical drying of fodder lupine in I t was concluded th a t adsorption o f herbicides under field condi the western U kraine. Chernobai, X . K h.; Proskura, I. P.; tions, where the soil aggregates are relatively large, may not he M efnichuk. P . D .; Bugaiskaya, Z. Y a. (USSR). K him . Set. as high as those estd. under slurry conditions. There may be Khaz. 1970, 3(2), 133-3 (R uss). P reh arv est defoliants and v ariations in th e exten t of adsorption of a herbicide within 1 field desiccants for fodder lupine were te ste d in 3-year field expts. if aggregate size d istrib u tio n varies. Peter Coad T he most effective chems. (dose in k g /h a given) were: X H .SC X (2. 3, 10, 25), X aSC N (2. 5, 10, 23), dinitro~a-:rcsol (1, 2, 3, 10), N aOH '3 . 10. 23. 50), Reglone (0.5. 1.0, 2.5. 5.0), and Gramovor.e 0.5, 1 .0 ,2 .3 .5 .0 ). S praying -if X aSCN or XH.SCN" 10-20, diniiro-o-oresol 2.5-5.0. and Cramox-jne or Reglone 1.5-2.5 kg. ha accelerated seed m aturation by 3-17 days, in creased seed germinating by 9-13% , and increased seed crude protein by 0 .4 -l.6 % . J . Malek 120333u Results of testing biennial weed control in wheat with new herbicides based on s-triazines, substituted ureas, and fluorenol. Chiapparini, L .; Soldini, G . B. (Sez. Patol., Osserv. M aiattie Piante, M ilan, Italy). Kalis. Mat. Pianle 1969, Xo. 80-81, 209--43 (Ita l). Excellent weed control with min. phyto toxicity were obtained with terbutrin [2-((er.'-butylamino)-4- (ethylamino)-6-methylthio-s-triazine] applied preemergence a t 1J kg of activ e ingredient,1h a , m etoxuron [Af-(3-chloro-4- methoxyphenyl)-iV',iV'-dimethylureaJ, neburon [X -butyl-Y '- (3,4-dichIorophenyl)-.V-methylurea], and prepn. 3633 (X-benzo- thiazolyl-.V,2V'-dimethylurea) postemergence a t, resp., 3.2 or 4.3, 3.0 o r 3.6, and 1.4 kg active ingredient/ha. / Felix Saunders X r - 120334V Toxic effect of som e h erb icid es on fish an d aquatic /'in v e rte b ra te s . Belyavskaya, L. I .; Konstantinov, A. S.; K onstantinova, X . S. (U SSR). Vidovoi Soslav, Ekol. Prod. Cidrobiontoo Volgograd. Vodokhran. 1 9 6 9 ,9 3 -8 (R uss). E d ited by K onstantinov, A. S. Izd. Saratov. U niv.: Saratov, USSR. For the prepn. of huge water-reservoir beds, woods and shrubs 120336Z P henothiazines as p lant growth regala r t s . Farben- fabriken 3-zyer A.-G. F r. D em ande 2,901,531 (Cl. A Oln. C 07d), 26 Sep 1969, C er. Apfil. 08 Feb 1968; 7 pp. Pheaothia- zines (I) <R, R* - halogen, alkyl, alkoxy, C Fj, alkylcarbonyl, r*O 3 - r'* w T ' w' ANRIP o 2 ^ A represents alkylene, R l, R* -- alk y l or form together wjthr.the X atom a 5- o r 6-m embered rin g ; n -- 0 , l , or 2) and th eir salts, are used as plant growth regulants. Compns. with v a r io u ^ ^ concns. of I can act as growth stim ulants, growth inhibitors, herbicides. T he com pns. generally contain (>.1-95% I and i r t ^ applied a t the rate of 0.001-100 k g /h a. I may be applied t o r " dicotyledons (cotton, beets, carrots, beans, potatoes, coffeejJO m ustard, w ater cress, bedstraw , camomile, and galingosa) monocotyledons (maize, rice, oats, barley, w heat, millet, sugar cane, cat's tail, spear grass, ray grass, and panic grass). G . P. Xatus 120339a Im idazoles as herbicides. Draber, Wilfried; Falbe, Jurgen F .; Buechel, K arl H .; K orte, Friedrich W . A. G . K . (Shell Oil C o .) U .S . 3,501,286 (C l. 71-92: A O ln), 17 M ar 1970, Appl. 29 Ju n 1964; 4 p p . Im idazoles (I) where R is H, Ci-w alkyl, cyanoethyl, or allyl, a n d Y is H , Ci_ alkyl, C X , halogen, are often destroyed with herbicides e.g., the Bu ester of 2,4-D, the Bu ester of 2,4.5-T, Foredex 75, and Fortex (aq. emulsions of 2,4 -0 and 2,4,5-T, resp.). After 20-30 min of contact of these emulsions a t 60-120 mg/1., crayfish (Simeeephalus vetzdus) died; on co n tact w ith aq . emulsions contg. 10 mg/1., th ey survived for 5 days. Of all herbicides tested on invertebrates, which serve as food for fish, 2,4,5-T was the most toxic, and it lcilled them all a t 2 mg/1. T hese herbicides a t 4 and 6 mg/1. were toxic to 7 ty p es of fish w hen added to Volga w ater a t 14-16* for 1 d a y . Solar oil was also toxic to th e fish and th e fish feed. A t th e ir present level o f use 6 k g /h a of shrubland, the concn. of herbicides ia w ater in th e studied reservoirs will soon reach fatal levels to the fish an d th eir food. However, if the herbicide treatm ent were lim ited to 0.01-0.1% of the total bed-area of th e reservoirs, th e resulting concns. in the w ater will be harm less to the fish an d th e ir bio-food. S. K. Raman 12033Sw Combined effect of trifiuralm and MSMA [mono sodium m ethanearsonatej on Johnsongrass control in cotton. R o r XOt were activ e a s general preem ergent herbicides a t 10 lb./acre, while they selectively controlled broadleaf weeds a t 2 lb/acre. W ettable pow der, d u st, and emulsifiable cone, compns. are given. A d u st contained 4,5-dibromoimidazole 4, Mg stearate 1, kaolin 58, and gypsum 37 parts by w t. In field tests I(Y - Br, R - H) gave 99% control of broadleaf weeds a t 2 lb/acre, b u t only tem porary burning of grasses. Germination o f weed seeds was prevented by incorporating 2-8 lb I (Y Br, R m H ) o r I (Y " I, R * H )/a c re in th e soil. L . Tetzloff 120340u Dialkylaminoalkyiene sulfide herbicides. Bordenca, C arl (SCM C o rp .) G e r. O ffen. 1,929,390 (Cl. A Oln), 19 Feb 1970, US Appl. 27 Ju n 1968; 24 p p . d- Dialkylaminoalkyiene sulfides RSXXR'R* (I), optionally as stable salts thereof, are used; R -- linear or branched C_u alkyl, preferably octyl and K leifeld, Y . (Volcani In s t. A gr. R es., B e t D agan, Israel). Weed l ' 7 ' ,l 8 ( l) . 16-13 (E n g ). P rep lan tin g application of trifluralin reduced the no. of mono-Xa m ethanearsonate (M SMA) applications needed for th e control of Johnsongrass {Sorghum htuepense). T he combined effect enabled weed control to be completed before cotton {Gossypium h inuium ) began to bloom, and hence w ithout causing damage to the crop. RCKZ 120336x M etabolism of ehlorbromuron-"C la corn and cucum ber. X ashed, R . B .; K a tz , S . E .; (luicki, R ichard 1). (Coll of Agr. and E nviron. Sci., R utgers Univ., Xew Brunswick, M -J.). Weed Sei. 1970, IS U ), 122-5 (E ng). T he metabolic che ro o t*aPplied, " C -carbonyl-labeled, 3-(3-chloro-4-broino- decyl branched or n o t, X lower alkylene, preferably ethylene, R 1and R' - same or different lower alkyl, preferable E t. d-Diethylam inocthyl n-octyl sulfide. 3-diethylainmoethyl 3,7-dimethyloctyl sulfide, and d-dicthylamir.oethyl 4,6,3-trim ethylnonyl sulfide, optionally as hydrohaiides. are claimed. I were tested as ore- and post-emergence herbicides. ......M anfred K naack 120341V H erbicidal C ym etrin [2-m ethylthio-4,6-bis(ethylamino)--tnazine| and benzyl alkylthlocarbamate m ixtures. Kimura, Ichiro; Sugiyam a, Hironari; Kado, Masaru (K um iai Chemical In d u stry C o ., L td .) G e r. Offen. 1,943,983 (C l. A O ln), 12 M ar 1970, J a p a n . A ppl. 28 Aug 1968-07 Ja n 1969; O CJ ^ L , : . yl;V-.m. " ho'> " l - ^ h y l u r e a (chlorbrom uron) was investi- cucumber ma?` ? ? 4nd ^ ePtible if*trlv Thm ,a **-*. v ar M ark eter) m z tune-course *u r ' c u b o liie found in corn shoots an d roots was the uonphytotoxic S -a -c h to ro -g -b ro in u p h e n ^ .l-m l^ " 24 pp. A m ixt. of C ym etrin (I) and thiocarbamates (II)II (R - R` - E t. X - 4-C1) (b ,.,, 127-31*) 7% . 1 1.5%. Xa lignin- sulfonate 2 % , wool ashes 5 % , a n d bentonite 34.5% by w t. were ground and mixed to homogeneity, kneaded with w ater, and granulated to granules of 14-32 m esh. T he In rbiculal mi <t. ZtZ90NN *Bfc06TM O a DOW C H E M IC A L U.S.A. -SEATTLE SALES OFFICE Bellevue* Washington April 23, 1980 J. R. Beachell K. D. Bohlander A. J. Herrera R. F. Kincaid J. C. Mitenbuler W. H. Walker P. H. Williams cc:V C. TfM&k L. E. Warren, Davis M. L. Smith D. M. Frederick, 9008 Bldg. SUBJECT: ESTERON 245/ESTERON BRUSH KILLER/KURON As you will note on the attached specimen labels for subject products, the latest revisions, dated January 1980, clarify the Environmental Protection Agency's most recent rulings on use of these products. As the brush begins to leaf out in farmer's fence rows, this would be a good time to remind your distributor salesmen-- and the dealers you are calling on-- that growers may purchase and use these products legally. Of course, additional specimen labels are available for distribution as you determine appropriate in your sales territory. District Sales Manager Agricultural Products pmg Attach. AN OPERATING UNIT OF THE DOW CHEMICAL COMPANY 5665 0001741 5666 334 rnKOM.47S3 MN07 2 6 4 9 notes Simultaneous gas chromatographic determination of 2,4*0 and dicamba r human blood and urine t dichloTrohbecnczoomicpoaucnidds(d2ic,4a-mdibcah)loarroephhcenrboixcyidaerscticcomamciodnl(y2 ,4u-sDed) and 2-methoxy-i to control a variety of broadleaf plants. An acute poisoning case was recently investigated bv the Hawaii o Q Community Study on Pesticides in which the victim intentionally ingested a forma, la a tion conta need for ining a rap i2d,4 -D an a d nd dicamba. sensitive m The occurrence of th ethod for detecting is attempted these comp suicide created ounds in small amounts of human blood and urine. o WcohnisliedSegereevndeerarfalolrlmytehsteehnoasdnistaivlfyeos,ristdheeotsfeec2tmi,4n-egDth2o,a4dn-sdDwdreeircseaidmnuebeisathiiennr a variety of substrates wer* hraupmidannobrloaopdplaicnadblueritnoe't-h*e relatively small samples available. C.H ZD CO CO to The Hawaii Community Study on determine pentachlorophenol (PCP) Pesticides uses the method residues in human blood. c:fnBaenveexvualeuaetliaoln* done by this laboratory*, the procedure was shown to be rapid, accurate, sensitive sainmdilarreptorotdhuocsiebloef. PBCePc,authseese2 ,c4o-mDpoaunnddsdaicreamalbsao ddeitsepcltaeyd bchyetmhiecaplroccheadruarcet.eristics This note describes the application simultaneous determination of 2 ,4-D and doifctahmebma ienthhoudmoafnBbelvooedncaendetuariln*e.to the Experimental Solvents and reagents. The following solvents and reagents were used: benzene, hexane, isooctane (Mallinckrodt Nanograde Reagents), used as received; 0.1 .VH.SO,. benzene extracted; 2 ,4-dichlorophenoxyacetic acid; 2-methoxy-3,6-dichlorobenzoic acid; N-methyl-N'-nitro-N-nitrosoguanidine, supplied by Aldrich Chemical Co., MilwaTuhkeeed, iWaziosem. ethane solution* was prepa Nof-m2 0et%hyla-qNu'e-noiutsroN-Na-OnHitrowsoergeuacnoimdibnienewd aisn raaeddd5ea0ds-mfiolnlleosrwlmesna:mllheeiyxneacrnreefmla(2es5kntmsa.nl)dWa0hn.ed5n 2g ml of the reaction was complete, the hexane layer was decanted into a small glass bottle equipped with a Teflon-lined screw cap. The diazomethane solution was kept in a freezer for up to one week. Because diazomethane is a skin irritant and a carcinogen, its preparation and use was confined to a well-ventilated hood. wasvposoriletliuhupttsmaiiormSseenottdearoa,tincchndetyfdaaallncarahedeslk,sltocssteowoh1rnl..ueu0tsdFtaimriogtnoonliimvson.sifgntteaSgha1ntecodssmhocekflgsuostorotosilcofo1uk5nltcuissootmoimnwolisunnph,t.osiiwcouThoonnhfrdwecko2ipasnn,soe4gtlr-taurDsimetntiaaoelantndeondsdfda1w0rwsdodei/slirt/vchegwea;0tnmemh.rt5e.belnmaIopnfldrienoiselpfeauadpbctrehiaeeadnrdzaczottoeioemnmneet1apo0tpohwo0pua-ernnmmordeeJl priate concentrations. MT 0.32 2c2Am0ppgIa.aDrsa.tcuxhsr.o1 Gas chromatographic determinations were made matograph equipped with a *H electron capture S0 cm borosilicate glass column packed with a on a MicroTck detector and a mixture of 4 *, J . Chroma! .,g . , jo (1970) 334-337 5667 \ 8. . *fJB . . * l SOTES 335 SSoE--31000 amnedsh6. 0 QI:-i on Chromosorb W (AW-DMCS), high-performance grade. The instrument conditions were: inlet temperature 220a, detector temperature 20S0, column temperature 190s; nitrogen flow rate S5 ml/min. The 25-ml evaporative concentrator tube was Kontes No. 570050-2526, the evaporative concentrator column Kontes No. 569251-0324. Extraction. One milliliter of blood serum or 5 ml of urine were combined with 20 ml of 0.1 X H-SO4 and 12 ml of benzene in a 125-ml glass-stoppered erlenmever flask equipped with a i-in. Teflon-coated magnetic stirring bar and mixed for 20 min at 50 on a heated stirplate. After cooling, the contents of the flask were transferred to a 35-ml centrifuge tube and centrifuged for 15 min to effect phase separation. The benzene (top) layer was transferred to a 25-ml evaporative concentrator tube, and the aqueous phase was re-extracted three times with 5-ml portions of benzene. .Yith each wash the mixture was shaken vigorously for 1 min, and the phases were separated by centrifugation. The benzene extracts were combined in the 25-ml evaporative concentrator tube, a boiling chip was added, an evaporative concentrator column was attached, and the combined extract was concentrated to 2 ml in a waterbath at too3. .1/ethylation. Diazomethane solution (0.2 ml) was added to the concentrated extract, and the mixture was allowed to stand for 15 min. Excess diazomethane as then removed by gently bubbling dry nitrogen through the extract until the yellow color disappeared, and the volume was adjusted to 10.0 ml with isooctane. Gas chromatography. The methylated extract was diluted as necessary, and - to S /I were injected into the gas chromatograph. Following each extract injection, an amount of standard which gave a response of similar peak height ( 10 %) was injected. Results and discussion The 2,4-D and dicamba levels in the poisoning case samples are given inTable I. The results of a recovery study to determine the applicability of the procedure to the analysis of human blood and urine for 2 ,4-D and dicamba residues are given n Table II. The samples were fortified, prior to extraction, with 2,4-D and dicamba acids at levels which compared generally with the wide range of concentrations found in the poisoning case samples. In all cases responses to 2,4-D and dicamba (as their methyl esters) were fluantitated by comparison with standard responses in terms of peak height. By comparing responses with [>eak heights differing by 10 % or less, quantitation problems associated with lack of linearity and changing sensitivity, sometimes encountered w'th electron capture detectors, were minimized. The limits of detectability in 1 ml of blood serum were 0.03 p.p.m. for dicamba 0.05 p.p.m. for 2,4-D. In 5 ml of urine the limits of detectability were 0.01 and 2 p.p.m. for dicamba and 2,4-D, respectively-. Dnder the conditions given, dicamba and 2,4-D methyl esters had retention hmes of 1.2 and 1.9 min, respectively; I*CP methyl ether had a retention time of 2,2 min. Because the three compounds are well separated on the gas chromatographic r,,Iumn. the procedure is useful for detecting anv or all of the comixmiuls in the same 'ample. LLOo TA IH.K I J . i - 1 A N II IIICAMIIA l.l - V I - l s IN l'O lK U N IN fi CASI'. SAMI'I.ICS Sam hiiug W o od ser inn Pah T n te U rine Ita le Tim e lo Ita li 7 MItf f o t . (m i) J . l U (/ / 1 ) .Sm ini U rine Sellin i (ft. f>. in ( lin i' i H '/l 1 7 :oo a m M i/l 1 11 :n n a .111. m in im o p.m . 71" 1 no li 1 15 2 ,|f. 1 21 y * i ">/' 1 1: imi J ni. M/' 1 1 :(h>p ni. Ml/l 1 11 :(Mi p.m . 575 in jl 27 I-" 22.5 -s .1 1 No Sillliplt I.ikcn M>/i 1 11 :<m> p.m . 10 /15 9:00 a.ni. 1 250 515 17 i 1 M l'l 5 o:oo a m. M '/'S (jan) a ni. M '/l5 .| :oo p .m . -- (H 1-9 ;f i2 .o H J 12 s 5 IN /IS :on p m . M /l. j :on p.in. M '/l5 11 :oo p.m. 775 911.2 5<si S fi. 12 95 < /I.1 l i : 15 p iu. I0 /15 11 :oo p.m . 1 0 /Mi 7 : -15 11 70 7 5 1.1 7 1 5 (MM! 1 7 Ml/Ml 7 : so a ni. Ml/l<> 7:1.5 a.m . Mi/ Mi I : 5 1 >' 720 fi |S.l |" S 1 117 MS H lll/lll I .1 5 p.m . m >/M 1 : 1 5 1* 1,1 Mi/ 1 fi 11 :oo p .m . 55 l'ilo 1555 J 90 5 9 io /III 11 :on p.m . I/M i 1 1 :0 0 p in. M l/l 7 9:0 0 a.in. 7 |o 5*5 1 MIO" t -5 1 IH IO 11 11' / 1 7 io / i ; 9:011 a.in . 5 :hi p .m . IO /I? I0 /17 9 :00 a.ni. 5:01 p.m . M1/17 Mi/ IN 5 :0 0 p.m. in :n o a n i. 5 mi Mi.|<) 5015 7 2 -I 1 1 12 1 (17 l.p i ni 0.S11 1 mi/ i M 10:00 a.ni. lo /lH iu :o o a ni. I0/1N .|: .P> p 111 il in 5 25 7 7 <l " 17 .SI Vi lo /) IVI l-m. mi/ i H IV1" 1 in . m i/ mi 9 :1 5 a mi. l.|00 >1 Mi/MI 1 : |5 a in . Ml/M) 9 : . 1.5 a .in . mi/ m j :no p.m . 15" 5 N o k;iim |iIi t a k m i / m j mm) p in . 10/20 a ni. 2 150 Hi M i/io i i :ih) a n i. M>/21 9:0 0 a.ni. 10/21 fi: jn a.ni. 2 1 20 1 .fui 1 -7 1.20 H.hO .1 SS 15' 2.1)0 0 2.1 0 .15 0 .15 n p> " VS o. 1 2 ( oli 7 |S HI I * il lo /il io/.*J lo / i t/2.| M i/iJ S : p> a n i. 9 : p a .in . 7 : 15 ' s ; |5 a n i . 1 ;oo p.m . lo /il MI111 M>/2 . io /i J lo /il K; pi a.III. 9 : 50 a ni. S:no a .n i. S:.|5 a .n i. 1 :oo p ni. 10/22 IO/2.I 1 " / 2.| Ml/25 M l/27 0 : pi a.ni. fi : no a .m. H:.|5 a n i. 1 :no p.m. io :.|5 a ni. 5 7 M* 21.00 11**5 299(1 )Kni) 5* 0.1 fi 0.50 fi. SS 0 . pS I.5S 71 05 1 11.71 0.25 0 15 0 nj 0 t7 11.nl . 0.(1 \ . i m i .. n n i n u| 1 U \ n n1 r i VI 1 V5 i" /i 7 io :.|5 a.ni. No sam ple taken Ml/.JI 1 :oo p.m . " / 7 9 :00 a.ni. 10 /2 7 to/2<) '" /.Il 1 / 7 10 :^ 5 a ni. fi: nei a .n i. 1 : p i p iu. 1: 00 a .m. 1 0/2*1 10/51 1 / 5 M /S Sino a .111. 1 :<m> p.111. 7 :5 0 a.in. ri:no a.ni. i l HI vv*n 1 pio 0. fi - il 0.59 0 12 1 'S li 5 S, .,0 o | *: o.n 1 ' . l nul n1 *0 <l ' n.n 1 '.i: . _ .... _________ .... ..........-- .. . . . -- -- . . . . . . ------- . _ ---------- . _. ----------- _. . - - . . . .... i 1' ; S8GS09 MOQ ri S'-U0.\` DOW 605986 .VOTES 337 TABLE II RECOVERY OF 2.4-D AND OICAMUA ADDED TO HUMAN BLOOD SEKUm' aXD URINE Sample* Fortijication level [p. p. 1111 Dicnmba 2 .4 -D Found (p. p . I I I . ) Duamba J .4 -D b recovery Dicantha 2.4-D I'rine Blood serum 65.0 13 1.3 0.13 O.OI3 0.000 _ 65.0 .50 0.5 0.005 0.000 6.S5 137 *3-7 37 0.137 O.OS -- 0.S3 6S.5 0.S5 0.6S5 0.066 * Each sample a single analysis. s Detected as their methyl esters* Limit of detectability. < 0.01c 6S.S 12.5 1.15 0.1 r 0.01 < 0.0! < 0.03' t.o 5 91 035 0.07 < 0.03' < 0.02c 779 *3 - 11.6 ` 35 0.15 0.07 < 0 05' 636 6>-0 5.90 0.00 O.On _ too " 4 96 9 66 5 >5 9S 77 109 -- 103 -- 94 93 9 ` tot 5 37 to-s MS " howevNero, tihneteursfeeroifntgheredsipaoznosmesethwaenree seonlucotiuonnteinreadmionunatnsygroefattehrethsaanmtphloesseainnadliyczaetedd; in the procedure can lead to interferences due to impurities in the reagent. sixteenUssainmgpltehsips erpdroacye.dure, an experienced analyst can process approximately The Hawaii Community Studies Pesticides Project is supported by Contract XProo.d8u6c-t6S5a-7f9etyw, ith Fo o th d e a n Division d Drug A of Community dministration, Studies. Office of Pesticides and Public Health Service, Department f Health, Education, and Welfare, Chamblee, Ga., U. S. A. Hawaii Community Studies on Pesticides, Pacific Biomedical Research Center, t diversity of Hawaii, Honolulu, Hawaii qhSjJ (C. S. A.) J erky B. R ivers W illiam L. Y a cg e r . J r . H o w a r d W. K i.e mm ICR * b . E. Cla r k , I'. C- W k io iit ano I.. M. H u n t . J . Agr. Funit Ch. " . 15 (m ; i 171. 1 b. J . Lisk. \V. H. ( u tm nm ann, C. A. B a c h e . K. Ci W a rner vno I). ( 1. W.A<..\hK. J . Dairy 4*> do'M ) M.I5t o G. Cro siiy a n d J . H. Bo w e r s , H ull, liu v irn ti C n n la iu iu . Tun. /.. I fin'>u| 104. A. Re v e n u e , .M. L . K mf.knon. I.. J . Ca sakktt a n d \V. L. Y.w c k k . J k . y . C hrom a/"*., j * (uji.-S) {J B. RIVKks, rrMilts. C*anmkk a n d J . I 'r k a l . |HTsnai c o m m u n ic a tio n . ^reived April 14th, 1970 /. I unit. . JOIH17111 I H - . U 7 TV I BACKGROUND Thompson-Hayward. These compatii: The herbicide 2,4-dichlorophenoxyacetic joined to form the Industry Task Force acid (2.4-D) was developed in the mid Phenoxy Herbicide Tolerances (ITFPH; forties and was the forerunner of a group of The chronology of major events associate phenoxy herbicides that have Jbeen instru with this has been as follows: mental in the control of broadleaved weeds in food crops and undesirable brush species April 13, 1966 on industrial rights-of-way. In addition these USDA announcement to abolish no res products have contributed immeasurably to due status--industry must comply by oir beef production by controlling weed and/or taining tolerances for residues in all treats brush on pasture and rangeland, resulting food and feed products and byproducts fe in increased grass production and corre December 31, 1970. sponding increase in carrying capacity for livestock. Besides 2.4-D , 2.4.5-trichlorophenoxyacetic acid (2 ,4 ,5 -T ). 2-(2 .4 ,5 -tric h lo ro phenoxy) propionic acid (silvex or 2,4,5-TP), and 2-methyl-4-chlorophenoxy acetic acid (MCPA) are major phenoxy products of similar chemical structure, as shown be low. but with unique characteristics of their own with respect to species controlled and crop selectivity. CHRONOLOGY OF EVENTS Over the years there has been consider able improvement in phenoxy herbicides and their use. Development of new formula tions, performance information, crop safety, timing of application, spray equipment, toxicology, use hazards, and environmental implications have contributed to both better product and specific directions for use. These herbicides are not protected by patents, so seven commercial companies were manufacturing one or more of the four phenoxies (2,4-D, 2,4,5-T. silvex. MCPA) at the time when the USDA announcement to abolish the no residue status was issued on April 13. 1966. Basic manufacturers in cluded: Diamond. Dow. Hercules. Monsanto, Rhodia (then Chipman), Thompson, and August 23, 1966 Industry Task Force on Phenoxy Herbidd Tolerances was formed to handle 2,4-C 2,4,5-T. MCPA and silvex. | Jt December, 1967 | Submitted petitions to FDA for tolerance of 2.4-D, 2,4,5-T, silvex and MCPA cove ing all food crop uses listed at that time; the USDA Summary of Registered Agric tural Chemical Uses. Extension of registn tion was requested for uses of these herb cides in pasture and rangeland. | April, 1968 f 5 Industry Task Force advised of inadf quacies. in the petitions plus requiremeri for information on all metabolites of herbicides that might occur as residues) food. 4 September, 1968 'j Review of literature on metabolism sii mitted to resolve metabolite question. | During 1968 programs were establish^ to determine on which crops additions work would be undertaken and what sped? projects would be done by each comps in the Task Force. October 29, 1968 Industry Task Force requested extensif A. 2.4-dichiorophenoxyacetic acid B. 2,4.5-trichlorophenoxyacetic acid C. 2-{2.4.5-trichlorophenoxy)-propionic acid D. 2-methyl-4-chlorophenoxyacetic acid The status of 2,4-D , 2 ,4 ,5 -T , Silvex and MCPA Herbicides for continued use of 2,4-D, 2.4.5-T, silv and MCPA on pastures and rangeland. tension was granted until January 1.191 January 31, 1969 Use of 2.4-D, 2.4,5-T and silvex on aqu sites extended to January 1. 1970. Crop samples for residue analyses collected during summer and fall of 19$ Protocols for milk and meat studies w* established during 1969. It was decided^ analyses of animal tissues would include phenol corresponding to each phenoxyc pound. Dow handled dairy cattle feeding! milk analysis for all four phenoxies By C. S. Williams R & D Herbicide Tech. Specialist. The Dow Chemical Company, Midland. Michigan. began feeding February. 1970. USDA was scheduled to handle feeding phenoxies to beef cattle and sheep durfc winter. 1969-1970. J 12 r '.October 29, 1969 The Office of Science and Technology lied statement on teratogenic hazard of 4.5- T based on work by Bionetics Re arch Laboratory. December 22, 1969 Phenoxy registration for rangeland use fended until January 1, 1971, except for 4.5- T. Dow undertook feeding of 2,4.5-T beef animals. December 3 1 , 1969 Petition for tolerances of 2,4.5-T in food oopswas withdrawn by Industry Task Force !tolerances could not be established by January 1, 1970. the deadline set by the Office of Science and Technology. r January 19, 1970 [Registration of 2,4-D . silvex and MCPA 'tended by USDA for use on food crops Sjn'til January 1, 1971. \ March 4, 1970 ! Registration of 2.4,5-T was also extended until January 1. 1971 for use on apples, blueberries, grains, pastures, rangeland. !and sugarcane. .April 15, 1970 Results of additional work on teratogenic properties of 2.4,5-T prompted suspension by USDA of 2.4,5-T for aquatic and home -uses. May 1 ,1 9 7 0 Cancellation by USDA of 2.4,5-T for use onfood crops. Uses on pasture, forests and industrial areas not affected. May 28, 197 0 [ Dow appealed the cancellation of 2.4.5-T for use on rice Hercules and Amchem also appealed cancellation for rice usage. Each company could appeal only crops listed on their labels for 2.4,5-T products. The appeal istobe reviewed by an Advisory Committee appointed by the National Academy of Sciences. June 16. 1 9 7 0 USDA began the beef feeding studies scheduled to have been done the previous winter. November 2 4 , 1970 ' Crop residue work completed. Milk anal ysis completed. Meat analyses underway. Completed data will be submitted as amend ment to petitions prior to December 31. 1970. ' To date, no official notice has been received concerning appointment of Na tional Academy of Science Advisory Com m ittee to review the appeal on the cancellation of rice.* REGISTRATION STATUS Tolerances have been established for 2 .4 - D in applies, barley, grapefruit, lemons, oats, oranges, pears, rye and wheat. There is also a tolerance for the sodium salt of 2 .4 - D in asparagus. Data originally submitted with the peti tions in December. 1967 is expected to be sufficient for 2.4rD in blueberries, cranber ries. grapes and raspberries; for silvex in apples, pears and prunes; and for MCPA in peas. Data on residues in grass includes that from previous work and from 1970 residue samples additionally analyzed by Dow for support with respect to pasture and rangeland usage. The Industry Task Force supported work for determining residues as shown in Table 1. For virtually all of this work, new analytical methods had to be developed that would permit analyses down to 0.1 ppm phenoxy acid in crops and to 0.05 ppm acid or cor responding phenol in the animal tissues and milk. For present registered uses residue anal yses indicate < 0.2 ppm for all phenoxy acids in all crops at time of harvest. The phenoxies were fed at levels of 30. 100 and 300 ppm for two weeks and 1000 ppm for three weeks in the total diet of dairy cows. Milk was collected and analyzed for residues. Grazing restrictions compatible with levels of phenoxies in milk as related to levels in forage remain to be determined. There was no evidence of accumulation of phenoxies in the cream. At time of this writing, December 1,1970. analyses are being run on samples of mus cle. kidney, liver and fat of beef animals. Data are expected to be available prior to December 31. 1970. Amendments to the petitions for toler ances for 2,4-D, silvex and MCPA will be submitted to the appropriate agency in charge, including uses in pasture and rangeland. The 2.4,5-T petition will be reactivated --deadline for this compliance will be ac complished prior to December 31, 1970. TOXICOLOGY Negligible residue tolerances can be obtained based on information from 90 day toxicology studies in two species of mam mals. However, tolerances at higher (per missible) residue levels require two-year feeding studies on rats and dogs, plus fertility and reproduction studies on rats. At the time the phenoxy herbicides were * See update i t end of text ( t ). 13 [ 5674 developed these long term feeding studies were not necessary, since these com pounds were registered on a "no-residue" basis. FDA has conducted two-year feeding studies on 2,4-D including reproduction and fertility. Dow has conducted two-year feeding studies on silvex but not reproduc tion or fertility studies. Ninety-day feeding studies have-been run on rats and dogs for 2,4-D, 2,4,5-T, silvex and MCPA. The no-ill effect levels are shown in Table 2. Based on single oral doses in rats. 2,4-D, 2.4.5-T, silvex and MCPA are classed as "slightly toxic" with LDso values ranging from three hundred to seven hundred mg/kg body weight. 2 ,3 ,7 ,8TETRACHLORODIBENZO-P-DIOXIN The word teratology has recently become much more familiar. It was tied to 2,4,5-T when studies by Bionetics Research Labora tory implied that 2.4,5-T was teratogenic (producing malformed fetuses) in mice and rats. Subsequent studies have shown that a potential toxic contaminant, 2,3.7.8tetrachlorodibenzo-p-dioxin, is responsible for the findings attributed to 2,4,5-T. The sample of 2,4,5-T employed in the Bionetics study contained 27 ppm 2,3.7,8-tetrachlorodibenzo-p-dioxin. Additional studies have shown that oral administration of 2.4.5-T containing < 1 ppm 2,3 .7 ,8 - tetrachlorodibenzo - p - dioxin produced no teratogenic effects on rats; rabbits or mice. The obvious concern is toi produce 2,4,5-T without the contaminant* The 2 ,3 .7 ,8 -tetrachlorodibenzo- p-dioxiiL can be formed in the manufacture of the? precursor 2,4,5-trichlorophenol. The condk tions required for its formation are high) temperatures and basic conditions. This cant occur in the alkaline hydrolysis of 1,2,4,5)| tetrachlorobenzene to the trichlorophenol| No detectable dioxins have been observed] in 2,4-D. This is due to the fact that th precursor 2,4-dichlorophenol is made by) direct chlorination of the phenol and notbyj alkaline hydrolysis of 1,2.4-trichlorobeivi zene. ? To date analytical methods have been; developed and validated for a method sen-., sitivity of 0.5 ppm for 2,3.7,8-tetrachloro;; dibenzo-p-dioxiri in 2,4.5-T acid. With proper; manufacturing controls there is no problem.' in producing 2,4,5-T with no 2,3,7,8-tetra-'. chloro-p-dioxin as indicated by these ana-' lytical methods. THE FUTURE By December 31, 1970 the Industry Task' Force on Phenoxy Herbicide Tolerances will' have furnished to FDA the supplemental" residue data necessary for the continued; evaluation of the petitions to establish negli gible residue tolerances for 2,4-D, 2,4,5-T, silvex and MCPA on the appropriate foodcrops and meat and milk tissues. Registra tions (USDA) are expected to remain in force. jgTaftleBLtZiHisuhi from.90-day *UMrotfiwinn;stodi5W:wnoi5^heno*nie8ieiaes mvitfr* ^d isL -,, ii&0k [Data provided to date indicate no hazard no significant residues in food crops, toxicology data support the use claims. The er manufacturing of 2.4.5-T will allethe problems associated with 2,3. JjS-tetrachlorodibenzo-p-dioxin. The USDA has recently stated that pro- biting the use of phenoxy herbicides uld cost the U. S. farmers an additional I million to maintain current agricultural duction. In addition, farmers and their nilieswould have to work 20 million more to control the weeds without these Mcides. For this extra labor, the farmers ild obtain no additional income", veral hundred thousands of dollars been expended over the past several to prove the safety of phenoxy herbi;to man and his environment. From a ntific base the phenoxy herbicides can tribute economically, efficiently, and slyin the future for the control of broadI weeds and brush on food crops, pasB,rangeland, and non-cropland areas as [have for over 20 years. ICAffT CHRONOLOGICAL EVENTS PREPARATION OF MANUSCRIPT s.of January 2 3 . 1 9 7 1 several sig n ifican t rhave occurred. fy/The fTFPHT su b m itted am en d m en ts for D.silvex an d M C P A p etitio n s o n D ecem ber i1970. The E n v iro n m e n ta l P ro te c tio n (EPA) ackn ow led g ed filin g o f these nents Decem ber 3 0 . 1970. \Jhe fTFPHT also refiled th e 2 .4 .5 - T p e ti tion on Decem ber 22, 1 9 7 0 and EPA advised on December 31. 1970 a petition num ber had been reserved but they (EPA) w ished to k n o w w hich specific 2 .4 .5 -T formulations were to be covered b y the petition an d for industry to identify the ingredients (active a n d inert) contained in these products. (3 ) The EPA has also advised that a specific tolerance for phenoxies in grass is necessary; a tolerance in grass had n o t been requested by ITFPHT as it did n o t appear necessary. In compliance w ith the EPA request e level o f 3 0 0 ppm for a ll phenoxies in grass was re quested on January 6. 1971 b y ITFPHT. (4) On January 11, 1971 D o w was inform ed that the advisory com m ittee from the N ational A cadem y o f Science had been appointed on Novem ber 2. 1 9 7 0 to review the food crop cancellation o f 2 ,4 . 5 - T. A t this writing. D o w a n d Hercules had each been given an h o u r on February 1. 197 1 to present inform ation relative to the safety o f 2 .4 .5 -T as used on fo o d crops. 15 5676 5677 Reprinted Iront the Archives o l Environmental Health March 1971. Volume 22 Copyright 1971. American Medical Association 316 IS PESTICIDE PUBLICATION A H ealth S u rvey of W orkers I 4S2 in a 2,4-D an d 2,4,5-T P la n t W ith Special Attention to Chloracne, f rim iu iir n v n iii rTOKCIlOT Chamle, Gtorfia o Porphyria Cutanea Tarda, and Psychologic Param eters Alan P. Poland, M D ; Donald S m ith , M D ; Gerald M etter. Atlanta: and Paul Possick, M D , Cincinnati Ol V*l A study of 73 maie employes in a 2,4,5-T fac 0 01 rinuria and at least three with overt porphyria cu tory was made. Chloracne was found in 13 (18% ) tanea tarda had been found in a study of the same workers. Severity of chloracne correlated signifi plant six years ago, no clinical porphyria could cantly with the presence of hyperpigmentation, be currently documented and only one worker had hirsutism, eye Irritation, and a high score on the persistent uroporphyrinuria. Evidence of toxicity manic scale of the Minnesota Multiphasic Per in other organ systems was markedly less than sonality Inventory. Chloracne was not, however, that reportad In previous studies and could not be correlated significantly with job location within the shown to differ from normal populations in most plant, duration of employment, or coproporphyrin instances. excretion. Although 11 subjects with uroporphy- CO OO or -vT ,osmtippcnhTuxaohevrblyueomodhnnapulicdorbvvgEoesxerihbt.addyionlhceaeTuedecmiathrnecbeitstiihniisrdcctheterhi)oaidetwvpaeaae(rsotnpcoi1rdboribl)2doednl,2e)d.e4,cn,mu-hS4Dcl,eds(o5tave2ir-roecsTa()eca2rncarn,n(4ilhb2eo-wdaee,df4rpiadiisb,cdant5thihiteid-ntonleltroyrtcsawiomretrcoxiohumplirclyucchokoeosieretdmebunoyridess--, Submitted for publication April 27, 1970: accepted M ay 27. From the Division of Pesticide Chemistry and Toxicology and the Division of Community Studies, Hureau o l Foods and Pesticides, and Office of Product Safety. Food and Drue Administration. US Department of Health, Education, and Welfare. Atlanta (Drs. Poland and Smith and Mr. Metier), and Medical Services Branch, Occupational Injury and Disease Control, Bureau of Occupational Safety and Health, Environmental Control Administration, Cincinnati <Dr. Possick). All correspondence direct ed to Dr. Poland should he forwarded c/o Rockefel ler University Hospital. Rockefeller University, 6Gth and York Avenue. New York 10021. Reprint requests to Division of Pesticide Chemis try and Toxicoloiry, Food and Drug Administration, Public Health Service. 4400 BufTord Hwy, Chamblee. (>a :<0341. Tfancoapstawmaoaatrai((eircohyrsnptTell3hfmohotoiieonsCupr)aogsncCmetcraatawarcraehlnkos.hhp.tcooia--pPaxn,eecnseiellhoncmcrhtoot,dM)ordcdrrlorhndsCtrronoiiepeuahneeaaitnwatmmrdhnixhisrtSocacnjngonoheciulatayklncecdetovdr,nyaeutsnarirrd,heeleorioaaniulyctobmaudaanblwiboonnedtanvcnelraiotfbdzepecnpienvnehactitsi4ldwudoudseizhtserktyo*ytThi,0saotrtor,iosrmaiei'cecMsasmCiddenfrunn(rhg.senpnsikapeipeeeaepPtauyaieortdesstnfedtaherrpgsocxec=rraipaasmtasepcoemea)rircte.tcandinaeuurc4T2enmbiatpstcarhitnssse,eiTce,hcotrod4icteargo(hdoc,uipeMmrh,tiaadnTsne5eizdylsbeiotttenspe-Cysuse(shth2butnstiydhoePcmcTr,ia,ecD,etma4siauhetCcaecCiwbbmsil,noDtpaodnnnh5pnsteThrydDleignioeev-lita,od)tnasmooxT)rehnedgD.inm.tmroth1nnsiepeispuo:aineffcta1s2ectesaslpira,ivgcIedlego,,rsetsach3urKwreocu-nenaltlriez,saohnoalvit6tbbawihinioynteoolmnttre,opleeeoiuao7ggyaarnrerdndndne--ss-ll.- Arch Environ Health-- VoI 22. March 1971 00024: 5678 2 iS i,8 e M o a H E A LTH S U R V E Y IN 2.4-1) A N D 2,4,5-T P LA N T-- POLAND E T A L 317 ssPspSftchrutilrsyudeueogyHmqdedgdcruieepehuoecpestocsndontaatlilmintotvyooolaoge,yfsntnrio..ce--anxowxcAioifimoncIatlnircesttpkgedyyaoere,snrarontsadtNtetiaidmchoneidwennutiirc,csreto,iois2obgmamit,ngouh4npuxntt,do5sislcu-cnOoTitaunotsotyndlstha,.dpernlariTacsPnebSnhyhcrdlmtyoeeos2osbsspr,mtsel4aetheao,cimmr5annmiiv-nlaicsTycesl,, hMhodhooocwaTtpsblseawtt(heiiityauePflimffrnuaoohohavreleaacdpIPti2ndtyBsrnubniaeuyanlnrneecu.9olskesrins'nlsGrayyhMdslatoiraknlfooieiwnby1oipewstpsaie)ubf0tasoynin9nhenrmohn.ostociierrf16e;dso,iagsdcydrtiTer1rs,n4aarnkrn*cpeotmprtha1ohmal,rhrieoeuiip2;heoannyedamcfeirerarrBoxyrraiadaecpisaryelgcFpdcserlsvtlisowefaaaeitehinehroioedpnrnCnsoncneseisoogeyprlyibrtifodsxtlqsfmufraanrnaaiooeieAnkcigualtvilnncohrtwifnreisainr2selhnmegernadLtevgecinr,ieiencaetismie4ntetnsosAeiasedaeochmitt,xg-mgrghhsonslabol:qtpD)ehycaeoeenatsodueoraafrbapadcillnodynlecifadniidatitslaohrTdoncptillyhsn1ceyevtaioiiar3ffouiekratoncdcdaebaunnnuyeohsrlrhnihfsrbuienix,cp;scredpe2tpeilloco.nuiirrc1peaciohopa,psanfola*etices4tulrpooryr,yn.aloinaraeemap1--,lsavoagrp4ta5rsro,pdcehiiatfninityeotrh-ncaibgincredhgooPyTdedctyar,aoudeeebeiano{tedgrlortaxpluloifanut-iro,lyeialnsdachsaoarrmpil4adfrtanscaaneir,mloduouh0lltfbmeecltdohotiyruusgpudo,ytgbeohaiigauinnne.vsiorrirnehtolo1veenyndnouhioiceaerstdt1nnnaes.tf-.,,,, twtbppwtpthioyeeihcoietx.rrAutyargpihkLllrcssiiaoieiagzeintrauvrmeymtssteed,.,feamreuoalpnwrl1Mndpploa5toeydlahtpaawistoaoeinnrnffso-eunretyuinpsasntupst,ihcruon.otootyanodiffprnoFxiiePsnditnetchydChubwecierPrnThaiuaocaCli,salbarwceTrhtsnyrtohaeahiosrlrcdieoktven1eresda9hdaeyrtt6iiscmhsmr9oinskennpupesiwwslnett,tooiiwisohyttmffmhhfeaeoraspecB,.pgshtaaohlaiotnelriyorsiidny Methods apnlaAdclepldhvyoosnliucnoatlceceeuxrpsaamstuiiobnnamatiilot,tnes;dmso|t>okeicunigam,l edemdriincpkahlianshgiis,stawonrayds cwdtwaosetawpeffumberdbtPvscdmnestcdpocstrghiapwtwPw1p(iohrrrohaehiufnmtianctaomldlioorLeyee3elerteeBsoiahoaoeavaioleeeceseeeehoearbcciAtTFEddnun5t.nndrnrmTnslniyDrmnnthupnrrfeitotdprGealvhhpeoingrise.tithdoeoiefeheagcss-snlkelAdrdsilootacwiraiiocmptrHdoitbeoecsrpoarrricmisrcetd,v(.tniaaluseurtacaoeoyprrieboyandaaesasdemhaiyeccnohiBdoyeholi)crveapnanelrtsacTccnimutnyetsdnecoernuegie,itdrcfMioanlipetntleUhotodoilnproer,riltaeiiaeennwduocueialbnorlrmluatnineaoncearrncyio.atghebwlsndtosai(rtnnmentsNinpueeftahtmyrovplettrttbdidexcbrniMyyihtfceitaokseedbsoosshgmtdeigsniioosoieeo)aiitntones,rpnerpn7toeoSmhcreantrhpennyeai,h,nevtwiirnmureamhdMeyumin5alltrlnslimsrncbhicoscatewrineidwaowmaie,nssepfnlaevreetfeaa,tiaaahytyinaduoaypr,dohetnytiicwahotdeaonvPenrdta(ataenlloeplppcsnagernfrsatieitieiettSstsdeellresatehsiqntIaamoerdainqaihblrmn(ceriioasweyacnrnftGslenia)ddengWrepnugencopudeyonfoe0i,degaign.pti.ta1eakidaacdsohencrhsae,aiMhwsrvmri.sOlusdinihealtfwtopp0slenume;uteaovnennBltctaeooIIapySetsaemelerwo5zteTHlpadwvdremnuenm.csyalanawlesoftrem.nbwrCpmticn.ese-.connrliuairrtslops)enmundtrfahielaouowdenaerilewoieb,,gl)ifoattTwnehaaiatidmpdotvAonsse.crethirpro,WnoashipsMiamgeedndbtetlaevce,tueennchduratrnrlshshrgweelernlxlrgotldr,erlalTmontcsneroneoiaeeehaoeaafenTysliasabasahopegfriaedopcatnubtvcwosrlmphsoneroeialttasddeqsiose'ihtos(tsnyp,ugo.spivn.osoateit,edtrwhhtdirnleoneddtp1rmuteoeniattseeizpcnmlmime,bheretrainrciysiirfdjsrborumadoanlrsgiepslpgtsnuncrloeuitiisuoeensfoc-lcsannerpeidcerornnsPveohhgmghrboToisnitwtmerhciawldyote,tstatdtulecnciaetrroyatoiee.icernodihlalno.asohieuetneepoivfeealtltmhusalsrpoadrcstf(oaitnueyeoednalfTnlpedhrtcnshoaeteivIngoeGtdetagloodSntedsetdnrerotliotsndiyytrlbsiuaodilelcrerohsilao'isilduetdameuvuc1vcsndtdaiprbtlgcruxdyrooA,cwe7utneetxraicordwoehucinaerafehroafoylnuabctamnxrehrnadtoccoeooooblLmoleeaef7tlanatardooeoxlireotaygupy,gciietsiodwardmofrsylge.irppnnnAfr-lvtldvmannr0eetpeoiropldefa.i,lyaewenktochduesudediantt,,r1airlsontea)flcee1sntthhaacimacettsyiirdlbs1rteraheaiaaaaavmwafuwteenaoauanaaistorosttiootItuirfriastmlnninnanoeanhtseeinhecigcIlgenneslnlcuoiessoayotnmtobitsasstsltyinddnddddnhdneeehddoes)ecseeseegesslrr-:.f,t, Arch Environ Health-- Voi 22. March 1971 5679 0002495 318 H E A LTH S V IiV E Y IN 2.4-1) AN D 2.4 -T P LA N T--PO LAN I) E T AL results on a given continuous variable was not norm al logarithm ic transform ations were made before in fo rm in g /-tests or calculating correla tion coefficients. M eans and standard deviations are always rejiorted as untransform ed values. Results emmwpwWtesonwzalcac3mitrmmta1bb1rwmpp(fpp(lhyaeicaaoycTlafin93n48ealueuallaoieeeoaleciohenaertTcoAalugi.rtAd)rrDatissiairidd3csn,rrthrhsnieomozislnyynworosiel,hhsebe;ncotdtoaspNsyoneosnye,omentermntneela,ttsnyistesntrnedneiidsslsdpsaeehone(hrfncsceapcoipxasseeiaaei-stc2eonpanaigfugs1i.earnnmoeusldmalsvtaenra--tiafrngiricwa.eris)wll2c.fc1anandumpnosnescpntosoi,,ndtsoaoilt)khooie.iirntctrTeaehsrsauAousoec.1n(terheiilniea1itfseonetsupi,hevtlneneTdbnfhdfhmepmdije,mdr1eaanidspliasocaetfOegeseereecfphey.ln'tstvuerTaes9ipnundsdtaomatprtehrihm,bfieacifdohscharcDwpcontsblolinrrsaeaaretyvaieobttrot)oooee2aufdapexecmbshwinosaiisrso6a;ify6ycpte0mnnospoordmtilnpihsiso,lp5trhc4nvuxi3cm2xetoepenndssoanouceoiaf5ocrnyaeeeisama.oc:arufie..arhacesnssd6ymtnehenekci)--er1li;mslawiswltenkilaoeeuoenswneanepamed,h,,sl(otrwypdodsdtndadnurmeoliodtrehl1esSce2tdoteaeatdn.na,pso1neoh1lhraethrct8safenaer,weniagk1hwtcltnsoeiT.dpetilmuaeintthohrntvsynrdthpnutyeeo-hpahiesneaphm.tlsyeeooaceptnr,feoedsnsa7oad.eeres.if(adesoonevlxntbtrmprecnnF3mrbar4muTstaaoeTcsnr(tceaospeencrftiatioa)yse8dPpvlscohoqwldoooorchevmmoirehiewleivumr-.iaonlemnoksunn.nudlupo3eenmtenaveoomedwoPgnpreih,gedasrschueefsitwgrwreyseaegearfwlgpo.senwatrguitnuakoactarboddgie)weihstvist(ohlorsrgniidrteicuoh.ceiinaeotypaooeoanetiyne7oottrroeedetffssnngyeossfmdyenlh2tnkkitr.aarudpdnepruroigyo6eeneapteee.nclehakocpdeeeeet8ocmrniinsdoegesqeieaehurnmtiionnnadnnoshnrsaafoonureysaauocmobwrdfepdfi,d,esaintrcrgeerphf.eawaneauseeooamhdsirtttetebteccctaalnnpnnmnnrahhrhe2hh7ryIhrnuieyaartoaueerssltiinldniidyga2doeao3eeeeees-sssl;r.l,,,f sassrtpprsssittpStsowaumm4rcsltngflgsiosreheioaecniccitcocciicarafse)lrrcanvnaoesvrsoiouoteoaaamataTnArcrt"naec:eigtgiritneaairrin8aalrorerddokioceAvoerwsnntle,ee,eenrss3smlerniey,tyfneleliettprchio7scamoeod9sdesnossixst(otnaowoidbectlolncy.htctreofamnfgipbiansffgfhosfeo.taiomaatvnaeeapcetavveAooc,)oymsy2crrefaitrlcd0lhesv9enN.ere1nefereinwsek-r0lsceaosreopewscdfud6eeifa,lteopo)b(oayitnwlrmtElrooon(hfds.r0.csviaonbiootirsmyemorsetcegmrtcokegsant)cdaeceyryoah.mhsuehc.aatecrhamyaeatiaiagr8elfe.hnenomorttnew')hinyl"sttF.tlcTxpe1rido;rvessi,irdeehlogaoetsosoiioeeieeotoraeihwnhs,saStmmeffpn,nansrnrssdnsloaanhiicottenrstrcp,iaaokassaoeatndaefaeeaaoemrosod<Tiadplossentx(slnmerdrtlodtlnfadinrtiashswuahrysapoaigdtdshrsnppruessvir7h)etibd(tr,eiyawonbtrneeeiiraoge,eco1nmedreeboylwesr,fodig2mshieh3eeuthssgei3tlstppvnbiMhhst0onersceraalnsea5hoipierastueyaiieo3eomdswdobvaedhgercnwceesctuatrsdff,oresmhuenlsevoisroagtgyotrsreeseuaoruraehiriwrsaie)akaraesarrtvbapsn,it(lc.nemeoueadfnlotessrtoeisyigtlletfethsaeyswsacdlgstweefoeedTivismrehntmtaetcocavbsvelycneahareaae0iseahceci-ow)(retyenneoreedcmsserc,e;dseirhgsoese.rpdaegrreetnnneeaiica:aaer2siei(altooprslmtcamveaotpcnnlshioboosd9saootagnyrrmrsdneer0ogad.dfefalugetvlreersh:eslavleeherurxvastyeaisaooScastacescdmfaelcaetsiddehrseorowiat0dcmomuatawcoocrndniinhaeprifrrraeaatiamtncnmlbveecmeiateolaenahtees)rrlgat4aydeyoea4aeee1sslrrt.ly.^sQQ^C<ChGUjOC1C-j awthhwggri(icecyrarorihaelatAvTptnaienihmveetthcteereltoe0(pdyitoarfvh.aiwf4geweco-gssc4omniccrirga)reetceae0ek,hncxuaran.seinr3atrctftciterrienir9anhsonecret)dgenraiit.wenoiicn(donsce*en(Acauno=.reswNlrmol(a-eclorfletpereoftigev0l-hosraare.rtenconc8il)e0nono.0lo.taepoae3t)sfbmfA,rt8seooea)wipltPdpmvbgthlhaoeonaoep<swornuiylfpuctdeopii0nhctogeyreh.aoyersh0hmyrnstr0ieeetittre1hnallhnssnay.ielsuacirtltgtenirTcioninpdsosthnoiomeaeiirfexsnfrr Arch Environ Health-- Vol 22, March 1971 000249 5680 384574 H E A LTH SU R V E Y N 2.4-1) A N I) 2.4.5-T P LA N T--POLAND E T AL 319 Table 1.--Distribution, Age. and Educational Level of Employees According to Job Category Group No. of Employees Age Years of Schooling* Adm inistrators, lab technicians# ja n ito rs 20 34.0 12.2 13.8 2.3 S u p e rv is o rs 11 46.8 7.4 12.7 3.0 On-line personnel 28 40.1 10.0 10.6 1.7 M a in te n a n c e Total plant 14 39.1 10.5 10.6 1.8 73 39 .3 i 11.1 11.9 2.6 nepttp1ihhny4arloreeftMeHeliycsaooeyei,numfan"epcab(,smeaos3flrhanreu1apcelods.toiq5wigmocmuMs%meeonepvuvenn)eleocemajtnroufnei,tbnsnmattoaroehectafpidanetonilarslvobneiyntoniayudefIgletcasrh,ecr.niresaitideOtcfilnaegihhnjvmtheaiHieno(cdtup4gitnchroi.hno.os1yf--ouysn0s%aftip.".ic.S9e)bsatemrl%Thlvoehew.meoea)--nxddeieasanHyhehmatemayopoysid-tf, . * Mean 1 SO. Table 2.-- Duration of Employment in Plant No. of Years- No. of Employees 0-4 4-8 9-12 > 13 Mean 1 SO -- 8.3 7.6 years. 33 10 1 29 naaaphhmlpptbtttbfhcgdasaitfililoeehineanonirudnslccohiaaulroiaoceeneegocticacdotpetmvTdrepitsniaeocnnaiahmadhrnehliutoenevsstd.hvedeni--ttiledeirtpntcoiteiaiaonorioeivagborgghodaOdgtatxngfTmrinitaiwncreharnae'o(souensyie?gf7oo)ticxctCcstt,saortapathdanhi.)o3ruuanetiismanvrlnahac0oidmPnabunppsIyecctelrcet.ed(tmnnoeeshs,on0iea1eohentnopvenobtwia5aecpl4lgsaraaheeotcf(raldenicvwaehttaon,ldhyxoel1lsb)heneemteti<ei(0fdaopdtijihtartdoe6esojtosay0nh(eputscoeehtemr,e2utovDlrnPhciad%cstiaolmnshaDg%popdaaoettstetnacotptCgeehaisnrote<nmrpfha)nCwolhsdieeeteesrirePwstlns1epnafvs,pemvym:tohaP[e04dmi)hrpv,yopilwbasoync(tt.osaoohieeeh1,Thhsrtrlameuidefoldfayaeskecegt0peolehneeosCtrttshnunevltewho]truhtsaspatprshepekdaocchamtPnmc)siediao,3srtetsellztndp.awedhtoiciare.mw,aiahealfdo4odeukimnnnnewfeisinfttsal0oonctceoinihaofeerctipdginrooif;aret.rtenmrstvdfeegneihksremhntorrtesoTyeoaieh.1aaekee(anat,sotosaarnnsadhntahaeiihucainHfcpnwne)dptpdccmhatdtenibnbwsi,nynirtehsehovalaoaareaeesdeceeaitgsosteenncfsidwtpinnoneeise.o,nnrgoninrwnv,desaaridcevkwcifewgtrrosmieyntnnpreaohomveod2etxtottaefctlrerrmhaeuh,eherilyoloitrstioru4aaerrdinynynnoeapdekeeeer-ffl-,, sTpApteobTcptnmecepPccrnnntipbwvimtswuctii(onshoiiondcooeorrnloehloeuoaeenrl1aoshmL<eiiaesaaneguar,gipUprrrnfrSlttnyrttbi0ntrneibrdtAiyprnphoeennatm0herrbceiiecgr7lhaefieraoecoiianortteeteniwenae,v.e/ibilggoethareaapnbPnp0in,dnnoaoncdeaeeyaruxanmmsgslliipoe3mdohPtefsCe.ioaba5trsrpbcroe/tt,trbluPh3t,varlabeglrBetewlocphhearpihdoT)arBptetyor0yhlrdatnseen1macnmhpon.ehehteGuiaeeahf,tlsytiios6otntrroytouyBuuelEewcibhntvahyaeap.vpi,inAtpcoebosurnrruAedntngeazhostsrxlletoiehsipiinoetSiefselsgiruhlyeetdlonawsosicoirLrymnyyhreeshpioplseydhcbepph.rnscrnbttnlfoprnnfAoetymovfhrhueseripseijihrdeiete,rrexineegfrOeetnoibraetrescyl,slndahesrhsgaxpiicicsepUmaweegrmriergap.ovrnnuncaAleucentrmtaxoco--evtenhPavlanohssieearnasutlieoniylrriwexrtoaLctiyeiyuenBTftfersroedteenisprsptnticThituthaoopreanrAmniieotnsmcteeaGheararnaioaassileaffoiahavsotfnaae.odrnsllobwylghleeideottlyer,rineytPeibn.tl,puinghloldHralmsadigy)uvhdoirisbetywasoidootrarf.nmetndoliftnaraenieimaeanhinyeetewbarprHirdcpohrr7itay4brutyvnpopna.difrecssoteswsoweovn2ne5oniaayirhershthudiecefrccnsPeararaoaso6wrtoterteen.yoyvtwowoahepteuhttinlltafpiBreiphgttnhioerrirenrDsphweaogroOeo.ehnooorisiihnrrevacmeotGntaesrngygnenrerrtaoeennrrolyrdnoAeligecrfi.kml1o.reeispmgeme,mes.1lroraeppttni(95edvlsvawks,nhfimhbnt1uaarewroaaCvproBs,iesaawielyrw1eTenennlasnroslflcrmeepeludotlr-iocmlttrloivsephediutidfeahodstflyrhlcenwiieootrhewaoaitih.oennninhraaeipomod0rnmrwrcnPcnlkinybshycudtnuTesodulv.undrhhtti2inoeoeoedtaiurtitlusroerehehensghtehotyletiiaboi5eexryksireieietahdnhsynnsrrenegdhgaerrne,fs,tet., oo Arch Environ Health-- Vol 22. Mutvh IV71 5681 0002497 320 H E ALTH S W iV E Y /.V 2.4-1) AMI) 2.4A-T P L A N T --POLAMI) E T AL snwe1oxa4ancn8srsc.e3etswiiogimntnhie1f(nhi4^c.i4agvnh5vet0eslrrys3um6vse.ag7ll/eutg*vhemaest1e9fodso.f6ar,mciPcnreoep<at5rthio06nep.i0no2wmer5p,oah)riN.yknPretier-ensr eccbli0wtaud1tCc4rpjdamcrciwmatrchmbctietrwocwiunmhrhny5eoevlh.esopto2eilumiouiia4oeaawom.eaodagottsroindtenepCeeOAtu)pPnnr7sHrptn2tttorheers;ttppygrrrneucetieehrgorsd.oekua,lttdeanmnlomepvo8hleeeneeerssisaoohidrftasifrerHlogadnisnnfhecandnertpoaormedPyy,lppninrapayreyaapvetysliippodctgstsmaArsiimmlogl.udcnaehenertsgrtaroo,maaoorusyt;tph<sooasabhatemee(dLyelpidmilmnnveyrgdmoieutu2efsncbpgsrlslsoivsattl1EaAinhdaaeyesihvsppll0ileos7ortlonaiakgi6soinyvsnmFsretactyh,anceuihsao.uyct.toinneoieey0ckgdeosHhnv4aifseoucnlryuterfaeotorci0uiiumieusrerhle%(deownhhg(twesscnignsx(ok1bFmotu<r8lcae^m(ldeltpasc5aeeeh.aBseoscraeltyanoircrt)0s-a.rm)ivttnroxosah7rnxirieeortsaheti.arailo%ano-meeet-lmohnOooai.cecdgrrbisaafcanvFte5naput8nndemtfdeunireainuolng,io)neneoni1n%5olnacn0a-etotcsrv.pnmrdardmfkrho5hllid-o0.sigbr.ogentdrnteceoiyaw1aiptndd--4)eppe)ihtcercmig.oiusaiayysgoul.a2iooe2(nshrneauat.saArtbuangtcoo2bfenbratehn--e,nnp.es6ygapfbrtpTianeushgsrec0ens.lhicocgreorratsrlu,eI(lpitvPtrcsesa1aohee1o.dTouiirwri22nnhub,rtF6.ueslnaoaser0erlts8nPoregrun--w05eaeawnyetmhdtue%y<rta.ortdrtaahto1omcnt2.nsv%terersialtee6whhwueev<crsep8ueeaaefyerPct,eet)nrnrf0ixTa.bnoleryernrnplneamome)oebrpuyw.tudwcesrt0sreer0ngctsthner1aueOily7sArsuma9rohrmaeadenkq.an0hssageoetes2mcoanttoe8t0ts0gfreaaesmsfu1heLvouirfneleksr6fottnostk%5smhmgakaodrokiiaroeAteidA-u.k)sbotfgoosamatmt)rraair6epne(kyesen.ohiross.ecaekLribasnnTstlconeerieeen7lttfomaiegdtkmtttceshslhosioArTeno,ai.boae3etpuxeshraiteoafrtxerrgbndoofcuwiemcwhlcpwvihdiorTy7sn1dlacarpaHmianeskilbaieie2.eebn5geiegterregnnihtrmunie2itbrihobx.sn-gelxeoi.oraiaoifruashand8teed4ynlya0naceyfeee5e.,flf woTTatlmKiaeiwtmsooKueopismo[llvbophaofttgtcfho(tuLh(enhnnoehiaelndotnwuarfn3rcfnlflof9fhoarhiervnnkaahaiecaeosgjiowarsaeetgusoobdenGnNv0mire.mseoelstevrgiealelewos,"vltg6teenujhlrgufgete2mech%lnoxlrse(stseoaedupmasiausrhb1%dniprlaeuleed,tw-ae/eotuSrdp(causnwi;oe4leilsi2rewnAiia1brsxnoy)eve2nmetaoncrttbanennlvaGtdr,e1in)0enadcsraednmaios5retnbroseo.eilnlfrngjeggnolen7tpietr0mtiiiw2oeerOmoor-rcxeytlliKstsehed,nwcvnaieupmvldTes%odo,cifmemoasitnmfr,ooe4oeilisTeesm"dxhdanarctgiitnwaussrnhofatrafnt,ymtatadaxaee7)dithrieoft5pinicee.rhnllnaaaptslenfwepmhirsohomtctat1a(svoies.hlls-girdtjeacesfnrtdouheat6eafnSiohoauu9brpiTas)fbtnhoeaiooe/atnceeifodmbrxyli;dlnwtyr.rilimolhTytmugohrsn,rnwaryKsxoatnoatunwjpyseFfft4kirappehvsrehFfettwteoabageoneufaotelatoeeihou5nitediiaauacerei7oolliotpnutomcrnlacmwtntrt-srcooselv2foryl7ihtllswahnmohwfttmaieesvooetngprKnssoudRl2eechbee3eor,aee.eorineir.nofera-siiiwt,ateistntinoxoyyaestaereran.awottfeAmosnchtbostsotshsoihplrcakmtmtstshiwar'Ot1nx,/vtaeTgefl)erphnhe/ueroomainuhermaetn2nee.ti1,breasxmasiorpganlewpnfsrefcduovrhas1tel1erde,gd.ieml.0doSnieehptrsnslrlrlo8atlv--ucren1teTsssy,ukoobysl'seioluen0o'lheh!aGgfrnnets7e.ueivhlKayidyosaeeyawecsir--rne,resemttaveeiorlscnuenSep'oK-ereyonOmtOinoleumpvewvo,gsadrepocowsoueokrrdarssgnneeecidnsihoniwrrSmci(neTAsfnppnnmenavwaeudidvsxguenomedh1y,wirfpuinlpreleaestnehlagtiycsfneyraepeatiamgtrys8aosoeninisgvaucugwrsuleehntavkr;dtntloheritd-.rdndrmciltse/naibancthaee7a8nbeldcaAcaoroksupinewaeigmehotiarncrdlikvsneisnfitetnoje.tlifspnndeaaissaaxueattlehtre2fhtwtiupotaeeinpgtvaadu:gmylstariibnlnamincenntteeci%ueenlnrroncosehhe/dyhree.istldyo|tnsntcdgita1dcipSsesunhdgnhsenfesiteosrsav.)andaiamtgue)t,itm0ism'loetlri,Gest,ncolstbisu,oIb.arurttpcasetvtolTlewvf0,hhpely1gniotyshenihideimoaaaotviscOlob(safnaasvevtanWlooi9thaoswnn/sasoonec4iinvetht4cosginelulllseee.7beftemiesriTn&aooradeasglfci8ysrrdil0e8irsxethtiidytr.esyd(3dsnnOy^ass/f.,tstr,-Ci%.JA"fT^SDfO}f Arch Environ Health-- Voi 22. March 1971 0002498 5682 DOW 3 8 4 5 7 6 H E A LTH S U R V E Y IX 2.4.1) AXI ) 2.4J-T P L A N T --POLAND E T AL 321 in the neurologic examina Table 3.--Distribution oI Certain Signs, Symptoms, and Test Scores pseTjtdahTcopmftMmmdBmmdddtnlt1mmchtohr2tfcttto(p(wueeiihiuiiewiomeo2rb6eae9rroaeiurliaboolochxroUvrgdfeeonagglfgrsioaurTeRcdcomEa40sie8reksennnm)lmnaeepesttmgnqnra/i/tyerryetrde-m,Neho.iichcsnie1l1oeepelhWepehmebui6anxntenriigasino.lobrotsaosegon00adnvaennodlo--oeoegaaieo%d(cytthyptuelfdarlftgslsse0re0aanhnesduodnronrhraiwiniwteenvetle.tfdu/tascvyndtes)ac1onrawOsaurttdnehoeei1rds/dyaoa1h;nsoakrmimi)elToninedtrtssseocsaefc0sldviotnfdfc.0nretmd-re.od,ny.uintthstfosahinealwmeo0yvu0-ilssbnlthstri;nsaTeecmc(edioylsoh2rwpueoeoS.atfeeean1ycWwfioor"unhhssc,huiuemomsrlmwsnnoasanrluiua4sooo,urfosrrorodAsehOeaxunnthurcebe.krolosim5eefgbaarofnlnamllasibAomamhaittcroees)arne)eh,thstljfw(twhoteastlomdsmutouho.ereoantctsh5hmdisyimfcsaiairelsernh1enccwcclmastforonehru/eg0huteeLayneyTi.maeg4oymroeoteetsostrruraecr.teridcma.dpolryolDp86hnklrusahlrrhssibtmrhna,utNaoscuwlasrlnie,raimntalseLteioempeeirdn(s)acuoHodoaholyyon3maptacjeareioa1wwis1,wloaveeinulfptnspaysne.ceteselon(niiaoo5170othsbutbeenrruagdndeneaaekcn.upantea(yrhgv2dee5nmwi%fyvreoanrierlnnytuenebsooioretTr,eamoefbu-icee25ssnrttadsrotildaioceeigs)reca,dhry)shl;cnelyeiaw08ccot1dtsvim;oxroaementaebaaoooerat77irme6oarhng(vpgobpoommdbnlrnsbse1aa.d1lonatoetia9seflrentumae,ntlhicaTip0ptoNtoyierdtMhdohlnotoe7ngedirirlrmel1ra.rnadoedpaeueneHScHEsneaiae4r0advfearer.7galsoDncalbemyvesceef2aitlasyTngt%(,libeeooMr4anenetigoltaeefghepelropssnb4hbcml(gdfeo/leep,yiou3eraeravmitfueiplr.iho1ensssc)rgalxecdrteosinoaone9elaiatrreciereiteenconis0mmican,ipeedp,oiiips,neloftntnatctgtblfr^ycaeg5iers0ks,asimcrelhpertigslas1hemigdyborMeohb,oaveitAeslr)etmtmnoa5s3loiaatwoi/w(ohhcwmoynno.ssaoaecnMoomudi(ydf031caeodt6tn1eh1n(eefrvotiscdv)nfeietiePslhoen0e2donle72Ie7rn4ettseeyiionseatrteaMr))estIhainninddn1o90oea6eeee)0essaes:.f.i,oaawgtnrimoiintTthnahgawymTwccn9warafdbrwNmaireiltt1tg7ltBh(e,srwehewoheauaynvecelre5oi0l<5ohaheisaeoiscoaaonneresmnltaOceMd1%tro.nossnes/3n5osd5elol11ob3nhbdtmeorri9s-ei-Mo/5/tls/-h/fn1pgdsa%ett9rime22.oh22yhpn/Chy-boe.hhonn;aa,aPgiclet,4/y52575ac5ome0ciosNeaiyerlaanoanlldInugei(oev7niarour0d0byapt(((v0sh1r]nnsfg(loeeiaa244hnrhed0rrs,o;e0Pnyua2atytpagee0s..rsnmncrpfiekeeit7w00i2nE5d%llpa%oexuni3ahtohnhge.ml<rnun%%eadpv7,5mwmormryoeglng2bne0oigle))r(tioteiteoao%eso))0r1natiimesfg/0,eooes<lwwnnphgolt.ssifsrdt6oo.et0oi0uorold>gotac.c2dellpnrnaanwknfe5witgroeyerfv/ceea1lvtThAiS1cenhg,)n9ty1solowhtv2ce.naaapeed7cume,elohelohd30ogfre5elAhils2tettne15rai2locativecidif1uo(ee.0omsvfai0alms48p2s8sh5cesmcso3rcsyaeseudodiefb0s/i/h./lttt/11mkixeas,38/3m3ainni5<n73toiobtmateivcvAace46scmen5ot55yi4ttdg61%dylerewtphucilndafssrhn%0ps(gae-i(ynsilrne)(1o(o8pl1al/c4ana/2eeie6taaeoh.eAo)ueco1em14oafir1tdwoli4hwi3nb3ru.nngsnfhyu0iooy9cesipr0%nOf%isSnc%%nanecudm.mtan/tehspayamnTro2casr1lahdteams)nl)e))s1smte(ayyeoonaeaso,nthutah0oetfee2ve3rrhveseesbptstheSlansma0elgbgrn9ls2auas2etyiasbseohmuedle5wtohin/.lutlg%rpr7owy)2dunuf1yemmiphv2ofpaasgthmiloo.llistpeindc0rhl;164sfgtl1duty)naahleiytyetpreawic9yl064/hii03/%htthmidmaoveelavccnegi1tmnue/e.oe/ta//eythro91iehm11rwe1nhhnauaSdr>r0thlomsrpoise3a33i;2riafavalrdrgWitei09ifssap;asnhiees(ds(v(nenltsgafecgha(mri6t4s8ilioonm,1aaseiouaocalhxro.1ocuBr9e963mdfpu0cprvcen4a(pen0ielrhey%ren%w%mt.03TunoeCactleye.a,nlwedlh0hhumsl5eo0a%9ottaocuntl)t))ishoa%)ealaehhu0hranooooti.lyovorooeo)rids3-dnhyerransans0-eeaefefftf-2ff.., A r r h E n v iro n H e a lth -- V o ! 22. M a r r h 1971 C 002499 S k seA oa 322 H E ALTH SU R V E Y IN 2. /-/> AND 2J.5-T P LA N T--POLAND E T AI. Table 4.-- Urinary Porphyrin Excretion Test ALA PBG C o propo rph yrin U ro p o rp h yrin Plant Values, pg/grn of Creatinine* 1.021 i 406 444 2 3 7 39.3 19.0 Trcet N orm al Values'* 1.900 600 tig/gm creatinine 700 400 pg/grn creatinine 0*175 g/titer 0-15 ,,( /lite r U pper Lim it of Normal, ig/gm of Creatinine 3.100 1.500 175J is : Mean SD, N 72. t Measurable in only one person; value. 107Mg/gm of creatinine. Values derived from norm al by assuming a 70*kg man excretes creatinine at ^ urine. 1.5 g m /d a y and 1,500 m l of ssshptssvcdirstemspt7TsbpcjMtsmsnfmaowtp[enoeaiecctroeeuccecxialrnraNafyro6oroeagnunenooaoMpreovoaenorlobteplMrMaarn)eonfrtiedhddflirritranenefrndsvjmdeli,pe-ePnsdteepedie-esicrnyoteeopicsluswMtMnrcstsiehoacoaeactoilasiInxdt,hetrrKcatemogol,alta5feonluiyilsnarhnipyioetrntPsPictnod2fln,esinhoanaibthcalafobsfacodohfsnIIh]cxatHlmoikteleiosuuyeetcgeonifeyniaaohtcnoaepcasnfrrifxtpttihu)ayrtsdbvzaoemselsteeh.waluootRLabndpellthredheeholeownfnstratatlerrskerdire,oMeoaphhdieluesefrnsutntveseneyoabstsnsfwaerraepleuelsFtgrneudoinuinoyceudgmr6toteosphedslseaounbraosibk,tbllahwumr9,emoadoreacnlmttpdytyjbpersjealtteeypsetbdhaarrenhukepdyeeaihue,sorwhj(in.sinhassnnrcamejcdc--sr.sienliprTel,oiheedtdrnacattgswtedaiaifTMcishaasoscoapaaiissLaatdtersttcaT(bpgtnc.rehtoeeteoitentoidbuetrtdiwtKeeaonuoohcarcehrrilrdnehehcfnTdmlsaAlsasolesaaknosrdnnhaseolieucyleeeictusnbhihtelaydtiyvraanidslrflcoaiinhdlldsenaioeuu1hacgelvaibegbltcmarmrgiaiw6bMdiacesfiy3wsatdcinetgseroelentosirgiyebd.lob)oitsiaiieivhrc7aaetuogeniohi.mgsfnsheM;nauoeaursiraSondt3nesinuutenineantgeshmpcacdcpuh,a.sgneFstailtbpnftDLtatdaPkaatohusnds,,ssnefihooteTaemjaltasnttveowfhgoiiscIeeenubeiasdr.mdsormwnhtienarns[neerlcdectadnlfoaonaodeeorsNciaenAy-toifsobaeoantnidrotvsfotfneloouiacsehaatkseabrilrrbtdaeslicesgrmblgrnnoendealockutferreohuytnenliieebyhrecm,sireulf.dmcvmreldrwedibevnsyeeocfciessehqgnu(ritsFwwwicet1jisiaa.nidihsmeFpunyeredhtnraosc(nou7odlu.saurodterAtaheeceideeenemehihnieni]usortnnooa>ntreAastsrsslLFrndfiind)oeeeeeeessllsfrff.t dswctnnocreooheofrataearmdessntesimsagshilvniefwteioffnortiicthercohsaabnstntohestdiitehlndhyuierecadrdagseiitfrpdfifonfoeueueronrcrspaarsfosmtlrnioionlamiennl(v)aaa;eenlnlaty.htcluheiThsrnviehsewoselptpsishllecmleaaicornnent.rgtaedfnnraoeidrnsKmoes apsowc3ltmvo6ejHahucnvo0nifa)oytcoiesTer.0mdinrstpirtor;kuTehaeii,ponMpxsdlesremhbacltwrerrroeheosaMsaeooeefsfodtniatnofdrncphginunPdeMrlvaornaydec,ewIlbernibetrisnfmo5inisiasieoWiattt0citsnsnnahehghnsa;teelbsteaionendisststefrnwivgthoftfltsalsgoaradwsieyotcdcoclancnaarauemaauankdlnflWemenespleroeaidnesmcstossnrrras,oa,msmddfilorrsaeopworierwta(fdsedrrfersnfNilIsomeeaetwtoLphdovrhthe,rvirieeeh,mufiyeanvmtactnoaicplhieoattpucpsMtaiioentileSotveplsosocorssnetiMrmpaohynantool,htrkoieyfgcniaxonPfeevne1il.widvtIsldem0ah(mu,eLWorT)pmencai,rwhetsaoatkatohotaaitaecbepfaenoetennwoadlralurfsnndyeeysslfrlt obvious demographic differences exist be ptwopeeunlatthioenp,"lanitt pwoapsultahtioounghant dththaet "anomrmoarel mtioenaniwnogufuldl dbeefinoibtitoanineodf tbhye pselaanrcthpinogpufloar personality patterns appearing in high fre quencies within the plant. A high-point code system was used which grouped subjects according to the two scales in which they scored the highest The following high-point cmoidneisstrhaatidvethsetafgfr,eKatyesptomfraenqiuce-hnycsytcr(iFcn)l:, fad- 2psma3t%ahlil;c,pfrrfoed-quuc1etn2ioc%ine.swIottrhkiasetrsa,nphopyaprooenmnetanpfirecor-msposnytachlheiostyepattern tended to dominate eithes- adminis trative or productive group. Correlation coefficients were calculated Arrh Environ Health-- Vol 22. March 1971 0002500 H E A LTH SU R V E Y I S 2.4-1) AHI ) 2.4 -T P L A N T --POLAND E T AL 323 DOW 384578 s0ms0smacsstmwwffhiciooee..hsgg30ioeiooeuvrvlttnn53aorsrhhneeee,ntiirrdrhffs,aasio[lpiieyftscelTccttyoovbePpnhshaaarobrveeseoennepbofitre<,tmttrowahhslflmrecyeleyejpaet0ahovehcrnea.hc3eebiag0oenMcrintimr1n1erarhigeorv)mhl)Mihtetu.moceh.iswaelesparaeccrPataetolatanoenciwxItFclsorerhtneicncseyucingaert,oes,rserh<ntsrtcos(tsPehcwiawaauru.ttoaelnnhhcpaea<onl--Arddheeessssf, Table 5.-- Clinical Laboratory Data Test BUN (m g/100 ml) Uric cm (m g/100 ml) Cholesterol (m g/10 0 ml) Two-hour serum glucose (m g/10 0 ml) Alkaline phosphatase (King-Arm* strong units) LDH (Wacker units) SGOT (K arm en un its) B ilirubin (m g/10 0 ml) Album in (g m /10 0 ml) Hemoglobin (g m /1 0 0 m l) Serum iron (/jg /1 0 0 ml) Total iron-omOing capacity O ig/100 ml) Saturation of iron-binding capacity (%) WBC (cells/cu m m ) Mean 14.5 5.4 237 95 11.2 146t 22 . 0.44 4.6 14.9 97 348 27.4 7,160 SO 3.9 1.0 44 32 2.5 46 13 0.16 0.3 1.1 32 44 9.0 2.190 N* 72 72 71 581 71 70 71 71 71 72 70 69 69 72 Comment Variation in N secondary to sam pling d ifficu ltie s: tor N m 73. mean age + SO 39.3 * 11.1: 25 % of population are Negroes, 75% are w hite. ~ t For N 58, m ean age SO 39 .3 1 10.9; 29% of population are N e hpswwcwtendatahsumbtiewptwttps((iharhihhuicoyirnesoatsrr1grgooioog6tuiouoe5geneuerOm.TdpttitisA8nhrrrgrcm6hmstsdgo0vcaekkkksahi%uetoaepHc%e%ieynlvafcreteeeeteholernitcnslisupn)ii,rtnyhotsrrr),acna(unteeyotshssdsietswefiea.a1.wrt.sghieipnan--2svihyatnahdnr1lnwmiwheeed,,eteieatp'lcartt%i4sdanivvr*lorsml(sndhFriieycose,eybeiuTstedeue5uvrltueanaon)hnlpmosrirsherewmt-2ybagemctbgnu,vtelimie'wnaes!,abohsthiasioinhernao4o'pyatcatcmtty2etlcityituntac,dhmibfpeyleatght35eatnoofehedtdoeshhll-deoaeeos-oeondoya3eerafeTaesctsfcar.oerfan(efriit)nhhegteto(aon3gvyhttfee.ptAdyhericm1sctspndehe2deaprrietcshDop.enoe)dhssrltc.t%lelcpitewptcnoesouanropOsiteuhoapoIeoritrrajopsentiao)tognrvfhorrueenpitomoesmrvfotnrptfesihoeelmuce7nhtulsuitemarvaroycaeneivaapa3sgochlrperstosaifolarficctgelithelaaasohhrgailifattionnrn,ensdeeeovcyicvaflnyietigtoetennttlndnwhnooenvhgirsruhah,intciacelr,iterfemoy,aciedtnieobtenosers-8ecbserfplseigecetEusoot"w2fdaueeihs,asePohlcscuiitloocan'n%n.esmeaenntarbthfuoettCnocftvlejp7xldrtdhdioooirsyrueaospenaess1rc2ciirTrfeuth.cuambnncgp%nleeioher,cmoftpny4ntuqcldegtomMfBlhipowyhysgpaav,ttpecasuaraalvrue5iiylarerettoteoconieennovaeienyneeh-etrne1intilarisrlrisdTdnidhnyndueddneyeee-w3-stlteh ite. is probably due to poor sample handling. TtseoTdtfpabpfhaaeTmttptflwmposthhhoiheriohvcfdceetnryllohrCnohCoueaoleeaoaearneencAdplnymacesrsrsdtdndDnorsrePeehekectritr,spsaeciiewutttgsSidreodeeneaDhd)aroenid,cpsae(rsccnei.nfeese1msxoyntuinuinsnihe1otshetr0sgpAfonctgrriaietue)pibwtutacgyiiwmooomntahhntatcllnryasmhlhehhsfdtsintisoceeztekp,eotehatseuadueemtschedrh,idi.etrnietevotrsbnnenefsr2aniouetinecretxtceaac2oseooanan,.ieatpc,odahp4eictnvn5efxntntfttdtaieTsatienctaro,trineowivthymt5oucoraboranntvtsihiratetieetph-arntorgeeni,uceeioihe(neTiheaptymeplrnniydo2rynmeoapceladasedtmonipswenhir)etvcretefibootnoanotloyfrtndeoethimohmaofusiaciasotorntf,uncmehttnheocnnttdttstoaootluiThrtephhrvetnasephyedoenoetnoTlearleeecnChcrennnnahhsrslesdtvvesh1frenCoeit,thrtoatviDlpiiuveeeehruclrepgneooncsaa,arPaasilceoemldrDaheupfwdoelnat'tostctnostenwsymeelepiiun-eaichhi,tavdwnntolcahddsrwshm'sktiha1olrecgiutectnaea,aooecrtheorniepwsnncpn,oicstoytruoneeeoihterhtethTfanrnenligrnshe(roneveodcoKdeaueuasamsC,v2atavfufsan.fvneronntaptiva,pntefaeroiliiPeewe4mfemuwtdcenunoHolexoolpdpin,echedohanp5psmttsaaiwpeotntnlhtetiytttttinno-efnnonhheewhhhehccTcinaoetnooctrsatdrgdddhoedeoeeeeees-rtrff,t. C005r-01Arch Environ Health-- Voi 22. March 1971 5685 t 324 H E A L TH SUDVEY I X 2.4-1) ANI ) 2.4J-T P L A N T -- POLAND ET AL Table 6.-- Scale Scores on the MMPI 52 P roduction W orkers 17 A d m in istra tio n W orkers Scale L Ft K H y p o c h o n d ria s is : D e p re ssio n H y s te ria Psychodeviance M asculinity Paranoia Psychasthenia S c h iz o p h re n ia M a nia Social introversion Year* of schooling Mean SO 52.5 8.2 54.6 7.4 53.2 8.9 55.5 9.8 59.5 11.2 59.0 8.8 59.8 12.1 57.4 7.6 52.9 8.3 52.1 10.4 51.2 9.8 60.0 9.0 51.4 8.4 10.9 * 2.1 P* NS < 0 .0 0 2 5 < 0 .0 2 5 < 0 .0 0 0 2 5 <0.0001 <0.0001 <0.0001 <0.0001 <0.05 NS NS <0.0001 NS Mean SO 46.1 5.8 54.4 8.8 55.4 6.3 48.8 8.5 56.7 11.5 58.9 6.7 55.5 12.1 60.7 6.1 52.6 9.6 50.8 8.9 51.2 10.0 56.8 12.4 50.1 9.8 15.2 1.5 P* NS NS < 0 .0 2 5 NS <0.01 <0.001 < 0 .0 2 5 < 0 .0 0 0 2 5 NS NS NS < 0 .0 1 NS P 0 .0 0 1 1 .vpmtvmta1maetTt1(hhhoarMinsn0hcoeoieeortpTddneeeindCnpoeThrtcTrtuo2pimo,weuhAolefCcC,aaacissm4heace)tcDh2D-TtdikndptDaec5TlbaDCDdolahrceatcgCnoroDfiw.oedpiosroynttsPhdepohdDiTratsmto.eamiefechohn.bloinsiSoesnl.ensrertdoovteeemtioaudmferdaTAscpaoildrriocfrlsemCbrlietleeme2siaomdsst2dPesdl,huuesul4t,ooan4trehfl,aswttwvo5wat,yhhtc5udert-siilauemei-TinyoitetadnxnThdhsdf,--.. . CAtbTo2Naa2ntmcleptsapflttritooaoohhsiehatecch,m,nooonrrsectght|ta44ctwectneeeo.inpnIorrlexeChidsuSTSiSvTsre,k-hapsnori0gmccdxeee5sthaetDohhrraiiieasl1replggnodige.uspeeerc-peeeotoutnonnnpeannaniotrdmimiwsotcnohmrdioyeccncrtiiifictsvgmdaboffrwfnoiwtiitlenetiinrriupoomecapsccencinhoansmsbcgeosoedh-daaah,nsld,oaatenrardooesptlayoep,inncnespunjiplqrrn<stnorenclrrnrcoccncefceleasvesliraaugenrnaiogopeezeoy2ehirpatmrnao/elsnwcanrepoe,dogsmt.a,,smelvfttbibat--iclhsth4iponmoopiixonupatanayyTngrehobuglp,euctplcnreuctheuo5reXslASdhrmnOoniaioaostldostaesnedel1r-oefhistectuuiuusluTasuiwnenDtotenotsnpteiepsnedcccf(ymwthgbas33nvno6shsesooca2Ddednhyp8t6o.obdtet(tioie2aarne1oc.saehi./ctFuSifl.roei8srrd5tms,rtafohnauei'hsneegttsoi2usatd2sunTwtnmvg*myam2shettehsntbd,s9s-totahiuhrh4v,cefcatgrieoe.ido1i4egeear6oine,rheseeseanenhb6inr5svs,ceiteatnirrotnpls5c.eytfiht'ee-ep(0qofda)isc,.vourditci-Tcd2dfnecplaurlitettaartsfoiasbaiatrrn-ht)lraydienrtsthuimonecnriehheyccctitssilwteetnlxhvnssfswtltyaeihtitehyfioteytwmiraedti.olewdyeadiucgftnsoedloctutcrni1ahneiehehecfmirvortfilffhrnt,ntihxetovfeteamt2eaioosehlutteatpcasr(deraeos,noteropnmeo4r1e.yetriensruerdwdlscaenlfolo,ni)enaeicynh5yerthestftstnodh-sitcyh.3hT2s1oo-nt(tanloafn(tiPte7uhi.o0grwkPAbce5hhcgldo.ioooemc/aer9rtahnay1etyeee<esre.f-ffhtt,<7se*0u30.al2.0.tmwssdm2ltwlAbcanbtrtal2tb0s4ae1rihauyaelg,eoaca.r0oi.oo)aot4tearncaen7vefcaiA.ePnTi5tngvsrar,oldecoecatki,l)5chykkoecehisdl.reemtknpsneam-de,oeeiuvrlhTseeoT,oygnprsopmdrehysnopomtiaspNnNefNdhiirsatnhogtuftsuPsznsrpsSaSt(SsoynlueiottovlhaelawdPsfleae|!)hrdsopthcaedrceiatlnonaTiiCtieoeinr)esnneaai.uinlnyese.ndqssdCsrntvllecpTg.deseaessuA.aleCvTl--ePprlecet)oCpaaenbeisitsaehaurtpisntnbnhtmluTophalnnedreiDi2ra2otewteeteteaeot(aaiiienex.tlechr,,fTaoserlaenontylyCn44intletddrToaepntnyetvvtgecieThnue-t,meehhnttitPro5hheiD,hmixienhraoimhsnweetmfcos(-t.sleakelphthtegvthTMosggeeoanthhTiopeTrtloa.aeipoporeanSns.chanmsedaeryatdrwCiCDsaatllifmksuknxercrkeeaocpa)ewcnriieeAIabeDttuidnirCcdncodgeooanddrdenrrtsocwpc.oeetlsstiaDerimesPeooiieahshrtnp.eut.dhtsnaiq1kmtotA-rleet.oraapg2mrfihooech--9tuoiinasrcrfoa,mnlnpteafr5eseceete4dipmlhnolornAtohmonglre6tesn-opralodyhrtbeiDsaaosprtp2hsriipsnyawidseeeotonleasfvltrni,ieTsleyrnphaoaru4ptmmrodvneoheeareterrhczra-eepCedxdmwoiyineTgmaeeiDestecsalrultoiiebacoeetinPenhtoavslCnainadccuoecftvslenrtseobrdiedioethhtfeeatDtoaeuikno-xeiinuiaoutfrtalaaernono1eloubnteyhomotrtDcaryisktecnrndoddiinnnsnyet-eroel.s, 384579 Q A rch E nviron I f ra il h -- V o i 22. M arch 1P71 0002502 DOW 384580 H E A L T H SU llV E Y IN 2,4-1) A S D 2.4J-T P LA N T--P 0L A S1) E T AL 325 smcTatazDceuhkpe2pboiannyattenurmC,alyiCui4dumidupooblocnDs,sissalPwal5lleae.aoeZioatuBD-dscrindgtrTgitshcnyhile.oi,eynoceecageeAWhpidgrxlbbeopelatpaLlepehxeohaefrodgtnoAcei-rcrrsnrg,eagr0ahmreirpptnhintaueweasmhrahaantstyfvieyai)ennoteypdenpeoe.osrtlyrnrtondidfi1aehAoinntnrtvd9acebaotubPiel6amatonstlersiabCdl4se1fiscinee3psaeiinrrToznnxevasosegacesc,iaeuairlisosynonempdeinsptteppnenfheehrpioaoretoeircynPhooeinxtetfroternheserrpaChdpimdatnsiailoPieostTnitstrhaprsreffdCcepii.o.o-eponmhrsaTldrhu2eTlsitRegaaimyrenaei,rprhecu4tinnrsdadesmainte-iocetttestcinDtastatifvsoitnuw.laiinbcigtsnDgrctotrgih(rTayaoiltaxautuCbitieunlxohchntdilneonnaiyinPyhnedeedcfl 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tsibaptwtOterhiapattaeetlptprrcmotishhncttchioterdphaaecoleynxoeathprhoyoonoyriwoseopnoinntainRpaTrnanprcenieopnnrosrrenmutopuPtbsezetctarnkesturepdt-olhdeahrhlthgesrgeh.nloeebeoraerupar,hyhtwscorvrhet,eetpdnheflhniyhgtirrreboelfkiTeeyesaaaenltdsepsealoaoahcriepdnmseyonsit,tcpvarrchxsdierhncfentyssiaenosuritnwtortdnhesoesiahemttneklyeeivnlues)wnakaudiyonpncysestnshlmyrtmair;iutenvcdesggtihaenroifuenittii,iarpienirxstcorretaiitlievddiirslo)vhiemenbhtateocaasaaedcbxemtatnstethiisuefeseohrr.rhnieshnrnfe.icadorrutcyftaessxieft,clewaneiatasedchautenofohfcucoeanohw,tpanTunegndiitsretiftcnehhrlprcnsytohwoapermdriltepeehaosielitfcwplpyueudapisnnrionrecndnehiacraameterugmouaesettaridaoohaesehnn.ciontelttyini3rokihspsrrctetprkfaiemynofy,e3ennhic,igeeteeeaiopioe.ieacdaipe2e,nnnltninntpnn1aurrohliIacoidegn,tsehm3to,tdiirtrdnntt4ngpfantoohhrnasnnnfmhhatteiefsoe-eipdrreoasedowmleweedibarmetcDapduaiptsochahtnpihxlsnncrayiatggawnvepaaaiewsaert,eumiydiieufltcobraneptecdi,ahnolnomhaharrhiirlideacconsv(ederttncfitidnois(aetiboishno(hotna1,uslnlceiteshgyacieslpydxi2imeoucetdpbsnd)uernstnisnunmnue.op,mhoirri)taiibermPrechledetsydttddcaaorola2tErioyepllsxewesnCeoouephs,aygeysytrep,fec,tvsepxyrhr4rsrtwhue.seo,oTo.naaoeeieaxo3eyasnlao,eitoorynatrm0dar5xrenrcshdiu,nppcmendkflfr-rdpnruntii3re-nsdahge;epoeiisrlsdisene2TipohsepdminocenoyrseclgerrtrfatevotfyetahakppuwsgenturmehecnidoaAtobtveearsrnntoaeha(ciahercsereimohpoenedliitricesroiontdldtlrydonyrisneeyeeenrea-st.eenssfs--f, 1.2 .4.5 tetra chlorobenzene 2.4.5 trichluro phenate sodium 2.4.5 trtchloro anisole 2.3.6 .7tetraclilorn <1iben/odioin Arch Environ Health-- Voi 22, March 1971 0002503 5687 DOW384581 326 H EALTH SU R V E Y IN 2,4-D A N D 2.45-T P L A N T -- POLAND E T A L 0iadeltgicgtnitaol.xheurvy0scrrolHtaioe5rsoernstyrinershiehmsnvapsat.aceio'riaihvvnnren2tSpeyeeoesa,0pioii.4ttglmd.ohnaeon,1Bsu5texedoei.iea-pl"icckaTmctsua1aroerioes-tdltlCgopiryysetna/twlilf,s.ekoasi--ointopngxod2riaatnoieT,cwcen4lsrtatT.vewfmode,l5'inenlAaDdoya-ktoneTerrl,sDtaaknnds.eabscielc,dsnAaepnbreygrwvrspetuiangolhtelgthepsteifaroaehciaimfcphtnodaeflrhsieetrtseecywiapciinsodhsnltauoatnereavtgelddocaosteoveoeotatfsfeonhdsfbutwnaexties,gttliiieehhenicitoaindnndheees-f swnonltr7ftachceiasleehdpaaathpoclajuttreiroinioeuiihltaic3udcloxanonrineeheoaennnv*oohnauecglcmbsninl;lSpmstrndrpdd-dtnoleen,disvctshdarehr'jimmh.yeecasyiaargseeneeuraesoyep...ahtotOr,cbtnthtsp,acimpwamtafcodniweoebclflWotveastyros3clHoeltOhfaet(tvssniooseed-pnaaieihpooob0odpifm;tonbtwoefettsdloetrfnnu.drdexyaipehp%ahyoisitayncaluflrtoiTdgodaAgifsitsfaopielhtlmmecobci,clnciioroiroimuueh)umaasdhanpcehxeahmmlkulesgudrbsaosrantebeinrousmTacdaaodeabiiihaettinsnej.npikxonltrabltssnrrlcgeoibonsoldsvaeaicdenesaaooltlnbriancdneIixcuniiupet,hs*gtsaarr(snitsv,nnthoooiwniicieetdgyondhe1astrhyoaohmaamcee1ioffjuraoao;i)ehm,r*titeintsnenlnenimrre2s,eerittmlmtlrcttatirhohrhchdehbsdimyhtudhy,yissleTrikloay4ael.cdtosanauaeaoneten.oam.eesnnilConia,a--fcdlmvpnlaMtsmbtntx5eumns,yiasllfdochAheserdserfsnDxnltl-igalpeeotiurefAislyte,2iTeedwplwoplnhlhvfoeymcit,apdnnipDtn,lunoihsarlvo,eleh4rrafinaopotcoraemepnsfeuatacstorophret,yoihStgehroiitgyvhn5eaehdeocbcoriorjufaeeneuteebarsoGuuhnaneoro-xenyclimftfrnedpdaalgieTaantidtrridmhoottedpetsolOrhbctepanhh7ehhrpeylipgsraiflphydlldiweocsrisipan3aemavvTlosvtpemvn-seiytasafwiddisagnB)fwaa1gtioute,aeovnupleilpiu,eshrhymie,dtlcnnirarsbdtlnlaniardaiaiiusenefbvenseehkwfsatsgseostlilb.,oenbrofehxtrcesieotpeeree,eimvnnlueebtlunxDdisbdteatarrinnsiaooeacopnihssinepnlsrsoncriamcsfboccruhlnitnoororeycafcdeiereywhvwpwnwoeownegegcuesnfvmtmilmoemtvnelheuitlvunkmaheoneimoaoiiimoihan7rcbtuoyltaontbionpaarearciciissrthdnl3hehnoieceesess-lf,ftt, ocmptspottstwabcoenclaPAdyaacdpapMtitscwdrittiatttghhoesahnohnyuiheeeffaohubepesarnnileneecieloeodhtefeeenafecesx2artevrutoMnnsrlrTrslsMulMnaadtlhsecienhtyeanooditeoaieysonheyt,icw.iesucsthsrtectthoao4rhnaskgdetseor.rirndwferheeiertsMiagiretorPa.rcttembhdaasd,deoisviebennenerdaeio5yngBsbrhodniwsowccteidIossgoeenarCevlaliunaprctcPpi-tye7wremteounhihriyaarartffianreoittciTirepcahcegronokahdn,.Ihtedttalblelc.anuwrydtnhaerni"eno4ahaeoooi.rnnrctfoeedtageevhtoejode--bt-plsihelit,lbinrhengocer7rip2hmdlnctoenaeearuonatriwfalbsiyatalicjpinihrr,ofdeumctssdraivdtunolnleTbetsc4rnkHea,eeetpihcteatsoieostdherenoclnelissencdsn.hys-nthivilthaoliereohnecabihnpitiealoyDlnntatdseegmieoctihnesypssnaftwgtuaeeynoasyrsutswto.oasdiishrdaThheecntetttoreeoislsrfnolooilgshenuhhioiofatnboBrt7eysaoneotnahrna"MRogn,ncebsdramoryotsdseenntlirtsdvsccfintlnaweehiiuosoaanarc.eceaenianooiepfceusiyefattmnsonbdMguobelrkuldeiianihvubsdairoptrMiauudntrlictgrsagroenoentpshsti,etrnideoeedeootmtrmheeldeadnwiPt,pccetrenheuosroet2tasrlvgafiitidhesobeoshwdshemaonaienipnItwicatpvef.n,hufpdueyrtts.eosf,lx4honticosaaduiarsnna,wygeibteiopesgoeifles1snniup,atnyotucTeclfoosropaifeteri5tbl,,esomiiqhpayeiconbi,anhbttcecc"esueruogwabgnrnnshyr-tgetoinlttoutslsoihemoriapjhyaoTssesnhlrijnuNo.stmdedfanureueiimploea7-osoo,bnmshpwtctin,edxeytspteWcnlreccoeoiatynnuhuremnepacaiyadat,iyeiegcferaathphpteocsnsnedhcclwnrltpolatotspTeoiwcnlrwaeowdossateeieaondhvhsoefilvymrsesolcthpey.drtontmreiiiertrpwymoiastaaeowyyaicieisfntetolmaxrsfhonrbfgrntTorpnseeyrcucsrdtrvie,ofssiihoifrkbemopspeenunetikixnrckatedsosenthheauhlvopmeelxhoiolrnosashdctrearraepiWcrcngenonaeiooieeerftto2fpeefiesmhoorlisoinshstlc.tiyewntsssweeirwfsftdmes,un,)aton,eoarefmaiadper,aosaaie4t,siauediaitrclossneawbavnz,hasnnrcxswnplnT,ycerefnaudteaaaedwau5tglaifthoesinbadgagpiooeiidwinnaIitncrhnniinnilen-brotopvttavecaeoiooadatoinIecoergideghhTndihyddsdenndhgeafystf.eessls.ffr.f Arch Environ Health-- Vol 22, March 1971 gflW 384582 H E A L T H S U R V E Y N 2,4-D A N D 2.4S -T P L A N T --POLAND E T AL 327 cfBohauaunnedgrewseittwhahll7eicsmhs asemyvaehsrakevededisienaadnsuyec.emd apneicrsoenfafelicttys Thomns Ashby, MU, Augustin Gombart, MU, Jerome Schulz, MU, and Arthur UcPalma, MU, provided clinicni assistance. Patricia Hickman per formed the ALA, PBG. and porphyrin determina tions. Janet Gating, MS, Community Study on Pesticides, Washington State Gepartm ent of Health, assisted in preparing a subset of our data for com puter analysis. Frank Clark, PhU, Assistant Professor of Psychiatry (Psychology) at Emory University, assisted in interpretation of M M PI results. Jacob Bleiberg, MU, gave permission for entry of the factory studied and provided written records of past medical histories for comparison on some workers. References 1. Schwartz L, Tulipan L, Birmingham U: Occu pational Dictate* of the Skin. Philadelphia, Lea & Febiger Publishers, 1967, pp 336-345. 2. Hofmann HT: New experience with highly toxic chlorinated hydrocarbons. Naunvn Schmiedeberg Arch Pharm Exp Path 232:228-230. 1957. 3. Olivier NE: Chloracne. Arch Derm 99:127, 1969. 4. Kimmig J , Schulz KH: Occupational chloracne caused by aromatic cyclic ethers. Dermatalogica 115:540-546, 1957. 5. Kimmig J , 'Schulz KH: Chlorinated aromatic cyclic ethers as the cause of chloracne. Naturuiistentchaften 44:337-338. 1957. 6. Schulz KH: Clinical and experimental studies on the etiology of chloracne. Arch Klin Exp Derm 206:589-596, 1957. 7. Bauer H, Schulz KH, Spiegelberg U: Occupa tional intoxications in manufacturing chlorphenol compounds. Arch Gewerbepath 18:538-555. 1961. 8. Uugois P, Colomb L: Chloric acne: Case re ports arising from the preparation of 2,4.5-trichlorophenol. J Med Lyon 38:899-903, 1957. 9. Uugois P. Colomb L: Acne chlorique au 2,4,5trichlorophenol. Lyon Med 88:446-447, 1956. 10. Uugois P, Marchal J, Colomb L: Chloric acne caused by 2,4.5-trichlorophenol. Arch Mai Prof 19.-626-627, 1958. 11. Brunsting L: Observations on porphyria cuta nea tarda. Arch Derm Syph 70:551-564, 1954. 12. Goldberg A. Rimington C: Ditease* of Prophyrin Metabolitm. Springfield, III, Charles C Thomas Publisher, p 110. 13. Epstein JH . Itedeker AG: Porphyria cutanea tarda symptomatica (PCT-S). Arch Derm 92:286290. 1965. 14. I<undvnll O, Weinfeld A: Studies of the clinirnl and metabolic effects of phlebotomy treatment in porphyria cutanea tarda. Acta Med Scand i i u . i o i . m u iokx 15. Bleiberg J . Wallen M. Biodkin K. et al: Industrially acquired porphyria. Arch Derm 89:793797 1964. 16. Oahlstrom S, Welch G: An M M PI Handbook. Minneapolis, University of Minnesota Press, 1960. 17. Marver HS, Tachudy UP, Perlroth MG, et al: The determination of aminoketones in biological fluids. Anal Biochem 14:53-60,1966. 18. Schlenkler FS. Uavis C L Kilchell CL: Uri nary total, aqueous, and ether soluble porphyrins. Techn Bull Regitt Med Ttchn 33:57-66, 1963. 19. Haeger B: Urinary {-aminolevulinic acid and porphobilinogen in different types of porphyria. Lancet 2:606-608. 1958. 20. Klim t CK. Proul T E , Bradley KF. et al: Standardization of the oral glucose tolerance test: Report of the committee on statistics of the Ameri can Uiabetic Association, June 14, 1968. Diabetes 18:299-310, 1969. 21: Sharp C L Butterfield W JH. Keen H: Giabetes survey in Bedford 1962. Proc R oy Soc M ed 87:193-202, 1964. 22. Osgood EE, Brownlee IE, Osgood MW, el al: Total differential and absolute leukocyte counts and sedimentation rates: Determined for healthy per sons 19 years of age and over. Arch Intern Med 64:105-120, 1939. 23. Hofmann MF. Meneghini CL: Apropos of fol- liculoais caused by chlorinated hydrocarbons (chlo rine acne). G Hal Derm 103:427-450, 1962. 24. Cam C: Cutaneous porphyrin related to intox ication. Dirim 34:11-15, 1959. 25. Ockner HK, Schmid R: Acquired porphyria in man and rat due to hexachlorobenzene intoxication. Nature 189:499, 1961. 26. Cam C, Nigogosyan G: Acquired toxic por phyria cutanea tarda due to hexachlorobenzene. JA M A 183:88-91. 1963. 27. Schmid R: Acquired porphyria. JA M A 183:133- 134, 1963. 28. Schmid K: Cutaneous porphyria in Turkey. N ew Eng J Med 263-397-398, 1960. 29. Rimington C. Ziegler G: Experimental por phyria in rats induced by chlorinated benzenes. Biochem Pharmacol 12:1387-1397,1963. 30. Lam ent NM. Hathom M, Joubert SM: Por phyria in the African: A study of 100 cases. Quart J M ed 30:373-392. 1961. 31. Strickland GT J r Porphyria cutanea tarda in association with hemosiderosis of the liver Iron absorption studies and evaluation of therapy by phlebotomy. Amer J Gattroent 50:202-207, 1968. 32. Felsher BF, Redeker AG: Acquired porphyria cutanea tsrda. primary refractory anemia, and he patic aiderosis. Arch Intern Med il8:163-167, 1966. 33. Peters HA, Johnson SAM. Cam S, et al: Hexarhlorobenzene-indured porphyria: Effect of chelation on the disease: Porphyrin and metal metabolism. Amer J Med Sci 251:314-322, 1966. A tvh Environ H ealth-- Val 22, M arch ISTI Printed end Published in the United States ot America 0002505 5689 0 6 .9 c  210 K 'e z i l i K . PSMisem s w WOKLDssii ire M l-!9 "9^' NATURE VOL. 231 MAY 28 1971 3 , t Y-- i- J -- / . k : 'fud i f?7' f i by our Washington Correspondent mrwiu"tBdsttratnTactdcwpmrcercphwbpwotfparrmuaHmIaiChsDrtarpbrnohhahhewteeesheeonnonelxebaoeterodasenaneeusniiheoiaahtaaaooemeevsaruolaeegaosfapnApnlepdpornrtnorSesvld.ibtdesneapasipniiclomuteistpmbynwblVnuftO.doonomeocttcetaiofuooelhtelleopduallhsarauiioeloirmthusruDsrlcrtvwmtmdriTtlittebeccccaineaeinlohirsrrtfhrthtrtetiatnehdTeegirodfcnsnertiai.etieethnyicranesecdeofceomreateetiMEiditceotbtietrmgrii.gskapmartmnittcsvgsaehhdoiodpeeedcnsfsusridniebtfoiiPeldtylctriahsilaevpedtnTaeecmunsoaperbagoifriirenageonsoDSwtPdp"mdionleeccrpiretisl,rstsdenAirdsthhmirdxcDulgldahbicviraaltioeAet,htbceomroultcaao2netraseoanmaeseewtitueaerpeepponleheyelborr1nehatrd,csmiCfytoridftoit,obsmrswdiu4wsrppaftrndtoteev9eishteenelssrieo.enthfpiLaoshfeigtltwip,ybetohltcerg6vouaerfdnaeoptlhdao5hynlphfbgvhtneeepeEeayxelriloos9sepayihrwDdsetsefhn-reegeoieyiarsoevseetec.Mx.2ss.nw1ciiTcseoitnP2ysaulnsaahettaibtwpnnreiniahetAtwishc9,htihvwlDBttttr,tostunncStwetee4pfdttuiialhtefm4r'iehhaie7itAtsDaetahsc?llueephfodrddoIdiees.tAbhitlmoalhel,aemOt0eiDor5aitinonneare5dlouervorasynuruttvlotd,bpynoebbt,ehennie-aChhgtes.bt-myPciSdcifsieAfhsgrebilelitTnuerffteeecwdTespterOdsdeeesaeehot.v2ocrepiauer.hdastenwhaltboegltcvorn"hrrep,2ioh1,in,iahaTcfrpihusaoabeilawvDidsa4oeaceawbwirtogsftcoebo9t,swpatfloctsiersealsshtimisth4onsisgr,,siluliceoosivn7eelDvrtcrcowree5deuadoeoroddineototehc,eemeuhtrtaesvernt0guceesruPeaC5rnsprre-esfieeulsoefnerhuei,gocfaattfem--eTdeeryifoptlslnslt-dtSanreilnpam"nuooyedebatostoohcs1Taiotottpto.uecrshuntAcnppnu,uairdsshmsntarsrsv9ttnfil'cimdAatwnlueeconrswVhesretpisehlautenegrdomlypee6detpog"aCftdresceb.ttlraimtaeednoStosaiaeyrk9ettrwoshiitnbnsrmIfeucgsusmorbupetenstiensvtPildcrotc,tco"itcaehseagehucosnpeossmaoaflinnauetrtptiimfpiedSthivlgoeierelutont,dtteh,seatysr'rOcothitodptwdlrnhucia,huidoaAaionoaoensuaunattfnetttttel-tteftteneuseoitnahhdhnhhiihhhbeeehceh--edoonaarooicltiaomtmlcrlnlasewt.rsCistidodnhnndyyyddddedyyeeeeeeee"eeeeeessaesresstrrfrl.fltt,, .TcTdecSdTaowqwdehsDtigwwelacdstbSCasrPaaehttttitPtpfopDr2Dtf1hnihhhnhaaooeeritovofervnbnaebeuerboouaeur9o,tanfheSehchuaaoofigeoetre4usuvdaeeeoeplisrWsrcJialcIjsttea7torcsenwieiaeysAsiddudsuppd,eefee.htpbhtesibaufougrslobbeesc1nh5ntifiterchieaclyesefleasaresohrernhondfnaCefte,nrl,eutbnddt-sRwueautna.et"raniilaPnigisrrosastslirgaogTyhcguorniredvlddcdefnrotoltvttssylcviccitvoasSaeiirlbfcohnoomvebedtfetaogteeeogglsciewntnuieeerpeniifpaodAtod.dihekleenesanoadasstetoanodetfescemgshltGadept2daoinihniretghyentodt'dy.tfa'xittuitenCwnngesdnrasna"ssuhdt,uisnooietsvrepnyeseedev?bef4oudahnllAvcdiboulkeetttitrdanacannddevefc.enpnymoatihs,decfCleetbeypolsSlv.is5setthoirttoroehriheieettmocteapnehcmoeesyosrdheoetW,fl-hoposaindrnannonte"nrataPsrasuneuesOaoefTt2oroespehtafhsiDalerteerThforgfgmsetodatanbnsnlrBpSseeeh,rnvnpnehfoihefPsrlensydaba4smibiftttmnDeflmrhhed,p,eitcAiystvaoiheodoaeuofmrmelSe,ma,yboAsdeoidfmawdeeareiea5tssbitAeeuaDcO,r.tbecynSeeCwgbpAecuetefwdrmnepraeaa-aeednfohdisatphnsnponbohfwferivTpitptacreeadatmadtnstCetmctffnseaoplihuoaotstbrneeioinmhtftvun,eItnrhducycinehtoroic,eseivsildeoegcfamnyinoduitredtovdrihehsioirrmatiepoleieifsgalnlnupeiperdssrtrpraheviendmdeteueeyftnml.ehaubaaamroJholopaihtP'geotiteoa1fDsacrfeebmetstucntnoichmlseohegtlnnmctnapt.sheoysu5utestSoptoen.fioouteisetrfrchehheweihtstceeeeWotnapilanerldrnonemeAPrcuacnnehttogbPiislapoasetctodolarlrlihrhmtoSaoapbaonkm"fhecnSttameei;thtrnseetoSoCra.nettvtoetnelisaidrcjofofIhuatdehieosct.thAsfitt1glrmopsavealihbAatCtEooittiipono,dyaarmlociattyetdeu,me9,BelifelcpnnaeitdetrnemtChepdolfgcyshtom.pxtvhlhChpnign6w,tfdteeirr,ardirpivgnoeerifhiisOeanpop2i2tesi7iomsecohenLteetlMnveotigfleiinw.eargneclawowa-riar,,eeurotednldaangansnlbeieaept44trsfstsceJdirgrp,mwcrtetdeOeavinalceelaeitothstenhlmcpeoee,s,olesytg--laaatehodicnxr55nnttraurddmittuawetineeteietipcrSoiineoinnnnioanhhcehcbgrhchii--licooctcisdlmollnrendcgitaostisrrlTiTtiTonnndtdyyihiynphygndhenyeeeeeeeeeesssfsr,f,l..tttttt ` ,DcswcscnthcrpewepclyrPhrcbawbgttr"2psmbsscibMbMtthcbsiOebdsimuohnehmehiteeetuoitoxhoocoouoaaoascae,eoeorieeyeuSonudeaefia4glrreegacopiiaaoscscpeATuovnavnnnubntueEtlsfkvtamncffeiAsadspre,imdenoohtyuiliotisnsgtesnfueeee5stastrtttodceiehrocaotvaaysonntpoescmwariaiotlsneacrCtihoaanlsn-laherilwbbulimobgoaaeidstcitroeeodiemcTstnungtnmsimlsatas,emenithtmrneelaanyehsnesu-sbotsiielm-toaosceineowerddmehsat,fdtlooisprsitgrcsoaeoiistrevwfbensrciotnertncidcnnbfaiihfaocceeioieuoarpefesymnefsyfwsem"bnnuenwatnoeieattieihhimftsDriu?anknfmtrlanrnpporosoheinclmogdnomlpStdcncndhhaniaSipoeebsteeyttybdanoaaeiuoaffwceeglcaoeeachoalptye.sadmchlnte.uu,uoeeruxustnnlthtepaigiitusomnhahttieettrsymunnbttlxerimelaaftctcdxfssrussvatiePrehia.hsonitrataeeheoisngaheeicstealteeisdigeMiar,aaetlnosetevtbSnssnigtassoaegot2ccgqttodsvletset.entrehcrofhtpwhrtrimtoeileusditnhee,Atsr,ueeeniueniHemra,etieedttleftyi4stopooPTeieoe.tssssasealnsnhhslseoeoldtoo.aeioC.yhtseraresSatxriadbceitiehen5anemyaninrnntmte.elhs.hftosrackistnioeAfcnacliutah-ciaOgihtatsvtdshtvnbotoeintcoiwrrswoTigeeiniihdietohriiddagpvroregtcCwiuntercrei,bsaafhlnmmsmnnrshessd-oeBezofeyPecnrriaeeraaiegc2tcsrtootgaatTaatbhrtdneeTaiccurehc"n.neddiaeabaiiSuh.aao,uuhesd1t-pptennadaienr4attaosomxtaraee0drsesnmstessfciwrtondyAaaepslmsycsthew,yimusorhsl.tcptnbdheetpi5tmdofetntssaeaNtelvathp2ehdnoechresoehqypoeCooeosa-tript.nppnnmetlereo.,ntroniregroisohTfbnaouiuasltooyten4obiUeaslxaeoudierieoghd"thpouetotshhentetorsmfef,mtornseicielfim-bhrppphisrh5Fns,enseeedomritlwtcttonssdrltneatadr1epwienuodlaoee.aseouo-ielslsceatiu0soetawya"-annvgyotyTpwstiidoerenqsosrrmiaoegdtrscltdihngiot0dPdrsetopdooPyaoorselsoftetmci.gurmhdtisoyiyehalaurohloennpoe,rneSmttxdstisSf.atrateogtwhssihosthidhpatuefshscnnwrestTlsitsosiiaAAatoaahoafsuesclrhacetstcnthtoee<tt-fttut-tecwvl:chlhsyhhe-ahhh:ivibn.aotcosoo-itoh"LCjctiotiCs,tiaadttjr-|r:?|{ofnie)oeesoejeae*nocreinfieee..r,ff-ft:t-?'l'ftifffi?tFf.LC|,,-..>-;cOfOSn5l ~_59I 3? NATURE VOL. 231 MAY 28 1971 ' 0 0 N2 I55035 211 /fl J" 7J / & l/'* jrf--''-dlCs"em2i(itcadlabonftprdiashmesgaplnmnpdhsme2iwtleiaitmhxsibosstms2frmsie,ornsonitriaboMhgiertadonogiiasosto4hrtsedohacuxmihtt,nhoeahehoneep,eumodaraOuansviise4enuardonoormsir4tBa,sonxlipaiesuttdacoomisdxoerMneutub5nxcmttgenfuegpstv,ieiidrttxpluy.qfp.eidtefuruihaen5uua0futfiheur5yesel-iacliurmthdhloainecoinelaDslnriuoitelnnlT.erclriha-ildsdltt-nlteondtct1ffmytteeelthhdrndoeldiTne,whduTnsohinino.emhrmaeiar.au,owotatraDscsstoteninmeseaerywliccesettctdefB.eecnwahbPoiantsfboreniwhetlperegoleatogpieepnhdaicwoibTtnstiulsocbdeneneseanghSemuon.ilthmsheupundosn1loufdeoaeadonhotnysescycdpiedtcasgtiAnosei0itmfargrl0tvimroibrenri.oumuhncuncn)bhviuoisdnstTeosetat,eqrtbt.asedeom.eeeEClehnh0mtfohretco0eeeihlpiirddxfuxindladmePguofhhvsiocuvnoaxnpnlsr.saintoiotoomrn0adltneri1poyiapeunlndsarh.vecaaaeeSfteltiimiru-tsenalnltue0raTewwpgivhfeansnmhrcridsupfpylnoenseemmsawmcoA2cntosenlclec6aocuaaeeeoe.ufihrtmeiaseorrlgntdaniaoeo,nLroeeidossuogvfttptseorniseetdorshnuC4mta,;lcfestiMyrhideisotmhaorbresiinxnsileesinpasappDoerntrdtodan,typpobnadngrceiiilu)os5titannidiiteteurinlsctltaaomnetnitqinaooPfdmootrclnohocnleytaeoteNmpnnx-hsootrteiureatiJolhPrussnirunqmsdTSnaa2nnseuadoitcglgatfdrtpndal,dteteseb.itefsshedeocpchmsnaSate0muhus"reur,isnAtrnoendsuailtnnoeqiea4lzthsoooaccw.oudetbiicpasfpets.obaAdiBseh1ocpcaytiddctdvouetaf,iCoiibscnoicetpmrasnfnycn1retwoo5eniillfponteraonoseithtneaehnuehoiCluooeputaurte0ttrhatri(n-prntbotnisnoaceaurrirtnolbtorioTarataiDnamtveTinhttO2"rptrnpstyeirb,i,sntpenyhsmneanghtfygecsuhtmudonessnsieietfp,ehryatasaoBolgcmgefDeteutet4eahpJt.omaersosliencdocf,eae.nstieiinsdttltgdorntiushstsi,ltiauhcenoooctamdhenh1hnopf5h.TBa,enhdhuodipyexoiauppondsitreptiddmmlpieAenelotijc-afmLeotbedfaoahnnhwvptiasfilanpeefsudptAatTbihaolfthonehn2siaeusoteoTrnyiastome,nansDm,opitose2rfpnebsaPstacanccsienu,,tcteclPotlrtfsrattelwhsitger4fhiokn,sylmti-idrtshemiwSetaeeie,,emridtee4biloStoecvpttobpfauceh,ioOmiihb'ucpotu,ntapoyiavoa5bAptn,iinivstjteeinoeAiithitnfcO5eafhectlebfdihasuhudonrsttcroreSogtch-bueabodhborierortnioorgahilCesao-iaeoteeeToieltnesnieCisltseaeyadssilfersrfTaedt,reteTharseffyl*nsyds,,*rsesf*atftllt', .cbhsPoiteqpociCpmiigauo"orsh--srobtac--mhamoatnhtdAekseososmotipnehyxaiunueocenSThuahesnnemeoevdsnsfiaoegbeenrmeulaocsaevTttimntnreatyunotAAaicmapbdazmkocTemilluunowsalttgmisetpt2stststiedseodcwctihaspenaumietyiossPohehicetrpabwChctheli,opocsnieiim--e.ostcergsienldraelr4capurinleeSnlddvnotehoetubltduroiateiardintnhtbial,hnroagyvtiieirAsoaesotwetne5nrolosuttwbsloasltialetNaotobhioatuthheytoxnsitartsefnhtu-oiIuntrirPnaobPCotdnanrtharewerleflnhgsTerslbtnrtdiasehiiesdsysycnxreeSatedapdnstecgaclitcpoanwit.aufdetaheerchynscbrttepl1iseAignhhhttsdstweoteriouclcoseeimnD--risowhihadxee4snoehuuaomceesnaefrnoleiecohqxfptCtooogpnitegaarftsddrengsamtbitunoinoasscotbOpwurfiaoneotinugmmnarrnnrnaioehneemgdiesAaambwantonthmbrseieeirnoiorrealigsddvdtnisfesrbp.tcermuoessbpcktryiatswluoanalwtdekteiesemtinhiiatneetoAetahfnsanoadr'scsrsilttngdMsfteseramemslmnadaiopvmepmtltsklskniatnetcnt,ongai.esintfruwechldatrhschs"stienowhssyeeleeepwealnsonemueoas,baocedstfemst.tltsyeavtlrebsnteuwecnra.oautdstcnladhcshetStrp.iaotypcp,eeairdeoaTehmcrsvlteritdothrradfseusicaltcheguawynnghiseenunsaatxtcmeeoo1aeabrtitanambtihntiwscnoibensrswtdgieieunetntbhirrp9etcrehtnscoc,owsilsheahnineecsmeonidepeuPdasin6nyPliodotosiiaieotneefnegckireriuncdtebsi8ooaacrwiitgSstciSscfcra,laspnmntlnetiohiaomhlthnac,iesliuaynrofallAeuthlhnttAemabslgelosyttntaelefaarturnieiiyafrhoesleaadrtndaeefiateficnetntCaftfCeyitdepieshricasisydnezanpfrodetp.cashnAnedSCorsapcmsh,heCuesi-nattderlecolsab,opwnfoscotsyeoccuognhhad,rsnooredrtrtfnoninoioeongvaoiisraeiaroeielogdtaiededmrunaniwvsneynsttisiinesityvdddteeelxmneitseniefvle,esnsmptesltngmtaimsmtnrgeenrxheeyeiydciptx.sipetedicpcattrcilcavasnfrurtfemnheotaoawirtaoxgterteotofeeanateabeeoeimkonhoheeyrcortnlaonnusroaoabbddssitshrsnsialdsonosyaayarldiyeesderoneesnolsastrd,efteetf-eessftl,.s.lt OEFENCE SPENDING FASFlays Faster by our Washington Correspondent sSw"cthooMyeimmmsaateeihnneyyugrentw,oapirnnlt,Jeheaawtpos"aalssdnnpotoermisctnhecepgeu,rr"orcSppehelnoeeSncsareaeersntysdaeant.thoi--bininrgloewhvsMeSsairtotraaummrdbacolesrhy-;rt rhlssPdsSwtdgSsmbSdIycieeeppneeaoooaeeeee.tcseaeaeravlavnhlnnnurhirfrlnnrteFrenegtaeacelenca.tadltollaotgehahsoo,gogfoiagrfsstnltpapoisr"toouhTimagmiaetmnntgenonhanSohynrthseioaetfi',edteefdcyiesbhnns"taaomahttelt.tSdtlrhfhernyshdetfieeotnwiDedsenseeehsicqvggiemnunvqgWeoeanuoiisdcpteutfednSeoiooheoaieltviieeSstporrodnetvnrurheSt"eeehUvreoip'sianse.rco7onihaaievmltintnte,rnaegnsug,ir8otoitienxegcyghatm8orbtynftopsaiaa0nonsUbotmaninolwoeinrseayuondncfrfftiicueerfrvnnoemiCaattenrom5wdgauhhIu,tpscinoh3tehleletsesiiohdlnih,'floeaaa0iHsfneeidgoetnDyirt0indar,naoUascren,y0asceyerahrtflsntaoeyseaJpnstn.umssinoaatcatommtrt.hecwhrneoirhieoeweawlikat.dndeyneersfl., JaIoonnhdina'ne&.n)gFi(noBesyeterJrin,ogdh.inreLc(etFowrroisom,fd1eTf9he7en1ceWWaaressshheiiannrcggh-toman Magazine.) gdeDisPmmsmapsgicahbtr"vFpmtpb5antieulheeleioolecryoea3nsrseuoecaiisalsseatmTiietlTtsmgtll,eacoldptssrhaiatese0tirosseutcceemrhttuet.hserpaeaniecs0mdpheaatdtssdsmeneserrerheitee0rmirne,ulievic.etdpin,'PyrsocroambbsctabdhenmsgteregatbelfneyyiFmeScee,tbGypmnlnemrencb'TisvoceeseeealotdnmnolP,ateydteleomdiotanhfspahreoitotnnoiidmoreiihosenF'atg?enfnsreessnmmaftetraertnaoSayc,ouolgxile-efanrtednsdtln,sicensmnasaafhceiotfttsaeoifeneloitnegtoemhgnueuesdywdooeIershbdqttAdwntagrurtneantiiitrenShraeaieuohltn,asessr'schtmrrbyrlsvreyderseiiiuptoregmeeoi'scaoctyseoeoneae,mapeelpudmlinhtmcwnytcameffUasnrie,retstomhtdeinedeqhdaeAeecagaMrmAsdinoamcmlFdoinrelulelaomnmlasnenoAadyffmhmossgftemvemdSb.vcnghnsveasoaesbnsrmiieesseoeaatgcaantilammunnteLatiapcrrlesirhivwtlrhfubrbdgvndcsodbeuiiorcraieiieeoeeachoeoecintatsnhsrsrobdIcetnfgarcediusyntngiefadeabiuespudontieneinDwmtteS,beaeinssesth,trnpatswenllemeooeaeeSttoSwStsieuCrhasnfdvulefsca"tceseovoictc,oehekersrot.onxfoaaviniRviibisisrt5dec,otaimemnmnntsniciaeeehmnotoneu0eriesecrridhgdgddand-ast-<<QfttCOc/DOO^Jj 5692 5893 DOV026820 P* 7 J w " MIDLAND THE DOW CHEMICAL COMPANY April 27, 1971 J. E. Johnson, Corporate Research & Development, 2020 Bldg. C. W. Hinman, corporate Research & Development, 2020 Bldg. H. L. Gordon, Corporate Medical, 2030 Bldg. C. G. Kramer, Corporate Medical, 2030 Bldg. V. K. Rowe, Toxicology, 1803 Bldg. RESULTS OF THE PILOT STUDY ON THE ABSORPTION AND EXCRETION OF 2,4-D and 2,4,5-T BY HUMAN SUBJECTS A report of the results of the indicated study is enclosed. Considering the quality of the subjects, as well as the in vestigators, the data were quite good. I feel that this study should be expanded to include 3 more subjects for each compound. Recommendations for future work are given at the end of the report. P. J. Gehring Toxicology Building 1803 slk 0000497 S69S UL File : K-2372,p T23.14-11-17 K-4568,'7T23.1 4 - 2 3 - 2 3 (2,4-D) (2 ,4 ,5-T) DOW 026S2 RESULTS OF A PILOT STUDY ON THE ABSORPTION AND EXCRETION OF 2,4--DICHLOROPHENOXY ACETIC ACID (2,4-D) AND 2,4,5-TRICHLOROPHENOXY ACETIC ACID (2,4,5-T) FOLLOWING A SINGLE ORAL DOSE TO HUMAN SUBJECTS P. J. Gehring Toxicology H. L. Gordon Corporate Medical April 27, 1971 5698 0000438 026822 OBJECTIVE The objectives of the work reported herein were two-fold: (1) to determine the concentrations of 2,4-D and 2,4,5-T in blood as a function of time following oral administration, and (2) to determine the amount of these agents excreted in the urine during the first 48 hours following administration. METHODS Two human male volunteers weighing 76.8 and 81.0 kg ingested 5 mg/kg 2,4-D (Sample AGR 30653C) and 2,4,5-T (Sample AGR 86187), respectively. For ingestion, the samples, which were analytical standards prepared by The Dow Chemical Company, were mixed in milk. Blood samples for compound analysis, 10 ml, were collected immediately prior to ingestion and at 2, 7, 12, 24 and 48 hours following ingestion. Samples of the urine, collected 0 to 23, 23 to 24, 24 to 47 and 47 to 48. hours following administration, were used for compound analysis. Analyses for 2,4-D and 2,4,5-T (R. Papenfuss, AL28-109, 1971) were performed as described by Marquardt, 1970 (Dow Report ML-AM-70-95). RESULTS AND DISCUSSION The concentrations of 2,4-D and 2,4,5-T in the blood at various times following administration are given in Table 1. Before doing a kinetic analysis of the data, the concentration in plasma was calculated (see footnote to Table 1). This transformation was needed to derive a volume of distribution from the kinetic analysis. In previous studies conducted in this laboratory, as well as in other laboratories (Courtney, 1970,* Erne, 1966a), the dis appearance of 2,4-D and 2,4,5-T from the plasma of .rats, pigs, calves and chickens followed first order rate kinetics. There fore, the kinetic model describing the concentration of these agents in plasma as a function of time was assumed to be that given by the equation Apkf -k t -k t cs = (e e - e f ) Vd (*f-ke> 5.697 0000499 DOW 026823 -2 - where kf = the apparent first order absorptionconstant (hr ) ke = the apparent first order disappearanceconstant (hr V3 = the apparent volume of distribution (1/kg) AQ*= the dose administered (mg/kg) t = time (hrs) Cs = drug concentration (^gm/ml) ) The values of kf, ke and V3 which provide the curves of best fit for the experimental data were determined using a computer pro gram recently developed by G. E. Blau (NCM-10526, 1970). The concentrations of 2,4-D and 2,4,5-T found in plasma together with the curves of best fit are illustrated in Figures 1 and 2, respectively. The rate constants (ke ) for the elimination of 2,4-D and 2,4,5-T were 0.035 + 0.001 and 0.045 + 0.004 hr" ^-. These values correspond to a half-life (t^) of 19.8 and 15.4 hours, respectively. In other species (rat, pig, calf, chicken) the t% ranges from 3 to 12 hours (Erne 1966a). The volumes of distribution (V<j) for 2,4-D and 2,4,5-T were 0.114 + 0.001 and 0.066 _+ 0.002 1/kg. The plasma volume of man is 0.04 1/kg and the extracellular volume is 0.18 1/kg. There fore, these data suggest that the distribution of these agents is extracellular. Since these agents are bound to plasma protein (Erne, 1966b), a volume of distribution less than 0.18 1/kg is expected. In previous studies using rats, the volume of distri bution of 2,4-D was found to be 0.11 1/kg. The excretion of 2,4-D and 2,4,5-T in the urine is depicted in Tables 2 and 3, respectively. Within 48 hrs, 94.4% of the dose of 2,4,5-T was excreted in the urine of the individual receiving this agent. Only 72.4% of the dose was excreted in- the urine of the subject given 2,4-D. This difference is very likely an in dividual difference rather than a difference in the excretion of the compounds. In a recent study using rats, it was found that as much as 22% of the 2,4-D excreted may be excreted via the feces. Therefore, the frequency of defecation may influence the amount excreted in the urine as well as the rate of clearance from the body. 0000500 5898 o- 3 - D In conclusion, the following suggestions are made for future studies. 1. Additional blood samples should be obtained at 32, 56 and 72 hours following the administration of the compounds. o fo cn 00 r\o 2. The analyses for 2,4-D and 2,4,5-T should be conducted on plasma rather than on whole blood. 3. The plasma protein binding of 2,4-D and 2,4,5-T should be determined. 4. Urine should be collected from 48 to 72 and 72 to 96 hours following administration. 5. Feces should be collected from 0 to 24, 24 to 48 and 48 to 72 hours following administration. REFERENCES Courtney, K. D. 2,4,5-T in the rat: Excretion patter, serum levels, placental transport, and metabolism. Pesticide Symposia, Inter-American Conferences on Toxicology & Occupational Medicine, University of Miami School of Medicine, 1970. Erne, K. 1966A. Distribution and elimination of chlorinated phenoxyacetic acid in animals. Acta vet Scand. 7:240-256. Erne, K. 1966b. Studies on the animal metabolism of phenoxy acetic herbicides. Acta vet Scand. 7:261-271. 0000501 Table 1 CONCENTRATION OF 2,4-D AND 2,4,5-T IN THE BLOOD OF MAN AS A FUNCTION OF TIME FOLLOWING THE ADMINISTRATION OF 5 MG/KG Time (hrs.) post-administration 0 2 7 12 24 48 2,4-D Uqm/ml blood uqm/ml plasma1 0 .0 21.0 20.0 18.0 15.0 2.1 0 .0 35.3 33.6 30.3 25.2 \ 3.5 uqm/ml blood 0 .0 25.0 33.0 30.0 19.0 3.9 5-T uqm/ml plasma: 0 .0 42.0 55.5 50. 5 32.0 6.5 1The ratio of the concentration of 2,4-D between plasma and red cells at equilibration is approximately 1:10 (Erne, 1966). Since the hematocrit of both subjects was 45%, the concentration of 2,4-D and 2,4,5-T in plasma was estimated using the following equation: 0.55y + 0.45 (l/10y) = C where y is the concentration in plasma and C is the concentration in blood. o O cn O oo^3 CD cn O ro 228920 M Oa Table 2 URINARY EXCRETION OP 2,4-D BY A MAN GIVEN 384 MG, 5 MG/KG Duration of Volume of collection, Hr urine voided post-administration ml. 0 to 23 23 to 24 24 to 47 47 to 48 1320 56 1400 180 Volume of urine following dilution ml. 15001 56 15001 180 2,4-D concentration 2,4-D Excreted uqm/ml Total,mq % Cumult: 79.0 98.0 99.0 29.0 118.5 5.5 148.5 5.2 30.9 1.4 38.7 1.4 30.9 32.3 71.0 72.4 1 Sufficient water was added to the undiluted urine sample to give the volume indicated. o o o o QI CM o O CO 928920 Moa DOV ' 6 8 2 7 Table 3 URINARY EXCRETION OF 2,4,5-T BY A MAN GIVEN 405 MG, 5 MG/KG to U1 1 Duration of collection, Hr >ost-administration Volume of urine voided ml. 0 to 23 23 to 24 24 to 47 47 to 48 1680 103 2340 204 Volume of urine following dilution ml. 20001 103 2340 204 concentration 4,5-T Excreted uqm/ml Total, mq % Cumulative 105.0 97.0 68.0 17.0 210.0 10.0 159.1 3.5 51.8 2.5 39.2 0.9 51.8 54.3 93.5 94.4 1Sufficient water was added to the undiluted urine sample to give the volume indicated. o o o cn o OT cb o to S O ai CO O en is 5705 1r ( M 0 6 9 797 T36.25-66681-14 3 >, Iai A PROBE STUDY OF THE ACUTE ORAL TOXICITY OF 2,3,7,8TETRACHLORODIBENZO--DIOXIN IN THE CANINE By: G. L. Sparschu, J. E. Bourne, P. J. Gehring and J. M Norris OBJECTIVES This study was conducted to define a range of toxicity from a single oral dose of 2,3,7,8-tetrachlorodihenzo-7 dioxin (TCBD) in the canine. In addition, clinical and pathological evaluations were conducted in an effort to further define the toxic properties of TCBD. This study is intended to he a probe study and the data must be interpreted in this con text. EXPERIMENTAL METHODS 2, 3, 7,8-Tetrachlorodibenzo-]>-dioxin (TCBD), reference number was 340-2-54B, /administered to unconditioned pound dogs. The study was conducted in two parts; first^male dogs, 2/treat- ment level, were given 3 mg/kg or 300 ug/kg TCBD and 2 months later female dogs, 2/treatment level, were given 100 or 30 l^g/kg TCBD. The TCBD was suspended in an 8:2 corn oil-acetone solution and administered as a single oral dose in gelatin capsules. The dogs ranged in weight from 8.0 to 17.5 kg and were given from 8-17 ml of the TCBD corn oil-acetone mixture. In the male dogs^ evaluations of blood urea nitrogen (BUN), serum alkaline phosphatase (AP) and serum glutamic pyruvic^ ^ q ^ OOGd.' DOW 749678 i -2 - transaminase (SGPT) were made on test days 1, 8 , and 15. Hematology studies consisting of packed cell volume, hemo globin, total erythrocyte count and total leukocyte cotint and differential were conducted on test day 18 for the two male dogs given 300 ug/kg of TCBD and on test day 64 for all the female dogs. Body weights were taken on all dogs prior to dosing and weekly thereafter. Complete gross and histopathology were conducted on all dogs. RESULTS AND DISCUSSION - MALE DOGS The clinical signs for all four dogs, reg.ardless of dose, were essentially the same. The clinical signs consisted of anorexia, dehydration, depression, emaciation, and gastroin testinal hemorrhage. The two dogs given 3 mg/kg of TCBD died after 9 or 15 days on test. Those dogs given 300 ug/kg of TCBD were killed after 18 or 22 days on test due to their extreme cachexic and moribund condition. The clinical chemistry results are given in Table 1. All dogs showed slight to moderate elevations in SGPT and dog 70-3171 had a terminal elevation of BUN. r ,, AKl| 0004345 749680 The hematology results are given in Table 2. Dog 70-3170 a. had a decreased PCV and both male dogs had/(leukocytosis. Table 3 contains the gross and histopathology results. The digestive tract appears to be particularily sensitive to the affects of TCBD resulting in focal areas of inflammation, necrosis, hemorrhage and ulceration. The major organ affected was the liver; focal areas of necrosis, usually central lobular with only a minimal number of hepatocytes involved. Another treatment related observation is the vasculitis that is most prominent in the omental and mesen teric vessels. An adjacent steatitis accompanied the vas-. culitis. The profound anorexia that the TCBD produced resulting in extreme emaciation and dehydration no doubt was a major contributing factor in the death or moribund t condition of the male dogs receiving- 3$ mg or 300 jjtg/kgtTC'& RESULTS AND DISCUSSION - FEMAT.F. nnfls The first several days after treatment the dogs appeared clinically normal. By eleven days they were dehydrated, anorexic, depressed, and had intestinal hemorrhage. Dog 71-424 (30 pgAg) appeared near death and had a generalized partial alopecia. Dog 71-425 (100 iig/kg) had minimal shedding of hair and both dogs given 100 yg/kg TCBD had unsteady . 5708. CG04G4G DOW 749681 gaits. At fourteen days the dogs still had evidence of intestinal hemorrhage; the condition of dog 71-424 (30 ug/kg) \A/ was unchanged. Dog 71-425 (100 ug/kg) was not showing extensive alopecia and dog 71-423 (30 jjg/ k g ) was starting to shed hair. At 18 days and thereafter, there was no evidence of intes tinal hemorrhage and all four dogs appeared more alert suggesting partial recovery. The dogs treated with 100 gg/kg TCBD continued to lose body weight the first 30 days after which they began to gain weight; by 64 days they were approaching their pre-test body weight (Figure 1). The dogs given 30 ug/kg TCBD also lost body weight, but after 15 days they began to gain back their weight. The hematology results, Table 2, show a decreased PCV, total erythrocyte count, and hemoglobin with a leukocytosis. The decrease in erythrocitic values is probably a reflection of the intestinal hemorrhage clinically observed. Terminal hematoxylin and eosin stained sections of bone marrow were unremarkable; there was no evidence of bone marrow depression. The pathology results are given in Table 4. Various degrees of alopecia were seen in all dogs, with the 100 u g A g treated 5709 GG04347 0QW 749682 -5- dogs showing the most hair loss. Microscopically the skin lesions consisted of a folliculitis with epidermal inflam matory cells and focal ulceration. There was no marked . hyperkeratosis or keratotic cyst formation to relate the skin lesions to chloracne. Dog 71-426 (100 pg/kg) had' scattered inflammatory cells in the hepatic sinusoids and dog 71-425 (100 ugAg) had a mild chronic colitis. ( \ 0DW 749683 -6 - SUMMARY Two unconditioned male pound dogs/group were treated with 3 mg/kg or 300 ug/kg of 2, 3,7,8-tetrachlorodibenzo-]3-dioxin (TCBD). The TCBO was administered as a single oral dose suspended in an 8:2 c o m oil-acetone solution. All male dogs became anorexic, depressed, emaciated and had gastro intestinal hemorrhage. The dogs receiving 3 mg/kg died on test days 9 and 15 and those receiving 300 jjg/kg were killed on days 18 and 22 because of their moribund condition. Lesions noted in the digestive tract included ulceration, inflammation, necrosis and hemorrhage. All dogs had minimal to moderate amounts of hepatic necrosis. There was a vascu litis present, especially in the omental and mesenteric vessels, with an adjacent steatitis. The profound anorexia and dehydration were important contributory factors as to the cause of death or moribund condition of the male dogs treated with 3 mg/kg or 300 ng/kg of TCBD. Two unconditioned female pound dogs/group were treated with 100 jjg/kg or 30 ug/kg of TCBD. Several days after treatment they became anorexic, dehydrated, depressed, lost weight und had intestinal hemorrhage. Approximately eighteen days after treatment the dogs became more alert, began eating and ; 5711 CG04343 t.896i* AlSP^c -7- intestinal hemorrhage stopped suggesting the beginning of recovery. The dogs receiving 100 ug/kg started to show weight gains 30 days after treatment and those receiving 30 ug/kg showed weight gains after 15 days. All female dogs had various degrees of alopecia with the 100 \jg/kg level being the most severe. Histopathologically, the skin lesions consisted of inflammatory cells in the epidermis and a folli culitis. The female dogs were killed 64 days after treat ment and at this time one dog at the 100 u3 /kg dose had a mild chronic colitis and the other dog at this dose zjc had scattered inflammatory cells in the hepatic sinusoids. It is concluded that single oral doses of 300 ug/kg and above of 2,3,7,8-tetrachlorodibenzo--dioxin are lethal in the dog. Single oral doses of 100 ug/^g and 30 ug/kg of 2,3,7,8tetrachlorodibenzo-ja-dioxin are severely toxic to the dog, but recovery does occur. 5712 0G04G30 Dose and Animal Number 3 mg/kg 70-3167 70-3168 300 \jg/kg 70-3170 70-3171 TABLE 1 STUDY: Single Dose Oral Probe TEST MATERIAL: 2,3,7,8-Tetrachlorodibenzo--dioxin TEST ANIMAL: Unconditioned Pound Dog - Male CLINICAL CHEMISTRY. BODY WEIGHT AND MORTALITY DATA Day of Test. ...B1UN (8mq%) 15 AP SGPT (1K.A. Un8its)15 (Ka1rmen Un8its/m1l5) Body Weight 8 Loss (kq) 18 22 11 25 -- 18 18 -- 10.9 32 -- 10.9 6 -- 56 92 -- 68.7 53 -- 2.1 -- 0.6 -- -- -- 14 21 15 8 15 17 90 11 7 5 58 15 20 53 32 60 1.3 2 .8 3.4 56 100 1 .2 4.2 -- Test Day of Death 9 15 22* 18* Killed because of moribund condition. C G 0435 or S896H M.0 CO 'A ' TABLE 2 STUDY: Single TEST MATERIAL: Do2s,e3,O7r,a8l-TePtrorbaechlorodibenzo--dioxin TEST ANIMAL: Unconditioned Pound Dog HEMATOLOGY SUMMARY Dose and Animal Number DiiPferential Couni (%) ,______ Sex Test Day PCV % xRB1C0 Heqm/ 1o0g0lobcicn xWB1C0 s Neut Seq. B/J Lymph Mono Eo3in Base 300 ug/kg 70-3170 M 18 39 5.9 14.2 27.1 70 10 16 46 0 70-3171 loo u g A g 71-425 71-426 30 p g A g 71-423 71-424 M F F F F 18 54 64 32 64 33 64 35 64 30 7.3 4.9 5. 3 5.9 5.1 19.8 11.6 11.9 12.6 10.8 31.2 22.1 24.2 79 33 51 24.8 20.9 63 53 51 9 0 15 3 21 1 22 3 31 70 10 42 11 14 77 67 0 0 0 0 0 CJ1 <1 n 9 9 B W 4 M D Q 0004332 im al riber -3167 3168 OI <s h* Ol TABLE 3 STUDY: Single Dose Oral Probe TEST MATERIAL: 2,3,7,8-Tetrachlorodibenzo-|)-dioxin TEST ANIMAL: Unconditioned Pound Dog - Male GROSS AND HISTOPATHOLOGY SUMMARY Dose 3 mg/kg Gross Pathology Died cachexic; buccal ulcers; liver-- congested with lobular accentuation; kidneys-- swollen; omentum-- white lobulated mass; adrenals-- enlarged; gastric ulcers; GI tract empty and congested. 3 mg/kg Died cachexic Dirofilaria, liver-- pale and granular; perirenal edema and renal swelling; mesenteric lymph node hemor rhagic; omentum-- lobulation of adipose tissue; GI tract-- edema, congestion and focal hemorrhage. C G 04353 His topathology Thrombus in renal arcuate artery; splenic lymphoid depletion and focal hemorrhage; diffuse pulmonary congestion; Vasculitis of vessel in coronary groove; liver-- j central lobular congestion with diffuse focal hepatocyte necrosis and lekocyte infiltration; enterocolitis; omental stea-! titis; a nonspecifiq vasculitis mainly in-* volving the arteries in the omentum and I mesentery; focal necrosis and leukocyte j infiltration of adrenal gland adjacent to necrotizing vasculitis; mesenteric lymph node-- lymphoid depletion and perilymphad- enitis adjacent to the vasculitis of the surrounding adipose tissue. Chronic prostatitis; acute inflammation of adrenal with periadrenal hemorrhage; chronic nephritis; lung-- vasculitis with Dirofilaria: mesenteric lymph node-- lymph oid depletion, hemorrhage, edema and vas cular necrosis; spleen-- lymphoid depletion; liver-- central lobular necrosis and fatty metamorphosis; omentum-- steatitis; acute enteritis with hemorrhage and ncrosis. ----------------------- -- -- [mal nber -3170 Dose 300 p g A g CONTINUATION OF TABLE 3 Gross Patholoqv Killed, cachexie; moribund; buccal ulcers; intestinal hemorrhage; liver-- mottled. Histopatholoqy Acute prostatitis; liver-- central lobular necrosis; acute myosititis of adductor group; enteritis with ulceration and necrosis; omentum-- steatitis; chronic colitis. -3171 300 v g / k g Killed, cachexie; moribund; Dirofilaria; urinary bladder-- serosal hemorrhages; gall bladder-- edematous and distended; omentum-- lobulation of adipose tissue; GI tract-- edematous with focal serosal and mucosal hemorrhages. Spleen-- lymphoid depletion; lurtg;-- vascu litis with Dirofilaria; acute esophaqitis. focal myocarditis, renal abscessations, liver-- fatty metamorphosis; omentum-- stea titis and vasculitis; serosal utface of abdominal viscera-- chronic hemorrhage with fibroblast proliferation. en GG04354 i -vJ 8 8 9 6 p M O Q TABLE 4 STUDY; Single Dose Oral Probe TEST MATERIAL: 2,3,7,8-Tetrachlorodibenzo-p-dioxin TEST ANIMAL: Unconditioned Pound Dog - Female GROSS AND HISTOPATHOLOGY SUMMARY limal imber -425 Dose ioo vigAg Gross Patholoqv Skin-- symmetrical bilateral alopecia of abdomen, sides, and legs with erythema tous papule^ and focal scab formation in areas of alopecia. Histopatholoqv Chronic colitis-minimal; chronic lymphad enitis of mesenteric lymph node; skin-- focal ulceration with acute and chronic folliculitis. -426 loo pgAg Skin-- symmetrical bilateral alopecia of abdomen, sides and legs with erythematous papulen* and focal scab formation in areas of alopecia; focal ulcerations on hard and soft palates; renal capsule thickened. Focal ulceration, necrosis and hemorrhage in palatine tissues; chronic interstitial nephritis with focal parasitic granuloma; liver-- acute and chronic inflammatory cells in sinusoids; skin-- focal Ulceration with acute and chronic folliculitis. -423 -424 30 (jgA g 30 vg/kg Skin-- ventral alopecia with minimal pus tular scab formation; Dirofilaria, intes tinal tapeworms. Skin-- minimal alopecia of front legs with slight erythema and papule formation. Focal chronic interstitial nephritis; focal dermatitis with folliculitis-minima]. Dermatitis with focal ulceratiort-minimal. OT 0004355 Xi r r m - m m n T a n 18 # 71-426 (100 Ug/kg) 17 16 15 14 13 12 # 7 1 -4 2 5 (100 p g A g ) # 7 1 -4 2 3 ( 30 \ / k g ) 11 10 9 # 7 1 -4 2 4 ( 30 ggAg) 8 7 6 DAYS ON TEST noor * f /AGO 5719 y / r/ Reprinted from BioScien The Public Health Implications of Widespread Use of the Phenoxy Herbicides and Picloram Julius . Johnson t This paper is concerned primarily 2,4-D, 2,4,5-T, siivex, and ^rJoram-the pmh_en_oxies because VoHf spread use; picloram, 2,4,5-T, and D more specifically because of their ^ an military defoliants. The purpose discussion is to explore possible f* f j l i c health consequences of wide- use. This presentation will be g * yrj/iei primarily to information as it Table 1 shows a condensed version of tolerances proposed for 2,4-D, 2,4,5-T, MCPA, siivex, and picloram. Those marked with an asterisk are for toler ances at a permissible level for 2,4-D in rice and flax seed and for picloram in forage grasses and meat. The remainder are negligibleresidue tolerances. Table 2 demonstrates the infre quency of residues of phenoxy herbi- Martin and Duggan, 1968;Comeliussen, J 1969). *V Air and water are potential sources of exposure of m m to these herbicides. Manigoid and Schulze (1969) have re ported on pesticide residues in selected streams in the West such as the Missouri, Yellowstone, Colorado, Arkansas, Rio Grande, Snake, and othen. Phenoxy herbicides were detected as shown in Table 3. Of the 320 samples taken, Tolerance proposed to FDA fractional parts per billion concentra tions were reported in 78 samples. The G ra in s ftic m F is * S * t(j P otato** 2,4-D X X X X* X* X X 2,4,5-T X X X X MCPA X X X Siivex X X Pidorem measurement of picloram residues in water has been reported following direct spraying o f a static pond in Texas (Hoffman et al., 1969) and a watershed experiment was conducted in North Carolina using 2,4-D, 2,4,5-T, and picioram (Sheets and Lutz, 1969). The results of these studies are summarized S u g a rc a n e X in Table 4. It is noted that picloram HPeayrage Gramme* ,MM(Aeilikalnt egligible fo titb a tolerances except those marked*) X* X* persisted longer than 2,4-D or 2,4,5-T. Air samples collected in spring and X summer from two wheat-growing areas in the State of Washington were ana lyzed for phenoxy herbicides by Bames- ,,3te to the health and safety ddes in foods as confirmed by the F D A berger a n d A d a m s (1966). The results are summarized in Figure 1. Phenoxy may t r i `r n e data presented will empha- market basket survey of pesticides in herbicides are widely used for early of ju^' -ctirar toxicity, and chem_i_st_rcyo.mposites of 12 categories of food- postemergence control of weeds in these l " I uriiJ cuom,?m"e`nt on exposure to stuffs. Of the 1548 samples analyzed, crops. There were detectable levels of zecFirts5t1, *1 f a,,fnidd air-. There is very little only 36 positive identifications were 2,4-D and 2 ,4 ,5 -T in the air up to 0.06 food, toa to ** Phen*y herbi- e*PlOStfte ogfffrh food. The principal uses made. All but one was less than 0.2 ppm. 2,4,5-T was found in three of the Mg/m3.M a n will inhale about 30 m 3 of air per day. A t a level of 0.06 ng/m3, cides ttirftTrbicideS *rowing fod surveyed samples (Duggan et ai., 1967; this would a m o u n t to an exposure of of these "'ooginaliy registered on a crops * ert,,cis. A change " po,icy now no-dul! f e registrations be supported ^quires residue tolerance. An i residues 1965-68 hV a negilgJv force has formally re- Number of Semples with Residues of 1548 S am p le A n a ly z e d industry to establish these toler- quested FD^ uest for 2.4,5-T has been ances. (The * qitJ|dfawn without preju- nporaniy fjJing,) ' ,e to futur* TKehseeaaructhh,oQr oi twVC hi c "M.1sidmenmt o*a"n4y.Director emt - f Ticca*l C.- - om pany, of 2,4-D MCPA 2 ,4 ,5 -T Siivex PPM 0.001 - <0.1 23 o .l - <0.2 4 21 2i 20 0 -4 O 1 o 0 I PoPufrbelHsieecnrHtbeiedea,alt#h((ICm/thipealiircmSaytaimon:npDsoosr.iufAWmrtihdugrBr**x.1*aiub*u* ABmloocmncatnig toinns.intitduetento.2f6BAtoulpouptcnl 9?Q tn c ts ' Duggan. B. E., H. C. Barry, and L. V. Johnson, 1967. Parr* Martin, ft. J,, end R. E. Duggan. 1968. A*-- Corneiiuswn, 9. E. 1969. A-- Total 27 4 3 2 / / O r* 5720 8o 5727 Q & 'A lC A L BIOLOGY RESEARCH /S C d M l T T C O B V CHARGE 01269-000-026 DOW C H E M IC A L U.S.A. DATE 10/25/71 -U-. FILE .1 NB T36.25-66681-22 K NUMBER K-66681 EYE IRRITATION STUDY CONDUCTED ON: 2,3,7,8 -TETRACHLORODIBENZ0-p-DIOXIN REPORTED BY J. M. Norris c h e c k e d b y : P. J . ~Gehring Ct) IN F O R M A T IV E SUMMARY W ITH C O N C LU S IOiNWSs BASCEOD OCN T H E S A M P L E R E C E IV E D . A D D IT IO N A L IN F O R M A T IO N INCLC) SING T H E E F F E C T S R E P E A T E D E X P O S U R E MAY B E R E Q U IR E D AS S P E C IF IC USES A N D F O R M U L A T IO N S A R E D E V E L O P E D OR IF P R O C ESS C H A N G ES O C C U R A sample of...2,3,7/8-tetrachlorodibenzo-p-dioxin, identified by #`340-2-54B, was submitted to Chemical Biology Research "for evaluation of eye irritation properties. Twenty-four hours prior to use, the eyes of an albino rabbit were examined and established as being involved instillation of w2itmhgouotf defects or irritation. the test compound into The test procedure the conjunctival sac of the left eye. The right eye was used as an untreated control. The treated eye was examined for conjunctival inflammation, iritis and corneal injury immediately after instillation, at 1, 24 and 48 hours and 13, 22 and 27 days post-instillation. The behavior of the animal was observed for indication of pain or discomfort at the time of instillation. * The compound caused discomfort on instillation and very slight irritation f of the conjunctival membranes which persisted for 24 hours. Delayed ( conjunctival chemosis causing the eye lids to be more than half closed was observed on day 13 and by day 22 the condition was severe. On day 27 the chemosis had subsided, however, the eye lid rim was thickened and encrusted. rT n T J. E. Jehnton H. H. Mclniyr* C. E. Kiminel L. K. F r .v .l D. D. McCol!' (2) B. E. Burger* H. L . Gordon, MD E. H. Blair A. J . Schwan, MO S . M. MacCutcheon DISTRIBUTION D . K ilia n , MU B. H o rva th H. Edward* R. J . Shavor V. B. Robinien B. INolder, MU L. S ilvontoin L. Pftchforth C. A. Goring CHI (9) 0004353 e*" ri* 0 ( s\ M 8\  D O W t 478694 12.021 BACKGROUND The herbicide 2.4-dichlorophenoxyacetic acid (2.4-D) was developed in the midforties and was the forerunner of a group of phenoxy herbicides that have been instru mental in the control of broadleaved weeds in food crops and undesirable brush species Thompson-Hayward. These com: joined to form the Industry Task Fc Phenoxy Herbicide Tolerances (IT> The chronology of major events assc with this has been as follows: April 13 , 1966 on industrial rights-of-way. In addition these products have contributed immeasurably to beef production by controlling weed and/or brush on pasture and rangeland, resulting in increased grass production and corre USDA announcement to abolish nc due status--industry must comply t taining tolerances for residues in all t food and feed products and byprodu December 31. 1970. sponding increase in carrying capacity for livestock. Besides 2.4-D . 2.4.5-trichlorophenoxyacetic acid (2.4.5-T ). 2-(2.4,5-trich lo ro phenoxy) propionic acid (silvex or2.4.5-TP). August 23, 1966 Industry Task Force on Phenoxy Her Tolerances was formed to handle . 2.4.5-T. MCPA and silvex. and 2-methyl-4-chlorophenoxy acetic acid (MCPA) are major phenoxy products of similar chemical structure, as shown be low. but with unique characteristics of their own with respect to species controlled and crop selectivity. December. 1967 Submitted petitions to FDA for toler of 2.4-D, 2.4,5-T, silvex and MCPA ing all food crop uses listed at that t the USDA Summary of Registered Ac tural Chemical Uses. Extension of rec CHRONOLOGY OF EVENTS tion was requested for uses of these Over the years there has been consider cides in pasture and rangeland. ci Cl able improvement in phenoxy herbicides and their use. Development of new formula tions. performance information, crop safety, timing of application, spray equipment, toxicology, use hazards, and environmental implications have contributed to both better product and specific directions for use. These herbicides are not protected by patents, so seven commercial companies were manufacturing one or more of the four phenoxies (2.4-D. 2.4.5-T, silvex. MCPA) at the time when the USDA announcement to abolish the no residue status was issued on April 13. 1966. Basic manufacturers in cluded: Diamond. Dow. Hercules. Monsanto. Rhodia (then Chipman). Thompson, and April. 1968 Industry Task Force advised of i quacies. in the petitions plus require for information on all metabolites herbicides that might occur as resid food. September. 1968 Review of literature on metabolism mitted to resolve metabolite question During 1968 programs were estab to determine on which crops add; work would be undertaken and what sp projects would be done by each cor in the Task Force. October 29. 1968 Industry Task Force requested exte A . 2.4-dichlorophenoxyacetic acid B. 2 .4 .5 -trichlorophenoxyacetic acid C. 2-{2.4.5-tr<ch/orophenoxy)-propionic acid D. 2-methyl-4-ch/orophenoxyacetic acid for continued use of 2.4-D, 2.4.5-T, and MCPA on pastures and rangelan tension was granted until January 1. January 3 1. 1969 Use of 2.4-D. 2.4.5-T and silvex on ac The status of 2 ,4 -D , sites extended to January 1. 1970. ro p samples for residue analyses 2 ,4 ,5 -T , Silvex and collected during summer and fall of Protocols for milk and meat studies established during 1969. It was decide MCPA Herbicides analyses of animal tissues would indue phenol corresponding to each phenoxy pound. Dow handled dairy cattle feedir milk analysis for all four phenoxies By C. S. W illiam s R & 0 Herbicide Tech. Specialist. The Dow Chem ical Company. M idland. M ichigan. began feeding February, 1970. USDA was scheduled to handle feec phenoxies to beef cattle and sheep c winter. 1969-1970. _ . \a nnn>*i4 a m I I L TABLE I The EHact 1 R a iiita a t Variety end Crop R oution on W ilt Control and Pirform anct IM S (P itt Walt and Vanca Counties) V M stf fle Remise WRt< Veite /Acre Remise w a r Valse M u /Acre 1 Mrsase By Retatioa Cater 8 4 AC. B it l# ti|k t S-B Cater 111 It 1 74 41 734 n in M 120 a IMS a IM I i t M4 as 1 B7 1M <W ilt M a x indicate! extent of root damage 0 - no damage. 100 maximum disease development TABLE S Tho EHoet of R oaiitant Variety and Chemical Soil Traatmam w ith TELONE C an WIR Control and Parformanca (P in . Walt and Vanca Counties) Variety Re Tostasti H it' latex Wee (/Acre ta sta sti WRt1 ta te t Was 1/Acts tacrsase ly fe u ifitie e Cater 2S4 a c . a it le s iti e-ts Cater 111 17 171 74 147 a 1M 71 111 I t 1227 tt M l a 107 n 744 at ta to 1M 1W ilt Index indicates extent of root damage. 0 no damage. 1 0 0 - maximum disease development. TABU 10 Tka Effect of Resistant Variety, Crop Rotation and Chemical Soil Treatment w ith TELONE C on W ilt Control and Performance (P itt Wake and Vance Counties) ] Variety W ilt M as1 te Re Retati Retati A Fatefatiw Wae t/Acra 'R etati 1 Rs Retati f sayni tarresta Cater 2M A C .812 tastata G-I t Cater 311 17 I I 74 47 33 11 n 1 17* ito 3M 147 1RM M2 734 1212 471 IH IH 3M ' W ilt Index indicates extent of root damage 0 - no da>`H > 1 0 0 - maximum disease development. TABU 11 The Effect of Crop Rotation & Chemicsi Soil Treotment w ith TELONE C en Control of Root Knot and Stack Shank in Two Resistant Varieties (Columbus, Edgecombe and Jones Counties) IIM Cresi Variety -S fatata G-H Seat lu t M u1 Hack Stete la te x ' a VW Variety-Cater B k Rest Kata la te x ' Back Skate la te x ' a Vatas la ta ! fin is Fame New M a 1137 Cara Maas 71 B IM2 Can Meat 41 f i l iia le y te a n Meat 41 i l 11M Fossa He 31 14 I1H Street Famen Meat 11 34 11-2' Atem al tal Retati Ne M 24 i i a Tatarts Nan 11 4* U1 Tettate TetaaeC M 24 1H7 1 1 1244 1 1 12M 1 1 ia i 1 1 USI 1 1 1223 1 1 UM 1 7 1271 3 7 IB I 2 t UM ' Root Knot and Black Shank Index indicates extent of root damage 0 - no damage. 100 - maximum riisrias* develnfm icnt 'V alone or continuous tobacco, plus TELONE C. These results suggest that a complete control program involving several practices is needed where resistant varieties (regard less of level) that are susceptible to the southern root-knot nematode are used. They also suggest that the use of a variety with high resistance to wilt and root knot might permit the omission of crop rotation or the use. of a multi-purpose chemical soil treat ment from the control program. Results are presented in Table 10. A second series of integrated disease con trol tests was established in 1968 at three locations. These tests were located in Co lumbus. Edgecombe and Jones counties in fields where root-knot nematodes and black shank were present at low to moderate levels. Sixteen treatments involving seven two-year cropping systems were compared with continuous tobacco culture. Alternate crops included in the different cropping sys tems were corn, cotton, small grain fescue, soybeans, sweet potatoes and peanuts. Two disease resistant varieties were used within all cropping systems including Coker 254 (high resistance to black shank, bacterial wilt and root knot) and Speight G-36 (high resistance to black shank and bacterial wilt but susceptible to root knot). Two soil treat ments (one using TELONE C soil fumigant and one using no soil treatment) were used in plots where continuous tobacco culture was practiced. No chemical soil treatment was used in rotated plots. Results from these tests provided a comparison of sev eral alternate crops a'nd TELONE C in re ducing root knot and black shank and their effects on performance. Disease development was low in the Coker 254 variety at all locations. Rotation with such crops as corn, cotton, peanuts, small grain fescue, soybeans and sweet potatoes failed to provide additional con trol or increase in performance of this variety. (Table 11). The Speight G-36 variety was damaged severely by both root knot and black shank in most testis where tobacco was planted continuously. Black shank root damage was reduced by all cropping systems and root knot by all with exception of corn and sweet potatoes as compared with continuous tobacco culture. (Table 11). The use of TELONE C in plots where tobacco was planted continuously gave equal disease control and a slightly higher value increase as compared with most crop ping systems in both the Coker 254 and Speight G-36 varieties. (Table 11). 0007109 5 7 3 1 1 October 29, 1969 m ittee to review the appeal on the The Office of Science and Technology cancellation of rice.* issued statement on teratogenic hazard of 2.4.5- T based on work by Bionetics Re search Laboratory. REGISTRATION STATUS Tolerances have been established for 2 .4 - D in applies, barley, grapefruit, lemons, December 22, 1969 Phenoxy registration for rangeland use oats, oranges, pears, rye and wheat. There is also a tolerance for the sodium salt of extended until January 1, 1971. except for 2.4- D in asparagus. 2.4.5- T. Dow undertook feeding of 2.4.5-T to beef animals. December 3 1 , 1969 Petition for tolerances of 2.4.5-T in food crops was withdrawn by Industry Task Force since tolerances could not be established by January 1. 1970. the deadline set by the Office of Science and Technology. Data originally submitted with the peti tions in December. 1967 is expected to be sufficient for 2.4-D in blueberries, cranber ries, grapes and raspberries; for silvex in apples, pears and prunes; and for MCPA in peas. Data on residues in grass includes that from previous work and from 1970 residue samples additionally analyzed by Dow for support with respect to pasture January 19 , 19 70 Registration of 2.4-D. siIvex and MCPA extended by USDA for use on food crops until January 1. 1971. and rangeland usage. The Industry Task Force supported work for determining residues as shown in Table 1. For virtually ail of this work, new analytical methods had to be developed that would March 4. 19 7 0 Registration of 2.4.5-T was also extended until January 1. 1971 for use on apples, permit analyses down to 0.1 ppm phenoxy acid in crops and to 0.05 ppm acid or cor responding phenol in the animal tissues and blueberries, grains, pastures, rangeland, rice and sugarcane. milk. For present registered uses residue anal April 15 , 19 7 0 yses indicate < 0 .2 ppm for all phenoxy acids in all crops at time of harvest. Results of additional work on teratogenic The phenoxies were fed at levels of 30. properties of 2.4.5-T prompted suspension 100 and 300 ppm for two weeks and 1000 by USDA of 2.4.5-T for aquatic and home uses. ppm for three weeks in the total diet of dairy cows. Milk was collected and analyzed for residues. Grazing restrictions compatible * May 1. 1970 with levels of phenoxies in milk as related Cancellation by USDA of 2.4.5-T for use to levels in forage remain to be determined. on food crops. Uses on pasture, forests and There was no evidence of accumulation of industrial areas not affected. phenoxies in the cream. May 28, 1970 Dow appealed the cancellation of 2.4.5-T for use on rice Hercules and Amchem also appealed cancellation for rice usage. Each company could appeal only crops listed on their labels for 2.4.5-T products. The appeal is to be reviewed by an Advisory Committee appointed by the National Academy of Sciences. At time of this writing. December 1.1970. analyses are being run on samples of mus cle. kidney, liver and fat of beef animals. Data are expected to be available prior to December 31. 1970. Amendments to the petitions for toler ances for 2.4-D, silvex and MCPA will be submitted to the appropriate agency in charge, including uses in pasture and rangeland. The 2.4.5-T petition will be reactivated June 16, 1970 --deadline for this compliance will be ac USDA began .the beef feeding studies complished prior to December 31, 1970. scheduled to have been done the previous winter. TOXICOLOGY * Negligible residue tolerances can be November 24, 19 70 Crop residue work completed. Milk anal ysis completed. Meat analyses underway. Completed data will be submitted as amend ment to petitions prior to December 31. 1970. To date, no official notice has been obtained based on information from 90 day toxicology studies in two species of mam mals. However, tolerances at higher (per missible) residue levels require two-year feeding studies on rats and dogs, plus fertility and reproduction studies on rats. At the time the phenoxy herbicides were 5732 received concerning appointment of Na tional Academy of Science Advisory Com *Sm upd*M w *nd o* t u t (| V 0 0 0 /1 1 0 s TABLE t. Cray* and tistM S m nplri lor mid u/yjii { t ) ttniir th i fTTPHT prsgnm. Corn Rico F lu Small Grains (Barley, Oats. Ryo. Wheat) Sorghum - Sugarcam M ilk Moat 2.4-0 + + Tolerances Granted + + + + 2.4.5-T + + - -- + + + Silva* / + - 0c MCPA c r cc + + + + + + ........" + + developed these long term feeding studies were not necessary, since these com pounds were registered on a "no-residue" basis. FDA has conducted two-year feeding studies on 2.A-D including reproduction and fertility. Dow has conducted two-year feeding studies on silvex but not reproduc tion or fertility studies. Ninety-day feeding studies have been run on rats and dogs for 2.4-D. 2.4.5-T. silvex and MCPA. The no-ill effect levels are shown in Table 2. Based on single oral doses in rats. 2.4-D. 2.4.5-T. silvex and MCPA are classed as "slightly toxic" with LDso values ranging from three hundred to seven hundred mg/kg body weight. 2.3,7,8- TETRACHLORODIBENZO-P-DIOXIN The word teratology has recently become much more familiar. It was tied to 2.4.5-T when studies by Bionetics Research Labora tory implied that 2.4.5-T was teratogenic (producing malformed fetuses) in mice and rats. Subsequent studies have shown that * a potential toxic contaminant. 2.3.7.8tetrachlorodibenzo-p-dioxin. is responsible for the findings attributed to 2.4.5-T. The sample of 2.4.5-T employed in the Bionetics study contained 27 ppm 2.3.7.8-tetrachlorodibenzo-p-dioxin. Additional studies have shown that oral administration of 2.4.5-T containing < 1 ppm 2 .3 .7 .8 - tetrachlorodibenzo - p - dioxin produced no teratogenic effects on r. rabbits or mice. The obvious concern is produce 2.4.5-T without the contamin. The 2.3.7.8-tetrachiorodibenzo-p-dic can be formed in the manufacture of precursor 2.4.5-trichlorophenol. The cor tions required for its formation are h temperatures and basic conditions. This < occur in the alkaline hydrolysis of 1.2.* tetrachlorobenzene to the trichloropher No detectable dioxins have been obser in 2.4-D. This is due to the fact that precursor 2.4-dichlorophenol is made direct chlorination of the phenol and not alkaline hydrolysis of 1,2.4-trichlorob zene. To date analytical methods have be developed and validated for a method s* sitivity of 0.5 ppm for 2.3.7.8-tetrachlc dibenzo-p-dioxin in 2.4.5-T acid. With pro: manufacturing controls there is no probi in producing 2.4.5-T with no 2.3.7.8-tet chloro-p-dioxin as indicated by these a; lytical methods. THE FUTURE By December 31. 1970 the Industry Ti Force on Phenoxy Herbicide Tolerances \ have furnished to FDA the supplemer residue data necessary for the contini evaluation of the petitions to establish ne< gible residue tolerances for 2.4-D. 2.4.5 silvex and MCPA on the appropriate fc crops and meat and milk tissues. Regist tions (USDA) are expected t<^rerpain force' o o o v m 5 7 3 uw u TABLE 2. R nottx frw i IB -A iy n k a c o tii fa rtin g i t r t i n 4 n tim a p h tn o ir k tfb ic M tt a ra ti aad Baps. Bits Dags Approximate No-Ill Effect Levels (Mg/Kg/Oay) 2.4-D 30 10 2.4.5-T 30 5 .TTCJn Silvax HO 't , ]* Ib \- 4 i CPA 18 0-10 Data provided to date indicate no hazard and no significant residues in food crops. Toxicology data support the use claims. The proper manufacturing of 2.4.5-T will alle viate the problems associated with 2.3. 7.8-tetrachlorodibenzo-p-dioxin. The USOA has recently stated that pro hibiting the use of phenoxy herbicides "would cost the U. S. farmers an additional $290 million to maintain current agricultural production. In addition, farmers and their families would have to work 20 million more hours to control the weeds without these herbicides. For this extra labor, the farmers would obtain no additional income". Several hundred thousands of dollars havH been expended over the past several years to prove the safety of phenoxy herbi cides ;o man and his environment. From a scientific base the phenoxy herbicides can contribute economically, efficiently, and safely in the future for the control of broad leaved weeds and brush on food crops, pas ture. rangeland, and non-cropland areas as they have for over 20 years. Si SINIGCNEIFPIRCEAPNATRACTHIROONNOOFLOMGAINCAULSCERVIEPNTTS A s o f January 2 3 . 1 9 7 1 several sig n ific a n t events have occurred. (1 ) The fT F P H T s u b m itted am en d m en ts fo r 2 .4 -D . silvex a n d M C P A p e titio n s o n D ecem ber 2 2 . 1 9 7 0 . The E n v iro n m e n ta l P ro te c tio n A gen cy (EPA) ackn o w led g ed filin g o f these am endm ents D ecem ber 3 0 . 1 9 7 0 . (2 ) The IT F P H T also re file d th e 2 .4 .5 -T p e ti tio n o n D ecem b er 2 2 , 1 9 7 0 a n d EPA advised on D ecem ber 3 1 . 1 9 7 0 a p e titio n num ber h a d been reserved b u t th e y (E P A ) w ish ed to k n o w w h ic h sp ecific 2 .4 .5 -T fo rm ulatio n s w ere to b e covered b y th e p e titio n a n d fo r in d u stry to id e n tify th e in g red ien ts (a c tiv e a n d in e rt) c o n ta in e d in th ese products. (3 ) The EPA has also ad vised th a t a sp ecific to lerance fo r phen o xies in grass is necessary; a to lerance in grass h a d n o t been requested b y IT F P H T as it d id n o t appear necessary. In com p liance w ith th e EPA re q u e s t a le v e l o f 3 0 0 p p m fo r a ll p h en o xies in grass w as re q u ested on J a n u a ry 6 . 1 9 7 1 b y fTFPHT. (4 ) O n J a n u a ry 11. 1 9 7 1 D o w w as in fo rm ed th a t th e ad visory c o m m ittee fro m th e N a tio n a l A cad em y o f S cien ce h a d been ap p o in ted on N ovem ber 2 . 1 9 7 0 to review th e fo o d crop c an cellatio n o f 2 .4 .5 -T . A t th is w ritin g . D o w a n d H ercules h a d each b een g iven a n h o u r on F ebruary 1. 1 9 7 1 to p resen t in fo rm a tio n rela tiv e to th e s a fe ty o f 2 .4 .5 - T as used o n fo o d crops. 5734 0007112 Sx 5735 8X 543831 Acta pharmacol. et toxicol. 1971,29, 81-86. From the Department o f M icrobiology, Dental Faculty, University of Oslo, Norway The Herbicide'2,4-DichIorophenoxyacetic~Add2 I :/Effects on'L Cells1 By Jan Kolberg, Kristen H dgdam l, Jon Jonsen and Olav TJeltreit (Received M ay 19, 1970) Abstract: The effect o f 2,4-dichlorophenoxyacetic acid (2,4-D ) on L 929 cells in monolayer cultures has been studied. I t was found th a t^ A D in the range of-50 o-500-ug/m i had --a-dosa-dependeat. inhibitory- effect.on cell-growth. With~350 and 500 pg/m l-com plete.inhibition-of growth. occurred.after.about 24 hrs- s t.in tu b a tio n . On removal o f 2,4-D a rapid resumption o f cell m ultiplication took place. This occurred even after exposure to 500 pg 2,4-D /m l fo r 12 days. In the presence o f 250 to 500 pg/m l, ecuoies, evhn.ii siaineU "W ltlrlipid* oluble-dyes, appeared- in-the-cytoplasm . On prolonged incubation w ith the herbicide, these vacuoles disappeared. K iy -w o rd s : Herbicide - L cells. OO'* ^1 *o . ' 2.4- dichlorophenoxyacetic acid (2,4-D) belongs to the phenoxy group of herbicides which at low concentrations induces growth responses similar to the plant hormone auxin. At higher concentrations of these herbicides an excessive, uncontrolled growth leading to the death of the plant is observed. This is believed to be based on an abnormal metabolism of RNA (H anso n & Sl if e 1969). 2.4- D is probably used to a greater extent than any other herbicide, and descriptions of its toxicity and hazards to man, domestic animals and wildlife have been summarized by W ay (1969). The acute toxicity following oral administration appears to be moderate. The LDS0 values found for a number of experimental animals range between 300 and 800 mg 2,4-D/kg body weight. In a recent, yet unpublished, study commissioned by the National Cancer Institute (U. S. A.) this herbicide was labelled as being a potentially teratogenic compound needing further study (Science 1969,166, 977 news and comment). Since 2,4-D might be a teratogenic agent, studies to elucidate the mecha nism of action on mammalian cells is of importance. For this purpose, cell cultures provide a suitable tool. The only study in this field has recently been ( A cta P haim acolofica. vot. 2>, (ate. I % f. i ' * rt I LI! II I null I..............m i l I II! H I 1.1 J rI J . I . H II IL -- M'l - f f , W . i ^ l I U " L II l 5736 000740 JAN K O L S E R G ET AL. made by Li &. Jo r d a n (1969) who found a transient growth inhibition in suspension cultures of L cells exposed to 5 and 10 pg/ml of an ester of 2,4-D. The present paper describes the effect of different concentrations of 2,4-D Na-K salt on the growth and morphology of L 929 cells in monolayer cul tures. Materials and Methods Cell culture techniques. Monolayers of mouse fibroblasts, strain L 929 (San fo r d et al. 1948) were. used. The cultures were grown in Eagle's Minimum Essential Medium supplemented with 10 % calf serum, streptomycin and penicillin. Plastic petri dishes, 60 X 15 mm (Falcon Plastic Inc.) were seeded with 0.4 X 10**cells suspended in S ml medium and incubated at 37* it* a humidified atmosphere of COs in air. After incubation for 24 hrs, the medium was replaced by test media containing 2,4-D. The media were renewed every third day. In reversal experiments the 2,4-D containing medium was removed and, before ad ding the control medium, the cell layers were washed twice with 2 ml of the latter medium. For growth measurements, the cultures were trypsinized and counted in a Biirker haemocytometer. Each point on the growth curves represents the mean of the counts from 2 cultures. Cytochemical analysis of lipids were carried out with Oil Red 0 in 100 % isopropanol or Sudan Black B in 70 % ethanol and counterstaining with Hams haematoxylin and neutral red respectively. The cells were not fixed before staining. Cultures cultivated in Sykes and Moore chambers, volume 0.7 ml (Sykes A M oore 1960) were photographed at 2 frames/min. by means of a Reichert inverted phase-con trast microscope with a 16 mm Beaulieu camera loaded with Kodachrome II A film. To reverse the effect of 2,4-D, the chambers were perfused with 23 mi control medium. H e rb ic id e . A stock solution containing 25 mg 2,4-D/ml was prepared by dissolving the acid (Eastman Organic Chemicals) in aqueous NaOH-KOH (0.1 N : 0.1 N) and adjusting the pH to 7.3 with HO. The solution was sterilized by filtration through a 0.22 p Millipore^nembrane filter. Results On~exposure-of L-929 cellsin monolayer cultures to-2,4-D in concentra tions from SOto-500 pg/ml,a-dose dependent inhibitionof growth was-found (fig. 1). Complete inhibition-was found^after 24-hrs in the-presence of-350 and-500-fig-2,4-D/ml. Cytopathogenic changes such as rounding up and detachment of the cells were not seen, however, in the treated cultures. When 2,4-D was removed from the cultures after incubation in the presence of 500 pg/ml for 3 or 12 days, inhibition of growth ceased (fig. 2). On removal of 2,4-D after exposure for 3 days there was a rapid increase in the number of cells concomitant with a disappearance of the vacuoles, whereas after 12 days there was a lag period of about 24 hrs before cell qvr- * 0007409 5737 V inhibition in es- <f2,4-D. ntions of 2,4-D Tionolayer cul- 2,4-D AND L CELLS 83 DOW 1 543833 >were used. The cnted with 10 % IS mm (Falcon m and incubated for 24 hrs, the e renewed every and, before admi of the latter ted in a Burker m of the counts 0 % isopropanol tematoxylin and irx E S &. M o o r e -rted phase-conf"ne II A film, rol medium. \\ oiving the acid ) and adjusting rough a 0.22 i -l 2 3 .4 S TIME (OATS) Fig. 1. The effect of various concentrations of 2,4-D on growth of L 929 cells. in concentsath was-found sence of-350 ding up and dtures. ation in th ased (fig. 2). d increase in he vacuoles, s before cell 7? Fig. 2. Resumption o f cell growth after removal o f 2,4-D. -------- untreated cultures. -------- cultures containing 500 pg/ml. --------- cultures from which 2,4-D has been removed after 3 or 12 days of treatment. 0007410 mm 5738 '' ' . 7 ' f : \\ ^ 1}- V -*%>_ v?. *si>^ H L -V >,T '--'art A ^*W 1 N V-S , v ***..'->* v .v %- * ' *1^. ' i k - A / V h " ;v ^ W l ^ f- f r & ^ J r A v . v.It ' ^ ^ ^ ^ h ^ ***** /..'& v- r--*V*j 0007411 multiplication sta prolonged incuba tion to this compc In the presenc. which stained wit after about 3 hrs 21 hrs. On furthc for 4-5 days the vacuoles were ob incubation. No a with 50 and 150 The observatic cn U time-lapse cinen 2,4-D/ml no cell w was removed a r or disappeared with The cultures wer no indication of pound. No obvic of the cell memb A concentrati layers of L cells hrs, and with 3 cells was found however, during tions which mit stage in the cell a synchronizing When cultures control medium synchronized ce likely. The rapid re for 3 and 12 da t(iMonueulsluearlly19r6e`. During a per the presence o: and/or a prolor 5739 ~fast microscope. 30 hr*. V. 2,4-D AND L CELLS 85 multiplication started. This experiment also shows that the cultures on prolonged incubation in the presence of 500 ng 2,4-D/ml exhibited an adap tion to this compound and some growth occurred. In the presence of 350 and 500 ng/ml, vacuoles of uniform size (fig. 3), which stained with Oil Red O and Sudan Black B, appeared in the cytoplasm after about 3 hrs of incubation, and their number increased during the next 21 hrs. On further incubation, the vacuoles disappeared, and after treatment for 4-5 days the cells were free fromlipid-containing particles. At 250 ng/ml, vacuoles were observed after 24 hrs, but they disappeared within 2-3 days of incubation. No accumulation of vacuoles was found in the cultures treated with 50 and 150 pg/ml. The observations made in the previous experiments were confirmed by time-lapse cinemicrography. During 33 hrs in the presence of 500 ng 2,4-D/ml no cell division was seen, and vacuoles were formed. When 2,4-D was removed a rapid resumption of cell growth took place, and the vacuoles disappeared within 22 hrs without being extruded intact into the medium. The cultures were followed for 32 hrs after removal of 2,4-D and there was no indication of a synchronizing effect from the treatment with this com pound. No obvious effects of 2,4-D on pinocytosis and the undulating activity of the cell membranes were found. Discussion A concentration dependent inhibition of growth was found, when monolayers of L cells were exposed to 2,4-D. The inhibition was evident after 24 hrs, and with 350 and 500 jig 2,4-D/ml no net increase in the numbers of cells was found during the next 5 days of incubation. Some growth occurred, however, during the first 24 hrs in the presence of these two high concentra tions which might indicate that 2,4-D was selectively toxic to a particular stage in the cell cycle. As this inhibitory effect of 2,4-D was readily reversible, a synchronizing effect would be expected if such a mechanismwas operating. When cultures treated with 500 pg 2,4-D/ml for 33 hrs were transferred to control medium and examined with time-lapse cinemicrography, no burst of synchronized cell division was seen. This makes the above explanation un likely. The rapid resumption of cell growth on removal of 2,4-D after treatment for 3 and 12 days seems to exclude a state of unbalanced growth. This condi tion usually results in cell death on prolonged exposure to the inhibitor (Mueller 1969). During a period of 21 days, the L cells exhibited an adaption to growth in the presence of 500 pg/ml. This may be due to a selection of resistent cells and/or a prolongation of the generation time. 0007412 f !LOj.ijB p i p p w w w a i i j t f U UM H IJljaill Hj I,. w .) H .11 ' I f * t DOW 1 543835 DOW 1 543836 <w 86 JAN KOLBERG ET AL. It has previously been shown that changes in the extracellular environment cause accumulation of lipid-containing particles in cultured mammalian cells. These changes include a decrease in the extracellular pH, the addition to the medium of phenol or aliphatic polyalcohols, the replacement of horse serum by rabbit or human serum (Mackenzie et al. 1967), and the addition of an unsaturated fatty acid such as oleate, linoleate and linolenate (Moskowitz 1967). These vacuoles, like those induced by 2,4-D, stained with lipid soluble dyes and exhibited little or no tendency to fuse. However, these investigators found no growth inhibition with lipogenic factors in the medium, except for some growth retardation caused by the polyalcohols (Mackenzie et al. 1968). On continued incubation with 2,4-D, the vacuoles disappeared. Moskowrrz (1967) observed a similar phenomenon in oleate-treated L cells. He suggested that the particles-might have been extruded intact into the medium by a secretory mechanism or, alternatively, that an intracytoplasmic hydro lysis might account for the breakdown and removal of the lipid particles. In favour of the hydrolytic mechanism, he demonstrated an increased lipase activity in steatotic cells. Whether a similar mechanism operates in our cells . has not been investigated. We found a transient lipid accumulation in 2,4-D treated cells and a release of growth after changing to the control medium. These findings sug gest that the occurrence of lipid vacuoles in cell cultures is not necessarily indicative of degeneration. REFERENCES Hanson, J. B. St F. W. Slife: Role of RNA metabolism in the -action of auxin-herbicides. Residue Rev. 1969, 25, 59-67. Li, M. F. t C Jordan: Use of spinner culture cells to detect water pollution. J. Fish. Res. Bd. Canada 1969, 26, 1378-1382. Mackenzie, C G., J. B. Mackenzie St O. K. Reiss: In: L ip id metabolism in tissue culture ceils. Ed.: G. H. Rothblat <k D. Kritchevsky. The Wistar Symposium Monograph No 6. Wistar Inst Press, Philadelphia, Pennsylvania 1967, pp. 63-81. Mackenzie, J. B., C. G. Mackenzie St O. K. Reiss: Regulation of cell lipid metabolism and accumulation VII. Increase by glycerol of the polar lipid and triglyceride content of cultured ceils. Proe. Soc. E xptl. B io l. M ed. 1968, 128, 42-46. Moskowitz, J ' S.: In: L ip id metabolism in tissue culture cells. Ed.: G. H. Rothblat St D. Kritc .-sky. The Wistar Symposium Monograph No. 6. Wistar Inst Press, Philadelphia, Pennsylvania 1967, pp. 49-59. Mueller, G. C : Biochemical events in the animal cell cycle. Fed. Proc. 1969, 28,1780- 1789. V Sanford, K. 1C, W. R. Earle St G. D. Likely: The growth in v itro of single isolated tissue cells. J. N a t. Cancer Inst. 1948, 9,229-246. Sykes, J. A. St E. B. Moore: A Simple tissue chamber. Texas Rep. B io l. M ed. 1960,18, 288-297. Way, J. M.: Toxicity and hazards to man, domestic animals, and wildlife from some commonly used auxin herbicides. Residue Rev. 1969,26,37-62. 1 * f 000741a J 5741 83 5742 83 THE DOW CHEM ICAL COM PANY M ID LA N D F e b ru a ry 19, 1971 H. L. Gordon, M. D. C. W. Hinman J. E. Johnson C. G. K ram er, M. D. V. K. Rowe - E nclosed is a suggested p ro to co l for the consum ption of 2 ,4 -D and 2 ,4 , 5 -T by J. E . Johnson and C. W. H inm an. The p ro to co l for the proposed study "A bsorption and E xcretion of 2,4 -D and 2 ,4 , 5-T " to be conducted in India is also included. The la tte r p ro to co l includes background toxicological inform ation, m etabolism data, and sam ple sp ecificatio n s. A ll of the inform ation should be studied p rio r to co n ducting this study. Since I have the sam ples of 2 ,4 -D and 2 ,4 , 5 -T , the study can be in itiated w henever feasib le. I suggest that the chosen guinea pigs and w itn e sse s com e to 1803 B uilding w h ere the m a te ria ls w ill be w eighed out and consum ed. DOW049272 r-. j . u e n r in g , D .V .M . , Ph-. D. C hem ical Biology R ese arch 1803 Building , 6-1089 A ttachm ent CONFIDENTIAL - SUBJECT TO INJUNCTION D .C , E D . M l. 4 -4 -7 8 ; D O W /E P A AGREEMENT 9 -7 9 0000795 5743 (K Oo r^io 00 oo I / PROTOCOL: HUMAN TOLERANCE OP 2,4-D AND 2,4, 5-Tv..',; OBJECTIVE: v To establish that the doses of 2,4-D and 2,4,5-T recommended for use in the study entitled "Absorp tion and Excretion of 2,4-D and 2,4,5-T In Man" are safe. v DOW049273 SUBJECT: J. E. Johnson- Director of Corporate Research and Development C. W. Hinman- Director of Chemical Biology Research COMPOUNDS : f Samples of 2,4-D (Sample AGR30653C) and 2,4,5-T` (Sample AGR86187). These samples sure analytical standards. The same materials are being used in the study "Absorption and Excretion of 2,4-D and 2.4.5- T In Man". PROCEDURE : A copy of the proposed study "Absorption and Excretion of 2,4-D and 2,4,5-T In Man" is attached. In the study proposed here, J. E. Johnson is to consume 81 mg of 2,4,5-T (1 mg/kg) and C. W. Hinman 79 mg 2,4-D (1 mg/kg). One week following this treatment, J. E. Johnson is to consume 405 mg (5 mg/kg) of 2.4.5- T and C. W. Hinman 395 mg (5 mg/kg) of 2,4-D. For ad ministration, the compounds are to be mixed in a glass of milk. The milk is to be consumed in the presence of witnesses and a sworn affidavit obtained. MEDICAL SURVEILLANCE: .The information provided in the accompanying protocol "Absorp tion and Excretion of 2,4-D and 2,4,5-T In Mam" should be studied by Doctors Harold Gordon and Charles Kramer. << Prior to administration of the agents, it is to be ascertained whether there are any medical reasons for not using the chosen subjects. For a minimum of 24 hours following administration, a physician should be readily available. Physical examination prior to and following administration of the agents will be per formed at the discretion of the physician involved. ANALYTICAL WORK: It is suggested that a 10 ml sample of blood be obtained at 2, 7, 12, 24 and 48 hours following administration of the 5 mg/k dose. These samples should be analyzed for 2,4-D and 2,4,5-T. It is requested that all urine voided during the first and second 24 hour periods following administration be collected. A sample of these composite samples is to be analyzed for the agent in question. 574000706 I DOVP049274 ABSORPTION AND EXCRETION OF 2,4-D AND OF 2,4,5-T IN MAN The extensive use of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 , 4 , 5 - trichorophenoxyacetic acid (2,4,5-T) as selective herbi cides ir. many areas of the world has increased the possibility of exposure of animals and man to these materials. This has created the need for creater knowledge and understanding of their fate after ingestion by man, as well as other animals. Such understanding is of great value in using data obtained from animal experimentation to assess safety for man. SUMMARY OF TOXICOLOGY LITERATURE There are a number of reports in the literature in which the acute and chronic oral toxicity of 2,4-D and 2,4,5-T were determined Cor a variety of animal species. In 1947, Hill and Carlisle reported the acute oral toxicity of 2,4-D in mice, rats, rabbits; guinea pigs and monkeys. Of these species the monkey was most sensitive showing no adverse effects at 214 mg/kg dosages but definite signs of toxicity at 428 mg/kg. The toxic signs ob served were nausea and vomiting, stiffness of the legs with some muscular incoordination, lethargy, hanging of the head and ptosis ol c.ht? eyelids. Ln 1948, Bicrn and N'orthen reported the acute oral lethal dose of 2,4-!': for chickens to be between 380-765 mg/kg. In 1953, Drill, and Fiiratzka reported on studies with dogs indicating the acute oral LL\, value for 2,4-D to be about 100 mg/kg. When 2, j o r '.f; mg/kgfduy of 2,4-D was administered 5 times/week for 90 days, no adverse effects were noted; however, 20 mg/kg caused the death of j of 4 dogs on days 18, 25, and 49 of the test. The toxic signs which w e r e 'observed varied from mild ataxia and svi.ffrt.-ss of the hind logs to a definite myotonia, weakness, dif ficulty ir. swa 1Lowi:k ;, anorexia and occasional bleeding from the g ur s . r: 1: 4, -nvo.and Hymns reported the acute oral toxicity of 2,4-D to rats, mice, i u l u -m plus nr.d chickens. The acute oral LD^ m f:es .id the;r c,r; confidence limits were as follows: rats, : ke - >2-4v ': - :i.i ?e, >f.8 mg/kg (312-434); guinea pigs, ::u k > i; and chicks, 54 L mg/kg (358-817). The o x .ci ty ;:.cu':t.d v i :epeated oral doses of 2,4-D in rats and 5746 0000797 ( b .z f i t 'O M o a - 2- chicks was also reported. Rats fed 2,4-D five times a week for 4 weeks at levels of 3, 10, and 30 mg/kg/day showed no adverse effects of the treatment, whereas rats receiving levels of 100 mg/kg showed varying degrees of gastrointestinal irritation, slight cloudy swelling in the liver, and depressed growth rate. Animals receiving 300 mg/kg failed rapidly and died with severe gastrointestinal irritation being the principal effect observed. In L963, Palmer described the effects observed in cattle treated with varying amounts of 2,4-D five times/week for varying periods of time. A steer receiving 112 daily oral doses of 50 mg/kg showed no adverse effects. Another steer receiving 100 mg/kg/day developed a digestive disorder characterized by extreme tympany after 86 treatments. Another steer given 200 mg/kg/day developed muscular weakness in the hindquarters and a staggering gait after 34 days. A steer given 250 mg/kg/day developed moderate tympany and meiena which was first noticed after 15 days on treatment. A limited number of reports have appeared in the literature dealing with unusual exposure of humans to 2,4-D. In 1959, Goldstein, Jones and Brown reported 3 cases of exposures to an unspecified ester of 2,4-D. Severe sensory and motor symptoms necessitated hospitalization in each case. The disorders began some hours after the arms and/or legs of the patients had be come wetted with unknown amounts of the 2,4-D preparation being used to kill weeds. The symptoms progressed for a period of days until pain, paraesthesia and paralysis were severe. Dis ability was protracted and recovery was incomplete even after a lapse of years. Electromyographic examination supported the *. diagnosis- of. -peripheral n e u r o p a t h y . In 1962. Todd, described a patient with peripheral neuritis lasting almost two years which was presumably caused by the same agent. In 1963, Berkley and Magee reported the development of a neuropathy in a farmer following exposure to the dimethylamine salt of 2,4-D. In 1'h 5, Neilsen, Kaempe and Jensen-Ilolm reported the only known fatal case or j ,4-D poisoning. The suicidal patient had in- :ested approximately )'-'0 mq/ka o f 2,4-D b u t the actual lethal Jo so could not estanl ished. I:i 1 *70, Berwick >_oporr.ori o:i a farmer who accidentally inrod about -D . 1. of a concentrated weedkiller formulation c o - '...lining . . . 4 - b isooctv lester, 4 9 % S - e t h y l d i p r o p y l t h i o carb.in.ar.'- (ig.'ar , )' .--.erosone, 0.5'i epichlorohydrin and 5% 0000703  -3 - f:. -- . -a--i DOW049276 emulsifiers. The amount of 2,4-D ingested was calculated to be 110 mg/kg as the free acid. In addition, 230 mg/kg of S- ethyldipropylthiocarbamate, and 2.3 mg/kg of epichlorohydrin must have been ingested. The patient was hospitalized and his progress through the acute stages and subsequent recovery were closely monitored. Symptoms associated with intoxication in cluded nausea and acute gastritis, breathing difficulties, , skeletal muscle injury and weakness. The levels of IDH, SOOT, SGPT, aldolase and creatine phosphokinase in the serum were elevated. No symptoms of peripheral neuropathy were detected. The patient was discharged from the hospital after 2 weeks. Laboratory studies one month after ingetion of. the material showed normal values for the serum enzyme levels, the complete ' blood cell count and the urinalysis. The most meaningful data relative to establishing, the safety of the doses of 2,4-D suggested foruse in the present study were reported by Seabury in 1963. He reported on the administration of 2,4-D as a treatment for two terminal patients with dis seminated coccidioidomycosis. One patient received 12.712 g of the sodium salt of 2,4-D during a period of 34 days without , observable toxicity. The drug was administered both intra muscularly and intravenously. On the 34th day, 2 gm were given intravenously without detectable untoward effects. On the 36th day, a dose of 3.600 g of sodium 2,4-D was given intravenously over a period of 2 hours. Untoward effects associated with the administration of the latter dose were stupor, hyporeflexia, urinary incontinence and muscular weakness. Within 48 hours after treatment the patient had returned to his prereaction status and no further evidence of neurologic or muscular change appeared in the subsequent two weeks of his life. Another study in which th dose of 2,4-D was accurately quantitaed was that of Assouly (1951). He ingested 500 mg/day for 3 weeks without experiencing adverse effects. The survey of the litaratiare revealed only limited information on the toxicity of 2,4,5-T to animals. No cases of unusual human exposures have been reported. In 1953, Drill and Hiratzka reported the acute oral LD^ of 2,4,5-T in dogs to be in the range of 100 mg/kg or higher. No marked toxic effects were noted but there was some wieght loss, anorexia, ataxia and slight to moderate stiffness in the hind legs in dogs receiving higher doses. Dogs receiving 2, 5 or 10 mg/kg of 2,4,5-T five days a 0003739 Do w O-1927? week for 90 days showed no adverse effects. The daily admini stration of 20 m g A g produced death in 4 dogs after 11, 49, 59 and 75 days of treatment. In the dogs that died, toxic signs were weakness, stiffness of hind legs, difficulty in swallowing food and bleeding from the gums. In 1954, Rowe and Hymas reported the acute oral toxicity of 2,4,5-T in rats, mice,guinea pigs and chickens. The acute oral ID values and the 95% confidence limits were 500 mg/kg (391-640), 389 m g A g (245-619), 381 m g A g (307-472) and 310 m g A g (211-456), respectively. The toxic signs at the higher levels of treatment were anorexia, weight lose, depression, rough hair coat and muscular weakness. Recently, a S'tudy of the effects of 2,4,5-T consumption for 90 days has been conducted using male and female rats. (Study conducted during 1970 by The Dow Chemical Company, Midland, Michigan and as yet unpublished.) In this study, 2,4,5-T was included in the diet such that daily doses of 100, 30, 10 and 3 mgAg/day were given to groups of male and female rats, 10 animals/group. Parameters evluated were appearance, behavior, growth, food intake, hematology, urinalysis during the 12th week of the experiment, terminal determination of blood urea nitrogen (BON), serum alkaline phosphatase and SGPT, terminal organ weights and gross pathological and histopathological exa mination of the tissues. There was 100% survival in all groups. At the 100 mgAg/day level the following signs of toxicity were observed: depressed body weight, depressed food intake, a two-fold increase in serum alkaline phosphatase, a 20% increase --in SGPT (observed: only in males), inconsistent paleness and accentuated lobular patterns in the liver of some rats, and cloudy swelling of the hepatocytes. At the 30 mgAg/day level, the only changes associated with treatment were increased liver and kidney weights in males and a slight elevation of serum alkaline phosphatase and SGPT in the females. No changes were detected in rats given 10 or 3 mgAg/day. Studies designed to reveal whether 2,4,5-T is teratogenic have been conducted by The Dow Chemical Company, Zionsville, Indiana (In Press.) In these studies, rats were given 1, 3, 6, 12 or 24 mgAg/day 2,4,5-T as a 0.25% suspension in METHOCEL" by oral gavage on days 6 through 15 of gestation and rabbits were given 10, 20 or 40 m g A g / d a y orally in gelatin capsules on days 6 through 18 of gestation. At the doses indi cated, no untoward effects were detected in either the mothers or the fetuses. 5749 0000200 DOW 0 4 9 2 7 8 -5- PHARMACOLOGY OF 2,4-D AND 2,4,5-T Absorption and Distribution Orally administered, 2,4-D or 2,4,5-T is rapidly absorbed from the stomach and distributed to all tissues. The compounds have been detected in blood and organs as early as 1 hour after dosing, with peak levels occurring between 4 and 8 hours after administration in several mammalian species (Khanna and Fang, 1966; Shafik et al, 1971; Erne, 1966; Courtney, 1970). The greatest concentrations occur in plasma, liver, kidney and lung. Metabolism and Excretion 2,4-D and 2,4,5-T have been shown to be eliminated completely and intact in urine of cattle (Lisk et al, 1963; Bache et al, 1964; St. John et al, 1964) and sheep (Clark et al, 1964). Courtney (1970) reported that greater than 90% of an administered dose of 2,4,5-T to rats was recovered unchanged within 72 hours after treatment. She suggested that 2,4,5-T was apparently not metabolized to any extent in the rat. However, Shafik et al (1971) found that treatment of rats wi t h 50 mg/kg 2,4,5-T re sulted in the excretion of at least three metabolites in addition to the parent compound. In the same study, rats dosed with 2,4-D excreted the major portion of the administered herbicide in the urine. No metabolites of 2,4-D were detected. Khanna and Fane (1966) administered l-C**-2,4-D to rats and found no C1' in expired air during a 3-day period following dosing. In the latter study counter-current separation of urine and tissue extracts revealed a very small quantity of an un identified metabolite(s) (0.25% of total radioactivity in urine). I The excretion rate of 2,4-D and 2,4,5-T appears to be somewhat dependent upon the dose administered. High dose levels (>250 mq / k q ) are excreted at a slower rate than low dose levels (Khurinn and Fang, 1966; Shafik, et al, 1971). The plasma ha if-Life values are about 3 hours for rats, 8 hours for calves and chickens and 12 hours for pigs (Erne, 1966). The major excretory route is via the kidneys in all mammalian species studies. Following administration o f a tracer dose of C1+-2,4-D to sheep, 96';' of the activity was excreted in the urine and 1.4% Lr. the feces in 72 hours (Clark et al, 1964). Only low levels of 2,4-D were found in feces of rats and pigs and in bile; of pigs g Lven 2,4-D orally (Erne, 1966). Renal excretion of 5750 0000801 m on -6 - phenoxyacetic acids has been reported to be the predominant mode of excretion in cattle and sheep (Lisk et al* 1963j Bche et al, 1964 and St. John et al, 1964). Shaflk et a)L (1971) found that in rats, orally administered 2 , 4 -0 and 2 , 4 , 5-T were almost completely excreted in the urine within 2 days. Khanna and Fang (1966) reported 94 to 99% of 2,4-D is excreted in urine and feces of rats within 48 hours following the admini stration' of 1-10 mg 2,4-D per rat. Of this, 93 to 96% was ex creted in the first 24 hours; almost all was excreted in the urine. EVALUATION OF THE SAFETY OF THE PROPOSED STUDY ON MAN Acute and chronic toxicological studies on various animal species (absorption, distribution and excretion) indicate that there is no significant difference in either the toxicity or metabolism of 2,4-D and 2,4,5-T. In the most susceptible animal, the dog, the LDm for a single dose of either 2,4-D or 2,4,5-T is at least 100 mg/kg. In this specie, a dose of 10 mg/kg/day of either compound is without adverse effect when given 5 times a week for 3 months. Data from various studies on other species show all of them to be less sensitive than the dog. Reports of accidental ingestion and therapeutic administration of 2,4-D to man strongly suggest that man is not uniquely sensi tive to 2,4-D in comparison to other species. Based on the similarity in biological effects of 2,4-D and 2,4,5-T in animals*, it seems reasonable to expect 2,4,5-T to be similar to 2,4-D in .its effect on. man. Thus, based on those experiences in which the ingestion of 2,4-D by man was quantitated arid on the biological similarity of 2,4-D and 2,4,5-T in animals, a single oral dose of 5 mg/kg of either 2.4- D or 2,4,5-T is believed to be safe for man. PRE-EXPERIMENTAL SAFETY CHECK Although the available data suggest that a dose of 5 mg/kg 2,4D and 2,4,5-T will not cause any untoward effects in man, it would be prudent to establish the subjective tolerance of this dose before conducting the experiment. Therefore, it is recom mended that two test subjects per material be given 1 mg/kg 2.4- d and 2,4,5-T. When it is established that this dose does not produce untoward effects, the dose should be sequentially in creased t o 3 mg kg and then to 5 mg/kg. If the experimentalists believe tr.nc selected clinical studies such as those proposed 5751 0 0 0 0 8 0 2 082610 M O Q -7- (Paragraph 6 of Experimental Design) are desirable, they should, of course, conduct them. The test subjects used for this study should not be used in the absorption and excretion experiment. EXPERIMENTAL DESIGN 1. Subjects: Five male volunteers between the ages of 20 and 40 years will be used for each compound. Prior to exposure, the individual will be examined and judged to be of normal health and nutritional status and should not be on medica tion. Clinical parameters to be assessed at this time and during the experiment are indicated below. 2. Samples of 2,4-D (Sample AGR30653C) and 2,4,5-T (Sample AGR86187). The samples of these material will be analytical standards supplied by Dow. Specifications fb r these samples are included. Administration: 2,4-D and 2,4,5-T will be given in a single dose of 5 mg/kg in a gelatin capsule. The test material is to be given one-half hour after a standardized light breakfast. Further meals for the first 24 hours should also be standardized. >\ 4. Sampling Schedule a. Blood: Blood samples should be obtained as indicated below. 2,4-D or 2,4,5-T Time Analysis 0 (Pretreatment) 20 ml 2 Hours post-treatment 10 5H 10 8 II 10 12 II 10 16 II 10 24 1 20 48 II 20 168 II 20 - Clinical Chemistry 30 ml - - 20 20 20 Thu blood samp Les for analysis of 2,4-D and 2,4,5-T are to be mixed w i t h 2 mg potassium oxalate per ml of blood and cciitr.fuued. Plasma is to be collected for compound analysis. Duplicate analysis should be made if possible. 0 0 0 0 3 C3 5752 -8- The blood samples for clinical chemistry should be col lected without anticoagulant. The clinical chemistry determinations will be made on serum. b. Urine: A pretreatment 24-hour urine sample is to be collected as a control. Following treatment, all urine is to be collected. During the first 24 hours, the volume of the urine voided and the time following treat ment at which it was voided should be recorded. A 50 ml aliquot of each urine sample is to be taken for compound analysis. Clinical chemical evaluations are to be con ducted on another aliquot. After the first 24 hours, a composite urine sample for each subsequent 24-hour period is to be collected and an aliquot, 100-200 ml, retained for compound analysis. c. Feces: All feces voided on each of t h e 7 days following treatment should be collected. The time collected and the weight of each sample is to be determined. A repre sentative aliquot of the feces, 100 to 200 gm, is to be retained for analysis. (All samples - plasma, urine, feces - are to be refrigerated as soon as possible after collection and prior to analysis. The analytical procedures to be used will be submitted with the protocol.) Analysis: The procedures for the analysis of 2,4-D and 2,4,5T are included. Prior to attempting analysis of specimens obtained ..from .test subjects, known amounts of 2,4-D and 2,4,5T should be added to plasma, feces and urine and'the percent recovery should be established. Analysis of samples obtained from tlie test subjects should not be conducted until a re covery of greater than 90% has been established. This is necessary to provide a basis for the evaluation of the vali dity of the analytical methods in the hands of the experimen talist. The evaluation of results from the first test subject for each compound si.ould be completed before using subsequent test subjects to confirm that the dose was appropriate and the sampling times were appropriate. Adjustments can be -ado as tie-.ano-d necessary. 000030,1 5753 7 0 v d Fin aa o n -9- 6. Physical and Clinical Evaluation of Subjects: In addition to obtaining a history, a thorough physical, examination, includi;:-: a neurological evaluation, will be conducted on each subject. Any evidence of abnormal nutrition, gastro intestinal, urinary, cardiac, muscular, or neurological conditions will exempt the individual from use in this study. If currently feasible, the clinical tests should include the following: electrocardiogram, electroencephalogram, electromyogram, urinalysis (color, transparency, specific gravity, pi: and concentrations of protein, glucose, acetone, m yo globin, biood, bile, as well as microscopic examination of the sediment , SGPT, SGOT, serum LDH and component Isozymes, serum aldolase and serum creatine phosphokinase. Renal function is to be evaluated by determining the clearance of .''Ah and creatinine. Hematological evaluation is to include . complete blood count and hemoglobin determination. in addition to the clinical evaluation prior to treatment, it. should be repeated 12, 48 and 168 hours following the ministration of the test compound. Expected beta: Examples of the character of the data to be obtained from these experiments are shown in the attached hypothetic ii. figures. Once the data have been obtained, v.e ha-'c a computer program which will allow us to determine tr.e r-ite oj absorption, the rate of disappearance and the vciume of distribution for each experimental subject. These alu'.-s will Then be subjected to a statistical evaluation to determine the mean and standard deviation. 0000805 5754 ! ' f i ' o u ^ ajr -j 7 ^ G o m s g % 5755 c*q ?*-; D i a a o h o o; T 826M}iV\O T M E D o v ; e x c m :c a l c o m p a n y MIOLAKO MtCl'tOAN ANALYTICAL LABORATORIES REPORT oate__October 13, 19G9 ( AL kumse* ___ .7r23-2_____ CHARCE .12 G l-02 6, PR08LEM _5602322. TOUS KUMP CR___ OEJCR'PTlO:. FULL DESCRIPTION OF ANALYTICAL STANDARD 2,4-D _______(2 ,4 -DICHLOROPHENQXYACETIC ACID)________ Analytical standard 2,4-D, AGR 30653C, was found to be 99.7% pure. Identity was confirmed by data obtained by elemental analysis, mass spectrometry, and nuclear magnetic resonance spectrometry. 1. Specifications for analytical standard 2,4-D: (Batches not meeting this specification will be reworked until they do or will be dis carded). 2,4-D Volatile Impurities (100C) Impurities determined by Differential Scanning Calorimetry % Minimum % Maximum 99.0 100.0 0.0 0.5 0.0 1.0 2. Identity confirmed by: (a) Elemental Analysis (+0.3%): Carbon 43.2 Hydrogen 2.45 Chlorine 31.8 - (b) Mass Spectrometry (c) Proton Nuclear Magnetic Resonance Spectrometry 43.8 3.05 32.4 3. Purity of the analytical standard will be determined as follows: (a) The purity of the analytical standard will be calculated as follows: % 2,4-D = 100.0 - A - B Where A - % volatile impurities B = % impurities determined by differential scanning calorimetry (b) Determine volatile impurities by the following procedure: ine. 1 nm 23 A!': o v ' ; o ;:: ocr 1-1 AR 823 sieves R. Hummel PmO.sE M E*__3543____ BLOC.1- .574_______ 5 7 5 7 000CSG3 98'36l?CW\oa O c t o b e r 13, 19UU -2 - Alj l-3o<i Weigh one gram of the standard into a dish and place the dish in a 100C oven for 30 minutes. Cool the dish in a desiccator and reweigh. Calculate the percent volatile impurities from the loss in weight. (c) Determine impurities in the dried analytical standard by analysis of the melting curve obtained with a Perkin-Elmer Differential Scanning Calorimeter: (1) Accurately weigh three mg of the dried analytical standard into a sample pan and obtain its melting curve by heating in the calorimeter through a range of 405 to 420K using a chart range of 2 m e a l./s e c ., a heating rate of 0. 625C/min. and a chart speed of one inch per minute. (2) Calculate the mole percent impurities in the sample from the melting curve following the directions given in Anal. Chem. 41, 330 (1969). 4. The identity of the analytical standard will be confirmed by the following procedures: a. Elemental Analysis: (1) Determine carbon and hydrogen by "Rapid Multiple Microdetermination of Carbon and Hydrogen", Mikrochimica Acta 1964, 631, or equivalent. (2) Determine chlorine by method MLS. 66.1 (semi micro oxygen flask combustion, potentiometric titration) or equivalent. b. Mass Spectrometry: The mass spectrum obtained with a suitable spectro meter should show: (1) A parent ion at mass 220 with isotope peaks indicating the presence of two chlorine atoms. (2) A fragment ion at mass 175 (loss of -COOH). (3) Fragment ions at mass 162 and 161 (loss of -CHCOOH and -CH2COOII). - - (4) A fragment ion at mass 145 (loss of -OCHaCOOH). c. Proton Nuclear Magnetic Resonance Spectrometry: The 60 MHz proton spectrum obtained by scanning a 20^ solution of the analytical standard in hexadeuterated acetone with tctramethylsilane as internal standard should show: (1) A singlet at -4.82 ppm (methylene protons in -OCH2-). (2) A doublet at -7.04 ppm, J = 8.8 Hz, a doublet, J = 8. 8 Hz, of doublets, J = 2.5 Hz, at -7.26 ppm and a doublet at -7.42 ppm, J =2. 5 Hz. (Chemical shifts and coupling constants characteristic of a 2 ,4-disubstiluted phenoxy ring). (3) A singlet at -9.15 ppm due to an exchangable proton, (carboxylic acid). (4) Integrals of the ratio: 2 mclhoxy protons: 3 aromatic protons: one acid proton. * 5758 0000.809 October 13, 1969 -3- AL 7-932 5. Synthesis, purity and identity of the 2,4-D standard now in use (July 17, 1969). Purity and identity established by methods in sections 3 and 4 respectively. a. Synthesis and purification of analytical standard 2,4-D, AGR 30653C: Technical grade 2 ,4-dichlorophenoxyacetic acid was purified by dissolving in sodium hydroxide solution, precipitating with acid, wasliing with water and drying. b. Tests for purity of analytical standard 2,4-D, AGR 30653C: 2,4-D 99.7% Volatile Impurities 0.20% Impurities determined by Differential Scanning Calorimetry 0.06% c. Tests for identity of analytical standard 2,4-D, AGR 30653C: (1) Elemental Analysis: (a) Carbon 43.8% (b) Hydrogen 2. 9% (c) Chlorine , 31.8% (2) Mass Spectrometry: The mass spectrum showed: (a) A parent ion at mass 220 with isotope peaks indicating the presence of two chlorine atoms. (b) A fragment ion at mass 175. (c) Fragment ions at mass 162 and 161. (d) A fragment ion at mass 145. (3) Proton Nuclear Magnetic Resonance Spectrometry: The proton spectrum showed: (a) A singlet at -4.82 ppm (methylene protons). (b) A doublet at -7.04 ppm, J = 8. 8 Hz, a doublet, J = 8. 8 Hz, of doublets, J = 2.5 Hz, at -7.26 ppm and a doublet at - 7.42 ppni,' J = 2; 5 Hz-,- "(aromatic protons). - (c) A singlet at -9.15 ppm due to an exchangable proton (acid proton). 1 (d) Integrals of the ratio: 2 methylene protons: 3 aromatic protons: 1 acid proton. o O .i i o CO ! SO-* CO ` k 5759 0000310 R R 17. R Vll M O O Wj AL 7-932 References Proof of Structure: Chemical-Physics Research Lab. combined report IR 203894, MS 205431, NMR 205275 by S. T. King, L. Shadoff and T. E. Evans. Elemental Analysis: Analytical Laboratories AL 9-150, J. Simpson, C. Mendoza. Preparation of Analytical Standard: L. White, Process and Formulations Research Lab. Additional Analytical Work on Sample AGR 30653C - It was necessary to dry the 2,4-D standard in order to obtain a meaningful purity result with the differential scanning calorimeter. Without drying a result of 99.0 mole percent was obtained. Karl Fispher determination of water in the standard showed that the drying loss was due to water (0.19% water, AL 9-645, R. A. Dutcher). To confirm the suitability of the calorimetric purity determination 2,6-D was added to a portion of the standard and satisfactorily delected using the differential scanning calorimeter. A 0.4% unknown found on gas chromatograpliic analysis after mthylation with diazomethanc is thought to be a result of the diazomethane reaction as it is found in about the same amount in all phenoxyacids methylated in this way (AL 5-666, J. Russell). This unknown was not found after esterification with methanol-sulfuric acid. ajg i 0 0 0 iV\ Qo 5760 SH y**'py KJ \J JL i By Or. Julius E. Johnson Vice-President and Director of Research The Dow Chemical Company safttv in the Development of Herbicides The consideration of this broad topic can well be divided into three parts--past, pres ent and future. The portion entitled "past" will deal pri marily with 2,4,5-T and other phenoxy herbicides. The chapter on the "present" will give you a portrait of the events currently involved in establishing the safety of a new pesticide. The concluding remarks on the "future" will present some of my opinions on what is needed to encourage and sustain a healthy weed control enterprise for the future. Past A controversy surrounding 2,4,5-T emerged on October 29,1969. The results of a National Cancer Institute study on tera tology conducted by the Bionetics Labora tory were made known through an an nouncement by the President's Science Advisor, Dr. Lee Dubridge. Immediately thereafter I was personally involved in re porting to the Government the possibility that the sample used in the Bionetics test was contaminated. The suspected contaminant was 2.3.7.8-tetrachlorodibenzo-p-dioxin. inese suspicions were later confirmed 'byAnalysis of the Bionetics sample. Efforts were immediately instituted to test the ef fect of commercial 2,4,5-T containing less than 1 part per million of the contaminant. Tests were conducted in Dow laboratories after thorough discussions with government scientists concerning the methods to be used. Sprague-Dawley rats were the rec ommended test animals. Repeated daily oral administration of 2,4,5-T by stomach tube to the pregnant rats was the recommended route of administration. The first series of dosages ranged up to 24 milligrams per kilo gram body weight, the second series to 100 milligrams per kilogram. None of these dosages produced the symptoms of tera tology claimed in the Bionetics report. The tetrachlorodibenzo-p-dioxin, on the other hand, was toxic to embryos at a dosage level of 0.125 micrograms per kilogram per day; 0.03 micrograms per kilogram was below the no-effect level. Only by combining 2,4,5-T at a dose of 50 milligrams per kilogram with the tetrachlorodibenzo-p-dioxin in a third ex periment. was it possible to produce cleft palate in a few of the test rats. (Figure 1). This is as close as we were able to come confirming the Bionetics work in rats. Later tests with 2,4-D and silvex also failed to produce terata at elevated dosages. It is noteworthy that the tests which were conducted by Bionetics Laboratories and by the National Institute of Environmental Health Sciences that were interpreted as showing 2,4,5-T to be teratogenic were ad ministered by subcutaneous injections of 2,4.5-T dissolved in dimethylsulfoxide. The doses were up to 113 m iilig ^ f^ g ^ k ilo - DOWN TO EARTH. VoL 27. No. 1. Summer 1971 1 FIGURE 1 --Teratology Study in Rats: 2.3,7,8-tetrachlorodibenzo-p-dioxin Added to 50 MG pure 2,4,5-T/KG Body W t/Day. Treatment Control (Vehicle) 50 mo Pure 2.4.5-T/kn +0.0 MB TCOBD +0.03 MB TCDB0 +0.06 MB +0.125 MB +0.5 MB + J0 MB TCDBO TCDBD TCOBD TCOBD No. of Litters 17 17 15 17 17 15 14 15 No. of Fetusos 194 172 150 173 155 134 76 46 Litters with Cloft Palato 0 0 0 0 1 0 4 5 Littore with Intstnl. Hemorr. 0 0 0 0 8 11 13 8 FIGURE 2 -A c u te Oral Toxicity, Single Oral Dose. LQi*XIflig/VgSedyWeigh -Bat Gointa r 2.4-0 2.4.5-T Silvex Pidoram DDT Parathion 23.7,8-Tetrachloro- dibenzo-p-dioxin *Varies according tovehicle or sex 375.0-666.0* 5000 500.0 8.2000 1500-8000* 1.7-30.0* 0022-0.045* lOOOi so: 8517 3000: 400: 93-31! OOOOf gram body weight in the Bionetics test', There was ample justification for cor and up to 100 milligrams per kilogram in the by government officials over the results NIEHS te sts2. covered with the original sample use. These procedures are not at all relevant Bionetics. This sample, however, (colle to actual exposures and it is questionable in 1964) was not typical of carefully whether the demonstration of teratogenicity duced 2.4,5-T. 2.3.7.8-tetrachlorodibe by these methods is meaningful. Golberg3 p-dioxin is an extremely toxic subst: has recently pointed out several complica (Figure 2). This is good and sufficient re: tions worthy of citation -- to maintain the tetrachlorodibenzo-p-di "In checking on potential teratogenicity of concentration as low as.possible. With trace chemical contaminants in food, the analytical methods and production t- height of absurdity is achieved by the niques available in 1965 a one part per combination of a maximum tolerated dose, lion level was a realistic specification, a parenteral route of administration, and the application of zero tolerance on the basis of the results. In this area we know full well that any one of a host of adverse in has since been reduced to 0.5 parts million. A few comments on the characteristic 2.3,7.8-tetrachlorodibenzo-p-dioxin wili fluences on the mother is reflected in fetal deaths, resorptions and/or abnormalities. Teratogenic effects are elicited by: trans port of mice by air on days 12 and 13 of pregnancy (Brown. Johnston & Niswander. 1970): fasting for 24 hr. or less at a critical stage of gestation (Kalter & War- of interest. This compound can be forr during the manufacture of 2,4.5-trichlc phenol where two moles condense to fc a dioxin (Figure 3). The following information is known at the stability and properties of the 2.3.' tetrachlorodibenzo-p-dioxin: kany, 1959) which may be brought about (a) in solution it is rapidly degraded by inadvertently or unsuspectingly by in >, f traviolet light at wavelengths that ducing somnolence, lethargy, muscle pear in the spectrum of the s weakness or ataxia; a diet of raisins for (Figure 4). one day (Peters & Strassburg, 1969): se (b) The tetrachlorodibenzo-p-dioxin i vere limitation of movement or avoidance one-fifth the solubility of DDT inwe behavior (Rosenzweig & Blaustein, 1970): and one-sixteen hundredth the sc and many other nonspecific factors prob bility of DDT in benzene (Figure ably acting through a stress mechanism, Thus, there is probably less tender as well as hyper- or hypothermia and to concentrate in fat. but the quest: endocrine influence (Kalter & Warkany, is really academic because the quar 1959). Even subcutaneous sodium chlo ties of tetrachlorodibenzo-p-dioxin ride is teratogenic in mice (Nishimur^ & the environment are exceedingly sm Miyamoto, 1969). Concern has.been expressed that thecc Here, above all. is a situation th at de bustion of 2,4,5-T (even though the pare mands the utmost care in selecting doses material contained no tetrachlorodibenzo that do not render the mother sufficiently dioxin) would cause dioxin formation, ill to produce even transient inappetence. laboratory experim ent w as conduct: The use of a maximum tolerated dose over wherein agent orange (a defoliant contai looks the possibility of non-specific toxic ing equal quantities of the n-butyl esters stress ... 2 DOWN TO EARTH. Voi. 27. No. 1. Summer 1971 --5 7ujbn oo 2,4-D and 2.4,5-T) was applied to 18.5 c Iter paper at rate of 24 lb. per acre (Fig- 6). This is equal to approximately 10 lb. 2.4,5-T acid equivalent per acre. The /eight."S lerwas burned and combustion products Guinee F iji 1.000.0 380J)f S50J hisair drawn through an absorber packed glass beads cooled with liquid nitro- . The combustion products plus the ash iassolvent extracted and analyzed by gas 3.0dO00O-H0*i 9.3- 310*3 natography. The burning temperatures countered are shown in Figure 7. There bsjio 2.3.7.8-tetrachlorodibenzo-p-dioxin ILOOOS* letectea in tne combusion products at A Similar experiment (as tried using woo'd as a substrate but were substances in the combustion products which interfered with the analy- . Speculative claims are widespread that 2,4.5-T residues on vegetation might be [inverted to 2.3.7.8-tetrachlorodibenzo-p- Dn for concej he results dj! mple used? ver. (collect! carefully pi hlorodibenzt ic substani ficient reasorfl enzo-p-dioxmS ble. With tjiff uction teci p er mi@ fication. Thij 1.5 parts pej ioxin if the dead foliage is burned. All availKableevidence to date indicates this conver Ision does not occur. The alleged precursoi [isindilute form oh the substrate, the reac tion is bimolecular--the molecules must be [freed close together to react. Concern has also been expressed that [traces of preformed tetrachlorodibenzo-pIdioxin contaminating 2.4,5-T sprayed into Itheenvironment would accumulate. The eviI(fence to date is skimpy but nonetheless [worth mentioning. Cu -labeled 2,3,7.8-tetradilorodibenzo-p-dioxin dispersed on clay loam containing 3 percent organic matter gave off a very small amount of C,4C0j in acteristics: oxin will n be former [twoweeks when incubated at 24C. (75F.). [This evidence suggests that degradation occurs slowly but it will require further ob| sen/ations to quantify the rate. 1,5-trichlorcS arise to foi Kearney4 has reported that | tetrachlorodibenzo-p-dioxin is the 2.3,7,8quite immo- cnown aboi the 2.3.7.8J | bile in soil. Even in sandy soil there is little | tendency to leach into the soil profile. Al though persistent in soil the dioxin is not readilytaken up and transported to the aerial graded by ul| ;ths that ap3 of th e sun? portion of the plant. | Silvex is also made with 2,4,5-trichloro!phenol as a starting material. A total of 8 lots of silvex have been analyzed, obtained -dioxin has| from our 1967, 1968 and 1969 production. )DT in waterj No detectable amounts of 2.3,7,8-tetra- h the solu-i chlorodibenzo-p-dioxin have been found (Figure 5 j| using a method having a sensitivity of 0.1 as tendency ppm. Before leaving the subject of con he question the quantj -p-dioxin irra lingly smallj lat the com!" th arenf! adii. ,zo-p3 rmation. As conducted) nt contain^ yl esters of| taminating chlorodibenzo-p-dioxins it is noteworthy that no detectable dibenzo-pdioxins (including the 2.3,7.8-tetrachloroor the 2.7-dichloro-) have been found in 2.4- D samples analyzed. The sensitivity of die analytical method used was 1 ppm. The formation of 2.7-dichlorodibenzo-pdioxin does not occur in the manufacture of 2.4- dichlorophenol since it is made by di rect chlorination of phenol and not by alka to 18.5 cmj line hydrolysis of a trichlorobenzene. Re- F1GURE 3 -F o rm a tio n of Z3,7,8-tetrachlorodibenzo-p-dioxin. - FIGURE Solvont , . , ' Water - Benzene . :' ; -. Solubility at 25"C TCDBD ' DDT ml g/100 FIGURE 6 --Burning Agent Orange Applied to F ilter Paper FIGURE 7 --Combustion of Agent Orange on F ilte r Peper. Papor S o rtici Body of Flame From of Filma ~ boo- boo \ 1200 ; C 300- : 550-65 700-75C URE-8--Safety Evaluation For New Pesticide--..Minimum Registration Requirements. ' ' - *1950 ' ` I960* 1970 V.. Acuta, V * - - ' --Lib. Ammali " . - Lib. Animals 30-90 day. rat 90 day. rat 90 diy, dog P :* year, rat: 1 year, dog Acuta, Lab. Animals 90 day, rat 90 day, dog . 2 yarn, rat 2 yiar, dog Reproduction. 3 gen. rat Teratogenesis. rodent Fish, shellfish, etc. Birds TAB0USM (ALYTCl: T y -. ' ' Animal (min.) - cwp*,:1PPM* . Food craps 0.1 ppm** ~ Meat 0.1-ppm.' Milk 0.1 ppm' Rodent and/or dog Plant Food craps 0X1-0.05 ppm** Meat 0.1 ppm Milk 0X05-0.05 ppm -T.",-'-- . - V .'. ' - , \ ology; esticide only - u;; -i:/'. - ** Pesticide plus toxic metabolitels) vr' ENVIRONMENTAL Stability Movement Spectrum Accumulation IGURE 10--Sunk Costs7- Stage II. > : v y - 0 '! Stages.. It* *-i * .1*'l i. M I.i`I ,i I a 1.1 ' I , . I . IX r Cjimlcthrt Costs, M$ 1 2X - 7 ' -f ' . - - J ' 3X t- ' . " ` mr. action conditions do not favor bimolecular decision by j. condensation to form a chlorodibenzo-p- functions. dioxin. Moreover. 2,7-dichlorodibenzo-p- In brief, Ste dioxin is more difficult to form than the Stage 2 idem 2,3.7.8 - tetrachlorodibenzo - p - dioxin even : by limitations when reaction conditions are favorable. ' can be identit Iwould conclude these remarks by stating commitment the firmly held opinion that commercially money coverii produced 2,4,5-T containing less than one residues, m part per million of 2,3.7.8-tetrachlorodi- studies. Stag benzo-p-dioxin does not present a hazard to research outs health. It is unfortunate that tests on one im ' essary confirm pure sample coupled with emotional concern and (b) the re over the use of 2.4,5-T as a defoliant in Stage 5 is th Vietnam have created unwarranted public The costs c fear concerning the appropriate use of this ; 9. A half milli valuable herbicide. 1 the end of St? 2 may have . Present 10). In this c Let me now turn to the present and de out on the ba scribe some of the experiments primarily : work with ac relating to safety as they occur in the de lion dollars. ; velopment of a new pesticide. For the pur _ by the end c poses of this discussion we could substitute years of time the word herbicide for pesticide because production pr most of the safety considerations involve tation. The pr handling hazards, residues, non-targel expensive los organisms and environmental considera .Stage 4 (Figi tions. I will briefly describe the stages tion limitatio through which a candidate progresses and Another exam then present some of the safety and cost ure 13, wher considerations in each respective stage vested at the I In a study reported by Von Rumker, Guest ; nated. Incide and Upholt*. it was claimed, in 1969, in . vested at 8% i a survey of 13 companies that one com-' Imposition of pit pound out of 5.000 tested emerged as a r plus interest, marketable product. Testing required ap million dollar: proximately five years prior to marketing hand a winnir and a cost of approximately four million e still substant: dollars. This reported cost did not include -'cash position process development or pilot plant studies *15) compared I would estimate today that one new pestK (Figure 16) ha cide emerges per 10.000 tested. The time { invested at 8 from discovery to market ranges from eight interest are si to ten years and the cost is ten million A winner is dollars or more. This cost includes all re i market lasting search. development and technical service; i sales level of process development, and pilot plant costs and a return It includes losers as well as winners. Tm .tax. A good other research-intensive companies re remember the cently surveyed have privately given com the losers. The parable estimates to us. a mixture of The challenge is to identify and eliminate some "real life the losers early. Conversely, the expensive Project A wa way out is to have the second best hang 5600.000. Pro in a poker game. You may be interested 000, Project comparing the minimum toxicological spending $4,0 quirements for registration in 1950. 1 ing $1.5millio: and 1970 respectively (Figure 8). In the 50.5 million, r ceeding discussion. I will refer to Stage I: tinuing and he 2. 3. 4 and 5 as steps in the process The time to development of a new pesticide. Promoti and hopefully to each higher stage represents a formali; 2. The present 5765 bimolectila [decision by personnel in many interrelated adib^zo*! Ifunctions. adit ,0?j | Inbrief. Stage 1 is the exploratory stage, n tharvrtra tstage 2 identifies the key questions where dioxin-eve! by limitations of a new discovery hopefully yorable;^ lean be identified early. Stage 3 involves the s by statin] Icommitment of extensive time and lots of jmmerciail [money covering efficacy, toxicity, process, s than-or [residues, m etabolism , and ecological :rachloro98 [studies. Stage 4 Involves (a) cooperative a hazards? [research outside of Dow to obtain the nec; on oriein lessary confirmation of efficacy and safety nal coricer [and (b) the registration of the new product. defoliantTM Stage 5 is the initial sale. ited pulili? I' The costs of Stage 1 are shown in Figure use of tms |9. Ahalf million dollars may be involved by Ithe end of Stage 1. The end point of Stage 2may have cost a million dollars (Figure 10). In this case the product was flunked rnt and de out on the basis of residue and metabolism s primarily work with accumulated costs of one mil in the de lion dollars. $1.900.000 could be invested or th e p'urj by the end of Stage 3 and consume four i substitiiti years of time (Figure 11). In this case the le becaus | production process was the deciding limi3ns involy|i itation. The product cost too much. A very non-targefl Iexpensive loser is one which flunks out in considers !Stage 4 (Figure 12). In this case registrathe stages] Ition limitations were the limiting factor. ret an] Another example of a loser is shown in Fig y and cosi ure 13. where 2.1 million dollars were in- tive staged [vested at the time the project was terminker, Guesfj Inated. Incidentally, that same money in n 1969, :f^ vested at 8% interest would result in a cash one corn! position of plus 3.4 million dollars, principal erged a s ;^ plus interest, as opposed to a loss of 2.1 quired a p | million dollars (Figure 14). On the other marketing hand a winning product for which there is our million? still substantial hope involves a negative tot include! cash position of 4.6 million dollars (Figure rnt studies^ 15) compared to a plus 6.1 million dollars new pesti^ (Figure 16) had that money been alternately I. The time invested at 8% where both principal and from eight; interest are shown above the line. ten million^ A winner is defined as a project with a ides all rej market lasting at least 9 years at an annual :al service^ sales level of 10-20 million dollars per year )lant costsl and a return on investment of 40% before oners. Tw? tax. A good investment all by itself, but oanies regl remember that the winners must support given conSrii*3| the losers. The total agricultural business is a mixture of winners and losers--and for d eliminate! some "real life" examples note (Figure 17). i expensive! Project A was terminated after spending best handf $600,000. Project B after spending $1,000.- terested iris 000, Project C. an expensive one, after >lo< re-i spending $4,000,000. Project D after spend 950. 1960| ing $1.5 million, and Project Eafter spending In the sue $0.5 million. Projects F, G and H are con o Stage tinuing and hopefully will result in winners. process og The time to kill a loser is in early Stage 1. Promotion! and hopefully no later than the end of Stage formalized! 1 The present costs as they relate to safety .' FIGURE 1 4 - Cumulative Cash Position <rf,a " L o s e ^ . B . Y ' r/gjfe ... Millions ct Doll |- v'.; f P^'act | '. v : ; U*a*d. ,~;Tnninitid > .1 ;* ' -_jl r I" n ---r ' r->. r t - --T 1360 `61 .62 :;*63 .'64r:651.,6 6 -`7 , '68 'SO '70 . S 4 111 42.1 * SURE 1 6 -- Cumulative Cash Position Of A " W in n e r " - B. ________ Cumulati Cash Position Of A " Winner" 'WUtoKOf Hatten '4 *6.1il 443SI ' ; ~T I ' l' :, I _J_ L. 1__ I l 1___ I__________ . ;^ r 7 >1360-61J82.-3 '34 `65 -66 `87 '68 '63 1370 GURE 1 7 -E xpenditure Pattern, Eight Typical Agricultural R & D Projects 1360 1362 1364 1366 1368 1370 fPBn-iFgT a XS - m | | PROJECTBIT S-II.OB 4. ' .1I : .. i''L: PROJECTT CCV 1 - 44.0 Bf I /-V. PROJECT p Y z - l i i S l jlaI.s..Sl, -m^'.;L' -i- j , ------------- PROJECTf ' I ts.463M I :- II ~ f 1 PROJECTC"' T 16.7 M iji.iijHjmiim.'.i JGURE 19--Safety Evaluation For New Pesticide--Miniinuni Registration Requirements, : ^ v ; ? a s t P rese and Possible Future :: .> V -1350 - 1360 - --? '1370 r- ' 1371-'8Qffl Vr f=-l- F>; Aaite',K . - Ub Auinnb lull. Animals' Acu,.; 'LatLAnimals V: Acuta,; tub. Animals S;- <-.30-30 day. n t - {JXICQLOGY -30 iy.nit'^iiiSB.dey;rat _ ' - 'SOday. dog - =-r '30 day, dog .2 year, ret ...1.:year, dog ' '*'2.year, rat- 2 year, dog y; !.; 'Raproduclion, y'.s'. ^5^JCx,<3 gaiCrat -g. -Taiatogenasis! ' rodant '30 day, rat 90day, dog 2 year, rat 1 year, dog 'Reproduction. - 3gon. rat .y -/. :Taratogansis. rodent Fish, shellfish, ate. Fish,shollfish. ate. Birds Birds v 2 yr, hamster... y(carcinogenesis) Primate " Human viv? V-.' ; Muttganasis ietabousm . Animal (min.) -rg r Rodentand/ordog Vlent.. Rodentand/ordog Plant Human ^<v''.v- Foodapsii ' : . Foodwpoppm*.*v<i^'V#K-i'iF0oJo)Sd;cprpopms;*0*3*1*-'#.- Foodcrops. 031.V035 ppm** . ;: .. MiwA;LYnCAL . ; "\ MMiolta0t.a1ippppmm<^.fM^Millt0.t000.15p-pamQS ppm Meat0.1ppm .Mint 0.005-0.05ppm `ENVIRONMENTAL;;; c-oio.cY s~. -A ,^5Stability **- " r v_,v. j : .. Bloamutty ^.v Movement Spactnm '..-.`~^l~^4ia..tieVi-d;eplust o x i\`c'-iVm, a t aNb:- o.:..'l.'if-`t t ) T- "Accumulation FIGURE 18-P ro je c t Technical Manpower For Safety R & D ...1 9 7Q Agricultural Chemicals lESLOaj government c fhade with goc on fact. 4220.000 ,2300 'te*22.000% - f p u ---- U Another sug [bedeveloped fc laboratories. T 'ratories, u niver TOXICOLOGY METABOLISM ANALYTICAL laboratories ar Greater public iopedfortheres ECOLOGY stories if cert ^qualifying boa ^adequacy of pi and ecological considerations in Resear ttment and hous and Development are shown in Figure II ^Improve and m Early toxicological work co sts abouft `of the data use $22.000 through Stage 2. Other safety costs! A ssum ing th (metabolism, analytical and effect on er ifjed, then on vironment) increase toxicology expenses /certified labore Stage 3 totalling $220.000. Further ex ETor the suppo panded work continued through Stage j|for the suppo resu lts in an accum ulated expense ^further sugges $692.000. But the most important consider! feof- a pesticide ation is time. The time value of money and I patent expires the effective time of life o f a patent a^ ^certified data often underrated. ^either from the from a govern Future jjjective would I Let's compare .the toxicology requin |lier so that ments in 1950, 1960 and 1970 with the ^competitive a tential needs that m ig h t become requ one who has ments between '71 and '80 as presented! development. Figure 19. These would include addition With a prov testing for carcinogenesis, primate toxic ^tHe only type ogy, human toxicology and mutagenea ible for suppe Perhaps human metabolism will also be~ requirement tr quired. plus intensified information Required to pa environmental effects. You will recall! possible to lc $692,000 figure for the cost of toxicol 'Tolerance petit metabolism, analytical and ecology tests to encourage t in 1970. The added requirements cTM ^ata at this p involve another $400,000-$500.000, jections. Toda\ cost is not as important as the added ti lator nor the p Once 5 million dollars has been invest^ they want th a three year delay could cost an added!] ijcious. Morec million dollars in terms of interest exper* ipected to he and losses of earnings while the proji luct, do not ha is held up. Developing pesticides is tr ion informat ic a high risk business. Changes and imprr ^(tension spe ments will have to be made in the way ie validity c manage our destiny. loreover. the I would like to submit some suggestiq [registration at for consideration. In the first place, 41 under pressun government should do s better job of 'Trbids him fr fining te s t requirem ents, yet the ii jpn-; available methods or protocols need flexibility^ :itioner. The individual judgment, a n d provision for''" erate change. Change - in require should not be capricious but should iblic distrus remot out in iWe should r from advances in science and advance^ new knowledge. Any change, howr of the ex listration sc should involve understanding and pai pation by industry, not by sudden units ^ ile can be ac ^sion. This w (government decree. Changes should be [madewith good and sufficient reason based [onfact. Another suggestion is that a mechanism bedeveloped for certification of toxicological laboratories. This means industrial labo EEEHSEE" ratories, university laboratories, commercial laboratories and government laboratories. [Greater public confidence could be devel- j-opedforthe results issuing from such labora tories if certification required th a t a qualifying board periodically rule on the [adequacy of personnel, procedures, equipions in Research ;ment and housing. The objective would be to w n in Figure.noJ improve and make more uniform the quality rk c o s ts abo? ofthe data used for support of products. )ther safety cosul Assuming that laboratories could be certi id effect on J ffi fied, then only toxicological data from >logy e x p e n s e s ^ certified laboratories should be admissible )00. Further exT for the support of product registration or :hrough Stage^ for the support of residue tolerance, it is ted ex p en se-o further suggested that the second supplier portant consider-! of a pesticide after the original petitioners ue of money anSi patent expires, be permitted to purchase of a patent arej certified data. This could be purchased either from the original supplier or perhaps from a government agency. The main ob oology require 970 -ith the bet e require ) as presented.^ iclude additional^ primate toxicoj-, id mutagenesis^ 1 will also be 1 information < u will recall _ st of toxicology^ ecology testing ? lirements could' -$500,000, but; the added time,; been invested,^ )st an added 12 t nterest expense lile the projects nicides is trulyj es and improve^? 2 in the way we;5 jective would be to burden the second sup plier so that he does not have an unfair competitive advantage compared to the one who has borne the original cost of development. With a provision of certified data being the only type of toxicological data admis sible for support of tolerance, and with the requirement that the second supplier be required to pay his way. then it would be possible to lay the registration and the tolerance petitions open for inspection and to encourage publication. Openness of the data at this point would relieve many ob jections. Today neither the qualified investi gator nor the public have access to the facts ifthey want them, hence the public is sus picious. Moreover, the experiment stations, expected to help support the use of a prod uct. do not have access to certain registra tion information. Experiment stations and extension specialists need confidence in the validity of the back-up information. ne suggestionSs^*i irst place, theg ;tter job of de^i] yet the test 1 flexibility for /sin- *or delib- i\ rements t should derive, id advances in ige. however, ig and particidden unilateral Moreover, the public official charged with registration and tolerance proceedings is under pressure because the present policy forbids him from making certain informa tion available w ithout co n sen t of th e petitioner. The whole situation promotes public distrust because supporting facts are not out in the open. We should more aggressively extend the use of the experimental labels prior to full registration so that typical, though limited, sale can be achieved under qualified super vision. This would permit the development of meaningful use experience with limited exposure of the total population. The ob jective would be to discover unexpected phenomena difficult or impossible to ex p o se under -laboratory or limited field test conditions. One of the weakest links in the chain is in diagnosis of pest problems and applica tion of pesticides. I do not wish to discredit those competent professionals who do a good job. nevertheless, in the country at large the process of diagnosis and applica tion involves people, usually unskilled, poorly paid, and nonprofessional. We have many elegant tools but poor craftsmen. To overcome this difficulty I would suggest that we classify pesticides in two categories --fo r professional use o nly and fo r non-pro fessional use. Those for professonal use could be applied by licensed professionals only--skilled in diagnosis and application. Moreover, since there is a scarcity of this kind of skill, a training of a new kind of pro fessional should be developed by our agri cultural schools, th is will take time, but it's needed and the professional licenses should be granted only to those qualified. The grower would then pay for results and the whole process of pest control, includ ing weed control, should be less poundage oriented and more result oriented. This will require some drastic rethinking on the part of industry on how they can make their contribution and reap their share of the reward. I am convinced that the American consumer can well afford to pay a higher percentage of the shelf price of food or fiber for pest control if the job is profes sionally done with well designed products. In other words, there is economic room to maneuver if everyone is compelled to com pete by equitable regulations. As a final note--I would strongly advocate that we worry less about conflict of interest and see to it that government, industry and university personnel communicate more effectively. REFERENCES 1. National Institute of Environmental Health Sciences. Research Triangle Park. North Caro lina. Summary Teratogenic Study NIEHS. Type script draft of record of hearing on 2.4.5-T before the Subcommittee on Energy. Natural Resources and the Environment of the Senate Committee on Commerce. Philip A Hart, Chair man. April 15.1970. pp. 225-232. 2. Science. 168 (864-866) 1970. 3. Brief of the USDA. In The United States Court of Appeals For The District of Columbia Circuit (No. 24.434). November 2. 1970. 4. Kearney. Phillip C. Report Presented before a joint meeting on Pesticides. United Kingdom. Canada and United States. Sponsored by the Council on Environmental Quality and Presi dent's Cabinet Committee on the Environmental Working Group on Pesticides. Washington. D.C., November 5. 1970. 5. Bioscience. 2 0 (1004-1007) 1970. 5768 DOWN TO EARTH. Vol. 27. No. 1. Summer 19 0 3 0 NORTH LASALLE STREET CHICAGO, ILLINOIS 6 0 6 0 2 - 2 5 0 7 (312) 7 6 2 - 5 6 8 0 R i v k i n ,R a d l e r ,D u n n e & B a y h EAB PLAZA UNIONDALE, NEW YORK 11556-OIH (516) 3 5 7 - 3 0 0 0 TELEX: 6 4 5 -0 7 < 4 T E L E C O PIE R : ( 5 1 6 ) 3 5 7 - 3 3 3 3 CABLE! AT LAW 1 5 7 5 EYE STREE T, N.W. WASHINGTON, O.C. 2 0 0 0 S - I I 0 5 (202) 2 6 9 -8 6 6 0 2 0 4 0 CENTURY PARK EAST LOS ANGELES, CA 9 0 0 6 7 - 2 5 9 2 (213) 2 0 1 -0 5 1 0 August 10, 1989 r 6 0 5 THIRD AVENUE NEW YORK, N.Y. 1 0 0 2 2 - 7 5 1 3 (212) < 4 1 8 -5 2 0 0 W R IT E R 'S O IR E C T DIAL Mary E. Alexander, Esq. Cartwright, Slobcdin, Bokelman, Borovsky, Wartnick, Moore & Harris, Inc 101 California Street, Suite 2600 San Francisco, California 9411 RECEIVED AUG a 4 ^989 CARTWRIGHT ET AL Re: Davis v. The Dow Chemical Company Dear Mary: In Dow's continuing effort to comply with the Court's directions, Dow has searched its files for records of shipments or sales between 1954 and 1973 of products containing 2,4,5-T to the United States Forest Service. Dow has recently discovered certain records which may be responsive to the Court's most recent order and encloses them without conceding their relevance or admissibility at trial both in terms of product identification and time frame. Please feel free to contact me if you have any questions or comments. Very truly yours, Rivkin, Radler, Dunne & Bayh JHK:lc Enc. TH E P A R TN E R S H IP INCLUO ES O NE O R MORE P R O FE S S IO N A L CORPO RATIO NS 5770 -W -X U *-< 0<Z~ . M*|"4 C U S T O M E R ORDER N*rs1,ew,,CMt* **-4 m o o u c t . *AX 372 ORDER 603 KPLTQ . M lM IM IIII I I I I I I I II , ------------ Us Afl j7 5 6 -0 g -7 2 0 63169 1136863 US D A ; FOREST SERVICE DISTRICT RANGER JiFPgR-LA&j al CAL-._&34$3. v.) 1 1 1 1 1 1 1 " I DOW NO. i '* it i i* <i n ii r n I n h V n m n ____ I l i UJ 75 S63j6gJ '"***. *CT. MO to. I lea SHippimOPOimT 21270 [ to u C W O OATS J OAtC TO ( SmiMCO 3 H i E L A M ? ____ MICH IDEL 03/03-17/----1 7642002 1370977 INSTRUCTION* US D A MARX ALL PXGS4 ? A P tR S WITH PO FOREST SERVICE NOR. SHOW U*S, GOVT PRICE FREEZE CERT P 030X 431 ON INV* mLQVSt?nr amt a t CAL 93983 TCAM * M OATS N k J OA tit.: COutCT 0 TRIP LEASE SECE rn tm o OUANTlTY OAOCACO ANO OeSCAIATION n<i lot iwiM *o*fua$wo*tiomo* UNIT RAICE INVOICING QUANTITY SHIPPING W EIGHT AMOUNT 34 5 GL PAIL ESTERON 99 CONCENTRATE 3*23 1141514/28682-04-1945 GL VEED KILLING COMPOUND FOB SHPG PT FRT PPD 23- 3 GL PAIL ESTERON 243 CONCENTRATE 1080314/28758-04-193 9*19 GL i VEED KILLING COMPOUND FOB SHPG PT FRT PPD FSN 6840-924-5361 CONTRACT GS*07 S*02728. t t I I i I I SPQCH^OfAtiifSSl* INVOICI TOTAl_____ f 5771 UO ru t 17U-5U-7i f ic PURCHASE QRDER . MtM l P .CI*.6 M i* ,,P .rilt. U.S.DEPARTMENT op agriculture OfdtrM. r a n n R IV K I 777-02-71 - . . u1 1 5 1 2 1 0 3 5 3 2 1I Fifth venu* San Diego, CA 52103 o*. ifar 21, w x r. s Tfc Dow Chenical Co., t <i i 9.S. Goveraont Marketing, 2030 ARC ? V. 5. Foroat Servie r Descanso Ranger Station i Midland, Michigan h& 6k0 I T O Descanso, CA 52016 hn'iiiN., C.-'OON* . tin.hr0*>im, 0'K.uaIt.rni 03-073-00281 (AO) ' : 'within one week Hot #. o. i h; Destination ih ; , vi. O r r 't 1 A .M .. Kam N a. ArChtu bn** Oxo.% IMl Unith*. A*wml I B$ ir 37. 68U0^52U-536l Herbicida, FOES, 2 ,U D - Itf a c id e q u iv a le n t (R e g is tra tio n k^k-yn) 8 - 5 g a tte n s te a l p a li Uo gal 2,67 106.80. 5U# 6 8 U O -5 2 U -5 3 6 3 H e r b ic i d a , FORK, 2 ^ ,5 T , 6jf a d d q u iv a le n t (Registration /U6U-3C2) U-5g*ttn steal pail 20 gal. 8.50 170.00 9 1* >NV * U C T '0 N V A | * . n r f a r .f c .* n W k . m4* M * .4 ,*>.<< n a i l , * WI ,|r* T M N l O' .iC V ilU lt -- N Forest Supervisor 3 2 II Fifth Avenue San Diego, CA 2103 06t'*4 k y i S^not*. rrn. J0H J. CA3TIS ADMETISTftATIYS OmCER. total 276,80 Z 5773 34-10 /w-'i J * II * Na.^`<1 67^-0 2 -7 1 PURCHASE ORDER ' i !'/ u.s.department of agriculture 1 0 2 - $ 70- * j i - 54- 703. (5010 4 P*"' S rOlHTUKViCI 3211 F i f t h A venue 1 1 0T * ' 1.5^AK J!i3<5eg^M^ k " f a a D ie g o t C a l i f o r n i a 92103 Wtf*c'ltf-Al(Hl<ICMl*tM***l*... .>../.||M| 0 >d*r Mi. 300- 02-71 Oai 11/ 30/70 si i ^ T h e Cow C h e n ic a l C o / ^ ' ^ / J ? , U .S . C-ovem cat M ark etin g i I 2030 ARC A ttn : H r. P .y u M id la n d M ic h ig a n 40640 _J W iifiJ N a. /a Canitad Na. C S - 07S -21939 I. O. I. Fa* o *e la n tIo n via n/s IfM Na. AM >$ * U .8* F o rest Serttcw [-, D i s t r i c t B an ger 'T P f B e e c a n a o , C a l i f o r n i a 92016 T A ____________ Tljjj U Oalwaty Oitcawnf Tara* 25 d o y s " Oa*`i.IANa.n e t - ` 30 d a y s n/a O ii >% U n it f r it * la 2. U e r b i d d e , o s ta r o n 245^ .2*4 , T l a {5 g a l l o n p a l l s > ( B u t o a y E t h a n o l E a t e r o r [P ro p y le n e G ly c o l B u t y l e s t e r s ) (4# a d d e q u iv a le n t p e r g a llo n ) ' - ' `t:? U? H erbicid e e a tero n |4? a d d e q u i v a l e n t c- 99, co n cen trate p e r^ fe a llo n c> 204-D f`4' *.*ur. `Vr -V . S'*. *-`7**VW3 * #. n 110 6 ,1 4 gain 6 7 5 .4 0 110 gain 2 .6 3 311.30 I ' U IM C IN4TU_CT'QNV f m a a t fa t ta ra - i l l ka -a k a 0>tfit Ur - a n a t ,* *> < a w it t la i _ V I H M ItM IM W M W M n ,ilfiJ91!i,,Si!2rvloor t 3211 F i f t h A venue S o n D l o r o . C a l i f o r n i a 92103 Nona :--i /:,/ r~ ^ J 0WI J . CASTLE na J M d n i s t r a t i v e O f f i c e r i o u i $ 9 8 6 .7 0 . . 5774 .. J * )O;'-I | purchase order YJL*--v^ f*'C1xi*H1:m>. - --*. vtf. 4\>A-------------------i DEPARTMENT CP AORlCULOi.* 0<f* N*. us-isi-os-'S (631) **#*. JLQisSIO.rA 2 r5 i= 7 ilL _ {3 73 Kiazp,3211 F ifth Avenu C a lifo rn ia 92103 -WA.lSJrSLJL ju i3 o /in _ \ s The Ccv C le ric a l Coopay Li V .8 . Covam eant H arletlng 2030 ARC A ttn : Mr. Syn C re d ln d , Michigan 48640 J *1*. CtMiim. n/a CS-07S-21959 P.fr. PiM SlipVi. , D is tric t Hangar pM U.S. Forost Servie* 1440 2 . F irs t S t., Roob 408 Santa Ana. C a lifo rn ia 92701 1 1 OU.^MT** 25 A7 not * 30 ya G*.')- HNk I h destin ation In* N*. n/a A/liU>. Jmkn o/e Quantity Utrt UMfti*. 1 ' Ap/ 1 . H erbicida, esteren 24 3,f` 2 ,4 ,5 -T , ia r ^ / g a l. 6.14 122.30 ^ 5 r.allon p ello (Sutoxy Ethanol Ester or Frcpyleno Clycol B utyl esters) (4# a d d equivalent per gallon) 2 . H erbicida, csteroa 99 concentrate, 204-IX. 50 *) g a l. 2.83 1 4 1 .5 (K (4# aeld equivalent per gallon) . */*r a ^TiN^ hfM M kf H", ,* m.I^ *: u.i. cenar** or 3211 F ifth Avenu* Sen ko. C a lf A m ia wll i . 92103 >- o.*.**,: S teven s * r- 2 & / o ...... T0TAt U 264.30 'Teresa C. Duprey r y ? T*t Purchasing Agent 5775 'S lty 8 V 5776 TOXICOLOGY Some Effects of 2,4-D and 2,4,5-T on Ehrlich Ascites Tumor Cells In Vivo and In Vitro Ernest M. Walker, Jr., PIlD., Richard H. Gadsden, Pli.D., Loretta M. Atkins, B.S., and Glen R. Gale, Pli.D. n z) E '..T c .'T :r,.j: -r.c/ C:i:nis:s.*. C r;ia 20351 m2,4e-nDt oafntdhe2E,4h,5rl-iTchsahsocwitessatpupmreocriainblme iicneh.ibitory effects on the in vivo develop* q t- INTRODUCTION T he application of 2,4-D, 2,4,5-T, and similar herbi cides to plants impregnates plant cells with a persis tent, spurious auxin which prevents the fluctuations of physiological auxins required for normal growth and differentiation. Hanson and Slife1 have made detailed descriptions o f the chemical and morphological changes in plant bodies and-cells following exposure to 2,4-D and related chlorophenoxycarboxylic acid herbicides. Such changes include: little or no root elongation, abnormal growth in the basal stem, abnormal leaf development, and a number o f other disruptions that may lead to alteration or death o f the plant. The fact that the effects o f 2,4-D, 2,4,5-T, and similar herbicides on plants apparently depend upon an auxin-like action led to the assumption that these agents would not be expected to alter or damage mammalian cells. Certain developments, which center largely about the potential toxicity and teratogenic activities o f these herbicides in animals, par ticularly 2,4,5-T, have led to serious reconsideration of this assumption. The Bionetics Research Laboratory o f Litton Indus tries, under NTH contract, tested various pesticides and related com pound: f-" *<'rntogeii:e activity.2 2,4,5-T. ad ministered in honey or DMSO, proved to be teratogenic in mice by causing increased num bers o f abnorm al fetuses and a statistically higher proportion o f litters affected. The chief anom oilcs observed in affected off spring were cleft palates and cystic kidneys. Similar re sults were seen in Spraguc-Dawley rats given various oral The following abbreviations are uved: 2.4-D. 2,4-dicliloropncncxyjcctic acid; 2,4,5-T, 2.4,5-trichloroplicno\>acetic acid; MEM. Eaele's minimum essential medium with flanks* balanced salt solution; DMSO. dimethyl sulfoxide; TPCV, total packed cell volume of Ehrlich uscucs tumor: DNA, deoxyribonucleic acid: RNA, ribonucleic acid; i.p., intraperituncally. doses o f 2,4,5-T on days 10-15 o f gestation. The highest * CD dosage of 46.4 mg/kg/day resulted in 60% fetal mor- * 4 tality and a high incidence o f abnorm alities in the sur- C? vivors. These investigators reported th a t 2,4-D also pro- ^ *1 duced an increase in the proportion o f abnormal litters $9 in mice. An anonymous report summarizing some studies of cz#? Q VI the Nationai Cancer Institute covers, in part, the poten tial teratogenic activities o f 53 com pounds, with the most conclusive results being found for 2,4-D and 2,4,5-T.3 Doses o f 113 mg/kg o f a b u ty l ester o f 2,4,5-T, administered from days 6-14 o f gestation in mice pro duced a total o f 54% m alformations in the mice born from treated m others, as com pared to 1% in those born from control mothers. Malformed m ice were seen in the litters o f all six treated mothers. A dose o f 98 mg/kg o f the butyl ester o f 2,4-D, given during the same interval o f gestation, produced an increased incidence o f malfor m ations in the litters o f fo u r o f the six treated m others. Several entries concerning the anti-tum or activities o f 2.4- D (NSC 2925) and 2,4,5-T (NSC 4 3 0 ) are listed in the Negative Results o f the Cancer Chemotherapy Screening Data o f the United States Public Health Ser vice. Entry No. 22683 reports that tw ice daily injections of 250 mg/kg o f 2,4-D for seven days caused 43% inhibi tion o f Sarcoma-180 in Swiss mice. In addition, the same entry reports that II daily injections o f 75 mg/kg 2,4-D caused 63% and 42% inhibition in tw o d ifferen t experi ments using Adenocarcinoma 755 in CBFy mice. How ever, the entry reports that daily injections o f 250 mg/kg 2.4- D had no effect on L-t 210 leukem ia in BDFy mice (mice were treated daily for 30 days o r until death). cC ^ *7 T Entry No. 37090 reports that twice daily injections o f )'* 4 S8 mg/kg of 2,4.5-T for seven days caused 60% and 43% inhibition o f Sarcoma-180 in Swiss m ice in t w o separate experiments. The same entry reports that 2,4,5-T had 0 0 0 2 4 8 1 Dose mx/kglday0 TABLE 1. Effect of 2,4-D on Development of the Ehrlich Ascites Tum or In Vivo tXtttnhcr o f injections'* Average weight Mortality Average TPCVd change (gm) T/Cc C T TIC. ml r/* inhibition S.D.C TIC 45 6 - 0 .4 / l.S 0/S 0/8 1.061/1.549 32 0.330/0.355 65 6 -1.7/1.6 0/10 1/3 0.924/1.744 47 0.431/0.320 75 6 0.2/3.2 0/9 2/9 0.728/1.572 54 0.460/0.26S 77 5 -0.1/5.3 0/9 2/9 0.983/2.000 51 0.393/0.330 Dimethyl sulfoxide was the vehicle ^Injected intraperitoneally, once per day cTrcated/Control ^Total packed cell volume cStandard deviation of the treated/control groups P <0.05 <0.0005 <0.0005 <0.0005 DOW 37578 TABLE 2. E ffect o f 2 ,4 ,S-T on D evelopm ent o f th e Ehrlich A scites T u m o r In V ivo Dose mg/kg/da}'a Number o f injections0 Average weight Mortality Average TPCV4 change (gm) T/C0 C T TIC, ml % inhibition 62 6 1.7/3.7 78 6 -3.3/1.6 SO 6 -1.5/5.S 85 5 -0.3/3.2 Dimethyl sulfoxide was the vehicle hjnjcctcd intrapcritoneally, once per day Trealed/Control dToul packed cell volume cStandard deviation o f treated/control 0/8 1/8 0/10 4/10 0/9 3/9 0/9 3/9 1.235/1.772 0.485/1.744 0.728/2.000 0.580/1.572 30 73 64 59 S.D.e TIC P 0.341/0.250 0.321/0.320 0.266/0.330 0.286/0.205 <0.005 <0.0005 <0.0005 <0.0005 little effect on Adenocarcinoma 755 in CBFi mice (11 daily injections of 44 mg/kg) or L-1210 leukemia in BDFi mice (mice were treated daily for 30 days or until death). Consideration of the foregoing studies of 2,4-1) and 2,4,5-T, especially the implications of their teiatogenic potentials, prompted us to investigate the effects of 2.4-D and 2,4,5-T upon Ehrlich ascites tumor cells, including certain in vivo and in vitro parameters. MATERIALS AND METHODS 2.4-D and 2,4,5-1" were provided in a highly puuficd form (99.0%) by the Pesticides Research Laboratory. Perrine, Florida. Thymidine-methyl-311, uridinco-Ml. L-leucinc-i4C, and sodium Gormate-tJC were obtained from New England Nuclear Corporation. Ascites tumor cells were maintained in BALB/c mice (Flow Research Animals, Inc.). The investigation included the following parameters which have been described in earlier reports as indicated: rates of synthesis of DNA. RNA, and protein4-5; de novo synthesis of RNA purines0-7 ; development of the Ehrlich ascites tumor in vivo.8 In addition, the survival times of mice bearing ascites tumor cells and treated with each herbicide were assessed. DMSO was used as the solvent system for all parame ters in the investigation of 2,4-D and 2,4,5-T except the survival experiments in which 20'."-DMSO in 0.9% saline was used. The volume of DMSO solution injected was 0.1 ml per 40 grams body weight of each control or treated mouse. In the in vitro experiments, the final con centration of DMSO was l%(v/v), and the same concen tration of DMSO was contained in all control vessels. RESULTS AND DISCUSSION Effects o f 2,4-D and 2,4.5-T On Development o f the Ehrlich Ascites Tumor In I'ivo The inhibitory effects of 2,4-D and 2,4,5-T on de velopment of the Ehrlich ascites tumor in vivo arc shown in Tables 1 and 2, respectively. Both compounds were effective inhibitors of tumor development, with 2,4,5-T appearing to be slightly more effective than 2.4-D atf ) 7 7 8 equivalent dose levels. Increasing the dosage of 2,4,5-T from six injections of 62 mg/kg/day to six injections of S0-S5 mg/kg/day doubled the extent of inhibition of tumor development. Similar increases in dosage of 2.4-D 0002482 TABLE 3. Effects of 2.4-D and 2.4,S-T Upon the Average Survival Times of Mice Inoculated with Ehrlich Ascites Tumor Cells Dosage Number o f Compound m s/kg/daya Injectionsb Number o f death: during Treatment Control Treated Ave. Time o f Survival In Days Treated Control %o f Control Survival Time S.D. in Days Treatedc Control P 2,4-D 2.4.5-T 75 70 5 5 00 88 0t S8 18.4 14.6 20.6 14.6 126 2.7 <0.025 3.9 141 6.8 < 0 .0 2 5 3.9 a207S DMSO in 0.97 saline was the vehicle bn ie first i.p. injections of 2.4-D or 2,4.5-T in the above vehicle were given 24 hours after inoculation of each mouse with approximately 6.xl0b Ehrlich ascites tumor celts. Mice received one injection per day for five days. cStandard deviation of the treated/control groups in days. DOW 3 7 5 7 8 2 did not result in such marked increases in the degree of inhibition as was observed with 2,4,5-T. Effects o f 2,4-D and 2,4,5-T on A verage Survival Tunes o fMice Bearing Ehrlich Ascites Tumor Cells The average survival times of mice previously inocu lated with Ehrlich ascites tumor cells and treated with each herbicide arc shown in Table 3. Both compounds produced moderate but significant increases in the average survival times of tumor-bearing mice. 2,4,5-T caused a slightly'greater increase in the average survival time (41%) than 2,4-D (26%), but the 2,4,5-T group had a higher standard deviation for survival of mice in the group (6.8 days) than seen in the case of the 2,4-D group (2.7 days). Effects o f 2,4-D and 2,4,5-T on RNA Purine Synthesis In Vivo Data presented in Table 4 demonstrate the effects of 2,4-D on de novo RNA purine synthesis in vivo. The effects of 2,4-D on RNA guanine synthesis were not consistent. Dosages of 75 mg/kg and 100 mg/kg ap peared to produce slight stimulatory effects upon RNA synthesis when injected i.p. 24 hours prior to the injec tion of sodium forinate-i4C. Averages of 16% increase in RNA guanine and S% increase in RNA adenine were found when 100 rnnlk?. 2.4-D was injected i.p. 48 hours prior to tiie injection of isotopically labeled foimatc. Table 5 shows the effects of 2,4,5-T on dc novo purine synthesis in vivo. 2.4,5-T appeared to produce a slight stimulatory effect upon RNA adenine synthesis when injected i.p. 24 hours before the injection of sodium formate-i4c by the same route. The effects on RNA guanine synthesis 24 hours after injection were not consistent. However, 2,4,5-T, 48 hours after i.p. injec tion of 100 mg/kg, produced an average of 28% inhibi tion of RNA adenine synthesis and an average of 30% inhibition of RNA guanine synthesis. Thus, the rates of synthesis of RNA adenine-i4C and guaninc-i4C were inhibited to about the same extent 48 hours after injec tion, indicating that the inhibitory effects may be exerted on the common pathway of purine synthesis (prior to synthesis of inosinic acid). Effects o f 2,4-D and 2,4.5-T on the In Vitro Incorpora tion o f Radioactive Precursors into DNA, RNA, and Pro tein o fEhrlich Ascites Cells The effects of 10-* M2,4-D on the in vitro incorpora tion of radioactive precursors into DNA, RNA, and pro tein are shown in Table 6. Incubation of the cells for periods of an hour or less with 10-* M 2,4-D caused a small and probably insignificant increase in the incor poration of thymidine-methyl-3H into DNA. Similar periods of incubation produced no notable effects on tiie incorporation of uridine-5-3H into RNA or on the incorporation of L-lcucine-i4C into protein. Incubation of the cells with KM M 2,4-D for periods of two hours resulted in slight stimulation, producing average in creases of 17%, 37%, and 22% in the rates of incorpora tion of radioactive precursors into DNA, RNA, and pro tein, respectively. The effects of KM M 2.4,5-T on the in vitro incor poration of radioactive precursors into DNA, RNA, and protein arc shown in Table 7. Incubation of ascites cells witii the compound for intervals of an hour or less pro duced slight increases in die incorporations of thymidine- 3H into DNA and uridinc-5-3H into RNA. Increasing the incubation period to two hours produced no further increase in the incorporation of isotopic pre cursor into DNA and resulted in the loss of the stimula tory effects of RNA synthesis seen after shorter incuba tion periods. Incubation of ascites ceils with KM M 2.4,5-T for an hour or longer resulted in stimulation of the incorporation of L-leucine into protein. Increases of 23% and 28%. were obtained after one and two-hour incubation periods, respectively. In summary, 2,4-D and 2,4,5-T showed appreciable inhibitory effects on the in vivo development of thc^ q' 0 2 4 S 3 TABLE 4. Effect o f 2,4-P on RNA Purine Synthesis in Ehrlich A scite' T u m o r Cells In Vivo3 2.4-n mg/kg I/nurs after hifectianh CPMimg Da++ RSA Guanine Adenine % Change Guanine Adenine Guanine Adenine 0 24 25 24 50 24 60 24 75 24 100 24 0 48 100 43 0 48 100 48 4608 3645 3552 4S76 5087 4036 3336 4712 33S7 3074 3064 2558 2725 2739 3464 2804 1354 2246 2369 2221 -21 -23 +6 + 10 -12 -- +41 -- -9 -17 -n u +13 +9 -- +21 -- -6 1.50 1.42 1.30 1.74 1.47 1.44 1.80 2.10 1.43 1.3S a2.4-D in DMSO. at the doses indicated was injected i.p. to groups o f 3-5 mice per group. ^Represents interval between i.p. injections of 2,4-D and l'ormate-l4 C. Animals were sacrificed three hours after the i.p. injection of formale-l3 C. Cells were removed and processed as described in reference 7. TA B LE 5. E ffect o f 2 ,4 ,5-T on RNA Purine S y n th e sis in Ehrlich A scites T um or Cells In Vivo3 2,4.5-T Hours after mg/kg injection* 0 24 25 24 50 24 60 24 75 24 100 24 0 24 25 24 50 24 60 24 75 24 i00 24 0 100 43 0 '48 100 48 00 CTM/mg Da ++ RHA Guanine Adenine 3657 3419 4563 3646 3363 4212 4377 3597 4019 5370 4160 2992 3336 2207 3387 2513 1256 1147 1336 1277 1401 1443 2593 3448 2777 3813 2636 2795 1S54 1067 2369 2038 % Change Guanine Adenine -- -7 +25 0 +6 + 15 -- -18 8 +23 -5 -32 -- -34 -- -26 m,, -9 +6 +5 + 12 +15 -- +33 +7 +47 +2 +8 -- -2 -- -14 oc ri Guanine Adenine 2.91 2.98 3.42 2.76 2.91 1.69 1.04 1.45 1.41 158 1.07 1.80 2.07 1.43 1.23 a2,4.5-T in DMSO, at the doses indicated was injected i.p. to groups o f 3-5 mice per group. ^Represents intercal between i.p. injections of 2.4.5-T and forinate-l' C. Aninuls were sacrificed three hours after the i.p. injection of form ale-l'C . Cells were rem ised and processed as described in reference 7. INDUSTRIAL MEDICINE, VOL. 41. NO. 1. JANUARY 1972 CO cn <r CO 5780 0002434 21- TA B LE 6. Effects u f 2,4-D on th e In V itro Incorporation of Radioactive Precursors into D N A , R N A , and P ro tein in E hrlich A scites Cells Synthesis Hours o f Average Average 7 o f Sampleb Ineuhationc CPMd o f Control DNA RNA Protein Control 2.4-D Control 2.4-D Control 2,4-D Control 2,4-D Control 2,4-D Control 2,4-D ' Control 2.4-D Contrcl 2,4-D Control 2,4-D 0e 0 1 I 2 2 0 0 1 1 2 2 0 0 -1 1 2 2 69138 70207 62294 65667 48211 56266 75788 69457 50149 49799 31856 43528 7644 7424 7250 7111 5567 6771 102 105 117 92 99 137 97 98 122 aThc cells were incubated with the appropriate radioisotopes for periods o f 20 minutes, at the end of which the reactions vere terminated by the addition o f an equal volume of 10% TCA (trichloroacetic acid). Radioactive precursors and final activities were: for DNA. thymidine-mcthyl-^H (10 pCi/ml); for RNA, u r id in c - 3 ]( (]() pCi/ml): and for protein, L-leucinc-lC. uniformly labeled (2 uCi/ml). ^Concentration of 2,4-D, 10~* M in incubation medium. Time interval between addition of 2,4-D and addition of appropriate radioisotope to ceils. ^Average o f two similar experiments in which duplicate tubes were included for each sample (average o f four tubes). *CcUs were added to incubation tubes containing 2.4-D and radioisotope. y TABLE 7. Effects of 2,4,5-T on th e In Vitro Incorporation of Radioactive Precursors into D NA , R N A , an d P ro tein in E hrlich A scites Cells Synthesis Hours o f Average o f Sampleb Jneuhationc CPMJ Average % o f Control DNA RNA Protein Control 2,4,5-T Control 2,4.5-T Control 2.4,5-T Control 2,4,5-T Control 2.4,5-T Control 2,4,5-T Control 2.4,5-T Cunt rol 2.4.5-T Control 2 2.4,5-T 0 0 1 1 2 2 0 0 1 1 2 2 0 0 1 1 2 2 37425 39599 28877 33568 2449S 26944 39846 44453 34 953 33714 29962 28765 135SS 12149 74 K8 9181 5379 6875 106 116 110 112 96 96 89 123 128 The cells were incubated with the appropriate radioisotopes for periods of 20 minutes, at the end of which the reactions were terminated by the addition of an equal volume of 1IV,7 TCA (trichloinucctic acid ). Radioactive precursors ami final .n ioiiies were: for DNA. thymidinc-mcthyl-?II (10 uCifml): (or RNA, u tid in c -^ H (10 uCI/ml): and for protein, I.-leucine-I^C, uniformly labeled (2 uCi/ml). ^Concentration of 2.4.5-T. 1(H M in incubation medium. cTime interval between addition of 2.4,5-T and addition of appropriate radioisotope to cells. ^Average of two similar experiments in which duplicate tubes were included for each sample (average of tour tubes). 'Cells were added to incubation lubes containing 2.4,5-T and radioisotope. o J CO cn CO 5781 0002485 Ehrlich ascites tumor in mice and caused mild to moderate effects on selected parameters of nucleic acid and protein syntheses as assessed by the preceding in vivo and in vitro studies. However, the extents of these effects may not be adequate to explain the relatively marked and significant degrees of inhibition of tumor development in vivo. These findings, therefore, tend to indicate a need for determining the effects of 2,4-D and 2,4,5-T on a number of additional biochemical and phar macological parameters of cellular metabolism in mammalian cells. REFERENCES 1. Hanson, J.B. and Slife, F.W.: Illinois Res. 3: la . 1961. Quoted from Oberbcck, J. Van, Survey in Mechanisms of Herbicide Action, in: The Physiology end Biochemistry o f Herbicides, edited by L.J. Audus, New York: Academic Press, 1964, p. 3S9. 2. A Report of the Secretary's Commission on Pesticides and Their Relationship to Environmental Health, U.S. Govern ment Printing Office: 0-371-074, 1969, pp. 664-677. 3. Anonymous. Thalidomide Effect from Defoliants, Set Res. 4: No. 23,11-12, 1969. 4. Gale, G.R. and Hynes, J.B.: Effects of Certain Arylhydroxamic Acids on Deoxyribonucleic Acid Synthesis by Ehrlich Ascites Tumor Cells / Vitro, J. AJed. Chem. 11: 191-194, 1968. 5. Gale, G.R., Simpson, J.G., and Smith, A.B.: Studies of the Mode of Action of N-isopropyl-(2 mcthyihydrazinc)-ptoluamide. Career Res. 27: 1186-1 191. 1967. 6. Gale. G.R., Ostrander. W.F... and Atkins, L.M.: Effects of Alanosine on Purine and Pyrimidine Synthesis, Biochem. Phcrmccoi. 17: 1S23-1S32,1968. 7. Gale, G.R. and Schmidt. G.B.: Mode of Action o f Alanosine, Biochem. Pharmacol. 17: 363-368, 1968. 8. Gale, G.R., Smith, A.B., and Walker, E.M., Jr.: Selective Inhibition by Sorbylhydroxamic Acid of Deoxyribonucleic Acid Synthesis in Ehrlich Ascites Tumor Cells, Cancer Res. 30: 24-29, 1970. Tills investigation was supported by the following: Trust Fund LOU, Department of Biochemistry; S.C. Community Pesticide Study (FDA contract no. 5 SOI RR 5420-09); S tate Appropri ated Research Funds (College of Graduate Studies); and N11I Research Giant GM-1395S. Ernest M. Walker. Jr., Pli.D., Richard H. Gadsden, Ph.D., Loretta M. Atkins, B.S., and Glen R. Gale, Ph.D., Department o f Biochemistry, Department o f Pharmacology, and Veterans Administration Hospital, Medical University o f South Carolina, Charleston, S .C 29401. 1972 M.P.I., Inc. DOVi/ 375785 Reprinted from INDUSTRIAL MEDICINE AN D SURGERY VoJ. 41, No. 1 --Ft. Lauderdale, Fla. 33304 Printed in U.S.A. 5782 0002486 7 S (o a . 5783 I m a j ... .. Ajt.ia.I...,,i.. . , , ....- -~n n r" -.^t.'.ilaw.h .x-n1*. f -2 3 - .` 'a u rc s s y c h ia e n c Fm ditiqx in P .B .5 . Expo*d P.at*r.ts by J iic iin iy J c r o s s , M.D., U n iv e rs ity o f M ichigan tte c ie a l C e n te r. Ann A rbor, Ml; R obert K. N ixon, M .o ., K tnry Ford H c s p ita l, D e tr o it, Mt; M arvin 0 . A nderson. M.O., Henry Ford H o s p ita l, D e tr o it, Ml. F o rty -fo u r Michigan fa r aenbers underwent ex ten siv e c l i n i cal in v e sc ie jtic n for possible health e ffe c ts secondary to s ig n if ic a n t P .3 .a . ex p o su re. In d u c e d in the study were p sy ch iatric-p sy ch o lo n ic e v alu atio n s and neurologic te s tin g ir.cludir.e standard electrom yographic and nerve conduction d e te n tin a tio n s . Complaints c f d ep ressio n , m enorv-concentratio n d e fe c ts and p a re s th e s ia s were fre q u e n t, r e s u lts in d i c ate the presence o f a re a c tiv e ra th e r than an o rganic depression m th ese p a eier.ts. h 'eu ro lcg ic a liy , a s i g n i f i cant incidence o f p erip h eral n europathies, c h ie fly sensory, v e re found by nerve conduction s tu d ie s . There was no c o rre la tio n between these fin d in g s and tis s u e le v e ls of P .3 .3 . I t is concluded th a t there are neurologic changes w hich nay be due t o t i s s u e t o x i c i t y by P .B .B . The d e p re s s io n encountered in th e se p a tie n ts was n o t o f an endogenous organic c h a ra c te r. -2 4 - SEUF9PATHCLDGY CF "SPINNING SYNDROMS" INDUCED BY PRENATAL : ntc: ; : cat: on with a ?cb in mice by s . m. Chou, md. ,p h D ., t Mi i k e . MO. W.M. P ayne, MO., and G .J . O a v is, PhD., West Va Cniv Med C e n te r, M organtown, w .v a .. A s trik in g n o to r d istu rb a n c e, "spinning syndromer developed w ith a high frequency in th e weaning n ic e whose doss re c eiv ed o ral 3 ,4 ,1 ' ,4 'tetrachlorobiphenyl(4-C 3) during the g e sta tio n . The syndrome i s p erm anent and i s c h a r a c te r is e d by s w if t c i r c l i n g ovestent (SO to 150 t u m s / n i n ) , r e s t l e s s n e s s and h y p e rk in e sia . The sp in n e rs (24) o f v a rie d ages alo n g w ith age-rtaechsd c o n tro ls were su b je c te d fo r h is to p a th o lo g ic , h is to f lu o r e s c e n t and e le c tr o n m icroscopic s tu d ie s . The s e a t r e lia b le h isto p a th o lo g ic marker fo r the sp in n ers is the p re se n c e o f c y l i n d r i c a l CIS p en in su las(C C ?s) in th e s p in a l n erv e and c r a n i a l n e rv e r o o t s . The CC?s c o n s is t o f e i t h e r CMS-type m y e lin a te d f i b e r s o r a s t r o g l i a l b u n d le s and a re en clo sed ir. e basem ent membrane. A d is tu rb a n c e in sy n ap to o e n esis by 4-C3 i s thought a ttr ib u ta b le to th e developm ent o f th e CC?s. In te rfe rre n e e w ith synaptooenesis nay have also occured in the dopannergie system. This lik elih o o d is suggested by e le c tro n m icroscopic and h isto flu o re sc e n t catecholam ine s tu d ie s on the nucleus accumbens and th e n ig ro s t r i a t a l pathway and a lso by th e responses on a d m in is tra tio n of dopam inergic a g en ts, dopaminergic re c ep to r stim u lan ts and b lo c k e rs. -2 5 LCSG-TZSM TOXICOLOGIC STUDIES OF 3,7,3-TETRACHLQRQDIBENZO-p-OIOXIN (TCDD) IN LABORATORY ANIMALS by R .J . JCociba, D .V .M .,P h .D ., D.C. K eyes, B .S ., J .E . B ey e r, B .S ., R.M. C arrc o n , B .S . and P . J . C eh rin g , D.V.M., P h .D ., Dow C hem ical USA, M idland, M ichigan. R ats w ere g iv e n d i e t s su p p ly in g 0 .1 , 0 .9 1 , and 0 .0 0 1 Ug TCDD/kg/uay, e q u iv a le n t to 2200, 210 and 22 ppc o f TCDD f o r 2 years. Ing estio n of 0.1 ug/kg/day caused m u ltip le in d i cations of to x ic ity , including increased m o rta lity , de creased w eight gain. Increased e x cretio n of propbyrins and morpnologic changes in m u ltip le organ system s, e sp e c ia lly the liv e r . This high dose caused on in creased incidence of h v p aco cellu lar carcinom as and squamous c e l l carcinom as of the lung, hard p a ia te /n a s a l tu rb in a te s o r tongue, whereas a decreased in cid en ce was noted in the p itu ita r y , u te ru s , aamaary g land, pancreas sod a d re n a l. R ats given 0.01 ug/ kg/day had a le s s e r degree of to x ic ity , lim ited p rim arily to in c re a s e d u r in a r y e x c r e t io n o f p o rp h y rin s p lu s some liv e r and lung le s io n s . In g e stio n of 0.001 ug/kg/day caused none of the adverse e f fe c ts seen w ith hig h er dose l e v e l s . Thus, c o n tin u o u s d o ses o f TCDD s u f f i c i e n t to c au se s e v e re t o x i c i t y in -r e u s e d th e In c id e n c e o f seme ty p e s o f tu m o rs, w h ile d e c r e a s in g o c h e r ty p e s . No in c r e a s e in tum ors o c c u rre d in r a t s g iv en s u f t i e i e a c TCDD to induce s lig h t or no to x ic ity . ' * A Au. DOW37317Z -2 6 - LC::G TEAM EFFECTS OF TOCO MO HCDO IN MICE ANO PATS by ?. A. Holr.es, j . H. Rust, W. R. Richter ana A. M. Shefner. I IT Research In stitu te ana University of Chicago, Chicago. Tetrachlorodibenzo-p-dioxin (TCEO) and a mixture of isomers of hexachloroaibenzo-o-oicxin (HCSO) were given by gavane in corn-oil and acetone to r.ice ana ra ts ar.e skinpainted in acetone on mice fo r 2 years. HOOD in femaie ra ts slig h tly increased hepatic tumors but nerp lastic nodules were increased strik in g ly . HCED treated ra ts of botn sexes had a marxea m ultifocal pulmonary a d erc ratcsis. In male mice. HC3D and TCC3 by gavage increased hepatic tumors. In female mice both TCE3 and HCEE markedly increas ed e p ith e lia l tumors. In mice whose backs were painted with a solution of 7CC3 or HC33. skin tumors increased in numoers and in aggression. EM3A pretreatm ent had no Influence in e itn e r case. This study suggests th a t TCEO and HCD3 are weak carcinogens civei o ra lly ana are complete carcinogens when applied to th e ' skin. These studies were carried out under Contract NCI-CP- 12338 with the National Cancer In s titu te and sub-contract 74-22-106002 with Tracor J itc o , Inc. (Contract N01-C?43350 with NCI). -27- STUDIES WITH CHLORINATED DIBENZO-P-DIOXLNS. POLYGROMINATED BIPHENYLS AND POLYCHLORINATED BIPHENYLS IN A TWO-STAGE SYSTEM OF MOUSE SKIN TUMORIGENESIS: POTENT ANTI-CARCINOGENIC EFFECTS." David L. ie rrv .P h .D .. Thomas ). Slags. Ph.D;, Oak Ridge N at'l L ab.. Oak R:cae. T N .. JohnDiGiovanni. Ph.D., and Mont R. Juchau. Ph.D.. L'rjv. of Washington. Seattle. Wa. Topical pretreatment with 2 .3 .7 .3-teaachlorodibeszo-p-dioxin (TCDD) or Aroelor 1254 (PCS1 inhibited the skin tumor-imeating ability of the polycyclic aromatic hydrocarbons (?AH1 7 .12-dimethylbensfalantbracene (DMBA1 and benzo(a)pyrcne ( SaPl in female CD-I mice. TCDD inhibited skin tumor-lnicanon by DM5A in a time dependent relationship. TCDD and Aroelor 1254 were very weak skin tumor initiator] at doses of 2 ug and 100 ug respectively, topically followed by 32 weeks of promotion by 12-0-tesradecanovlphorboI-13-acetate (TPa). The potency of the anti-carcinogenic effects resulting from pretreatment with TCDD and PC3 correlate with the ability to induce epidermal monooxygenase enzymes that convert the PAH into reactive electrophilic intermediates and with the marked reduction in amounts of DMBA bound covaier.tlv in vivo to epidermal DNA and RNA but not protein. ' Rescarcn scunsorcu joinrlv by NCI Grant CA20Q76 and Div.of Biol, a Environ. R e s.. u.S.DOE under contract W-7405-ecg-2b witn the U.C.C. -28- REPRODUCTIVE EFFECTS OF HAIOGENATED AROMATIC HVOROCAR- SONS ON NONHUMAN PRIMATES by J.R . A llen, O.A, B a rso tti, l.K . lambrecht and J.P . Van M iller, Univ. of Wisconsin, Madison, UI. ^ Polychlorinated and nolvbrominated biphenyl mixtures and '2.3,7,8-tetrachlorodibento-o-dioxin have been fed a t low levels to female rhesus monkeys over an extended Period. Their general e ffe c ts on reproduction are n u ite sim ilar with modifications in serum e strad io l and progesterone being associated with changes in the menstrual cycle. D ifficu lties with conception, early abortions ana b irth of undersized infants were observed. Slow growth, tnymic involution, and increased su s c e p tib ility *.o infection were present in the postnatal Period. Altered learning and benavioral patterns of the Infants were also noted. nr I I I ...... . 0002357 ^___ 5785 iviutagenic Effects of TCDD on Bacterial System s . By S . Hussain, L. Ehrenfcerg, G. Lofrolh and T. Gct'vall, Radiobiology Department, Wallenberg Laboratory, University o( Stockholm, Sweden I TCDD, which occurs as a contaminant in the herbi-ide 2,4,5-T and possibly in other compounds derived from 2,4,5-trichlorcphcnol, is among the most toxic substances known. 2,4.5-T is widely used ns a defoliant. The authors of this report claim that TCDD c s/ is rnut2 genic. Their experiments indicate that the muugcnic effect of TCDD is caused by intercalation with DMA. '-t vn 2 ,3 ,7 , 8-tetrachlorodibcnzo-p-dioxia A^ Vi*4I ^S i 1. Reversion to streptomycin inde 2. Reversion to histidine prototrophy (TCDD) occurs as an impurity in 2.4.5- pendency in EschericiJc colt Sd-4. A in Salmonella typhim urium strains de trichlorophcnoxyacctic acid (2,4,5-T) test procedure which lias been described ficient in uv excision repair. A treat and possibly also in other compounds previously was followed for the most ment procedure similar to that used for derived from 2,4.5-trichlorophcnoi (I). part (9, 10). The required amount of E. coli was followed except mat cniy TC D D was early implicated as the cause TCDD (11), dissolved in 0.2 ml dime 0.1 ml DM SO was added to 0.9 ml of o f chloraene in workers subjected to thyl sulphoxide (DMSO), was added to the bacterial suspension. For the deter occupational exposure in 2.4.5-Y plants the bacteria in lag phase susoended in mination of survival, the treated bac (2, 3). TCDD ccntaminatio.i as high as 0.S ml of a 0.1-.M sc-dittm phosphate teria were plated on nu'.rien' agar. The 30 ppm in 2,4.5-T has been reported and potassium phosphate buffer o f pH minimal medium described by Hart (4), but 2,4,5-trichlorophenoi that is 7.0 containing 4.0 g;l sodium chloride man (12) and supplement according to currently produced is reported to con and 0.2 g/1 magnesium sulphate. The Ames (13) was used for the determina tain less th-n 3 ppm (1). In addition, treatment was carried out at 57C for tion of mutants. The mutant coicnies TCDD is formed when vegetation one hour and was stepped by diluting were counted_after four days. treated w'ith 2,4,5-T is burned (5). ten times with buffer solution at 4C . . i) S. typ h im u riu m strain T A 1520. TCDD seems to have an exception The results are summarized in Table This strain has a base substitution in ally high toxicity. An LDeo (a dose leth 1 and show that there is a high muta one codon for the first enryme o f the al to SO % o f tha population) o f 0.6 tion frequency at a TCDD concentra histidine biosynthesis. A one hour treat /ig /k g has been reported for female tion of about 2 /ig/mi and at a survival ment with TCDD concentration ci 1 guinea pigs (6). A single oral dose of which excludes a prc.srcu-'iai sclectio.; and 10 u g /m l resulted in a yrvival o f 10 /rg/kg in rabbits is lethal and 1 uSJ o f spontaneously occurring mutants. JO and less than 1 per cent. resp. N o \ kg causes serious liver damage and Parallel tests with acridine jotar^tj mutations above the spontaneous level chloraene (I), whereas rats seem to be showed that the mutation frequency were found in this ranee. less sensitive in view of the fact that could be raised to 50 per 1C4 at a con- ii) $. typ h im u riu m strain T A 1532. ten consecutive oral doses o f 8 ng;kg each, given daily during pregnancy, caused death only in one out o f eight treated animais (7). In the same study on rats (7), foetal toxicity was observed at a dose level of ten consecutive doses of 0.125 ftg/kg each. y Studies or! genetic effects of T C D D have not been published except for a preliminary r.ntc on 'Aline date -from the present report t.s). The re........ for the initiation of the experiments des centration o f <3--70 y. nil and a sur- v This strain is assumed to nave a base vivai o f about 50 per cent. J * pair in excess o f the aminotransferaw- ll?N-- \IwTV* locus. The effects cf-ncridine mu.-tari O rfTCR-ITOl^uiisolved in both DM SO TCDD \ Mutation Number o t concentration Survival (;caucn:y muianu and water, were investigated parallel C/ml U x I0~' ' counted to the tests with TC D D dissolved in DMSO (Fig. 1.). N o increased ir.uta- 5*0 (100) 22! 7 tion frequency was opserved at concen- 0.5 100 3 6 tiations o f TCDD and acridine nu'turd I# ! 90 \ 90 ] A i s ; 34 11 ..2 5 6 3 10 v 46. which caused a survival in the range cl' .-O r 50-- 100 per cent. i.c. at concentration less than 2--3 //g.m l. At higher con ' S ro centrations and lower survival w c mu .a 4 3 (201 I tation frequency increased, reach.i'-: cribed here is that the three rir.i; -tin c ture of TCDD resembles that of acri dines and oilier compounds w his.lt' in duce mutations through intercalation Table 1. Fieveriisn to '.eptomyci.i indepenuency in . co.'/ JM treated with 1CDD. *) D u u liu to ampie* ci'irtn*) treatment. A 10s-- ID* per 10' surviving bacteria for i to TCDD around one per cent survival. Acridine mustard, dissolved both m fw 5786DMSO and water, gave sbphily lower 10 19 v 100 50 10 5 SURVIVAL. PER CENT 05 p r e n c i a number of mutants per I0 1 surviving cells, versus survival in S. typhununum TA 1532. treatment with TCDD in DMSO treatment with cridir.e mustard In DMSO treatment with . acridine mustard In water pttdtccehDcrdmsDtcT0oTpwplp<ssttwwi1wawthQehierenihaineutriiuio-r5llnnorMeCosohr)TaogMceuavaaT5uaaaairTccoelpsn3ae3dntptntatesnashssstqqeseDuphdhhrS0naistr.aehSathhstec.ceprunuerebetieuediteOmaogoDtnetaseeofaomOrantaenPegaasrbcodorfeedwfsdldgn.lpFsesdbteterl/iutdniendfTt,deemepsagnmhopTaaaltsoieqyhnTefceiashtdt-ewcrltAftgptepwldlpcfouACahtedodcierrCoieerlwtltfae,ihnpwhenoeofiisroeinaerdnnpDfaltgtrvDlidadarniteaaewtcntrtmhottubinThtTpneh1ooahgercrhgDumtfctnasgtrDeftiha5naeoe,ftCpChwtoeiteecenonooiDbid0eio3greeeelI2rn.oecduioablgrDbntD1c.u3isrtfneihsuMlio9icntgineana.um-amty.fnhmtnhhgat9acDtDc0r,tucfohtfdchtitwoieSdbro5tmoefo.ocesooXwurinoflthe5veceaxnooiwurO.uuernettnfatt(D5dflfeeoelhsch1adivdcerrnTrtmtcaifohic.viesmdthmiiersgretMotdis0e3ueartutabcaatefideolao.ouKire:cqecfoD5ctyhittcbcupuoornrXaeleStonlnrumtdrtwecnspa-o-ialatufhuttsMcniiT0ibsOtie1pineraahihf.oerdnsthtvii1tec.,-euisdt9sCtiheoo3ib1keii0ennDfsiSccraeesi3oeunnnro7weietfeedmo'tDoeloJOrrii)tannMhcu.melSnn0tuive.nnneamTshukdohenfdmiwDlspe.bsCbdre(n.tgcoi>fiSlsncniiCahue1rliterScldeiiasdlgtj'howvh(noaOnTcwocros4mcwihrhsDdu1lttsioPteedwudDot)wciihhebia5meiiihuohhictp.bmnmDniilnlllowthwiv)efefnMsdsuemcpaoftolhhesK.bfrntspi.lTruaittrnueeaarhoaTonoe/fioietttSte-eititarsmcrnndhonhhhfemhooount3uooanoArtaniseOseikoto0nygnidneeeeefeft9tsfrrf.,l,-/ aSlwtopcbTcodctftdcedpvttyihoiiothohhheifiofrroioogCiesbbrrfordenldllBmeuneaneodimocooeeDuxninescsnteeannannerrreniysiyusdDaliioouamzezpqlknnirlwlc.rbaosnronfdseoutanaiaieeneise--etnpidltefpttonxeppteayiaswee(gonhehocsw--tpd-PiddtmcraddlhnehnohCistfnhel(niiiTldkesrdosteoo1ocoiiBepyrdueunCgirh6xnxrupsopholmc.r'ah)iissswpODcseoufeoentncict)iyeutntnrlsrfAi.snooDuldcamiulito,zwuecunttanoriitteg,oolycs-oetelptxstlaApgadIafieydn,tistoiounioeonroochlroktUpfnsertctelamiomracthuhseayttxa.siihssatyiseongmnmicsepleaoesmsearushimwyiy(slrbedifnait(ai1olghopciim2(necobaiomo7h1frcun0pefbhetlrcer)onpc9iiiernis)oe.tploannsni.ht))hvottctltoumhogghtanloyeyiirohormOroedmaitoedofcemimasestnretphcptsvhdaiineidhieheenclolninafchxot(wrnsaaeuarrlgtovIcartyonngttphtetSeireimbievehoomtonnayaaass)idhdaaeeaffll. Table 2 TCDD concentration pg/ml Bacteria surviving treatment, N o ./m l x 10- 0 (without DMSO) 0 (with DMSO) C5 1 IJ 25 4.4 4.2 4.1 2J> 2o2 1.7 Plaques N o ./m ! x I0- 7J . IJ9 5.4 1.8 2.6 1.6 (Hill ). Kimnua and K . II S-Su1/'. D erm alo- lofieis 11?. pp. J 4, -- S-t', <|-/?7|. It. Iliu.-r. K . II. S.he'.t a n j U. Spicsel- berg. A rel.ie li.r (it-* i V - i i * m ul G e*trl\m i c e re la. pp. 5)v--??? (tSS!). K. Dtar.c Courtney, O W. G.i>!:r. M. D. Horan. II. L. fa ir. K. It. II-.!-.- and I. Mitchell. Science li.J. pp. :M-- it-6 ( tv701. N. P. Ouu-lloi, C . Saint-Ruf. P. U.^ol and M. Mur.gane. Complet Rendus des s m e s d e l 'A e eJ em .e des Sciences Scries D. 273. pp. 7)3--711 iw :n . Sature 231. pp. 2 H --211 (1971). C. L. Sparschau. H. L. Dunn and V. K. Rom:. Food end Cosmetics Toxicology 9, 'p p . <95-- 112 (1971 >. S. Hussain. L. Ehrenberg. C . Lo.'roth and T. Gcjsall in Fen-n/snor (report by an et pert committee c i the 5>ed.h National Poisons and Pesticides Dard. Stockholm, .1971). L 7 unazky and L. Ehrenberg. Mutation Research S, f-p. 224--ls (I94I. C. Ahnstrm. L. Ehrenberg. S. Hussain and A. T. Natarajan. Mutation Research 10. pp. 247--2? J 1147)). A sample of TCDD c i 'ri per cent purity obtained from FDA. v.'ashi.-.aten D. C. P. E. Har.man tn Gcnette Studies uith Bacteria (Carnegie Institution c i Washing- ton. Washington. D. C - Publication 612. 19*67 pp. 35--6!. B. N. Atpa-s in Chemical M utrent. VoL c C 1 A Holbcndcr. Ed. (Pie.tum Press. New York. 1471) pp. 267--2s2- IL Markovich and R. Laiartet. Advances in Blolovcci and Medical Physics 6, pp. U3C 7S--94 (195S). V \cB. Hcinemnn in C hcnocn l .\fum em s Vol. L, A . Holber.der. Ed. tP:--num Press, New York. 1971) pp. 2:5--266. N. P. Buu-Hc-L Do-Phuoc Mien. G. Saint- Ruf and J. Servoin-Sideir.e, Comptes LPC Rendus de sances de rAcademie de Sances Sries D, 272, pp. 1447-- 1450 ' (1371). 17. G. R. Higginbotham. A. Huang. D. Fire- seoe. J . Verreli. J. Rees and A. D. IS.Cur.pbell, Sature 220, pp. 702--3 (I96). R W. Boader and H. 3. Bauer, Industrial Medicine end Surgery 20, pp. 2S6--2>0 (1321). 19. J. G. Vos. J . H. K oerun. H. L. ve.-i drr aas, M. C. ten Houver de Urat-w and JL H. de Vos. Food end Cosmetics Tosu- 20.&cdoKgyats8u. kpi p.(C62h5a--irm63a3n)(19F7u0k)u. oka Acta 21 Medico 60. pp. 4)3--553 (19691. .Tais investigation has keen supported by pants from the Swedish Atcmtc Research Council and the Hirrta-ll.-uius huun- duion. The authors are gratetul to Dr U. X Ames, University s( California. Berk- i y for supplying the S . r>ptnmurium ' o ain s. to Dr D. Tirevtone. Fo>;d and Drug Administration, Washington. D. C. far supplying the TCDD sample, a n j to Dr II. 3. Creech. Institute uf Cancer Research, Philadelphia for supplying the ICR-170. Received November 1, 1971. 0002480 Tab'2. Prophaijo activation*) in . co/> K -3 (/*) treated th 1COD. p H qh *) Ihe term "prepharys induction" is pDr-f O i poiely avoided here. DO on 00 co 5738  ''ll 1*1I: Vnjmr !>Sri l HI. 'unt-ilN if in*tivly. A't'ir <1 n f tilTrvrnt lr.7i ()(/<tryng l . r .lr. <-t nl: nnd fHlliliviilmi'tiilo. :o. r r 0.1 : M rcha- IMisint' *( Oli"" H ea lth l.V.1 1.1- h*m H A . c t nl: o( Inlmralnry rmm'iil 17:72l>- nlinn ln\i>itirn armaeol ctTrct.i o f nerni-l Ip m iio Up o n la Inoculala. C '.inn V I.: jlii- inai.tf nil- iiy iiliim i- form uli U-livilo oi^pniiisin. Gig i(. miiiiiolic T h cr 127:25- c t nl: AflrpnAreh Intern led nroloW p!CJ- I74:U-15>. ,led nrrlnlflph 21:354-337, T W . c t nl: Sensitive new :inni (inn nml Anal Chem enninp -nsidrnj'one lest in air. Anal s proditelion -litflips iisinf: ..338, 15)70. od of delcr>kc: A p p lin i'lieti/.ril nml ler It: Nnsnl pxpprimrnlfi: . moke nip i. 1371. Toxicity of Chlorinated Hydrocarbons and Related Compounds A Uevinv Including Chlorinated Dilii-nzodioxins :ind Chlorinated Dilien/.oruians Ileiiu .e />. h'imlirm igli. M l ) , C h m n h lrr, (la Various chlorinated technical compounds, Chloracnc namely, 2,4.5-trichlorophcnol, 2,4,5-trichlorophcn- oxyocctic acid (2,4,5-T), and European chlori nated biphenyls (Phcnoclor DP6 and Clophcn A60). have been lound to be contaminated with trace amounts of chlorinated dibenzoturans or chlorinated dibenzodioxins. Toxic tat which pro duces hydropericardium in chickens also contains chlorinated dibenzodioxins. These and other tech nical chlorinated compounds such as the tech nical pentachlorophcnol have been im plicated in causing chloracnc, liver disease, teratogenicity, x-discasc in cattle, and chick edema. The litera ture on the toxicity o( the chlorinated technical compounds is reviewed. It is mentioned that 2,4.5-T and the chlorinated biphenyls also induce porphyria. Whether the various disease entities arc caused by the contaminants, combinations o l the chemical and its contaminants or by the chemicals themselves needs further evaluation. tanvpaatzcnR(cdttromeohh2reuhnonyaoiafnesle,learslSfudlt'4elastoiue.slxniohopa(t.vreioddri5idsmeTnsieua2adaonint-erscccn,sefnur.nTeb.,a4soifriNdipsdeistoedcn,)ncoie5tsshF,irauidolTitudef.,seitidsao-scrh2acshpenLiTTetmmltnat,hahiccty.4eishYvtpsseobihicco,eleneot5laOsphnelihpeysaseoadl-aantloconcrtstarioiprrieshceicepfxfi(cimrrnecfnoadnchhesiibaioevectrishandueielieedm.rieacinwtImaneveodceurlaoAfajtndineanoidlxcocrialntodltctoaamedihrnttuliacldrupdedsynldpiaflotishynictcscbu.boseaymbchtehhoireeduginyhnpxlttlloapnutnrhohooaodhopsneoozrdeverrxereeeaonuiissixteeyrsnnnerinthdeentsanihanyaewabieimdcdctonafotnvltaieheseeeocsevxrersinedaldtacdrieinlindomapvolnu-c(tvtrccoebnsdsaPhoocfaaoel.soiilmcoeyCfrpubrdasooiTeruaidilscheerB.eambinnoehdiinaeneottldsd)dniyies-reft S iihm iltcfl for pnblim lion Oct 8, li>71; nrcejitrtl M n trh 3. 1372. From the Chnnthlec Toximlof-v Ijibom tory, F.nvironinenliil Protection Ai:enry, Clinnihlee. (In. Iteprint request* to Chnnthlro Tnxirolocv lolm m lory, Knvironmenlnl Protection Apenry, 4770 llnford Hwy, Chnmblcc, Qn fJtXMl (D r. KiitibrntiRli). of Tchoims eddisoenaesse iws idthescorirlxw'ditbhyouthte cfyosrmts atainodn wprsMwfcorSnefApfwfllslmGisnavlwAweecieeuooHiboyyRoetufaiatlenlirnhoasdalhlvalraspaeeruarotsushivttcCToaodeteousmhchmsotsteetilcrepttrnorel.fharlbucneauesetncmhtagrdhsotctorfsantihisiwenndelhtehlsldoalhotwsldW)nrfuaheesearoariooi'daeapeflmosonihneteswttksonct,labracfsrcsf,pasetowwhfarih.pbagadyuxfafytfiteibotehksmopoaubfteivsaepicaoaoeetce5peehironcerlffnTchsarcrlruncedrhtrsrntsncosakhodhra,tuyewaehrentide.ntc1atotehcecyesrltlss5lalnl1bn,ooeldailnine..uedaopsHaebmfon,vinttlduuei9lnetoysdysnpmiriioe(eiwaleOtEtaan.xsmssntadkearDaemlsfaceerleccrlftisoi,acayenei.nstctnorrweHtotxasoeauC.nrepidsonnc,eaumamhc1agdawdtsolipmtodeaT.ttiansencpao9ldnrnleaoosnboTisrdsacaetass,s1ehCtdee2kcedesei`miryaisxteciientc`8h"Cnynl,uso6aienpepohpuhylcuaeoc1etidewidssrl-ofrrgnfyooehterleosrknHsflaas1y5,da.k.eoeoorefoE,lea1ellreranser9ydone00elseidlylfrAetnlselanotocwnliwud4tiaelBacrdlrptuhsrtoe,rracuyw9imotbrarahcdaxFsusinuhcarejwmaoak-cowMtttrrndcrtae1hoseoTtilltseshsiauiyrbdp9liicei.iocuhnefd2aefultoa7fnttan)ceircoihriedosmsaneii8ontaym.sacnss!gtllwcnntmhnh,Jaocisier0hdstueeltikhnroaipcitseleoiosopfaakmslcynisnheylp'lVhrcf.nler,lawcosmfsprpjlyoarleolodohfxaaFieexattanJteifaao'heal1rnoodfewced>tlrsiomlanpmdmCaajnnrtccrsytedterpur>,ppfureaninidoc.ei*okdhihsterBtan'oramecenpaactUnys'eeceJie1clvrtiNanunteiooOpuocGra,ssemmqrTh9hatrnefncicdioldfos1.re1errri(octuf5loalleaarhhoilne"8avieaiAoxeoote9gaalet0pnelrantn0i.lHamennebetnis-rst2prrnshnloetrnl?hdaihkaiddyndiyiyotyeddye7l.ottns)nnetedxsst-sytoe"do,.---r 7 ' Arrh Environ Health-- Val 25, Aug 1972 7 7. Z Z L Z I A O Q i i 735 7 8 8 0 0 2 ; } POW 37 3323 r.!i; t o x i c i t y n r iiY i> i:n c A iiii(h \'S i<i m n n n tir .it n'cpnvntimol1Ihsoiseonlfo.li2eemlisdetpnn.snKi4tappricaii.i.aKios5vianhfinhl.nhf-ueTe)stemll,nrmemliccni'iddITtlaeatniemIircnietieynntihaeci.iahf3sitlisohel1r)h,er;d.wIaedIltotiolmI'irh(krituunaTloheewr'iasbpibiealhtnlyberrcteloo2aesIvinwndtkck,bezIr4cuisdnbaiccn,dlucb5ilnaeetrib-pehnlrsittlir,,nefeyrtoaTi.jradwprreiatmerhrenihehnri.nciareslerildpalknoriIinytf*eirfesennlsorntcrnopIcwnIeamlJhrhIihoineowthotel..ihsoeinnned:gaietlirogxseehlbarrynlpdaecime;emlnlrndddvio.rytcieinfiialnyddsls--.,- THftK.MtwndihwiwIioytfl Chemical structure of a chlorinated dibenzodioxin and a chlorinated dibenzofuran. pp2dtruteah.hhrc4rboeccti.slabn5nycl-ioowastlliuih.TsncswiaacHohrthtdh.ifiovciewhdIeh1taem,adw2vpwga,era4renaorse1n,s.uds'tuifuus-tcsat.eclocectcelednthrudcanhirclnieuydhcdpulatooeherlfrdireonk22lgpe>,t,44hrrcaa,o,an55ltdtzko--ucllasrrbnciiliycscictnihh-opelIownoorfhorerooordyte-f-h e cichallos.rnHaiec ciintews osderekvseecrrrsaillicexrsejxfewtrheeendcteooscctwhuhersriceehncchienedmoif cewtifaoThtneeil.cetthtoharte ddtoeirrxemiccathtiectpiosanttiisatcitstlircwesrituehsltusltthfreoofmcthhaeebmslooiccrapall, ..pifaeMncbcotnnrlxhhchfanaaocjoCnlelktdtploooenreeeohrsrredtMyudsanplleieoelmcccoesrdexirnnbtse,tranip.cceaipcocepe,tcTetrnngeihorohnnschridhecdltmeoelanious1hyncisryepfnc5eeoaihlne1tsnssdMl0gsctei.oaiat,ltmrp.rbiamelacwvohlsiepPjnByegnescateldllosliaonsncyrworpyywhm.slnchicelroeatoitEicedlshldgnhxtijstx1dhxaga1ai*eAjdwbjel4hltncr,oepelBrallhianxnsorucsms>mloun,edchpaordds1lenperh7coc,udoildear1encprdt3raceo(hotiHnwhadacbncbpldaioacnosoanPcscehforrihemCmtrkknlolhlsvopseobtBoaee.reoero1risindn4s)-df-tf - naatacCdtnbctphcnpzWaaihllhaoauoeoiocypohssmceKlmucnnndecsdotponolaosssoeizttiriplrleoaaitnemionfredoroobrlexciaoaanlcydofbnlmdlhyibcuhpneGeeiidnssb0slnbvntcyn.tieuoc3eise.gnceaidtprtst30crtrttrnoovrntrmeho0aeaazanxetTfwdrptna5nccohrandhkciithoehht%ocaaddnhahdyedcmlcrsenitwelripo,oi.SrntobamoerepxpifcorptooetotcsrhroeioouAlhpnhlfbrnakpihauccsdorusuzsbhennxheni0iideoltr(srscblio.ztnahhdirF0ufxacpnt3ivcrcieui.itacimp"tlcncoh5peoeswgmlteharnli%edslnhszuoedshtoly,tnitoharoaapdosropcepffhietocmrlcutnloeourna);hoahybnoscmrtmfrspaerxlpcetkmeaaootmtrhatiwpnwdednieemtbohcrcoczorrsrodeboehaswekuaceiu(blpeuhr,ailcellntFlrntkeyocvvhoccflaraciTdvooexreeecnacrsgb.ofesrcnaaslradiatnsubsy.csaorpllcDcrod,tskrlihs.tehtTeltowhsoieaoitnlccbI)esitwohectlfotdhnhntccrinrrmaeiannaaeiciehaoonnoridynans-etesy,.-l-t em1i0gn1ha4tp, hpmtehnantllaeccnheal.odruolntsaphhtyhnlaepnpel,yainngd hHcxnalocwhlnox- eborerscmowmoiretehhcichghlholloryirnateocnxacitcoamwlshso.e3n0dStehovemeyleocpoofnettyaheieniwrthroirrtenke-- eprhsBenaHouilen. rtInehtasnonPmdleerdoejf>ttoehrcethcsndeicwcahollrok2rea,r4cs,n5nc-elriunicrohwmIoourroks--cnfbmoulocueltapnnrphld,rwaoaredtloshaucokacontenoejchspucsynuu,pcrrjeelriervskdy2ie.tc,i4rhsaK,o,5tpoi-mnlnsrlinmihscdohiwglIoohglreniivoncnepndrahlpccSpnihncnlaivhlenoudglldvezties3od0-, pcnslpdniuoreoeuooiruainetdndrr,toiutdfanshrmcrgobeidoumnpyasutgao,csttduynoiiBtlsntrthaotedrreaxacmCti|utPcsrincyciscothy,omywa'cln*,opbh,t3rctosai0oanholcmocltrtongoopsniwcrl,tceaioircacttpanhhnlanto.cenecrtrphesaaohectltteysihoocnorehintaarsebslm.oct3hooc-1iapuhcAnrratoaoodclls----,, Arch Environ Health-- Vo! 25, Aug 1972 0002 X-disCMSC linphlhulcni*!;, bill also with petroleum prod Thin disease in raillc was described by adcsdtmschl(oichnohleiyoimaovondulapnocplwccise,iursiehndrsicolodnk.eianfsiawceea7(cte]erpi"'reahMnaxainordsnatlhaitiioolrvrsnmelgrsaptoieahniellunisa,eanalbi!iwlppnfaMupolohrihigtiioobKfsiu7lntobhlmuhc.iiynntellcaAhiuadcAallx(erreell-liu,onidpasdemdfsfeil,heesknaesouewngmsinaacwenomedusocxasnicyrastcrtnfeuorkeaeognriipasesslllauhesdlsredstoms.ivo(deiyvw.owevbsAeelnlsaeasra.tmtlll.ohatoaicoeuveMcpahShfrsart,iryreilgiloamiiaceidrhnpnrnvnirvielpaiansoieiiyddc---ll,r cbcpcttdattLiuhherhlfeaoioalbeetngnlnledsouksrceridneTMrlsrnoeieweeenfdsinpraoniaf.onhstoeftgthm,hdredfefCoedwisetbhbnychttairieisgrseloorsi,xtelrunsinaaharciaamnescdlonpsndhounshddowaslficn^ioslltoedhsirafcrnobqaoetpedoIfisunereocretatoadtgnthnhrmehplvceeeixehsnefcoo,e.pldeow-uhflrwr,.rni,sa1a'iaavtnitltlteatimeelhcIhoridnHien,nneopeaiductflnssaceeaabmhgpotltnmehelfollosdiraeleanhdeypsigdcineiod,ggaeitaruinianhtalnnnhglatotleldgoyegesf svAwhctwlmnptcoiwheahchavaiaelgDxledtpielsmlohnsinarsehlrnar.eicypoioivalDnhhloscrdwnaleskeolcafeea.rnoesenelgprdrrsoacraprtWeheoibnnratointnipetsadtcllnhehnsverfon,eroeer'fpikbdaovnldaldhiwcputifedtuiuntetlcdavlnSyctmnhcnios.heeaecirt-teygalddhcwooawchsoehvvfnohdrgcafnaiiacrleettetndncokhttacgnlsogudrecmte,ooeseeroknesrtnhrdsileeh7asenaaywrtterlpcpiaafiwyAooeohohteatifrnihnolurllnvehpo3pebne1oaltronlsdoacfalifeboenllsffsrersenmrtaittvasehhamdhetheciraeee,oadvheoadhnteplftlmloeeisaadiaottvvxrnnbihhtonoileeaiidieendfc-.lr- ucts such ns crank rase nil, 'Jim toxic prod ucts were excreted in the milk and produced x-disense in the (-lives drinking the milk. 'Jliey cited other authors who were able to produce the disease with a complex wood ppgclttUehhohrrileaoaIeulroslluntneeercphdnlrodhlivesn-'gi1lats3coahothprietodvoiherpofientsdrrhntcoaienpahdntndlhihludclooetenchtrtadeehinnp,elclauxcrnhotbn-anelddhrcobddiiucricslcixealinIneiatanraaynicstcdtp.ch(ehh.holdloelfoallrriniodvaiennnliaesaaacredtUpifpiaoTieh-hssu,edlcetclshoitpa,anvwecllaeonuaehprnmnnLieholdedie--, hntnhenaappplLhhcntilhchchan,lllocccnmnaccunsaawepndahdslhshleaecvslpesetnrateeco,hxdnlioicnsrdeoUanosiaadepn.ahchOthhclcaxotlaracoccnhnhclnl.ooprrhoo--- Chick Edema snscdeadctibwctattlonehueianasuaiheliohesodrce3rrIrAoercmiBmlnmkdendcinuautitlhnkeilbi3ucbdelgeuolu0ydey1eeusesciehd.fminrpi9e3nsrssdsnsddt75oescthcetocesto7Tadrapaiooxeofhvxit,fnnnloxavhnybpieiaifiddcrnine,ceeemcolmsrc-okr1drdehir1GdewafaaipaLfpi5efadefspinicelaears0avnactdbirkdaereteftlicftoeeweesddsietahaeclmlcfecwneaissewtiaaoashsitsafnseyssaaattmseue3esestisp.dwa*rnrerwtn,)seefsiarhedkdaatrhgoedhptdWesescaensiiydiueir.uidohvqpocnocndrulchcienhucpfeaedc(rtcenec,ces"ioalpktueadnohlwuinTocrpieedandctnidrethonocdniectutldrenthiytresxkdinhetcmhccnt.ekf,dhhiesdkhieacDibneiyrhSeclghricosifluccpdynunoiry.tfeffmokoteidseanccmddunacwgreIttrkhcpreetm"eodtdlooteissrdtwaslocrphiosopnaentttaktiwt.eahianpiertmshctcnoergrneirhhaoeaaiunndnieaendase-,yftl ctdtdtthhohourseeawoIcittpseskl.,eichiadnxlaiding-vnitdehenhyilrgesbyseevcamacihsnctaheaadonlnmnooigtfcfrhieeinlenussthtaAdsaeteiktendipisdonlkansfissnsernhmova,oomepafwtrhoaltetgelhthdhveeaeteshlhspldey,eeeinpsircseeeriwsaeeorsskpni.epteoeh.rrrIooaItnasft is possible that Uic vitamin A deficiency represents a manifestation of concomitant plirvoSedirkudecisesdeeat siaenl.3c5 aftotulendwtihtha t hxi-gdhilsyeacsch lcooruinlda tbe de oscuoncspjuafheclrKicboiccnc*otrcuthkreourmreeisrmtannieaautsdtnsvilo(oeaawogai)tosnfen,itcuntdohufhasi)el,fnsaatiepiud%rdswarreektreeoimvdeecrnfedyceadrvsuta,sho.ocfnadeteleWlhdodedaejicinhnlbrehtieieoidumctlnaxh.memsiaemceGnldadsatoul.faonbnacts|Fuotdmktt,ormteiaitcycaepiilansslsaols-issuspguea(sleleMrricnocuebenliaimlarandess-, of necrosis nnd degeneration were observed in the liver; and the bile duct epithelium winaesd fuonudnedr ttohebeealefcfetrcotendmwihcreonsciot pwea. sDeixlnatma- --Ar ch Environ Health Vo! 25, Aufi 1972 DOV/373324 9 5791 2M 2375 Tnblo 1.-- Dlscnso Rcsultinfj From Expoiuro Chlorjtcno C ertain ptMtnl'Mim p ro d u cts ChliuotM iililhtilniPs (mainly prid a, Itrxa, ami hoptn) ChlurnhiphonyJs C lilfiro tltp h n iy lo iltlc s 2 4 5Cltlo'ophnm ils (l*cfmicaf . . -lrichlorophcnol, prnlaehlorophftnol) Technical 2.4,5-T Pathological rinding* Oirlh Oefects and of Iho (.Ivor Lothal Factor 2 4 5CMIor!natr*i| n a p h th a le n e s C ertain lcclmir.nl , , -T Chlorinated hlphonyls Technical 2.4,5*T ow l Plldflod m aterial from toxic fat* othor chlorophonols Polychlorinated biphenyls In C attle Highly chhirm alrd naphthalenes Petroleum products * ''Toxic fa t" is a term iisnd lor tat found in chicken feed th at induces chick ndem a. ovCco(cpifeFeclaocpiaudEFtIltdoclIfafv1ciiaoonhhfxaocndfahadolucnoeoolxnunlhl,meatanwilTtregac2ciuiaIteeicnntIreiecncdlcorlrccdessdpnthnhlr,tsmmrrotbtotcdhdekvotjk3k,teiCesceesoslroidaierraxdeiao2i,rodhecaao.atvocdwyfiwum7omonrnsechi,1eefnrd.sy.cidemsc3omc,s4btonttdrzeh9dtaet8aohTnpri"ukewhm,neo*yfnedTetos6neea7m,tnffassloechepi9hetnhrto7nmcaetTaa,uucrtoAahllpiaae8-erdeeBmphtre,4ne4otrtnitsagthhmeylala4e,i0rholnlohfcecalfadg-wetdosegcsilcleedeoheCptotlxshlesbn1wisex/hteidneeahenfe0hitrwtwiehatafnieiinogfanalatosana,ntaxocicttopholomasj2cwopcscxctttnhasiogplalirkoprfcahoereo/rnrh,hcxhactialeetcei3ancocematoltidcdeeoenornnolnioriedh1toh,tdcrodcrteresnnprnp,fecw7ilesmtdi2iwlafhcci:feirltlhtrycrocscto-ld5omeoalwLenifiehaao,iaKatapehoiralectprh6wandrrtdmdhsdiilldnenhnoaroclfkosiecn'cts4eceaieiconsidoluesdkabpddbpe7uonanlkin(aiabtlahia/ericnwipvcnewclh0ihcrdniottctlilruslkirlfeohnprnalesfyhheneh.yocnlp-uhdxhatdaeyem6eAsdpa,il.dpzducrcaanirpitueilsec3r,selcdt%etocnoocxh4-uotdsdero*so.ertsclpareed#4sk(hpazsme-dondycltifcihlruh2rmdpeoofsioesswersoicartitpomoiiifaseilsiun-odrrse-r.c.naihelcmnbxerxaxdehphdpoonnes,hldotdcddoslkid'clWtdtTii:o-oerpojunppdiga,nieieiun4ouddhexo4undtnlwdsr0zochdhehhdtc.x1cihurhcinnihdyassPntopdut1crcyseorteiintceiynmeaeiuntt-ynknnroee.nsrCibxcnecnh0edpdanonnoicbddmHzsugoeidaoeondsepB%-rednetfdeoeratersllsddiofainycghaaceoc.uldc-nvrneegpilafph)ptrstrhmtcet.ecotpvaselgwThaagtebiulttdwhtiof-iieimohlnxcehirrhcigisnatdiencieldcyraedtacneoolnnoaneroliskede*edoiek-.--yasn.eer.e.t--f TdfftaohoisherrahemltegeecAatahrtstrrseoi,ttcurcAaalrosnfbrlorisusncifinleedo(drsFraufsOnlsfucdtoBirardi)nfasol.caprTarfrvpclhouaaedicucyduuisscteuaoedmrrfcsocprhaprailuichsnmmkoydadpuerrsdstia,elreuyadamlnniaaacsdss., dsepbetapsoidafccAwcbhnyuomsneleronhroiiareesopcnrcprtrvnmlopssesrolohttpitew,,tdcchhliamUfrreaironltricosieeehnloioncniygsenstictnsnitr,ezbgtariieueciisamcgcletdm,sdaiiholnei1anoneareuanferdssc2onrenenstlledcnveh4o.dfuad4adsae,ot2ecsSn2niti4ldTnersohcni0oDd%tlpcnehsalehboiooiA,gaDtaraprg.endscnferte,lsrsresryoaiulsTtMseocyonyrami.tiisvahlnchmnalniawitoinenllniptTetslnosonnepdhlisn,cothdeddrrgpraeaaruuireeel6erniptnrpim,nilnyiegc0sta0bvrieyapcdh1svhmnasbi%eiarespedeett.caneairoshcahnniatarntTx.ttsiledrsenttrcrsosdtieihh.ynaca.1rhvmonS0seeimydA9wl0abnicbn,mkyoadl4ereo,ruhaaitDrntu4aoanengtioohtwni,nanuefrcesmteaxakolaiddelrhtpcvritcomeauei,eohch,ieelhnrsilntnciiivnlenewattfsoedbwe41yadrtdentirrhehuutudp22viaagiheneibinr6niooe%xobelnneneyga0e-def,rfs 7332 OQ5^ 5 CJ"t sthdpildthonuoiuirwsselxocxtcmideo-mucmgudlasiorltcsnaaaygeei.dtnnisdeeccDcoroaiosifialubronlrrlsyymeis(tchitaessiotestntiuiemajoobtenpAfnhicctpl.retuatoliVlhitorcecasenolsknaotinsotieoreinsoOnoa.atusnrheniTlsfondicttehsoorineemnhagtpijwohospespfnkeocal,tiritl3cincxiks4ohbkar,na,llti'looei,kne3onr0sawnw0ipTco:er"tihnnhtoor,7hceseef, aaTlcPlfnohittaraeeelnornogautgeuiftccbastnelhraierotnlaierDnkdtaguhnonegHfdteyhlerdipervsrmot.eooircsosoackNrnohbitfilnhosoghnmirssiizni,tcwuhaemtoaeTartidokn5xi2dnibbcveimioopctllahosyvueenesnrkdevayeenlyoasddsft. Arch Environ Health-- Vol 25, Aug 1972 5792 POW 373326 . * to Technical Compounds Tnblo 2.-- Toxic Contam inants Found in Somo Technical Compounds* Chick Criem* luxlr, fat C hlorinated IWphonyl* Mfiliim of prnlnchloro* nnpldhnlcnfl a n d ItcxAchloro* nAphthateno 2f4t5*irlchlorophcnol 2. 4, 5-T Toxic (At European chlorlnnlcd biphenyls (Phcnoclor DPG And Clophen A 60) 1* Listed In Tablo . Tetrnchlorndihnn/nlurnn ITctrnchlorodJIjcnrodloxfn fTetrachlorodlbcnzofuran ITctrachlorodihcnzodloxin 1121,.32,.73. t7r,l8c h.9l ohreoxdai hc hlorodlbenzoenzo-p'dioxln p-d lo sln 12,3,7,8 tctrachtorodlbcnzo-p-dloxln fTctrnchlorodibcnzofuran <P cntachlorodibcnzofuran 1H o x sch lo ro n sp h lh alen e rmmhbliilIennexeonaaaegailnnksnenlevcitseustdnatdGfi(aetnaKtindXpcoiwda)niatnrgunptiipmoctrcenhiiecoineaexHhpwbngnjllicrdtbeeoualpoidrsnlrirreoenoi4nadocprc0boeiWoefl0nbisson)oistbier.pfht.rsrashhmTaatnAeeetnihtannrnasholnytoatlicwehsiJdnllg,xaeiaclrbcpstroashehenarykeaaronoiionnsbsbnJeughwsicapegeoopihrhhnfvnioadenetnsnanppdhtetyrim.iiahTMlksnglldese--- TasctoaepSolwwthioernnnfrokthhoievexisdVdletletisxe,yhilhcicohrictdsChdeqaaisoiE.htaE,irlulyxsllarmuowCauolfckipnirpaacihrrohs-aolodthnioibatsiitnplhgecwdrpyrayaKeogenkupnecetarpweeroaeodhr.neor,AednefnlodAdfoGemirrduesudcraObtodchmunniwocetmi)ounsncrepmmoalma.epe6dathtl6asdipntTeapewgEhosidtndoosmchneaetuuhyfuucedsnerooblnndtmotsndwniaahhdtitpptrseoeuceeseeih(ecdidltdPleaohoFes(xaUhnAnhtybmocCinnhcymonehurradmBenllswloicliiisotaolaodcoteesmcrrocnilmsidnd.axoo6n,lxlc4orimmriiincgfevSrchscihcl1peeonitiahe,taD2rgrawndnyeict6ruhicindsePiinen0rkoeeatesstgdgh)6--flr,. dtAbhuerrncoee/ce.dolofrpucoroar1mnp2,hGpyo0r.puicnanA.dtslCalchhertlemhovrrieeocaeadlliebdaccnonmazltopycftosuriuasrncadohnsfl,otrhpoaedrnsoide- hfActitrhnocaoredrxromdinicEcnicluauothttmhrrihloveoi1perso2oUec6ponacn0franue,ipsrtpsmetrhahaedlramdehalaSltpaoiitluqcocaenctnulshaec.aobssnai.ruto6ssi6tntiafnaSeelosicllntytoocitneifnhn-taaathcmhtytheotdiihnxscriaiakoscmnspwtfepfareialcnides bo2tey7fcEhtpenmpcimhcenarolsicofan22l,,c342t,,,754a,-,8l5T,-T-tTcflworiaunacnshrdlaoctareoo.tndeAtirabapmctponiagznroeae-ntnpeid-tcldyeio,fwfxteiihtcnhet. wwmItnaaaimcsstehifsneotaausatltnneeudddddytwotohpibnatuheetbltt3iehssr0theareatpodtiepngcbmehynnoiCif2cc,oa3iunrl,7ar2ttt,wn8s,4e-ol,yc5wsl-cmtTartsacawhilnI,hc6os6iorcoonihft- clTAdTec03aemgMnswncafceptdtthnif,n.ehuoehhoevanpeb71fihldosxdroKlaanniecsefomd,et2boevst8ooihlcultletcrnac5cryniks6ia1-otrrasypgotzvfntp/macii0idlwfticoa)oiofdndhagoywlrnagietw,nfiidoleniabitrnoodtptnda/btdndivirhcek.hnodrisoulAowegeccnnayoe1glhfafrnstAxdclygdzgerT1li0qhi/iazrmiefioplsoerdl00ctuniidoiotyaesttcwgnu-e0cccaoi.au-rsftnpehptdthpophynohraladeE-atlpcptffo-nsdnsodoetbwhilrodhmdmhwcpmtieybiotrlapeasibriioaroemaldtnmiocmhlrueieyecsixlosdnnnok1dxcelvinrbseitifeaitle2rniesannatezoooesycsnvatadod0n5loAyoltfirbi.relt.4sanaouatta-ope0(losrhl./.6nAtiynnt11rfaopgroe5Mfnoce0eoctsrcropcreIeitgtp0ve0tapdollao6hgmirmpteieoddre.npuatctmogrpr5coen.rdemeemollacahxsomtpod6tdjidntoe1tuiThaicCngegroufnnat2gs2rrcbr/2g/comclt4aoeco,2hik1k1eay4^tleuait2efmb,2i5nbegsTgna,,3nlrod57et4edfSltas/ct,eicrnte,,adnmw72steccnocp-a8tddrnyuteh,acTphfdtda-oaas8bceoyybreliaorto2si-sret1ecsursn,cp1tTlyfe,s.a5ack4acfatgtdu2rrsaioctrrhsld1ea,th2eni5hgtironoon5caeo*v0gaaetn,aud4eem-t---fif.dsf0s-t*sc, Teratogenasis Comment BeRtheecsednatl,y,MBdio(nucntpicusblRisehseedarcdhataL)a,bnosractoitreyd, ixiIunndssomaorcinrsetsapnocnessi,bltehefosramtheecohcecmuircraelncceomo-f /'' A rch Environ Ilcnltli-- Vnl 25, A ug 1072 5793 0002377 1:10 T O X IC IT Y or IlYD U O C A JtnO N S-- KIM IlllO IJC Il t ebdepsHetayortaolcpiaTmlothoipltnisnttt1iionineiithnwnrnaelmrorrenrnpedrfoiireeoaaoh)eaglscyaoslgemerovsotn:mtiiee.inobtnwlueaihtluolbisdhecidenucoatcnlSzoatriirlltescjeeanideueerlt.kiucfonnhnothioedomeonho.yarsnrpesovo.de-concecrlafeossneiWiiumIhzcpdooefueuliccmenmoea.,httddsCm.otiisooluridgddhnlrHdsoerwqstepeiixeo,h-coiwmearaaiii,licctdeIsxecupcid-msloinrnchenomslndteaoisednaionpbnc-cesipstcer.fcocst.indnaenloihutymhhoobieiisobwnf,shfw.trniSzost(trsseeldauy'oyaSezoucot(a1lhapwopohsaaenonr2in2to'butaoosooxnromIune-faocrnsrroissmie0selor'o-ctrpmiicaiIdmadin>nlludosthkdnpectlisc/aneilro-htlspcsieiekhnipinuie-pcekldodedlaseglofsneadaidlnaltlircrasioiinmcirormanfrhurndvcllhssonio1toljcocnoue"p,toreoyi-e0eisetxtrtagcnpowhmmcltm'nsplito2hxpda/ftirfnviistlaiht<otlnterrdfnlhgiie)rpnki.onohno|efpatgai5vneeua>sae.nosteohedictfmrvvedovcties0lnnrsdhoeabwdiuieotbiTeeateercsaiInrnn/ndclfelasoindnn,rcmiiihoseiaryahhinUraidcssedbtglaere2dymoyssenmsctotoniaeetienannhpiol,/nrbotusmmhvidenibs.tsdkecikihnpti3ednraczytthnotldiiteerslmdwgohdicabstrcs,puhonehecd1antG>t<onehvok/viatiotacl1fttdhrisaucdcnbwno|hveochie,aueewttelrtfe7wnonaeimeihio:ecnsmreeudri,mttcedrahno,ootnndethuu2idodadda-ofsissiosnfads(entnoret,ienfnreshiyfmtotch3tadcncna.anacaifeeleafmhsohlrvnnam,lantzbiioscflmidssc7mwe(elecwnfynoxsSeflecorhabfettciTitsf,asideymh.clugnrrsngs8dei,avoesneihkbinhchinacleptirit-ltottiNgerismlmailaostneeoI-hinaiaoiiobtagtgienoealrdoongcnl.unhhcoenrdvorrsuxonaoahvl"linao"rrrilintideLngcpdeeeyeseet2l---r---2tttft.t scotcwttcttreeihauhiohfoaaccmeglAnlns.hhootg2thinnnr2ranecmotieoiimshnahccbtatnlaaaahcWseidottnlltheneseraerhzo,dt.s2t2hmeoep,iu4H,onoatmh4nihb,rentcoc,5see5sianot-pr,-ttblTooloehTshvwoflfreeestmpthnhdQihttoinhy,hacpefWrlderpyehmopsoisurnsrhmeoswhcpcryebaceahhncelrnllvevyosireowcameeoepmmrtshissoniubiickrxcscoioieapnsshenaferlphoduintrntwyhostwhotdtehrefchnoedisieaa.etuothupj.hscx2bjs2Cru2eeojsoe,crcsrdre4ddeosrhpcTr,suiuw/vhobuihbbdcny-an1lle'yTeye-cs-t" dwccnooieitonteihodrtadnemivadnaliatrneotiaeoxcdtu|i>losaeenrprihiifmidyogeefhmtnhltyeitaohslleetosotpgxutiriecdoccaibhellssnesumitbicunsasdt.lnaiennscic,mheaesanm,ldsiwcaaaerdllesl nDntpilaooelpCsynruhahmrtillheno7arremlon5effe:ene6nrne8aees9nn.bh,icymea1ssa9Slo5hlsin7ee)lenel.nnexTydpphernemronidmedaunuecKtnehttdlooaiglrosmcxdhc|aelirnoctmerrii(anmaAladcrtendecxldih- References 1. Herxheiincr K: tiller Clomkne. M tlnch M ed W tcbr 40:278. 1899. 2. W aiter H: Gcwcrlilirlio Krkrnnkungen durrh g clilo rlo KoliIcnwmwcrNlnlTu. Xbl (lew Ily a G: UX), 1018. H. T ricky Ig Din Pemnkrnnklieil {O dors- c). Klin Wtr.hr r.:847-K4H-, K07-!X)I, 1!7. 4. .Innas iJW, Alrlrn IIW : An nenciform dcrmnlcr- posiM. Areh Drrm Syph 0:1022-10.14, lil.'IG. 5. Teleky Jg liber Ncurcre l-'orschnnKwiirl linden unit I'orsi luinccn nuf deni Gcliiot dor Gcwcrl>okrnnk- heiten. Klin W tehr 27:240-2.17. 1049. 6. Klinn KM, .Inrvik N14: Actions of rertnin rhlnr- inntrd naphthnlcncs of the liver. Proe Hoc K ip lliol M ed .11:118-120, 19.1. 7. Drinker CK, Wnrrcn M F, P ennell GA: The problem of pnssihlo systemic cfleets from cerlnin rtilorinntcd hydromrixm*. J Induttr lly g Toxic 19:282-111, 1917. 8. Jones A l': T lie etiology of note with *|iet-inl referrnee to nrne of nrrutHilionnl origin. J In d u ttr H ya Toxic 21:2:10-112, 1941. 9. Grrcnhtirg U Mayer* M il, Moss-Smith A: Tlic systemic effect resulting from exposure to certain rtilorinntcd Hydrocarbons. J In d u ttr Ily g Toxic 21:29-18. 1918 10. Culler l i t : Pcnlnrhlorinnlcd nnphllinlcncs in industry. J A M A 125:271-274. 1944. 11. M rljerhlrhic NGH. Rnlicrtsnn D : O ilorinnted naphthalene jioisoning. firit M ed J ldi!II-92, 1942. 12. Collier K: Poisoning by chlorinated naphtha lenes. Ijw c r t 244:72-74, 1911. 11. Von Octtingen WK: T h e Haiogenaled Hydro- enrhant. Toxicity and Potential Danger, publication 414. Public Health Service, 1911. 14. Seliwarlx L, Tuli|win I* Mirmingh.-im D-J: Oe- eupnlinnnl D iteatet of the Skin, cd 1. Philadelphia, last and l-'cbiger l 'uhlishcr*. 1917, pp 11-145. 15. Meigs W.l, Alliom .1.1. K artin MI.: Chlornene from and unusual cx|x?suro to arorlor. J A M A 154:1417-1418, 195-1. 1. Hofiunnn M I', Mcnegliini C L A prosixwito dclle follimlnsi da idmrnrhuri rlom snslituili (Arne clorirn). (! Uni Derm 101:427-410, 1902. 17. Mimiinglinm 1).I: Ocruimlinnat dermatology: Current prohiema. Skin 1:1H-42i, PM2. 18. Plcwig G: /.nr Kinctik der Coincdonenliildiing liei Chlornene (tlnlow axam e). Arch Klin K xp Derm 218:228-241, 1970. 19. It.-iuer H. Si-hut/. K H, Spiegellierg U : n eru - flii-iie Vergifltingcn liei dcr Iferslelliing vou Clilnr- phenol-Vcrliindungrn. Arch Gerteerbepath Geneerbrhyg 1H:i:18-111,PKlt. 20. Kimniig J , Srhulx K II: Meruflirlio Akne (Nog u O $ CO <E CO CO ro o.i.. A ll.. 21 re-:|r tlnv- 2:1 4 22 lion 20 2> 21 heal' 2.4.1 24 nteii. 21. 1970 20 tic . < 27 lion Imvi: whe: 25 (ion chi'. 29 Oilo Amr 10 ehlo 4:27 31 ICxp nd 12 men rail! pro:dr p a ir vine 197 3-- larv 131V: lion V diw pre! 1123 effc dS ci pro Di1n cn<:. u11n-i14- nii< kes 19. 4 per by. 4 nm /a v . K\ Arch Environ llrnlllt-- Vol 21, Aug 072 5794 oofeora  I wvll il ml* s nr ni'nf- A rr/i rultliil cx- uitpd M rd lurch 0:100. kncl. inic r im ini rnnk- hlorllio l The rlnin Vaxic It. The rlnin oxic a in nterl 12. Ihn- drnlion Ochin, me AM silo me vy- ing rm ni- lurTr- ki* DOW373328 rn s ic rrv n r nvnnncAHnuNs -KiMhitniir.ii 1.11 1*1 1] I I llu m in i* I l i m i l i r l l l l l l II* m * l|llilli'* r tll / v k ll'w 'llll I l l l t l l | r i l i ! i | . r l i i i I t r l l i l I I I i i m i x I l l M I I l f |>t' l I n . ,, m l P o o h S e t 7 1! Ath*'v. / i . j (i n f i i / t i l i . . i . io M i ; , i: , v . l n * x m - l i I m m i n t i l i l I i i i I i *i i i **i . 7M -424 C il), lp . ' . j `i ' . l . A it im i* K M . I i m I i M I ) , N | h *m i *i *i * I I I ! , r i n i : 'U n * 4 1 . | - * li r k 111*', l - ' i i r - i l i H i i * I ) , M i i i l i m * .1 J S l i n t i i - i n f m **i | m i i i *m* n f i n l i l n l a k i n I n i n i i i | t t i i i i n l ' i i r | n i i l i * i l I n l i l t * r l i i i ' k i v l t - t i n i i l i i H ' i i f i r ; J | . I ' r i ' I H i m l i i H l m i l l I I 11rr - Imvi* i*itn*i*il iirni'lnrni ilrriinililiH. //nlim lr A/ri/ Nnrg i r n l r l l r r l i i n f n i * r v i i ( i i l l ! i i i * i * l i i r k I* |||* I|||| f i ,, * | / i r 2:1 I. l!MI. I'lnin l i l r i i l r . Poult Sri *1 4 :1 2 1 4 *1 2 2 2 . IfX ifi, 11*.!. Iliiinlrr KW. Iliiitrr ll.l: !niln*ili*inl iiil<iKi*n* liitn ilu r In |M*iiliii'liliini|>lii*nnl. Im i M c d Sur/t Ip.M. 211. t'nliind A l1. Sinilti J), M r llr r l, e l ni: A hr.-illli aiti-vi-v a l wm krr* in n 2.4-1 ) pillili nini 2.4.3-T |iliinl. A rd i Kneirun llr a lth 22:.'llli-:)27, 1971. 21. llrnim W: Kliniarhr llrnlnirliliinitcn /u r Knlaliliiing ilrr O ilo n irn r. H nulnrzt 111:1211*1211. 1959. 25. Cimv K I): Clnrnriir. Urti J Derni 8*1:799.000, 11170. 2l. Olnfann )': lfv|>crkcnilnain (x-diacnac) nf cnl* Ile. 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N ature 220:702-707, 190A 49. RiarlirniiL'h RW , Rieelie 1', I'rnknll D ll, c l nl: I'olyrhlnrinntrd liiplirnvls in this global econyntctn. N ature 220:1098-1102, 1908. 60. I-iriw nslrin K l', Schulr. K R , Kuhrcm nnn T W , c l nl: Rinlngiml inlcm rlion liclwren plnalirizrrn nml innrrlirirlra. J Krnn K ntnm 02:701-703, 1909. rlilorinnlnl napltlhnlrnc. Scirncr 110:330-507, 111.72. 29. llroek \VK. Jo n ra KW. M nrV irnr 11, e t ni: dilorinnlril nn|tlillinlrnr inloxinitinn in aheep. A m ar J V r t U n 18:G25.(20. 19.77. .10. Hula*r W15, I jn k I l i ': T nxic pileria of hcxn- 61. M illrr .IW: Pnllmlngiml rltnngra in nniinnla ex|Kwcd to n coinm ereini rhlnrinnled liiphenyl. P ub lic H ealth Itrp 09:1083-1093, 1944. 52. N islii/um i M: U g h t nnd clcrlm n mii-roaropr nludy nf Hilombiphrnyl poianning. Arch Knuiroa Hilonninplillinlpne on aurine. T nxic A p p i Pharmacal H ealth 21:020-032, 1970. 4:2.77-21 >2.1902. 63. Tnki I, Miannngn S, Amngnae Y : R rim rla of ni. Schedile CK. 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W J, 1loilrlior RV , Cnllrnluirli 03. Crow K l): O iln m m e . T r e n t S i J o h n l l n t p l KW, c l ni: Suino pliyamlogicnl rea|iuitacn of New D erm Sue 60:79-99, 1970. Arch Environ Health-- Voi 25, Am : 1972 ooo:2379 579; Z%A 5796 SUMMARY TERATOLOGY il 10/6/72 The no-effect dose level of 2/A-D and 2 A 5 - T IN A VARIETY OF SPECIES IS IN THE RANGE OF 25 - 50 m g /kg/day t h ro ughout o r g a n o g e n e s i s . 2/3,7,8-t e t r a ~ and h e x a c h l o r o d i b e n z o -p-dioxin ARE HIGHLY TOXIC TO THE DEVELOPING EMBRYO AND FETUS OF THE RAT; 1/2/3^-TETRA^ , 2 , 7 - D I ^ AND OCTACHLORODIBENZO-P-DIOXIN ARE NOT. c { Z Z tp n n - AUTHORS TERATOLOGY - 2 > D FORM OF DOSE LEVELS SPECIES 2,4-n ROUTE mg /kg/d a y . Co lli ns & W i l l i a m s , 1971 (C.6.8) Kh e r a & Mc Kinle y , 1972 (c.6.6) Sc h w e t z E l AL 1971 (PP 8F0670) Ha m s t e r Ac i d Oral 200,60,100 Ra t Ac i d , Sa l t Or a l 250100,150 Rat EAScTiEDj Oral 12.5050,75,87 ) = No -ef f e c t dos e le v e l for embr yo and FETAL TOXICITY. 5797 0002855 TERATOLOGY -2,4,5-T AA t ~ \r \ AUTHORS. Co l l i n s & W i l l i a m s , 1971 (C. 6.8} * FORM OF SPECIES gA H Ha m s t e r Acid RQIE Or a l DOSE LEVELS - mg/ kg/ day 20j,80,100 Co u r t n e y ex al.. 1970 ( c . 6 . 9 ) Rat Mouse j ^ pt>M 2,3,7,8-TCDD Co u r t n e y & Mo o r e , 1971 (C.6.11)* (C.6.12)t Kh e r a & Mc Kinle y , 1972 (C.6.6) Mo u s e . Rat Ra t Ac i d Ac i d sc sc Acid, ^ Sa l t , l Esters j Or a l J , 100,125,150 10,22j@,80,250 SUBERT & DlLLMAN, 1972 (C.6.13) Mo u s e Mouse Poll, 1971 ( c . e . i 4 ) * Mouse Ac i d Buty l Ester Acid Or a l Or a l Or a l 135,60,90,130 Emers on EI AL* a * 1971 (C.6.3) * Sparschu EI AL., 1971 (C.6.4) # Binns S Ba l l s , 1971 (C.6.2) ft Ra b b i t Ra t - Acid Ac i d Rat Acid Sheep- Acid Ester Or a l Or a l Or a l . Or a l ) = No -effect dose level for EMBRYO AND FETAL See PP 1F1102, May 1973 5798 TY. 0002S56 AUTHORS TERATOLOGY - DIBENZO-p-DIOXINS -DIOXIN SPECIES ROUTE C rc DOSE LEVELS * ^G/KG/DAY Sparschu El AL 1971 (C. 6 . 5 ) Jf 2,3,7,8-Te t r a ~ Ra t Kh ERA & RUDDICK, 1971 ( C .6 . 7)* 2,3,7,8-Te t r a ^ Ra t 1,2,3,9-Te t r a ^ Rat 2,7-Di Ra t Oral (gTp,0,125,0,5,: 8 Oral Cffj2l),0,25,0.5,: 2 ,4 ,8 ,1 6 Oral 50^100,200,400, Oral 250,500,10004^ Sc h w e i z e i a l > 1972 (c.7.5)* - 2,7-Di ^ Hexa ^ Oc t a Ra t Ra t Ra t Co u r t n e y & Mo o r e , ( ( 1971 C.6 .1 C .6 .1 1 2 ) ) * 2,3, 7,8-Te t r a d Ra t Mo u s e NEU3ERT & D i l l o n , 1972 2,3,7,8-Te t r a -^ Mouse (C.6.13) Or a l Oral Or a l SC SC Or a l 0,3,3,4,5,9 ( 3 ) See PP 1P1102, May 1973 000285? 5799 8? 5800 i------- " IN S TR.*' ~"ET OR C TISE C r ' ^ -- NTTAL Summary of Teratology Studies with 2,4-D c o m p i l e d b y M a r g u e r i t e L. L e n g e Dow Chemical Company, Midland, Michigan O c t o b e r 18, 1972 Teratogenic studies have been conducted with 2,4-D and a n u mb er of its d e r i v a t i v e s in rats (S chwetz et al. 1971, Kh er a and McKinley, 1972), in hamsters (Collins and Williams, 1971) and in sheep (Binns and Johnson, 1970). In each case, a no effect level of at least 25 to 50 mg/kg/day was found.* In the st ud y conducted by D o w (Schwetz, 1971) 2,4-D and its propylene glycol butyl ether (PGBE) and isooctyl esters were g i v e n o r a l l y to p r e g n a n t rats at do se s of 12.5, 25, 50, 75, and 87.5 mg acid equivalent per kg of body weight each day on days 6 through 15 of gestation. The highest dose (87.5 mg/kg/day) was chosen because levels higher than this caused signs of toxicity in the pregnant rats. Fetuses were taken by Caesarian section on day 20 of gestation. (The day on which sperm were seen in a vaginal smear was considered day 0 of pregnancy.) Signs of fetotoxicity and embryotoxicity were noted in fetuses of mothers given the highest dose levels daily for half her pregnancy, and included decreased fetal body weight, subcutaneous edema, delayed ossification of bone, lumbar ribs, and wavy ribs. These effects were not considered teratogenic because they would not seriously inter fe re w i t h d e v e l o p m e n t or survival. No effect was noted at 25 mg/kg/day and minor effects were noted at 50 mg/kg/day. In the study conducted by the Canada Food and Drug Directorate (khera, 1972) 2,4-D and its butyl, isooctyl, butoxyethanol, and dimcthylamine derivatives were given orally to pregnant rats at doses from 25 to 300 mg/kg/day, with most tests at 50, 100, or 150 mg / k g / d a y on days 6 th ro ug h 15 of g e s t a t i o n f and the animals wore killed on day 22 of pregnancy. ( Spcrma- 5801 0002802 *See also attached tabular summary of teratology studies with 2,4-D, 2 , 4 , 5 - T an d v a r i o u s d i b e n z o - p - d i o x i n s (Schwetz 10/6/72.) DOW -2- tozoa in the vagina on the morning following overnight mating was considered day 1 of g e s t a t i o n . ) They r e p o r t e d t h a t e f f e c t s noted a t 50 mg/kg/day were not s i g n i f i c a n t l y d i f f e r e n t from those in u n t r e a te d anim als. Doses of 100 or 150 mg/kg/day o f 2 , 4-D induced f e t a l deaths and caused an in cre ased in cidence of s k e le ta l anomalies. Normally occurring anomalies included wavy r i b s , e x t r a r i b s , delayed o s s i f i c a t i o n of s k u l l bones and s e v e r a l changes in the sternum. They also observed a low incidence of anomalies a t the high dose le v e ls which were not p resen t in c o n tro l animals. These included fused r i b s and bent or d i s t o r t e d l e g bones. Some animals were allowed to d eliv er normally and the weight gain and v i a b i l i t of the o ffsp rin g were w ithin normal lim its . c c In the study conducted by the U.S. Food and Drug A d m in is tra tio n ( C o l l i n s , 1971), 2 , 4-D from t h r e e d i f f e r e n t m anufacturers was q given by o r a l i n t u b a t i o n to hamsters a t doses from 20 to 100 mg/kg/day on days 6 through 10 o f g e s t a t i o n and the animals were s a c r i f i c e d on day 14 of g e s t a t i o n . The incid en ce of f e t a l anomalies was low and c o n s i s t e d of delayed head o s s i f i c a t i o n or e a r abnorm ality in only two f e t u s e s from t h r e e groups given 100 mg/kg/day, and fused r i b s in a t o t a l of nine f e tu s e s from t r e a te d groups compared to th re e in the c o n tro l group. The au th o rs concluded t h a t the % a b n o r m a li t i e s , even a t 60 and 100 mg/kg/day^ were not s i g n i f i c a n t l y d i f f e r e n t from the c o n tr o l percentage. In the study conducted by the U.S. Department of A g ric u ltu re (Binns, 1970), pregnant ewes were given 2g of 2 , 4-D mixed with a l f a l f a meal each day fo r 30, 60, or 90 days a f t e r breed ing. (This is e q u iv a le n t to approximately 40 mg/kg/day in sheep). No s ig n s of c o n g e n it a l malformations in the lambs, or clin ical poisoning or histopathological lesions in the dams were noted. . 5802 0002S53 -3- Schwetz, B. A., G. L. Sparschu, and P. J. Gehring (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 Cosmetics Toxicology 9_, 801-817. (C.5.3) Khera, K. S., and W. P. McKinley (1972). Pre- and Postnatal Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4-Dichloro phenoxyacetic Acid and Their Derivatives in Rats. Toxicology and Applied Pharmacology 22_, 14-28. (C.5.4) Collins, T.F.X., and C. H. Williams (1971). Teratogenic Studies with 2,4,5-T and 2,4-D in the Hamster. Bull. Environ. Contam. & Toxicology 6_(6), 559-567. (C.5.5) Binns, W., and A. E. Johnson (1970). Chronic and Tera togenic Effects of 2,4-D (2,4-Dichlorophenoxyacetic Acid) and Atrazine (2-chloro-4-ethylamino-6-isopropylamino-striazine) to Sheep. Proc. N. Cent. Weed Contr. Conf. 25, 100. (C.5.6) Addendum, August 1973. Also attached are excerpts from two papers presented at the Eighth Inter American Conference on Toxicology and Occupational Medicine in Miami, July 1973. (a) Clegg, D. J. and K. S. Khera. The Teratogenicity of Pesticides, Their Metabolites and Contaminants. (Phenoxy Herbicides p. 270-273) (b) Durham, W. F. and C. H. Williams. Mutagenic, Teratogenic and Carcinogenic Properties of Pesticides. (2,4,5-T and 2,4-D p. 316-317) 5803 000 2S54 l' t ,* . Ai D O V /373315 Eil'ccl of Telraelilorodibenzo'p-Dloxin on Growth Rale and ihe Synthesis of Lipids ami Proteinsin Rais Ly II. M. Cunningham ami I). T. Williams li esecrili .abTiiimrnmtuyr'sffl, 'faostttuir!ra, mOiltDarnUaf: litrccturnrr Chick edema disease was first observed in 1957 when thousands of broilers were lost (1) but it was not until 1966 that chlorodibenzo-p-dioxins were found to be the causative agent (2). One of the most toxic dioxins was 2,3,7,8-tetrachlorodibenzo-p-dioxin (3). The most prominent toxic effects in chickens are hydropericardium and hydroperitoneum (4,5,6) while in mammals the liver appears to be the main point of attack showing enlargement and fatty infiltration (6, 7). In one report, enlarged livers in rats were not accompanied by an increase in liver fat (8). The present experiments were conducted to determine if dioxins affect the ability of the liver to synthesize lipids and protein, and the dosage and time required to produce maximum effects. MATERIALS AND METHODS In each experiment 12 to 16 weanling male Wistar rats were allotted at random into two to four treat ment groups. 2,3,7,8-tetrachlorodibenzo-p-dioxin (99% pure, courtesy of the Dow Chemical Company) was dissolved in corn oil and given by oral intubation at levels of 0 to 10 pg/kg of body weight. Both treated and control rats received corn oil at a level of 10 ml/kg. One to seven days later each rat received an intraperitoneal injection of 1.0 pc of 1-14C-Lleucine and 10.0 pc of 3n-Sodium acetate'. -Exactly 1 hr later the rat was killed by a blow on the head, the liver was removed and 1-g samples were homogenized with 4.0 ml of 0.9% NaCl. The lipids were extracted by a procedure similar to that of Folch et al. (9) and the radioactivity was counted with a liquid scintillation counter (10) . In some experiments the lipids were partitioned by thin-layer chromatography (10). .The proteins were separated from the aqueous phase of the Folch extraction by centrifuging, washing twice with 5% trichloroacetic acid and twice with 2:1 methanol: ether (v/v). They were dried under nitrogen and 20 mg aliquots were solubilized in scintillation vials with 1 ml of Soluene (Packard Instrument Company) at 55C in a shaking water bath. In some experiments lipids were arso extracted from adipose tissue and proteins isolated from heart and skeletal muscle. VHouil.lr7l,inNouf. IKuIVril.M!,tiytiMi >'ti|lniilhG|'oiriluiiiiii.itii\oii*iw\oisiinli. t 1 1,1 n 45 5805 00023G6 RESULTS Experiment 1. A comparison was made between levels of 0, 1.0 and 10.0 yg of dioxin per kg of body weight on the incorporation of labelled acetate and leucine into liver lipids and pro"sins. The dioxin signifi cantly reduced body weight gain (P<0.05) and the radioactivity of the free fatty acids in the liver (P<0.01) along with reductions in other liver lipid fractions (Table 1). The specific activity of liver protein, on the other hand, increased (P<0.05) when dioxin was given. The 10.0 ug/kg dose level appeared to have a greater effect on most measurements than the 1.0 ug/kg level but the differences between them were not large. DOW373316 O oH O O TABLE 1 Effects of Dioxin on Liver Lipid and Protein Synthesis 3 days after dosage Oral dioxin dosage (yg/kg) S.E. Number of rats 4 Initial weight, g 49.5 Weight gain per day, g 3.1 Liver lipids (DPM 3H/mg liver) total lipids triglycerides free fatty acids diglycerides phospholipids Liver protein 23.66 3.47 0.31 13.95 5.91 (DPM 14c/mg protein)234 4 51.2 0.4 15.46 2.09 0.13 10.68 2.58 330 4 51.0 0.2 11.46 2.17 0.08 6.63 2.66 330 2.1* 4.25 0.53 0.05** 2.67 1.26 28 *P < 0.05 **P < 0.01 I Experiment 2. The 10.0 ug level of dioxin was again compared with a zero control over a 3-day period. \ It again had a similar effect on growth rate and the incorporation of labelled acetate, and leucine into lipids and proteins but with more rats per group more of the differences were significant at the 1% level of probability (Table 2). The 14c-leucine incorporation by the heart was not affected by dioxin Aut there was a significant reduction (P<0.05) in the incorporation of 3n-acctate by epididymal adipose tissue. 46 000236? 5806 TABLE 2 Effects of Dioxin on Lipid and Protein Synthesis in Liver Compared with that in Heart and Adipose Tissue 3 days after Dosage Oral dioxin dosage (uq/kg) 0 10.0___S.E.____ Number of rats Initial weight, g Weight gain per day, g Liver lipids 8 76.1 2.25 (DPM 3H/mg liver) total lipids triglycerides free fatty acids diglycerides phospholipids Epididymal adipose tissue 15.62 2.69 0.08 4.02 5.55 (DPM 3H/mg lipid) Liver protein 215.8 (DPM l4C/mg protein) 166 Heart protein (DPM l4C/mg protein) 38 8 76.2 0.36 0.35** 6.92 1.31 0.07 1.62 1.98 1.86** 0.39* 0.07 0.95 0.64** 71.7 37.4* 223 6.9** 33 7.0 *P < '.05 **P < 0.01 Experiment 3. The 10.0 vg level of dioxin was used to determine the number of days after dosing at which it would have the greatest effect on liver lipid synthesis (Table 3). There was a slight reduction in ^H-acetate uptake by liver lipids (DPM/g of liver) at .2 days and a significant decline (P<0.05) at 7 days. TABLE 3 Effect of Dioxin on Liver Lipid Synthesis at Varying Intervals After Dosage Days after qivinq 10 uq dioxinAq Controls 1 2 3 7 Number of rats Initial weight, g Weight gain per day, g Total liver lipids (DPM/mg liver) 4 61.5 3.4 16.38 4 64.0 2.5 16.79 4 '4 4 65.7 62.0 '63.5 1.9 2.8 3.1 13.78 14.35 9.00* *P < 0.05 47 DOW373317 00023G8 D ow 3?33i8 Experiment 4. Graded levels of dioxin were used in this experiment to determine the smallest dosage that would have a noticeable effect on protein or fat synthesis 7 days later (Table 4). There was a pro gressive increase (P<0.05) in liver weight as the level of dioxin intake increased with a maximum increase of 21% at the 10 ug/kg level and a minimum of 9% at 0.1 pg/kg. There also was a trend towards reduced 3n-acetate incorporation into liver and adi pose tissue lipids with dioxin treatment but with only 4 rats per treatment the differences did not reach statistical significance. Part of the reduction in the incorporation of 3H-acetate per g of liver could be accounted for by an increase in the size of the liver. TABLE 4 Effects of Varying Levels of Dioxin on Lipid and Protein Synthesis 7 days after dosage Number of rats Initial weight, g Weight gain per day, g Liver weight, g Liver lipids, total (DPM 3H/mg liver) Epididymal adipose tissue (DPM 3H/mg lipid) Liver protein (DPM 14c/mg protein) Oral dioxin dosage (ucr/kg) 0 0.1 1.0 10.0 S.E. 4 63.2 4.6 4.3 4 64.2 5.0 * 4.7 4 69.2 5.1 4.8 4 65.7 5.1 5.2 0.43 0.21* 14.93 11.34 9.12 9.51 1.88 322.3 125.2 56.3 138.0 86.0 186 168 153 143 17.0 Experiment 5. This experiment compared protein and fat synthesis at 3 and 7 days after dioxin dosage (Table 5). The dioxins had little effect on rate of body weight gain but liver weights were noticeably increased 3 days after treatment (P<0.05) and slightly increased after 7 days. The increase in liver weight was accompanied by a large increase in liver lipids which also was more apparent 3 days after dioxin treatment than after 7 days. The total incorporation of ^H-acetate into liver lipids (DPM x 103/liver) was unaffected by dioxin but the increase in unlabelled liver lipids diluted the labelled acetate so that the specific activity of the liver lipids (DPN/mg lipids) was significantly (P<0.05) Reduced. * 48 0023G9 - 5808 !%J' ..*fS' ./ *V* ; : . Adipose tissue in the flank region of rats incorporated considerably less ^H-acetate into lipids than epididymal tissue in earlier experiments but dioxin appeared to have a greater ..'.nhibitory effect on incorporation at 7 days than at 3 days after treat ment, although neither of the differences reached i T statistical significance. The protein concentration in the liver (mg/g) was slightly reduced 3 days after dioxin was given, apparently due to the larg^ dilution of the liver with lipids. At 7 days the protein concentration in the liver was unaffected by dioxin. The incor poration of l4C-leucine into liver protein was significantly (P<0.05) increased 3 days after dioxin treatment and only slightly increased after 7 days. The specific activity of skeletal muscle was not affected by dioxin treatment. DISCUSSION AND CONCLUSIONS O CO CCOO cc Most of the animal experiments reported to date in the literature have used "toxic fats" or fractions of these fats containing mixtures of different chloro- dibenzo-p-dioxins. The tetrachlorodibenzo-p-dioxin used in the present experiments was 99% pure and the highest dosage used (10 yg/kg) was close to lethal for when this amount was given orally to rats each day in a preliminary experiment they all died within 2 to 4 ] , days. The lowest level in a single dosage that caused 'n"?7 an increase in the liver weights of rats was 0.1 yg/kg. 0 et . 1 The dioxin had no effect on the rate of incorporation } of 3H-acetate into liver lipids but since large quan tities of lipids accumulate in the liver it would appear that in some way the chemical restricts the j transport of lipids out of the liver. This storage . *1. reached a maximum at about 3 days after dioxin was given and was accompanied by a significant increase in the incorporation of 14c-leucine into liver proteins. i Seven days after dioxin was given the incorporation of 14c-leucine into liver proteins varied considerably between experiments being slightly higher than controls in Experiment 5 and slightly lower in Experiment 4. Weight gain per day showed an opposite trend being lower than controls in Experiment 5 and slightly higher in Experiment 4 but none of these differences was statistically significant. It is possible that th increased synthesis of liver proteins at 3-days was the j result of an induction of liver enzymes (11) in response to the dioxin and that the time required for it to decline aghin varied slightly between experiments. , 1 0002370 r.-J 5809 V.y /V%` *-7- ` ... ":: > r . \ ; i r v - r : . ; . TABLE 5 v\ Effect of Dioxin on Lipid and Protein Synthesis 3 and 7 days after Dosage Dioxin oral dosage (ug/kg) Number of rats Initial weight, gl Weight gain per day, g Liver weight, g Liver lipids mg/g liver mg/liver DPM 3H/mg liver DPM 3H/mg lipid DPM 3h x 103/liver Adipose tissue, flank DPM 3H/mg lipid Liver protein mg/gm liver mg/liver DPM Hc/mg protein DPM 14c x 103/liver Skeletal mifScle DPMl4c/mg protein 0 4 63.0 5.2 3.78 60 229 15.3 258 58 1.61 277 1046 135 141 42 3 days 10 4 63.0 4.9 4.80 132 640 12.1 98 58 1.53 241 1157 156 178 42 S.E. 0.7 0.4 0.20* 25 138 4.5 42* 22 1.33 14 66 6* 7* 2 0 4 64.0 5.1 4.03 56 226 21.7 394 86 2.30 252 1015 144 14 5 40 7 days 10 4 65.0 4.5 4.65 92 428 17.5 190 82 1.35 252 1169 152 177 39 S.E. 1.4 0.3 0.30 19 28** . 3.2 '62* 9 0.53 13 83 8 11 2 *rats on 3-day treatment were dosed 4 days after initial weights taken and were then killed on the same day as 7-day rats. q t *P < 0.05 no **P < 0.01 0 2 e e ie M a 0002371 ^ i H i r i< f l w l > f i i M ' i > r i i l i * r ' i k i DOW373321 Acknowledgments We are grateful to Mr. R.C. O 'Brien who assisted in the chemical analyses. References 1. ANONYMOUS, Am. Hatchery News, 33, 17 (1957). 2. CANTRELL, J.S., WEBB, N.C. and MABIS, A.J., Chem. Eng. News 45, 10 (1967). 3. HIGGINBOTHAM, G.R., HUANG, A., FIRESTONE, D., VERRETT, J., RESS, J. and CAMPBELL, A.D. Nature, 220, 702-703 (1968). 4. YARTZOFF, A., FIRESTONE, D., BANES, D., HORWITZ, W., FRIEDMAN, L., and NESHEIM, S. J. Am. Oil Chem. Soc. 38, 60-62 (1961). 5. SANGER, V.L., SCOTT, L. , HAMDY, A., GALE, C., and POUDEN, W.D. J. Am. Vet. Med. Assoc. 133, 172176 (1958). 6. FLICK, D.F., DOUGLASS, C.D. and GALLO, L. Poultry Sci. 42, 855-862 (1963). 7. FRIEDMAN, L., FIRESTONE, D., HORWITZ, W . , BANES, D., ANSTEAD, M. and SHUE, G. J.A.O.A.C., 42, 129-141 (1959) . 8. CAMPBELL, T.C. and FRIEDMAN, L. Proc. Soc. Expt. Biol. Med. 121, 1283-1287 (1966). 9. FOLCH, J., LEES, M. and SLOANE-STANLEY, G.H. J. Biol. Chem. 226, 497-509 (1957). 10. CUNNINGHAM, H.M. and LEAT, W.M.F. Can. J. Biochem. ' 47, 1013-1020 (1969). 11. CONNEY, A.H. Pharmacol. Rev. 19, 317-366 (1967). .J 1 0002372 5811 r 5812 / ' S . VH.,'>lair ` .J < . J ' C o n n o r M . W e s s c i y .aIcV(_/ v. R. i<- II "Howe Stehl -/-V7JJ -<T":--H r--Btrv rfedrrottin Transi. MID Ko, 9506 ICIIER ZZITJ'JG no.70 p. February 12, (1973) Dioxins-insidious poisons in our environnent, Author: Hans Paul Bosshardt Translator: P. Fisher Date: May, 1973 Requested by: M. Irons, 2040 Bldg. Translation no.: 73-3-8 18,140 ue,2CiH\oa r e c e iv e d J!!N 2 5 1373: registration 0000520 DOW32372 C 0 Neue Zrcher Zeitung 8' - 5 1 1 - 3 7 3 73- 3-8 Translated by: Phil Fisher Number 70/Noon/Tebrvay 12, 1973 Dioxins-Insidicus Poisons in Our Environment. Origin, properties and behavior of polychlorinated dibenzo-p-dioxins By Hans Paul Bosshardt* In the large scale production of chlorinated phenols as side products, very toxic polychlorinated dibenzo- p-dioxins are formed which are damaging to the fetus. These impurities in their environmental behavior are similar to chlorinated hydrocarbons: therefore one has to count on accumulations within biological material and consequently on irreparable damages. In the U.S.A. millions of chickens fall victim to these toxins. In order to keep our environment and our food and nutritional sources free of these dangerous toxins, according to a decree from the Research Institute of Wadenswil, the especially dangerous tetrachlorodibenzo-pdioxin must not be detectable in plant protection agents and weed des troying agents. It is to be hoped that the Confederated Office of Health will establish similar specifications for other polychlorinated phenols and all compounds produced from them which are broadly used outside of agriculture in industry and technology, for example in wood protection or that their further application will be forbidden on the basis of the toxic r;:hr.tp:*.ccc lav.. 0000S21 ciese iV A o a 2 Reliable analytical proof of polychlorinated dibenzb-p-dioxins is extremely difficult and requires the use of modern devices (gas . chromatography coupled with masspectrometry) which cannot yet be introduced into the municipal and Cantonal laboratories probably for financial reasons. In order to protect human and animal life and health, and especially in order to prevent possible birth defects, legal-industrial and technical steps are urgently required. On a worldwide basis, residues of chlorinated hydrocarbons which are used as insecticides (DDT etc.), fungicides (HCB), lubricants and insulation materials (PCB) or as solvents can be detected in biological material. By way of preventative measures for protecting possible irreparable environmental damages, the use of such substances in many countries, also in Switzerland, has been limited or completely forbidden. In addition to these rather well known impurities, with progressive development in analysis technology additional chemical environmental problem substances are beginning to become detectable. With respect to their amount they are indeed less, ecologically however'they can be of at least equal significance. By way of an example of such analytically, very difficultly detectable environmental toxin we will describe here polychlorinated dibenzo-p-dioxins in somewhat greater detail which have already caused serious toxic catastrophes although they are present in only relatively minute amounts. 0000622 5815 Impurities of Chlorinated Phenols to CO In the technical manufacturing of chlorinated phenols, by way of impurities due to the condensation of two molecules, polychlorinated dibenzo-p-dioxins form. This condensation reaction occurs at temperature over roughly 200C almost quantitatively and in an explosive manner; a few factory installations have a l re ad y fallen v i ct im to this. Under 180< in technically flawless installations these side products are formed only to a minute extent. Therefore, it was possible for the research agency of Wadenswil to establish for weed killers produced from trichlorophenol based on 2,4,5-T a degree of purity of less than 0.05 ppm of 2,3,6,7tetrachlorodibenzo-p-dioxin with respect to 2,4,5-T acid; in Germany a similar specification was established (0.1 ppm). Toxification Catastrophes in Chicken Farms Due to the dangerous toxicological properties of tetrachlorodibenzo-pdioxin, strict purity criteria have to be maintained. The acute oral toxicity (LD^q ) of 2 , 3 , 6 ,7-tetrachlorodibenzo-p-dioxin is: Rats, male female Guinea pigs ^ d 50 g A g (1 pg' = 0.001 mg) 22 45 0.6 r 000C623 5816 uo,w ; DOW The for the lower and for the higher chlorinated dibenzo-p-dioxins is somewhat more favorable: 2, B-dichlorodibenzo-p-dioxin rats orally "> 2,4,8-trichlorodibenzo-p-dioxin Hexachlorodibenzo-p-dioxins Octachlorodibenzo-p-dioxin 5000 mg/kg 5000 mg/kg 1 mg/kg 1000 mg/kg The acute toxification phenomena with chlorinated dibenzo-p-dioxins is shown in the picture of chloracne: breaking out of the skin, difficult to heal. . Especially dangerous are the polychlorinated dibenzo - p-dioxins due to their high damaging effect on the embryo (teratcgeneity): even 0.125 -7 yg/kg of 2,3,6,7-tetrachlorodibenzo-p-dioxin cause phototoxic damages in rats if they are administered daily from the 6th to the 15th day of pregvn.<a_ncy. Also in mice such tr ea tm en t wi t h 1 u g / k g leads to cleft pallets. The exact evaluation of these and a whole series of further pertinent investigations is in d e t a i l quite difficult, still no doubt exists as concerns the fact that tetrachlorodibenzo-p-dioxin is highly teratogenic. The same is also, true for the isomeric hexach lo ro di oxi ns which when administered to rats in 10 daily doses of 1 y g / k g caused the number of fetuses to drop by one half. Octachlorodioxin,on the other hand, does not seem to be teratogenic. Data concerning experimental investigations on chronic toxicity of chlorinated dibenzo-p-dioxins is locking. 0000624 5817 DOW03237G 5 1 The U.S. Food and Drug Administration has available a copius backlog of experience concerning the effect of these substances on poultry. In the years 1957 to 1969 a disease broke out repeatedly in various States in the U.S.A. on chicken farms and many millions of animals fell victim to it. Exhaustive investigations were able to trace this disease back to the chicken feed whose fatty components were made'impure with polychlorinated dibenzo-p-dioxins, namely the isomeric hexac,hlorodibenzo-p-dioxins. In addition to liver and kidney damages, the autopsy in the animals concerned showed a hydropericardium, that is to say an accumulation of liquid in the heart cavity. This disease was named "chick-edema-disease". Such toxification catastrophes in chicken farms are a warning to be taken seriously because dioxin contaminations do not necessarily have to remain restricted to animal feeds. Also, in addition to the various possibilities of ingesting the toxin through the mouth (orally), probably many possibilities exist that it can also reach the body through the skin (dermally) . As an example of this v/e should point out the toxification of infants due to pentachlorophenol; pentachlorophenol was used in an American drycleaning establishment for treating diapers and sheets and thus led to the death of two newborn infants. Large Scale Technical Production The question as to whence and in what way chlorinated dioxins reached chicken feed has not been decisively cleared up because several possibility exist for this. Chlorinated phenols and their derivatives arc consumed in count) ess area;: of application; by way of examples wo will me n t i o n here O00CS2S 5818 >O W U3237V 6 the use of pentachlorophenol in wood protection and of trichlorophenol for producing the weed destroying agent 2,4,5-T or the bactericide hexachlorophene. Although no production figures are known, it is easy to estimate that the world production of chlorinated phenols reaches very large dimensions. The production alone of 2,4-D and 2,4,5-T in creased in the United States from roughly 15 million kg in the year 1958 to 40 million kg in the year 1966, and for the destruction of the Vietnamese jungle in the years 1962 to 1970 some 22 million kg of 2,4,5-T were also used in addition to an essentially larger amount of 2,4-D. The annual production of sodium pentachlorophenolate in 1969 alone in Japan amounted to over 16,000 tons. The use of pentachlorophenol as defoliant on American cotton fields was mentioned as a possibility of contamination of chicken feed. The most probable source of contamination is considered to be however the treatment of cowhides with pentachloro phenol. This fat adhering to animal tissues is mixed into chicken feed, proved to be very toxic and showed a high content especially of hexachlorodibenzo-p-dioxins. Since technical pentachlorophenol, in addition to some 8% tetrachlorophenol and 0.1% trichlorophenol, contains larger amounts of polychlorinated dibenzo-p-dioxins, these compounds went directly into the fat on the one hand or were able, on the other hand, to be freshly formed in addition from the chlorinated phenols during the melting of the fat In addition to these acute oral or dermal toxications with chlorinated can scar ce ly bo :;K'a:.urcd by do ct or s and patient 5819 DOW32378 7 Only the damages can be measured' The chemical and physical properties of chlorinated dioxins make such chronic damages very probable. Polychlorinated dibenzo-p-dioxins are stable and principally fat-soluble compounds. Therefore, in the environment they behave according to the pattern of chlorinated hydro carbons. Tetrachlorodibenzo-p-dioxin was decomposed using ultra-violet o r sunlight, wh er ea s octachlorodioxin is stable un de r these conditions. Aside from this reaction, until today no degradation mechanism is known 7 >: which can make these dangerous compounds disappear fast enough. 2 , 2 , 6 , 1 - tetrachlorodibenzo-p-dioxin is heat-stable up to 700C and only decomposes completely at 800C. If paper which was treated with sodium pentachloro- phenolate is burnt, the concentration of octachlorodioxin even increases. Tetrachlorodioxin is extremely persistent in various soils and over the course of 160 days is only unessentially degraded. ' Due to the low water so lu bi li ty (0.2 ppb) one cannot anticipate its be in g washed out into the ground water, however, absorption into plants -- even to a minute degree -- could be detected. The absorption of larger amounts, however, is improbable due to the extremely difficult solubility even in apolar i solvents (best solvent: o-dichlorobenzene, 0.14%). These properties favor a broad distribution in the 'environment so that dioxin contaminations, in addition to the relatively larger contaminations with respect to amounts with chlorinated hydrocarbons in biological material arc very difficult to detect. Since dioxins do not all have the M...... Li clerical effect, the possibili Ly exists that this ur.:..a sural; la contamination coo]cyirnlly is as significant as the me as ur ab le one. In *< 0000627 5820 DOVW3237) this sense dioxin-contamination is a prime example of environmental damage: nothing is measurable and damages can appear however at. biological foci. The contamination only becomes measurable when the damages are irreversible. f Preventative Measures -- In order to pr ev en t such damages, in the n e ar future 'the technical production of chlorinated phenols and all subsequent products manu factured from them and formulations must be improved in such a way that no more polychlorinated dibenzo-p-dioxins are formed. Should this not be possible, then the use of these preparations should be drastically limited or completely forbidden. In a new Confederated Toxic Substances Law, which is in force now for 1 year, legal bases are provided which make it possible to rapidly control this problem area. The manufacturers of chlorinated phenols will probably lend a hand in bringing about a hygienically satisfactory control and do everything to make their produc tion units inert within a useful amount of time. It is understood that such problems cannot be solved in Sv/itzerland alone International cooperation is indispensable and fortunately has already been extensively realized. It would be quite proper however for our country in such matters to lead the way and work out proposals for possible solutions of problems of prevention and restoration.of chemical environmental contaminations. (A l i t : : ..ture i iv.'ck can l._- availab le upon request.) 5 8 2 !0^00S28 U 5822 J AN ANALYTICAL ME7HC. DL CTiMC DIOXIN* H` MRaorabvtetahrredtw :T;-;Mr;:ucv; ahmrsnin ty Cambrj d g e , M a s 5achusetts 02138 2,3,7,8-Tetrachlcrodibenzo-p-ciio:<ir. (TCDD) is an extraordinarily' toxic substance that is produced as an unwanted side product in the industrial synthesis of 2,4,5-trichlorophenol, an intermediate in the manufacture of the herbicide 2,4,5-trichlorophenoxyncetic acid (2,4,5-T). 1 2 ' Because of its chemical stability *nd its lipophilic nature, the possibility exists that TCDD released into the environment could accumulate in food chains. A direct test of the possibility of biologically significant accumulati/on in animal tissues requires an analytical method able to detect TCDD at leveis well below those knevn to be toxic. The lowest value known for the lethal dose cf TCDD is that observed in the guinea pig, for which the 3single cral dose L DjrnU is SCO parts *oer trillion (ppt) body weight. Allowing for sublethal toxic effects and pro viding for a conservative margin of safety, it seems desirable to have an analytical sensitivity of at least one ppt. For a on<- - 1 2gram sample this moans the method must have a sensitivity cf abwiil 10 * grams or one Dicogram (pg). The most common method for analysing chlorinated organic compounds in tissue samples is gas-liquid electron capture detector. Its chromatography (glc) v;ith an limit of detection for TCDD, 10- 9 o o ?<) 9 ? 9 3 1u&o3 O0 *Paper presented April 2, 1973 at the Conference on DAbunzodioxins and Dibenzofurans hold by the national Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. To be published in Envircnv.cntal Health Fersooctiv--.~. ~ .. . 5823 000CSS2 D o w o o 89 9 10 ft 9 -2 - gram, is inadequate. This method is also susceptible to intciference from other compounds and so is not very specific. Mass spectrometry offers better possibilities. It is highly sensitive and in the high resolution mode of operation it is highly specific. We have previously described a time averaged mass spectroscopic method with an adequate limit of detection. 4 However, full sensitivity could not be realized in most sample types because of interference from DDE (a major degradation product of DDT) and polychlorinated biphenyls (PCB's). In this paper we describe a cleanup procedure that overcomes this difficulty. Homogenized samples are saponified in alcoholic potassium hydroxide and extracted with hexane. The extract is shaken with sulfuric acid and chromatographed on alumina. Elution with carbon tetra.chloride-hexane removes most of the DDE and PCB's. Chlorinated dioxins are then eluted with dichloromethane-hexane. The TCDD containing fraction is further purified by preparative gas-liquid chromatography and analyzed by mass spectroscopy, using a multi-channel analyser (CAT) to average successive scans. We also report the levels of TCDD found in a limited number of samples of fish and crustaceans from locations in South Vietnam near areas heavily exposed to 2,4,5-T. 5824 00005G3 -3- EXPERIMENTAL SECTION A. ' Reagents and Apparatus. /666<y<70/M0(C. 1. Hexane: Pesticide grade (Fisher Scientific). 2. Dichloromethane: Reagent grade (Eastman). 3. Carbon tetrachloride: Reagent grade (Merck). 4. 95-97% Sulfuric acid: Reagent grade (Dupont). 5. Sodium carbonate (powdered): Reagent grade (Mallinckrodt). 6. Ethanol: Pesticide grade (Matheson, Coleman and Bell). 7. Activated alumina: Fisher No. A-540, activated at 130C for 24 hours. , 8. Gas chromatograph: Bendix Model 2200 equipped with thermal conductivity detector. 9. Gas chromatographic column: 5% SE-30 on 60/80 Chromosorb W, 2 m x 2 mm (id) stainless steel. . 10. Trap for preparative gas chromatography: 150 mm x 1.5 mm (id) glass tube packed with 30 mm of glass wool. 11. Mass spectrometer: Associated Electrical Industries MS-9 double focusing mass spectrometer. 12. A Varian 1024 time averaging computer (CAT), interfaced with the MS-9 as described earlier. 4 B. Cleanup Procedure for the Analysis of TCDD in Tissue Samples. 1. Weigh the sample and homogenize it with 1.0-1.2 parts EtOH. 5825 000CS04 ' * -' 2 ^ r ~ b = r < 1 -b VV /'/ F ">~-^, ------- fT u ^ x,r 1v t / '+ 'f Y I b t> b-8 O O M P < 3 o o o c oO.ry. / V 5826 Do Wo o P 9 999. -4 - 2. Transfer the homogenate to a round bottomed (RD) flask equipped with a reflux condenser (Teflon tape should be* used on the ground glass joint). Spike the sample with approximately 1000 ppt 37Cl' TCDD, add 2 parts 40% aq. KOH, and reflux for 2 hrs. 3. Let the solution partially cool and add 1 part hexane. 4. Transfer the solution to a separatory funnel, separate phases and extract with three more identical portions of hexane. Collect the hexane in the original RB flask. 5. Transfer the hexane to the separatory funnel, rinse the RB flask twice with a few ml of EtOH and then twice with a few ml hexane, refluxing solvent each time, and extract the hexane with 1 part 1.0 N NaOH. 6. Extract the hexane four times (or until acid phase is colorless) with 2 parts 95-97% H^SO^. Break up emulsions with a few drops of saturated Na^CO^ solution. 7. Extract the hexane with 1 part water and add several grams of Na2C03 to the hexane. 8. Filter the hexane through a column of Na2C03 (100 mm x 10 mm id for 300 ml hexane). Prewash Na2C03 with several ml of hexane. 9. Concentrate the hexane (Snyder column) to 3-4 ml. 10. Chromatograph the hexane residue on a column of activated A1203 (50 mm in a 5 mm disposable pipette). Do not prewash column. Elute with 12 ml of 20% CClg in hexane, then 1 ml of hexane, and finally 4 ml of 20% CH2C12 in hexane. 11. Concentrate the 20% CH2C12 fraction carefully to__ about 50 y u lt add 100-200 ,1 benzene and concentrate 582 7q 0009 *(, fc 6<S o Q /H -5 - agaJn to 2 0 ^ 1 . 12. Add a few/g of m-tcrphenyl in benzene to the residue and preparatively chromatograph. The retention time of m-terphenyl relative to that of TCDD should be determined beforehand and used to make certain that the TCDD collection is carried out at the right retention time. 13. Elute the glc trap containing TCDD with 60 /fl followed by 10 /tl of benzene. Measure the total amount of eluant collected and calculate the fraction size in /<1 for the planned number of fractions (typically ten). 14. Prepare the fractions for TC4DD analysis in the sample tubes described previously. Add a known amount of TCDD to three or more fractions for quantitation of any TCDD observed. The amount of TCDD added per fraction for quantitation should be approximately 3-4 times the amount expected to be present. 15. Analyze the fractions with the MS-9/CAT. Typical conditions are: source 220C, resolution 10,0G0 (based on a 10% valley between peaks), trap current 1.0 mA (rhenium filament), electron multiplier 700, ionizing voltage 70 eV, time averaging at four scans per second. 16. Measure peak heights at m/e 321.894. Compute the quantity of TCDD (grams x 10-12 ) present in the fractions to which TCDD has not been added from the ratio of their mean peak heights to the mean peak heights found with added TCDD. 17. Repeat steps 14-16, but add ^7C1 TCDD and measure peak heights at m/e 327.885 in order to compute the amount of 37Cl TCDD recovered. Calculate the recovery 5828 00003 D o w OO P9? 9V . through the complete cleanup procedure bared on the amount of 37Cl TCDD added to the sample at the beginning of the cleanup. 18. Correct the quantity of TCDD computed in step 15 by the recovery factor obtained in step 16 to give the final result. C. Sample Collection. Freshly caught fish and crustaceans were collected in South Vietnam in August and September 1970 from local fishermen. The samples were homogenized with a meat grinder, placed in' acetone-rinsed glass bottles with aluminum foil lined caps, and immediately frozen in dry ice. Later on the same day, samples were placed in a Linde LR-35 liquid nitrogen refrigerator where they remained until analysis. Water blanks were present in the liquid nitrogen refrigerator throughout the storage period and were analyzed with the samples. Fresh Cape Cod butterfish (Poronotus -tricanthus, 'family Stromateidae) were obtained from a local market, homogenized, and kept at -20C until analysis. Domestic beef livers were ob tained and treated similarly. RESULTS A. Methodology. The mass spectra of natural and 37Cl TCDD are shown in Fig. 1. The most intense signal for natural TCDD occurs at m/e 321.894 (nominal m/e 322), corresponding to the isotopic isomer with one atom of Cl and three atoms of Cl. The natural abundances of the Cl isotopes are 75.53 and 24.47 percent, respectively.- The... ' 5829 0000908 D o w o o P ? *9 jr -7- observed spectrum for the synthetic 37Cl TCDD corresponds tc an isotopic purity of 95.5 percent 37ci, the same as the value claimed by the manufacturer of the NaCl used in the synthesis of the labeled TCDD. The synthetic 37C1 TCDD contributes only 0.042 percent as much to the peak at m/e 322 as to its most intense signal at m/e 328. The contribution at m/e 320 is even lower, by a factor of nearly 100. This allows an excess of 37.Cl TCDD to be added to each sample before cleanup without interfering with analysis of natural TCDD at m/e 322 and 320. The addition of 37Cl TCDD provides a carrier and makes possible tfie calculation of absolute recoveries. An alumina chromatography step has been developed which, when combined with the cleanup steps described previously, makes possible the measurement of picogram quantities of TCDD in samples initially containing more than a million-fold excess of DpE and PCB's. Fig. 2 shows the effectiveness of this procedure. The calculation of TCDD levels described in steps 14-16 of the experimental section assumes a linear relationship between peak height and amount of.TCDD present in any given sample. Fig. 3 demonstrates that the response is indeed linear over the full range of TCDD amounts introduced into the MS-9 in the course of the analyses reported here. The reproducibility and overall recovery of the complete analytical procedure is illustrated in Table 1. A sample of beef liver was homogenized and divided into three portions each of which was then spiked with 20 ppt TCDD and 1000 ppt 37Cl TCDD. The three samples were independently put through the cleanup procedure up to the glc step. Each sample was then split into three portions before preparative glc and mass spectrometric analysis, giving rise to a total of nine separate values for the recovery of both TCDD and 37C1 TCDD. The average recovery was 3 4 + 7 percent for TCDD and 0000909 5830 9 fc 6 6 3 o o A i o (L 8 2 7 + 5 percent for 37Cl TCDD. When the slight background signal at m/e 322 in an unspiked sample of the same liver is taken into account, the calculated recoveries from the spiked samples become even more nearly equal. Experiments performed separately with each individual cleanup step established that the step with lowest recovery is preparative gas-liquid chromatography. We conclude from these and other controls that the present analyti cal method provides the sensitivity and reproducibility required for biologically meaningful analyses of animal tissue samples. The method makes possible investigations of such samples at levels approximately ten thousand times lower than those reported here tofore.^ B.! Ob s.e..rved TC DIID Levels. Signals at m/e 320 and 322 were conspicuously present in each of the fish and crustacean samples from Vietnam. The calculated levels of TCDD, summarized in Table 3, range from 18 ppt to 814 ppt, based on total wet body weight. No peak was observed at m/e 320 or 322 with Cape Cod butterfish. The background signal corresponded to a level of ^ 3 ppt of TCDD. No peaks were observed in water blank samples present in the liquid nitrogen refrigerator throughout the sample collection and storage period. Confirmation that peaks observed at m/e 320 and 322 are in fact produced by TCDD is routinely provided by the criteria outlined in part A of Table 2. All three of these criteria are- met by the mass spectra from each of the Vietnamese samples. 5831 OOOIOCO 3 o w o o 8") 9 9 7 -9 - The additional confirmatory procedures listed in part B of Table 2 were carried out on a sample of Vietnamese fish. This sample, carp from the Dong Nai River, exhibited a mean TCDD level of 540 ppt. The mass spectrum in the regi on m/ e 322 is shown in Fig. 4. The compound observed in this fish behaved identically to TCDD in each of the three additional confirmatory tests. We consider it extraordinarily unlikely that this compound is anything other than a tetrachlorodibenzo-p-dioxin. In contrast to the signifi cant amounts of 2,3,7,8-tetrachlorodibenzorp-dioxin known to have been disseminated as a contaminant of 2,4,5-T,^ we know of no mechanism by which other isomers of TCDD might have been introduced into th Vietnamese environment. We have ruled out the possibility that the- TCDD was formed during the saponification step of the cleanup procedure. It is known that chlorophenols and chlorophenoxyphenols can form dioxins vinder alka line conditions at high temperature.^ A sample of Dong Hai catfish was worked up omitting the saponification step. The sample was digested in concentrated sulfuric acid and carried through the rest of the usual cleanup. The TCDD level, 304 ppt, was somewhat lower than the 522 ppt observed after the complete cleanup but the dif ference was not significant. The locations from which the Vietnamese samples were obtained are designated in Fig. 5. The letters correspond to those in Table 3. Areas heavily treated with 2,4,5-T before its use was ordered dis continued in April 1970 are shown as stipled. The number of samples is not adequate to permit reliable conclusions concerning the dif ferences between various locations and species, although this cer tainly should be a subject of future studies. C Oo''-aoioot I -1 0 - Con sidering the limited number of samples we have analyzed and the fact that they were collected two and a half years ago, it does not seem appropriate to attempt any detailed evaluation of the possible toxicological significance of our results. Such discussion is made even more difficult by the complexity and incompleteness of the existing toxicological data. However, in order to provide perspec tive for such discussion, a tabulation of some of the principal toxicity data on TCDD is presented in Table 4. .It may be noted that guinea pigs consuming their weight of food contaminated with TCDD at <1 level of 600 ppt would have ingested a quantity correspondirnj to the lethal dose. In contrast, a far greater quantity of TCDD is required to reach the LD5Q cited for rats. The table shows that teratogenesis in the rat occurs at doses substantially lower than those required to kill. Feeding studies in monkeys show that dioxin poisoning is cumulative.6 Various levels of a toxic fat known to contain chlorodio^ins were incorporated into'the daily diet of Macaca mulatta monkeys. As pointed out by the investigators, the mean survival time depended inversely on the daily dose. A plot of their data (Fig. 6) conforms rather well to the relation (mean survival time) X/(daily dose) + K', where X and X' are constants corresponding, respectively, to the accumulated lethal dose and to the lag time between the accumu lation of this dose and the time of death. No departure from this relation is seen even at the lowest level of toxic fat tested, where the mean survival time was 445 days. The importance of this result is that repeated intake of quantities of TCDD individually equal to only a small percentage of the single oral dose LD^g may over time cause serious poisoning. Unfortunately, the LD^g for TCDD in these primates cannot be computed since all of the animals died (5/5) even at the lowest dose level and the concentration of TCDD in the toxic fat employed has not been definitely established. 0001002 1 Do W c o P 9P -1 1 - In South Vietnam itself we have little information regarding ho possible occurrence of toxic effects of TCDD in humans nor has it been established whether or not measurable levels of TCDD have accumulated in exposed populations. Certainly, it should be pointed out that while we were in South Vietnam in 1970, the medi cal member of our group, Dr. John Constable, Professor of Surgery at Harvard Medical School, did not encounter evidence of any severe and widespread unusual illness in visiting Can Gio and several other villages or in discussions with officials of the South Vietnamese Ministry of Health. However, it was felt that certain indications in birth statistics ought to be investigated further for possible connections with herbicide exposure.^ It is of obvious interest to.survey appropriately.chosen populations in South Vietnam more closely, especially if TCDD residues should be found in human tissue samples. Finally, turning from questions of environmental toxicol&gy to the biological mechanisms of action, we note that TCDD seems to be particularly toxic to proliferating tissues, as suggested by its effects on spermatogenesis and hematopoiesis and its apparent toxicity to the intestinal epithelium and the thymus. These indications are consistent with the effects of a mitotic poison, such as TCDD is known to be in the African blood lilly*0 and possibly in Drosophila melanogaster. We are led by these obser vations to speculate that TCDD may be able catalytically to dis rupt microtubules, the subcellular elements of which spindle fibers are constructed and which are ubiquitous in their structural roles in cell extension and cell movement. Voi <o o -0o .0 0 CO 000100 5834 12- SUMMARY ]. A procedure has been developed for the reliable detection of -TCDD in animal tissues down to levels approaching one ppt. It makes use of chemical cleanup, preparative gas-liquid chromato graphy and analysis by time averaged high resolution, mass spec troscopy. 2. A limited number of fish and crustacean samples.was collected in South Vietnam in 1970 near areas heavily exposed to the herbi cide 2,4,5-T. TCDO was detected in these samples at levels ranging from 18 to 814'ppt. TCDD was not detected in a sample of Cape Cod butterfish used as a control. 3. These results suggest that TCDD may have accumulated to biolo gically significant levels in food chains in some areas 'of South Vietnam exposed to herbicide spraying. DO W o O *o O oO 0 5835 0001004 -1 3 - ACKNOWLEDGEMENTS -This research was initiated by the Herbicide Assessment Commission . of the American Association for the Advancement of Science. The work has been supported by funds from the AAAS, the Ford Foundation, and the National Institute of Environmental Health Sciences (NIH grant no. 1 ROI ES0C851-01 TOX). He thank Professor Bui Thi Lang, Mr. Robert Cook, Professor Arthur Westing, and Or. John Constable for aiding in tho collection of samples, and Professor Lang for helping to identify the specimens collected. We also thank Kenneth Gross and Lesley Newton for their expert assistance in the laboratory. I Q o p 6 Q O f M O < 5836 000 *n 1 \J n,rr 'J 0 REFERENCES 1. "Report on 2,4,5-T," A Report of the Panel on Herbicides of the President's Science Advisory Committee, Executive Office of the President, Office of Science and Technology, March 1971. 2. "Effects of 2,4,5-T on Man and the Environment," Hearings before theSubcommittee on Energy, Natural Resources, and the Environment of the Committee on Commerce, United States Senate, 91st Congress, April 7 and 15, 1970. 3. Sparschu, G.L., P.L. Dunn and V.K. Rowe, "Study of the Terato genicity of .2,3,7,3-Tetrachlorodibenzo--dioxin in the Rat," 1971. Fd. Cosmet. Toxicol. 405. 4. Baughman, R. and M. Meselson, "An Improved 'Analysis for 2,3,7,8Tetrachlorodibenzo--dioxin," in press. Adv. in Chem. 119. 5. Woolson, E.A., W.I. Reichel and A.L. Young, "Dioxin Residues in Lakeland Sand and Eagle Samples," in press. Adv. in Chem. 119. "/ C o 0 0 6 0 0 n* o < . Allen, J.R. and L.A. Carstens, "Light and Electron Microscopic Observations in Macaca Mulatta Monkeys Fed Toxic Fat," 1567. Am. J. Vet. Res. 28: 1513^ 7. Neubert, D. and I. Dillman, "Embryotoxic Effects in Mice Treated with 2,4,5-T and Tetrachlorodibenzo--dioxin," 1972. Naunyn-Schmledbergs Arch. Pharmacol. 272: 243. 8. Verret, J. in "Effects of 2,4,5-T on Man and the Environment," Hearings before the Subcommittee on Energy, Natural Resources, and the Environment of the Committee on Commerce, United States Senate, 91st Congress, April 7 and 15, 1970. 9. Poland, A. and E. Glover, "2,3,7,8-Tetrachlorodibenzo--dioxin: A potent Inducer of -Aminolevulinic acid synthetase, .1973. Science 173: 243. 10. Jackson, W.T., "Regulation of Mitosis: III. Cytological Effects of 2,4,5-T and of Dioxin Contaminants in 2,4,5-T Formulations," 1972. J. Cell Sci. 10: 15. 11. Herbicide Assessment Commission, "Background Material Relevant 'to Presentations at the 1970 Annual Meeting of the AAAS" and "Preliminary Report of the Herbicide Assessment Commission," both reprinted in Congressional Record IIS(32): S3226-3233,. 3 March 1972. 5837 0001006 -1 5 - 12. Ruu-Hoi, N.P., Phairf Huu-Chanh, G. Sesquel, M.C. Azum-Gclaile and G. Saint-Ruf, "Organs as Targets of Dioxin Intoxication," 1972. Naturwissenschaften 59: 174. 13. Davring, L. and M. Sunner, "Cytogenetic Effects of 2,4,5Trichlorophenoxyacetic Acid on Oogenesis and Early Embryogenesis in Drosophila Melanogaster," 1971. Hereditas 68: 115. 14. Porter, M.L. and J.A. Burke, "Separation of Three Chlorodibenzo-dioxins from Some Polychlorinated Biphenyls by Chromatography on an Aluminum Oxide Column," 1971. J. Assoc. Off. Anal. Chem. 54: 1426. 15. Woolson, E.A., R.F. Thomas and P.D.J. Ensor, "Survey of Polychlorodibenzo--dioxin Content in Selected Pesticides," 1972. J. Agr. Food Chem. 20: 351. 16. Crosby, D.G., A.S. Wong, J.R. Plimmer and E.A. Woolson, "Photodecomposition of Chlorinated Dibenzo--dioxins," 1971. Science 173: 748. / D o W o o nr o o a 3 0001007 5838 FIGURE LEGENDS Figure 1. Mass spectra of TCDD (A.) and.-^Cl labelled TCDD (B.). The isotopic purity of the J/Cl is 95.5%. The asterisk denotes an impurity. The multiplicity of lines associated with each major molecular species results from the presence of various isotopes of Cl and C. Figure 2. Mass spectra showing reduction of DDE and PCB levels in fish residue by means of alumina chromatography. Following the sulfuric acid cleanup step, the residue in hexane is added to a column of activated alumina. The trace in part A of the figure is from the material eluted by 20% CH-Cl., in hexane after the column was first eluted with was obtained 20f%roCmC1a.4siinmilhaexran2e0.% The trace in part B CH.Cl. in hexane elu tion after the column was first eluted with 1 % CH-Cl- in hexane. Elution with 1% CH-Cl, in hexane was reported to be effective in reducing*the amount of PCB residues (Porter and Burke, 1971). Elution with 20% CC1. is clearly even more effective and was routinely used in obtaining the results reported here. Figure 3. Linearity of response for TCDD in the presence of beef liver residue. The TCDD values are the amounts intro duced into individual runs on the MS-9. Figure 4. TCDD signals observed in fish samples. A. Vietnamese carp plus 60 pg TCDD. B. Vietnamese carp. C. Cape Cod butterfish. The wet weight of fish represented in run A and run B is 0.18 g. In C it is 0.16 g. Figure 5. Map showing sampling sites in relation to rivers and principal sprayed areas. Sites A and B are located on the Dong Nai River, site C is on the Sai Gon River, and site D is on the coast at Can Gio. Sprayed areas are depicted only within the region bounded by the dashed lines ( __). Figure 6. Mean survival time of monkeys fed toxic fat (Allen and Carstens, 1967) plotted against the reciprocal of the percent of toxic fat present in the diet. 0 ( h O OQ b O O 0001008 5839 5840 VJ e address presented at the 2 5 th annual m eeting o f the Southern Weed S o c ie ty a t Dallas. Texas. Jan. 18. 1972) Teratological potential of 2,4,5-T By James G. Wilson. Ph.D. Children's Hospital Research Foundation and Departments of Pediatrics and Anatomy University of Cincinnati College of Medicine T h e herbicide 2 .4 ,5 -T has received m ore notoriety and probably has been the cause of m ore public concern than any other p esti cide except th e insecticide DDT. Since early in th e V ie tn a m e s e w a r, 2 .4 ,5 -T , to g e th e r w ith several other herbicides, w as e xten sively used as a defoliant to reduce th e likelih oo d of am bush in areas w here dense undergrow th in th e fo rests and sw am plands of V ietn am favored guerilla w arfare. During June and July of 1 9 5 9 , South Vietnam ese '* TO EARTH. Voi. 28. No. 4. Spring 1973 n e w s p a p e rs re p o rte d th a t th e re had been | an in c re a s e in h um an birth d e fe c ts in some p a rts of th e co u n try . T h e s u g g e s tio n was m a d e th a t th is in crease w a s s o m e h o w re la te d to th e u se by th e U n ite d S ta te s Armed Forces of defoliant chem icals. These rep o rts of possible teratogenic e ffe c ts in V ie tn a m m ig h t h ave gone un noticed had th e y not been fo llo w e d in Octo ber. 1 9 6 9 . by th e a n n o u n c e m e n t of Dr. Lee A. D u B rid g e , th e n th e P resid en t's Science A d v is o r, th a t use of 2 .4 ,5 -T in th e United S ta te s w a s s o o n to be re s tric te d by certain governm ent agencies. The announcement w a s a conseq u en ce of th e release a few d a y s e a rlie r o f th e resu lts o f a s tu d y of a n u m b e r o f p e s tic id e s and industrial c h e m ic a ls by B io netics Labo ratories which in d ic a te d th a t m ice tre a te d d u rin g early p re g n a n c y w ith large d oses of 2 ,4 .5 - T gave birth to m alform ed offspring. Dr. D uB ridge's announcem ent, together w ith th e V ie tn a m re p o rts th a t h u m an birth d efects had possibly resulted from exposure o f p re g n a n t w o m e n to th is h e rb ic id e , had w id esp read repercussions. D uring th e fol lo w in g m o n th s a v e rita b le e p id e m ic of high level d is c u s s io n s o ccu rred in th e fo rm of: g o vern m en t-sp o n so red panels o f experts: c o m m issio n s s e t up by scien tific societies; conferences o f scien tists from universities, chem ical industries and th e government: and even a subco m m ittee of th e U .S. Senate. These sundry groups discussed th e matter a t le n g th b u t w e re unable to reso lve the m ain issue of w h e th e r 2 ,4 ,5 -T . as currently p ro d u c e d an d u sed , re p re s e n te d a real risk for hum an pregnancy. T h e q u e s tio n rem ain ed u n a n s w e re d in J a n u a ry , 1 9 7 1 , a t w hich tim e it w a s inherited by th e new ly form ed Environm ental Protec tio n A g e n c y (E P A ). D u rin g th e p re v io u s year g o v e rn m e n t re s tric tio n s had b een placed on th e use of 2 ,4 .5 - T , both fo r m ilita ry pur p o s e s in V ie tn a m and fo r s e v e ra l ty p e s of use in th e U nited S ta te s th a t, it w a s thought, m igh t result in exposure of preg nan t women. . M a n u fa c tu re rs of 2 .4 ,5 - T as w e ll as agri cu ltu ral in terests th a t used it w ere entitled u n d e r la w to a s k fo r s c ie n tific re v ie w of a n d /o r pub lic h earin g s on th e bases for th e restrictive actions. Tw o m anufacturers. The D o w C hem ical C om pany and Hercules In c o rp o ra te d , had already re q u e s te d a s c ie n tific review , several m o n th s before EPA officially cam e into being. O ne of E PA 's earliest fu n c tio n s w a s to a c tiv a te in Jan u a ry , 1 9 7 1 . a S c ie n tific Ad viso ry C o m m ittee w hich w as charged with re s p o n s ib ility to review all a vailab le evi dence on the (capacity to caus and to recom m e that w ould seer scientific data. A selected from a I by th e National regarded by the . priate qualificatic The C om m itte m o nths in w h ic all in fo rm a tio n tt dence and to pr a form idable job of p ag es of rese and survey resL p articularly duri: N e v e rth e le s s , in port* w as submi haus. w ho had. appointed Adm: report represen; m e n t re a c h e d bm ittee except fo w as Dr. Theodor not a qualified asked to appenr and exceptions A lm o s t immer to tw o scientif and Science) ir regulations un. operated spec: lecse u n til de ta k e n b y th e Er articles appears alm ost exclusi' exceptions of * No m ore than ; to the conclusk the eig h t toxic an ists and bioc in th e m a in rep By all appear behind these c hoped to destr port and arous scientists and had an opporti jective w ay. n sidered action taken to date. Mr. R uckelshai *Copies of the Repor available by w riting : Mira. Betty Bazille Room 3125 Environmental Prot South A griculture t 12th Street and Inc W ashington. 0. C. 5841 -a that there had been! birth d e fe c ts in sorriep T h e s u g g estio n wasf^ >e w a s s o m e h o w r lj U nited S tates A rm edi m icals. Dossible tera to g e n ic ! ight h ave gone u n || ie n fo llo w ed in O cto|, uncem ent of Dr. Leej| P re s id e n t's S c ie n c e t I.4 .5 -T in th e U n ite d ! re s tric ted by certain^ The announcem ent^ f th e re le a s e a fe w ^ u lts o f a s tu d y o f a~L es and in d u s tria l^ ; Laboratories w h ic h |a rested during earty^fl oses o f 2,4,5-T gavejfl pring. >uncement. to g e th e r^ rts th a t hum an birth 3 suited from exposure" ! this herbicide, had. on? D uring th e fo lb l idem ic of high rrea in th e fo rm of: panels of experts; scien tific societies; ts fro m universities, id th e governm ent; ! ;e o f th e U .S . S e n a te , iscussed th e m atter able to resolve the 2 .4 .5 -T , as currently ^resented a real risk led u n a n s w e re d in tim e it w as inherited vironm ental Protecng th e previous year s had been placed oth fo r m ilitary pur er several types of tnat. it w as thought, of pregnant wom en. -T as w ell as agrised it were entitled scientific review of on the bases for Two m anufacturers, tpany and Hercules e? re q u e s te d a rai ,.io n th s before being. : functions w as to '1. a Scientific A d w as charged w ith all available evi dence o n th e te r a to g e n ic p o te n tia l (capacity to ca u s e b irth d efects), o f 2 ,4 ,5 - T and to re c o m m e n d a n y c o u rs e s of a c tio n that w o u ld s e e m to be in d ic a te d by th e scientific d a ta . A n in e -m a n c o m m itte e w a s selected fro m a list of s c ie n tis ts s u b m itte d by th e N a tio n a l A c a d e m y o f S c ie n c e , and regarded by th e A c a d e m y as h a vin g a p p ro priate q u a lific a tio n s . The C om m ittee w a s allow ed only fo u r months in w h ic h to co llect and evalu ate all in fo rm a tio n th a t had a n y s c ie n tific c re dence and to p re p a re its re p o rt. T h is w a s a form idable jo b in v ie w o f th e th o usan d s of pages o f res e a rc h d a ta , e x p e rt o p in io n , and su rvey re s u lts th a t h ad a c c u m u la te d , particularly during th e previous tw o years. Nevertheless, in M ay, 1 9 7 1 , a 7 5 -p a g e re port* w as su b m itted to W illiam D. R uckelshaus. w h o had, a fe w m o n th s e arlier, been appointed A d m in is tra to r of th e E P A The report rep resen ted a concen su s of ag ree ment reached by all of th e nine-m an C om mittee except fo r one m em ber. The dissen ter was Dr. Theodore S terlin g , th e only m em ber not a qualified bio-m edical scien tist, w h o asked to append a len g th y list of objections and e x c e p tio n s to th e m ain re p o rt. A lm ost im m ed iately th e report w as leaked to tw o s c ie n tific n ew s m a g azin es (Nature and Science) in s p ite o f th e fa c t th a t th e regulations u nder w h ich th e C om m ittee operated specifically prohibited any re lease until d e fin itiv e a c tio n had been taken by th e EPA. H ighly critical m agazine articles appeared during th e sum m er based almost exclu sively on th e o bjection s and exceptions of th e one d issen ting m em ber. No m ore th a n passing reference w a s m ade to the co nclusions and reco m m en d atio n s of the eig h t to x ic o lo g is ts , te ra to lo g is ts , b o t anists and b io ch em ists w h o had concurred in th e m ain rep o rt. By all a p p e aran ces th e p erso n or p erso ns behind these d isto rted m agazine accounts hoped to destroy the credibility of the Re port and arouse prejudice in th e m inds of scientists and th e public before the EPA had an o p p o rtu n ity to exam ine it in any ob jective w ay, m uch less take carefully con sidered action. The only action E PA has taken to d ate, has been a new s release by Mr. Ruckelshaus on A ugust 9 ,1 9 7 1 , stating. ` Copies of the Report of the 2.4.5-T Advisory Committee are available by w riting: Mrs. Betty Bazille Room 3125 Environmental Protection Agency South A griculture Building 12th Street and Independence Avenue. SW . Washington. D. C. 20460 " A public hearing w ill be held in th e fall to obtain additional facts on 2 .4 ,5 -T w hile the cancellation order continues on the use of the herbicide on food crops grow n fo r hum an consum ption..." Ruckelshaus w as fu rth er quoted as saying th a t "w hile the A dvisory C om m ittee Report significantly adds to an understanding of 2 .4 .5 -T , ques tions rem ain as to th e balance betw een benefits and risks from use of th e herbi cide." The new s release noted th a t th e Com m ittee w as not charged w ith assessing benefit-risk factors and th a t a public hearing w ould be desirable to resolve th is issue. No public hearing has yet been scheduled. If it w a s th e in ten t of th e persons w h o leaked th e report to d iscred it it before its legitim ate release, th is objective w a s only p a rtia lly ach ieved . In th e fir s t p la c e th e m agazine reports w ere so flag ran tly biased th at even readers unfam iliar w ith the sub je c t could hardly m iss th e selectivity used in describing th e c o n te n ts of th e report. The careful evaluation of data and ju stifi cation of conclusions presented by th e A d visory C om m ittee w ere neither quoted nor discussed. On the other hand, m ost of the objections and criticism s of th e one dissent ing co m m ittee m em ber w e re rep o rted in as favorable a light as th e w rite r could m anage, despite the fact th at these objections were based on little m ore th an personal opinions and subjective reactions. One of several un fo rtu n ate consequences m ay be th a t the re lia b ility o f scientific journalism w ill rem ain in d o u b t in th e m inds of m an y readers. A l though letters w ere w ritte n to th e m agazine principally involved, objecting to th e overt bias and unfairness of th e articles on the 2 .4 .5 -T Advisory C om m ittee Report, the only letter yet to be published w a s th at signed by th e entire m em bership of the Council erf th e S ociety of Toxicology. The protest of a panel so highly qualified to pass judgm ent could h ard ly be ignored, nevertheless, th e letter of th e Council w as delayed several m onths before it w as printed. (Science, Nov. 5. 19 7 1 ). B ut w h a t ab ou t th e m ain is s u e -- is 2 ,4 ,5 -T really terato gen ic? T h e an sw er is ves. but so are hundreds of other com m only used drugs, plant products and environm ental ch em icals (T ab le 1). T h e s e co m p o u n d s w e re given to p regnant lab o rato ry anim als in doses u sually- g re a tly in exce s s of th a t e q uivalen t to th e hum an th e ra p e u tic d ose, in the case of drugs, or of th e likely exposure level, in th e case of e n viro n m en tal chem icals. This list of chem icals n ow know n to be te ra to g e n ic in rats, m ice o r ra b b its is so DOWN TO EARTH. Vol. 28. No. I & ^SSelicylates (e.g, aspirin, oil of wintergreon) ^C ertain A lkalo id s (e .g , caffeine, nicotine, colchicine) -; I 7^-' Tranquilizers (e .g , meprobamate, chlorpromazine, reserpine) Antihistamines (e .g , budizine, meclizine, cyclizine) Antibiotics (e .g , chlorampbenacol, streptonigrin, penicillin) Hypoglycmies (e-g, carfautemide, tolbutamide) Corticoids (e .g , triamcinolone, cortisone) Alkylating agents (e .g , busulfan, chlorambucil, cyclophosphamide, TEM) ' Antimalarials (e .g , chloroquine, quinacrine, pyrimethamine) - ir-- Anesthetics (e .g , halothane, urethan. nitrous oxide, pentobarfaital) AntimetaboHtes ( e ^ , folic acid, purine and pyrimidine analogues) ' v Sohmnts (e .g , benzene, dimethylsutfoxide, propylene glycol) ,1 ili Pesticides (e.g, 2,4,5-T, carfaaryl, captan, folpet) . Industrial effluents (e .g , some compounds of Hg, Pb, As, Li, Cd) Ili. : Il Miscellaneous (e .g , trypan blue. Tryparanol, Diamox, etc.) . i Capian and Folpet .; ! > : . v ^ ; ;Carbaryl (Sevin) .-d j p " ; - rabbits and mice mice and dogs Dieldrin. Chlordane and Keptone -m ice- " Diquat and Paraquat v.;':-/ ' ; ; ' Various organomercury compounds rats mice k > 2 .4 3 -T * and 2.4-D j :L- *i*'..'.Tera'togenic effects may have be,e,nc.a.used by an im purity. ;ki ..-- i 1 6 DOWN TO EARTH. Vd. 28. No. 4. Spring 1973 rats and mica .exten sive t h a t it is q u ite n a tu ra l To ask if n o t all c h e m ic a ls m ig h t be d am aging to e m b ry o n ic a n im a ls u n d er th e rig h t condi tio n s . A s a m a tte r o f fa c t, in vestig ato rs in th e fie ld o f te r a to lo g y te n d to assu m e that th is is in d eed th e c ase. O ne of th e widely a c c e p te d p rin c ip le s in th e fie ld is called K a rn o fs k i's L aw , w h ic h s ta te s , "th a t any d ru g a d m in is te re d a t th e p ro p er dosage, at th e p ro p e r s ta g e o f d e v e lo p m e n t, to em b ryo s of th e p ro p e r sp ecies, w ill be effective in ca u s in g d is tu rb a n c e s in e m b ry o n ic de velopm ent." A lth o u g h K a rn o fs k i's Law s p e c ifie s only i d ru g s , it u n d o u b te d ly h o ld s tru e fo r many ; o th e r, if n o t all. ty p e s of ch em icals. All p e s tic id e s w o u ld c e rta in ly b e s u s p e c t be cause th ey, like an tib io tics, sulfonamides, an titu m o r co m p ou n d s and im m uno-suppres- sive drugs, w e re designed and developed e ith e r to kill living o rg anism s or to sup press th e ir m e ta b o lis m and g ro w th . Three y e a rs ag o a s u rv e y of th e lite ra tu re (Table 2 ) reve a le d th a t s e v e ra l p e s tic id e s had at th a t tim e already been reported to be terato g e n ic in m a m m a ls . T o d a y th e list w o u ld be a t least tw ic e as long. Thu s it is probably s a fe to a s s u m e th a t m o s t p e s tic id e s given in high dosag e to p reg nan t m am m als would, if th e y did n ot kill th e m o th er firs t, interfere w ith th e g ro w th a n d /o r m e ta b o lis m of the em b ryo s. N o b io lo g ical s y s te m is kn ow n to g ro w fa s te r th an th e m am m alian embryo d uring its ea rly d iffe ren tiatio n and organ form ation. W h y th e n , if m o s t p e s tic id e s are likely to be te ra to g e n ic at high d oses, did so much e x c ite m e n t fo llo w th e rep o rt in 1 9 6 9 th a t 2, 4 ,5 -T at high doses caused m alform ations in m ice? T h e a n s w e r is not e n tire ly clear, b u t it p ro b ab ly relates to all of th e follow ing co nditions: 1) th e high level o f e m o tio n that surrounded all aspects of th e V ietn am War, I 2) the unsubstantiated report th at human I birth defects had resulted fro m defoliation I in V ie tn a m , fo llo w e d w ith in a fe w w e e k s by 1 th e report of th e terato gen ic effects of 2,4. 5 -T in m ice. 3 ) th e w id esp read public con cern about m an y asp ects of environm ental change and th e ir effects on "ecology", 4) over-zealous and som etim es irresponsible 1 effo rts by som e agencies and individuals to | influence public opin io n fo r personal gain. | or fo r sincere but ill-founded reasons. | N o w I w o u ld like to speak d irectly to the f subject of th e teratogenic potential of 2,4. f 5 -T . T h e A d v is o ry C o m m itte e ag reed in a | ratio of 8:1 th a t acceptable d ata on th e em - | bryotoxicity o f 2 .4 .5 -T w ere available fo r 6 | m am m alian sp ecies; nam ely, m ouse, rat. | 5843 I '1 hamster, ra b b it, s h e e p , and rh esu s m o n k e y . None of th e s e s h o w e d ad v e rs e e ffe c ts a t a dosage of 4 0 m g /k g / d a y d u rin g th e p erio d of organogenesis. T h e m o u s e w a s th e m o s t sensitive of th e s p e c ie s s tu d ie d , s h o w in g a low level o f te r a to g e n ic ity , m a in ly c le ft palate, a t 1 0 0 m g /k g /d a y g iv e n th ro u g h o u t organogenesis. H a m s te r and rat req u ired higher d o sag e to o b ta in c o m p a ra b le e ffe c ts . It was th o u g h t lik e ly th a t all s p ecies c o u ld have been c a u s e d to s h o w e m b ry o to x ic ity ff. 2 .4 .5 -T d o s a g e h a d been raised h ig h enough. F o r e x a m p le , p re g n a n t ra ts g iv e n a single dose o f 2 0 0 m g /k g o n d a y 9 , th e m o s t susceptible tim e fo r te ra to g e n e s is in th is species, c a u s e d m o d e s t in c re a s e s in th e percentage o f m a lfo rm e d su rivio rs as w e ll as in percentage o f in tra u te rin e d e a th . The d io x in c o n ta m in a n t 2 .3 ,7 .8 - t e t r a chlorodibenzo-paradioxin (T C D D ) has also been show n to h a v e a lo w te r a to g e n ic p o tential at d o s e s in e x c e s s o f 0 .0 0 1 m g /k g . This dosage level is v irtu a lly im p o s s ib le to achieve w ith c u rre n tly p ro d u c e d 2 ,4 .5 - T , which rarely e x c e e d s 0 .5 p pm and u s u a lly contains m u c h less. Careful s c ru tin y o f th e re p o rts of a d v e rs e effects on h u m a n re p ro d u c tio n a fte r u s e of 2.4.5- T as a d e fo lia n t in th re e s e p a ra te locations, n a m e ly , V ie tn a m : G lo b e . A riz o n a ; and Sw ed ish L a p la n d , y ie ld e d n o a c c e p ta b le evidence th a t th e a lle g e d e ffe c ts w e r e re lated to e x p o s u re t o 2 .4 .5 -T . It w a s c o n cluded th a t, as c u rre n tly p ro d u c e d a n d as applied a c c o rd in g t o th e re g u la tio n s in fo rc e prior to A p ril. 1 9 7 0 , 2 ,4 , 5 - T re p re s e n te d n o hazard to h u m a n re p ro d u c tio n . This w as th e s itu a tio n as of M a y 7 .1 9 7 1 , when th e A d v is o ry C o m m itte e s u b m itte d its report. H a s a n y th in g h a p p e n e d in th e meantime th a t w o u ld m ake it n eces s a ry or desirable to ch a n g e th is conclusion? New data from G erm any (N eubert and Dillman) re p o rte d t h a t o n e s tra in o f m ic e showed s ig n ific a n t in c re a s e in. c le ft p a la te incidence a t a d o s e o f 4 5 m g /k g o f p ure 2.4.5- T on days 6 th ro u g h 15 of g e statio n . An increase in c le ft p a la te w a s a ls o n o te d after tre a tin g s u c h m ic e w ith d o s e s of TCDD e x c e e d in g 1 f i g / kg. M o re s ig n ifi cantly. p o te n tia tio n of th e te r a to g e n ic effects oif 2 , 4 , 5 - T by T C D D w e re o b ta in e d . but only w h e n th re s h o ld o r h ig h e r doses o f bath substances w ere use d T h is d e m o n strated th a t a T C D D c o n te n t o f < 1 p p m w a s unlikely t o c o n trib u te t o th e e m b ry o to x ic effect of 2 .4 ,5 - T . e v e n in th is h ig h ly s e n sitive s tra in o f m o u s e . Canadian w o rk e rs (K hera and R uddick) have s h o w n th a t W is ta r ra ts g iv e n 0 .5 ig/kg of T C D D on days 6 -1 5 produced young w ith som e em bryotoxic signs. No evidence o f dom inant lethal m utations w as seen in su rvivin g m ales after any dose. Kearney and associates at the U .S. D e partm ent of A griculture have reported th at T C D D is im m o b ile in soils, n o t readily ta k e n up by plants, subject to photodecom p o sitio n . p e rs is te n t in soils and s lo w ly d egraded in soils to polar m etabolites. "S ubsequent studies revealed th at envi ronm ental co ntam in atio n by T C D D is e x tre m e ly sm all and not d etectab le in bio logical sam ples." W oolson and associates from several governm ental agencies found no dioxin residues in eagle sam ples from various geo graphical regions of th e U .S. or in soil sam ples a fte r application of 2 .4 ,5 -T in to ta l am ounts of 9 1 2 Ib/acre over the span of 7 years. These and other data have becom e avail able since th e A dvisory C om m ittee sub m itte d its re p o rt. T h e n e w in fo rm a tio n in large m easure represents refinem ents and m oderate extensions of inform ation that w as already available, or could reasonably be deduced from data exam ined by the Com m ittee. In any ev e n t, th e new d ata do not indicate th a t a single one of th e recom m en d atio n s m ad e in th e original w o u ld need to be ch ang ed ff th e rep o rt w ere re -w ritte n today. T h is is probably reflected in a n ew s item in th e Ottawa J o u rn a l of D ec. 6 , 1 9 7 1 . to th e effect th a t th e Canadian Governm ent has begun to relax th e restrictions placed on th e use of 2 ,4 ,5 -T . It w a s s ta te d th a t "Starting next year, m anufacturers w ho produce 2 .4 ,5 -T w ith a dioxin content of less th a n 0 .5 ppm w ill be able to sell th e ir products w ith o u t the w a rn in g s --now required on all 2 ,4 .5 -T labels." A n in ter esting com m entary on th e situatio n in th e U n ite d S ta te s is contain ed in th e sam e article. "The controversy about the appli cability of anim al tests to hum ans still rages, p a rticu larly in th e U .S . w h e re th e herb i cide's use has political overtones." In fa c t, th e article m akes clear th a t th e Canadian G o vernm ent is ready to adopt e ssen tially all of th e reco m m en d ation s set fo rth in th e Report of the 2 ,4 ,5 -T A dvisory C om m ittee. U nofficial reports indicate th at restrictions on the use of 2 .4 ,5 -T m ay also be relaxed in 1 9 7 2 in S w ed en and Finland. Thus th e old adage ab ou t "a pro ph et w ith o u t h on o r in his ow n co un try-" seem s to h ave been o nce m ore exem p lified in th e Report of th e 2 ,4 .5 -T A dvisory Com m ittee.-^ DOWN TO EARTH. Vd. 28. No. 4. Spring 1973 1 7 TCDD residue disappears A toxic contam inant that once appeared in th e p e s tic id e . 2 .4 .5 - T , w a s n o t d e te c te d in re c e n t s tu d ie s o f soil sam p le s and bald eagle tissue and so does not appear to be a residual th reat to wildlife. The contam inant. 2 .3 ,7 .8-tetrachlorodibenzo-p-dioxin (TC D D ), can be form ed during m anufacture of som e chlorinated phenols if te m p e ra tu re s above safe lim its are u sed (A G R . R E S .. O c to b e r 1 9 7 1 , p. 8). Prior to 1 9 6 9 , tra c e s of T C D D w e re fo u nd in som e sam ples of the herbicide 2 .4 ,5-T. W hile regulations and industry quality control have been established to elim inate th e th re a t of T C D D co n ta m in a tio n in p esti cides, chem ist Edwin A. W oolson and te c h nician Peter D. J. Ensor of A R S investigated th e possibility th a t T C D D residues from old. extrem ely heavy 2 .4 ,5 -T applications m ight still pose a th reat to w ildlife. The research w a s done in cooperation w ith W illiam L Reichel. chem ist w ith the U.S. D epartm ent of Interior. Patuxent W ild life Research C enter, Laurel. M d ., and A lvin L Young, form erly a project scientist w ith th e U .S. A ir Force. Eglin A ir Force B ase, Fla. They analyzed soil sam ples from experi m en tal plo ts of Lakeland sand in Florida th a t had received m assive doses of 2 ,4 .5 -T by aerial application during an 8 -year period from 1 9 6 2 to 1 9 7 0 . a period during w hich TC D D traces of up to 4 0 parts per m illion (p p m ) had been d e te c te d in com m ercial* 2 ,4 ,5 -T . A n a ly s is fo r T C D D residu es bega: in 1 9 7 0 . A t o ta l o f 9 4 7 p o u n d s o f a c tiv e 2 .4 ,5 -T pe ac re w a s a p p lie d o v e r a 3 -y e a r period to th Lakeland sand. T his is a m assive dosewhe c o m p a re d t o n o rm a l a p p lic a tio n rates fc bru sh c o n tro l on g razin g land of 2 pounc per a c re , o r 6 p o u n d s in a 3 -y e a r spar U s in g a n a ly s is te c h n iq u e s c a p a b le of de te c tin g le s s th a n 1 ppm o f T C D D , th e sc e n tis ts fo u n d no re s id u e s in 3 -fo o t co: sam ples. N o T C D D re s id u e s w e re fo u n d a t th e lowlim it o f d e te c tio n --0 .0 5 ppm in eagle tissu B ald e a g le tis s u e e x tra c ts w e re obtaine fro m 1 9 carcasses co llected in 1 5 states ; w id e ly s e p a ra te d as A la s k a . M a in e . Florid and M is s o u ri. E agles w e re used as repr; sen tatives of th e to p of a food chain. The s c ie n tis ts su g g est several reasot w h y no T C D D w a s d e te c te d in e ith er tt soil or eagle tissue sam ples. For exampi dissipation from th e soil m ay have result from m icrobial degradation, photodecor position, v o latility, a n d /o r w ind erosion, th e c a s e o f th e e a g le tis s u e s a m p le s , t: resu lts su g g e s t th a t T C D D residu es frc past p e s tic id e a p p lic a tio n s w e re n o t ava able to e n te r th e food chain. These results, com bined w ith th e toleran lim it of 0.1 ppm T C D D assures freedc from health hazards due to T C D D contarr n a tio n . * From October. 1972 issue of Agricultural Research. 1 8 DOWN TO EARTH. Voi. 28. No. 4. Spring 1973 5845 Amendment to PP 8F0670 - 2,4-D - June 1973 C.7.0 Summary of Toxicology Studies with 2,4-D in Livestock, June 1973. .-- As discussed in Section C of .the petition submitted by the Industry Task Force on Phenoxy Herbicide Tolerances in December 1967, no effect was produced by repeated oral doses of 2,4-D salt and ester formulations at 50 mg/kg in cattle and 100 mg/kg in sheep, even when administered five days per week for as long as two years. Levels of 250, 500 and 1000 mg/kg caused anorexia and resulted in death after several repeated doses. The following tables summarize studies by USDA with various phenoxy derivatives in livestock. (Complete reports of the USDA studies can be found in Sections C.l, C.2 and C.3 of the December 1970 amendment and in Section C.7.1 and C . l . 2 of this June 1973 amendment.) These and other studies with 2,4-D in livestock are also summarized in Section C.0.2 of this June 1973 amendment. DOW C f The effects of exaggerated rates of intake were studied by USDA and by Erne in Sweden (C.7.2 and C.7.3). In the USDA study, administration of the 2-ethylhexyl ester of 2,4-D as an emulsifiable concentrate formulation or as technical material at 250 mg/kg/day for 9 to 56 days caused ill effects in all seven animals after 3 to 14 doses and death in one calf after 14 doses and in two sheep biopsied after 17 and 29 doses. A significant increase in plasma magnesium:calcium ratio was noted in the animals that died. These animals also had increased blood urea nitrogen (BUN) levels, and patho logical examination at necropsy showed kidney damage and swollen, blood-engorged thyroids from this high dose rate. In the Swedish study, various effects were observed in pigs given 2,4-D triethanolamine salt or 2,4-D butyl ester at doses of 50 to 300 mg/kg/day for 5 to 103 days (C.7.3). Lesions . 580407028G2 r- 7 2- - were observed mainly in the digestive tract and the respiratory and excretory organs. Persistently high 2,4-D plasma levels (200 - 400 yg/ml) were found in animals which developed definite signs of poisoning indicating that their threshold level had been exceeded. On the other hand, 2,4-D plasma levels declined to about 10 yg/ml within 24 hours in animals tolerating repeated administration of 50 mg/kg/day. The 2,4-D amine was also administered at 500 ppm in the diet of pigs (equivalent to 20 mg/kg/day) for up to 12 months. Growth rate was irregular and depressed and locomotory dis turbances developed. This exaggerated dietary level also caused difficulties in pregnancy and parturition in the one sow studied, and the piglets were underdeveloped. Surviving piglets fed 500 ppm 2,4-D in the total diet for 35 weeks grew more slowly than controls and also exhibited locomotory disturbances. The 2,4-D plasma half life was highly variable in the pigs in these studies compared to the normal value of 12-2 hours obtained after single oral doses of 50 or 100 mg/kg (C.6.1). In the USDA studies, 2,4-D salt and ester formulations were administered by drench or in gelatin capsules five days per week for prolonged periods. Although not representative of ingestion of declining residues of 2,4-D in treated forage, they show that spraying pasture and rangeland with 2,4-D for control of weeds and brush presents no hazard to livestock. As discussed in Section D.5.0 of this June 1973 amendment, residues of 2,4-D are not expected to exceed 300 ppm in grass immediately after spraying at 2 Ib/A. (Brushy areas requiring higher rates of application generally contain little or no grass and afford poor grazing for livestock.) The residues in grass decrease rapidly to less than 10 ppm within a few weeks after treatment, depending chiefly on 5848 00028G3 geographic location. This residue amounts tn * level of about 1 mg/kg/day in livestock ingesting 3% <<r their body weight daily on a dry weight basis. The lack of hazard to livestock from residues in grass was also demonstrated in feeding studies conducted by USDA and The Dow Chemical Company. Phenoxy herbicides were fed at levels up to 2000 ppm in the total diet of c a t (_ie and sheep for four weeks prior to slaughter and sampling of tissues for residue analyses. Based on measured feed intake, individual animals ingested 2,4-D at 55 to 62 m-j kg/day continuously for four weeks without apparent iii effect. Similar levels of 2,4,5-T and silvex in the diot caused anorexia in some animals. (Section D.6.4 of 11,13 June 1973 amendment is a complete report of this study 1 cattle and sheep and of a similar study at up to 1000 ppm in dairy cows. It also shows the residues found in milk, cream, muscle, fat, liver and kidney of animals during ingestion and following withdrawal from the herbicides. \ 509134 ( 0 0 0 2 8 G '1 'is 5850 Amendment to PP 8F0670 - 2,4-D - June 1973 C.6.0 Summary of Metabolic Studies with 2,4-D in Animals, June 1973. A review of available information on metabolism of 2,4-D and other phenoxy herbicides in animals was submitted as part of a supplement to PP 8F0670 in September 1968. Work at Cornell indicated that phenoxy herbicides were rapidly excreted unchanged in the urine of ruminants (Ref. 42, 43, 48, 49, 51, 52). Work at Oregon State University in rats showed that maximum residues occurred in tissues 6 to 8 hours after a 1 mg dose and 8 to 17 hours after an 80 mg dose with highest levels in kidney (Ref. 50). Traces of a metabolite were detected in liver. Total residues decreased rapidly to less than 0.02 ppm in all tissues except the stomach within 16 hours after a 1 mg dose. Elimination was also rapid following subcutaneous injection of 2,4-D, its butyl and isooctyl esters at 100 mg/kg in mice by Bionetics Laboratories (Ref. 53). The butyl ester was hydrolyzed more rapidly than the isooctyl ester. Only 5 to 10% of the 2,4-D remained in the carcass after 1 day and no 2,4-dichlorophenol was detected in extracts of whole mice. Oc on o co to Erne in Sweden has studied the distribution and elimination of 2,4-D in rats, calves, pigs and chickens (C.6.1). Single oral doses of 50 to 200 mg/kg were readily absorbed and peak plasma concentrations were attained within 2 hours after dosing in chickens and-within 4 to 7 hours in mammalian species. A dose of 100 mg/kg gave peak plasma levels of about 100 to 200 ug/ml in the species studied. The peak was attained more slowly after administration of the butyl ester in calves and was much lower after administration of the ester to rats. Plasma levels declined and urinary levels increased when rats 5851 0002358 - 2- and pigs were given repeated doses of 50 mg/kg/day, except in two pigs which exhibited signs of poisoning at this dose level. A similar decline in peak plasma level was noted in chickens given 2,4-D amine at 300 mg/kg/day. Erne also studied tissue levels of 2,4-D in pigs maintained on a diet containing 500 ppm of the herbicide and in rats, chicks and chickens given 1000 ppm in their drinking water for 2 months to 2 years. However, the levels found are not representative of normal tissue distribution because the threshold dose was exceeded as evidenced by anorexia and reduced weight gain. Only low levels of 2,4-D and no intact ester were found in tissues of pigs given the butyl ester at 50 mg/kg/day for 1 month. In all cases, the lowest levels were found in fat and muscle with higher levels in liver and kidney. Similar results were .found in feeding studies con ducted by USDA in calves and sheep (Section D.8.0, June 1973) O C -< Cl o cp to CO Erne also demonstrated that 2,4-D is bound to plasma protein and that a portion is excreted in the urine in conjugated form '(C.6.2). In his method of analysis for 2,4-D in biologic material, the samples were extracted with an organic solvent under acid conditions, followed by solvent partition, thin layer chromatography, or photometric estimation of the color produced with chromotropic acid (C.6.3). However, some conjugates may require alkaline extraction and hydrolysis (D.8.11) Recent work at Dow in pregnant rats has demonstrated that 2,4-D and 2,4,5-T undergo placental transfer both into and out of the fetuses (C.6.4). Placental and fetal levels were proportional to maternal serum levels in this study and in another with, labeled 2,4-D in mice (C.6.5). The radioactivity showed a slight tendency for accumulation in the visceral yolk sac, passed to the fetus and was rapidly eliminated (within 24 hours) from all tissues. 5852 0002859 -3- In related work with 2,4,5-T, it was shown that uptake, distribution and elimination depend on the species and on the dose level(see PP 1F1102, Section C.8.0). A single oral dose o.f 5 mg/kg was excreted rapidly unchanged in the urine of rats and man, but was retained longer and was partially metabolized in dogs. The distribution, metabolism and excretion of 2,4,5-T in rats was much the same following doses of 5 or 50 mg/kg but was markedly different following doses of 100 or 200 mg/kg. Thus detoxification processes may be quite different at high doses such as those which caused effects in teratology studies with 2,4-D and 2,4,5-T (Section C.5.0 of this June 1973 amendment). Oo cc O CO A gas chromatographic method has been reported recently for the determination of trace quantities of 2,4-D and 2,4,5-T and their respective phenolic moieties in urine (C.6.6). In rats given 2,4-D at 10"5 to 10~^ of the LD5Q (375 mg/kg), the major portion of the administered herbicide was excreted as the unchanged compound and no metabolized residues were detected. Detectable residues of 2,4-D were r present in urine within 24 hours after administration of 3.75 yg/kg. Urine samples from occupationally exposed people were found to contain 0.2 to 1.0 ppm 2,4-D by this method. 5853 0002860 % V Qiirnnn Amendment to PP 8F0670 - 2,4-D - June 1973 SECTION C. FULL REPORTS OF INVESTIGATIONS MADE WITH RESPECT TO THE SAFETY OF THE PESTICIDE CHEMICAL C.0.2 Summary of Toxicology Studies with 2,4-D, June 1973 The following tables and abstract summarize the data available on acute/ subacute and chronic toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in a variety of species. A more descriptive summary was presented in Section C of PP 8F0670 submitted by the Industry Task Force on Phenoxy Herbicide Tolerances in December 1967. It was repeated in an amendment submitted in December 1970, along with several reports on toxicity studies conducted by USDA with 2,4-D in cattle, sheep and chickens (Sections C.0, C.l, C.2, and C.3). An amendment submitted in September 1971 included a summary of two-year feeding studies in rats and dogs and of a repro duction study in rats conducted by FDA (Section C.4). A published report of the long term studies is included in this June 1973 amendment (Section C.4.1). Teratology studies were initiated in 1970 as requested in PR Notice 70-8 and a report of a Dow study with several 2,4-D esters was included in the September 1971 amendment (Section C.5.2). Section C.5.0 of this June 1973 amendment summarizes four teratology studies conducted with 2,4-D in rats, hamsters and sheep, followed by published reports of the studies by Dow~(C.5.3), Canada Food and Drug Directorate (C.5.4), the Food and Drug Administration (C.5.5) and the U. S. Department of Agriculture (C.5.6). In each case, no effect was produced by administration of 25 to 50 mg/kg/day during organogenesis. 0002840 Dow 5094 07 <D Amendment to PP 8F0670 - 2,4-D - June 1973 SECTION C. FULL REPORTS OF INVESTIGATIONS MADE WITH RESPECT TO THE SAFETY OF THE PESTICIDE CHEMICAL C.0.2 Summary of Toxicology Studies with 2,4-D/ June 1973 The following tables and abstract summarize the data availabl on acute, subacute and chronic toxicity of 2 ,4-dichlorophenoxyacetic acid (2,4-D) in a variety of species. A more descriptive summary was presented in Section C of PP 8F0670 submitted by the Industry Task Force on Phenoxy Herbicide Tolerances in December 1967. It was repeated in an amendment submitted in December 1970, along with several reports on toxicity studies conducted by USDA with 2,4-D in cattle, sheep and chickens (Sections C.0, C.l, C.2, and C.3). An amendment submitted in September 1971 included a summary of two-year feeding studies in rats and dogs and of a repro duction study in rats conducted by FDA (Section C .4 ) . A published report of the long term studies is included in this June 1973 amendment (Section C.4.1). Teratology studies were initiated in 1970 as requested in PR Notice 70-8 and a report of a Dow study with several 2,4-D esters was included in the September 1971 amendment (Section C.5.2). Section C.5.0 of this June 1973 amendment summarizes four teratology studies conducted with 2,4-D in rats, hamsters and sheep, followed by published reports of the studies by Dow (C.5.3), Canada Food and Drug Directorate (C.5.4), the Food and Drug Administration (C.5.5) and the U. S. Department of Agriculture (C.5.6). In each case, no effect was produced by administration of 25 to 50 mg/kg/day during organogenesis. 00028*1 2- - POF'finn 4a A review of information on metabolism of 2,4-D and other phenoxy herbicides was submitted in September 1968 and the portion on animals was repeated in Section D.6.1 in December 1970. Section C.6.0 of this June 1973 amendment summarizes a number of additional studies with 2,4-D in animals, including rats, pigs, calves and chickens (Section C.6.1 and C.6.2), pregnant rats (C.6.4), and pregnant mice (C.6.5). These show that 2,4-T is readily absorbed into the bloodstream and is excreted rapidly in the urine relatively quantitatively as unchanged 2,4-D. Placental transfer of 2,4-D occurs both into and out of fetuses. Residue studies have been conducted in dairy cows, beef calves and sheep which showed that levels of at least 1000 ppm 2,4-D in the total diet were required to cause detectable residues (0.05 ppm) in milk, muscle or fat, while low levels were found in liver and kidney at 300 ppm in the diet (Section D.6.4 of this June 1973 amendment). Low levels of 2,4-dichlorophenol were also found in liver and kidney of ruminants fed 2,4-D, partly in conjugated or "bound" form which require alkaline hydrolysis prior to quantitation (Section D . 8 .11). Several methods of analysis have been used recently for 2,4-D and dichlorophenol in biological materials. These include spectrophotometric measurement of the color produced with chromotropic acid (Section C.6.3), cleavage of the ether link with pyridine hydrochloride and gas chromatographic determination of the liberated 2,4-dichlorophenol (Ref. 45, September 1968), and gas chromatography as methyl esters (Sections D.7.3, December 1970; D.8.9 and D.8.10, September 1971; D.8.12, June 1973) or ethyl esters (Section C.6.6, June 1973H This amendment to PP 8F0670 also contains a number of 5857 additional reports on toxicology studies with 2,4-D salts 0002S42 -3- and esters in livestock. Section C.7.0 includes summary tables for studies conducted by USDA (Sections C.2 and C.3, December 1970; C.7.1 and C.7.2, June 1973) and a discussion of an extensive study in Sweden (C-7.3). Some animals tolerated repeated daily doses of 50 or 100 mg/kg by capsule or drench without ill effect while others showed symptoms of anorexia resulting in reduced weight gain. Similarly no effect was noted in calves and sheep maintained for 4 weeks on a diet containing 2000 ppm 2,4-D, equivalent to ingestion of 55 to 62 mg/kg/day (Section D.6.4). However# pigs given 500 ppm 2,4-D amine in the diet for 12 months did not gain as well as controls and developed locomotory disturbances (C.7.3). Toxicity studies with 2,4-D in birds are summarized in Section C.8.0. Repeated oral doses of 2,4-D amine or ester by pipette caused reduced weight gain at levels of 100 or 250 mg/kg/day (C.7.0). No effect was noted at dietary levels of 500 and 2000 ppm for 7 days in cockerels (C.8.2), of up to 1000 ppm for 3 weeks in chicks (C.8.3), and of 50 or 150 .ppm for 28 to 48 weeks in laying hens (C.8.4). Con tinuous ingestion of 500 ppm in feed or 1000 ppm in drinking water for up to 5 months caused renal hypertrophy in chicks and decreased egg production in laying hens (C.7.3 and C.8.5). Spraying of chicken and pheasant eggs with 2,4-D at normal and exaggerated rates had no effect on hatchability in two studies in the U. S. and Canada, (C.8.6 and C.8.7), but was reported to be very harmful in a French study (C.8.8). However, no data were provided on hatchability of untreated chicken and pheasant eggs under the inadequately described conditions used in the latter study. r ^ c c c c Cl 0002S<J3 ACUTE TOXICITY OF 2,li-D 0002844 Formulation Triethanolamine Triethanolamine Butyl ester r Triethanolamine Organism Swine Swine Swine Chicken Butoxyethanol eater Phytoplankton Dimethylamine 1 Ethylhexyl ester Phytoplankton Phytoplankton Propylene glycol butyl ether ester Phytoplankton Butyl ester Rats Unspecified Unspecified Unspecified Trout Trout Man Isopropyl ester Rats Propylene glycol butyl ether ester Bluegill - - - ' -- ----------------- -- ------------------------------------------ . * mg/l is approximately the same as ppm. Dose 50 m g / k g 500 m g / k g 100 mg/kg 300 m g / k g 1 ppm 1 p p m (U hrs) 1 p p m ( hrs) 1 p p m (U hrs) Effect No effect Lethal No effect No effect l6H decrease in COg fixation No effect on CO2 fixation 9% de c r e a s e in C02 fixation decrease in COg fixation Reference (a) (a) (a) (a) (b) (b) (b) (b) 620 mg/kg 3 mg/l* 2 .2 mg/l* 689 mg/kg 700 mg/kg 3 ppm l d 50 Lethal 2h hrs Lethal U8 hrs Lethal l d 50 U8 hr TLn Olf'GOQ M O C I (c) (d) (d) (d) (e) (f) OX GO CT> o o o fo CD >r.. Cl Formulation Alkanol&mine Isooctyl ester Bu ty l e^ster Isopropyl ester Dimethyl&mine Organism Bluegill Bluegill Bluegill Bluegill Bluegill Alkanolamine Isopropyl ester Isopropyl ester ' Isopropyl ester Butyl ester Butyl ester Butyl ester Propylene glycol butyl ether ester Acid Acid Chicks Rats Chicks Guinea pigs Rats Guinea pigs Chicks Rats Dogs Chicks Unspecified amine Ac id Acid Mallard ducks Pheasants Mule deer ACUTE TOXICITY OF 2,U-D (Cont.) 0tr\ Otr\ 99 Dose * 0 5 PPn 9 ppm 1 ppm 1 ppm 166 ppm 380-765 mg/kg 700 m g / k g ll*20 m g / k g 550 mg/kg 620 m g / k g 81*8 m g / k g 2000 m g / k g 570 mg/kg 100 mg /k g 5^1 mg/kg 2000 m g / k g U72 mg/kg 1*00-800 m g / k g Effect * 8 hr TL n 1*8 hr T L a 1*8 h r T L m 1*8 h r T L n 1*8 hr T L m UJjO ^50 LDjO ^50 l d 50 ED50 l d 5o l d 5o l d 5o l d 50 l d 50 T " ~T' *' A ~ f-* i \ Reference (g> (g) (g) (g) (g) (h) ' (h) (h) (h) (h) (h) (h) (h) (h) (h) (i) (i) (i) y t r n n c aa n ACUTE TOXICITY OF 2,U-D Literature Cited Bjorkland, Nels., and Erne, K. -- 1966 (See C.7.4) Toxicological Studies of Phenoxyacetic Herbicide in Animals Acta. Vet. Scand. 7: 36^-390. Butler, P. A. - 1965 Effects of Herbicides on Estuarine Fauna. Southern Weed Cont. Conf. Proc. 18: 567. Edson, E. F., Sanderson, D. N., and Nookes, D. N. - 196U Acute Toxicity Data for Pesticides. World Review of Pest Control U(l) Spring 1965 McKee, I. E., and Wolf, H. W. - 1971 Water Quality Criteria - Publication 3-A, April 1971. State of California, Water Resources Control Board. Hayes, Wayland J. J. - 1963 Clinical Handbook on Economic Poisons. U. S. Dept. Health, Education and Welfare. Hughes, J. S., and Davis, J. T. - 1966 Toxicity of Pesticides to Bluegill Sunfish Tested During 1961-1966. Report to Louisiana Wildlife and Fisheries Commission, Monroe, Louisiana. Lawrence, J. N. - 196U Aquatic Herbicide Data. USDA, ARS Agricultural Handbook 231. Rowe, V. K., and Hymas, T. A. - 195U (See petition, 12/67) Summary of Toxicological Information on 2,U-D and 2,U,5-T Type Herbicides and An Evaluation of the Hazards to Livestock Associated with Their Use. Am. J. Vet. Res. 19: 622-629. Tucker, R. K., and JCrabtree, D. G. - 1970 Handbook of Toxicity of Pesticides to Wildlife. Resource Publication No. 8U. Bureau of Sport Fisheries and Wildlife, U. S. Dept, of Interior. 00028 * II J Formulation f Triethanolamine Organism Swine Dose 50/mg/kg/day Butyl ester Trie thanolamlne Swine Swine 50/mg/kg/day 300 p p m in feed Triethanolamine Rats Triethanolamine Chicken 1000 ppm in water 1000 ppm in water cn Not specified CD cn Not specified to Dog Rat 500 p p m in feed 1250 pp m In feed o o Not specified o r0o0 Rat ... 500 pp m in feed * *See summary which follows. CHRONIC TOXICITY OF 2,U-D Duration Effect 8-10 doses Minor transient effects. 5 doses None 1 month Some locomotory disturbance, depressed growth rate, no gross pathology 10 mos. Depressed growth rate, no gross pathology Dally from hatching through first 2 mos. of egg production. Egg size normal, production reduced 30 Reference (a) (a) (a) (a) (a) 2 years 2 years 2 years None No effects on growth, survival hematology or tumor incidence. No effects in repro duction studies. (b) (b) * (b) r* T t r> <-i Formulation Ethylhexyl ester Organism Cattle Dose 250/ m g / k g / d a y Ethylhexyl ester Sheep 250/ o g / k g / d a y ' Et hy lh ex yl ester S h e e p & 100/mg/kg/day Cattle Duration lU days 17 days 10 days Effect Sick in 3 days, survive A recover from 9 doses. lU doses lethal. Sick in 3 days 17 doses lethal. None to minor effects Reference (c) (c) (c) 2,U-D in water Human taste threshold 0 -01m g / l * Bad taste (d) ai go en oo O O O r.i DO OO Alkanolamine Sheep Alkanolamine Cattle Propylene glycol Sheep butai ether ester 100/ m g / k g / d a y 50/m g/ kg /d ay 100/m g/ kg /d ay Alkanolamine Chicken Propylene glycol Chicken butai ether ester Propylene glycol Cattle butai ether ester Acid Mule deer 100/ m g / k g / d a y 50 mg/kg/day 100mg / k g / d a y 00 & 2l0mg /k g/ da y ng/l is a p pr ox im at el y the same as ppm. **8l days 112 days **01 days No effect No effect No effect (e) (e) (e) 10 days 10 days 10 days No effect on weight gain No effect on weight gain (f) (f) No effect (f) 30 days Minor symptoms no weight loss mcosMoa (g) - n HO 8 HnX a 0u t1r ti 00 artr 5m09*Od Chronic Toxicity of 2,4-D Literature Cited (a) BJorkland, Nels., and Erne, K. 1966 (See C.7.4) Toxicological Studies of Phenoxyacetic Herbicide in Animala Acta. Vet. Scand. 7: 36U-390. (b) House, W. B. et al - 1967 Assessment of Ecological Effects of Extensive or Repeated Use of Herbicides. Final report on Midwest Research Institute Project 3103-B under Dept, of Army Contract DAHC 15-68-C-Q119. (c) Hunt, L.M., Gilbert, B.N., and Palmer, J. S. - 1970 Effects of a Herbicide, 2-ethylhexyl Ester of 2,U-D on Magnesium: Calcium Ratios and Blood Urea Nitrogen Levels in Sheep and Cattle. Bull. Environ. Contamination and Toxical 5: 5^-60. (d) McKee, I. E., and Wolf, H. W. - 1971 Water Quality Criteria - Publication 3-A - April 1971. State of California, Water Resources Control Board. (e) Palmer, J. S., and Radeleff, R. D. - 196U The Toxicologic Effects of Certain Fungicides and Herbicides on Sheep and Cattle. Ann. N.Y. Acad. Sci. Ill: 729-36. (f) Palmer, J. S., and Radeleff, R. D. - 1969 (See C.7.0) The Toxicity of Some Organic Herbicides to Cattle, Sheep and Chickens. Production Research Report No. 106 - ARS - USDA. (g) Tucker, R. K., and Crabtree, D. G. - 1970 Handbook of Toxicity of Pesticides to Wildlife. Resource Publi cation No. 8U. Bureau of Sport Fisheries and Wildlife. U.S. Dept, of Interior. 5864 0002849 il \ ! J p DOW 090038 uV JAHICC ClXCCLAfJD AND KCVIfJ P. Ci:CA Is,' ' ' *\r \ 1 yon isuy rpt races ckm yon pay for o \ l - SO u f c::c!!)u.:ti:. toxic to luim.m hcmirs mil nns-.siijy !o Hie .nv'!''!iiiion? is Mic p ro d u c ts o f side reactions w liiv.li o c c u r Itinnu H:'\;:',.in i.icHux.' m' .urictiirur:il .tml industrial ch e m i cals. Twi- .x'cenllv p ia d ic i/c d ox.inip'eK ire '..V7..s-ietr:clilor'nlihi;n/o- p i l i n x m :< i)l) .r . i i i i i i i a u!iuv 'u is o n o u s chem ical found a: a cu n ia m iiu n r in Hie ..July used lcrhicide 2 4.5-T: and the chlorinated d i K m / o f , n n s . fou.fd is con ram in am s m so m e I'oreipn-inaile p n lychlpri- :ill led iMniie n v f ^' ^ `Minds, lio lh ch em ic a ls are far m ore t o x ic than the com p oun ds they contam inate and in tact are imomt the most . t o x ic chem icals k .a iv b . Pints while m e n tio n has been focused on the to M c ily i:'a persistent p esticide, .purities w hich mav :n fact he m ore t o x ic have one u n n o tic ed tor mar.', years. Xertcultural chemtca.s. widely dispersed m the environment, usually contain vary tilt:'.timutr.rs o f impurities. R ec en tly , workers in the l :.S. D epartm ent ot' Xdncuiture n . S I M i anal'./.ed a van elv o f .lyrieultural ch em ic als tor p ossible co n ta m in a tio n w ith d io x in s. The group ot' p esti cides w h ic h w ere a n a ly sed were ail derived, like 2.4..5-P from a iip'MP o f ch em ic als k n o w n as c h lor op h en ois. <h ie o f these chem icals, p entaeliioro- plien ol <pt p i turned >>ut to be o f particular interest, because it con tain ed significant q u a n tit ie s o f ih c d io x in s. O f further interest is the fact that it ' s been used as j replacement for highly toxic mercury com pounds for siim e c o n t r o l in the m anu factu re o f paper pulp Ironically. I'< | m paper mill w astew ater is turmn" out to be about as hazardous to atfiialic life as are th e m ercu ry c o m p o u n d s it is replacing and m ore t*< l' ili.m mereury is needed to counteract the dune. - PCI* has b een used extensively ind for i variety o f purposes o ilier than lor slim e c o n t r o l ver Hie sears Xs a i . n e i c i d e it has been used m the processing o f oils. leathers, paints clues, and 'cxlites and has K e n in corporated in to hair sh a m p o o s anphcui "ii i*< !> .v.ut provides p ro tec tio n against erm ites and w ood-hi rime insects is -sell is m old s.' I*' P in.iy also c o m e directly <nto con tact .vuh I'.-nd 1 has been used as a w oiui 'le se ix a liv e m crates used !> r . iw len.a.itui'.ii n .il e r u lv as a luneikitle in Iv e i >iis raj m the c.r.k.-' . , .rij : vat ' d .Miiauiers In i i i ..ue 4' os. o o ii (s, 1112^1 s ( |u * \ i p! i] ,^.ai 11itI m .-ic :hail oiU'-ti.d! *'l tills 1 I.ii w.js . cit as * ' il :* s'i'. at o e s i ,:us. the possilMl- us o i Muin.in ex p o su re i::.l 0 ee \:r. :i:.d . oiitair.i;i.itiiii with pi I* ami ils toxic im purities in great. In ihe i: u> \ slm ly. 12'* .aniplcs o i I " ilil lerent p estititles were analyzed liv ing ;ias chrom atography in u len lity the presence of die varnms dioxins.4 Pil'fcr- eni tliuxins can be tlisiininiisiictl from each .nher by the number n i chlnrincs winch they contain, there are eifiiit po sitions where chlorine could he attached lo the basic dihcn/o-dioxin structure, as shown in Figure t. This m akes a variety o f dioxins possible ranging from (lie mono form lone chlorine atom ) to Ihc oriu form ieight chlorine atom sI. Then, depending on the nrienlalion o f these chlorines in space about the m olecule, various isomers may also he form ed (il in all are possible for Ihis particular chemical structure. Although the dioxin found in 2.4.5-f is the letra form (fo u r chlorines are attached lo ihe basic di oxin stru c tu re at Ihe 2..1.7. and x posi tions! and this form i l i 'l i n i has re ceived m uch a tte n tio n , this is n o t (he only toxic form , as we'll see later. In Ihe i is i i \ investigation. 2.< o l 4 2 sam ples o f 2 .4 .5 -1' were co n tam in ated w ith dioxin as well as 4 o f (i sam ples oi inchlorophcnol. all 5 samples ol letrach lo ro p h en o l. and ID o f II sam ples m l*riv th e dioxins containing 4.i>.i. aim CD CD 00 LO S chlorines were found. Ih e contam ination of r<T by dm xir. was co n ltrm ed in a later study in w in d : the investigators looked at l'( I* alone Hoih dioxin and anoiher contam inant d ih en /o tu ran . were loiinil.. In parrs oer tnillioti.ip.ptni ..| llie m il were t.iu lid . l a s ppm ol la-ilioxill. and I f4 ppm .It th e all. i ' lie\a->l. Ihe hep. u t a !. O o o vm. Ilcta- m d hepl.it h l.ir.lib e n /. . 1 . tails were ..leiuitie.1 I'nt the 111.011:1 . ts -U' a . `I >1.-le iM iiu e.I * I he I'j s i . -I: .. I II ' ! I.l'fll/.ll II III s IIs. I s||. >wu o n- CD I gin I i While 'le* et e viinplc' I - l in litis slii.lv .vele pi.Hli.s.-d r e t i I o .o ilu- j iili.ir. pointed ml th a t i f 1 samples from a 1970 batch in another study xfac contained toxins - 0.5 to 37 ppm of the hexa-dioxin and t o to I3S ppm of the bcptatiioxin. The pres ence of the dioxins has also been con firmed by studies in other parts of the world - a recent study by Swedish in vestigators revealed the presence of SO ppm of octa-dioxin in a sample of Per.5 : r. Form ation o f D ioxins and Fusaas H ie dioxins found in contaminated a n , JJles of l,4 i- T and in the chiorophenols arc formed during the manufacturing process (Figure 11. During me process a reaction between two molecules of trichlorophenol may occur resulting in the formation of tetra-dioxin (Trunk* The formation of TCDD during the manu facture of trichlorDphenol and 2.4,5-T has been documented by a number of chemical companies both in the U25. and Europe. The same sort of conditions (high temperature, high pressure, and alkalini ty ) arc necessary for the production of P C P when hcxacfalorobenzene is used as the starting chemical (sec reaction 2. Figure I ). Under these conditions both / the dioxins -and the furans may be formed.5 Alternately, the cfalorophenuLs may be produced by the direct condensation if chlorine and phenol (see reaction 3, Figure I ). When too much heal is ap plied. especially during the manufacture o f tetra- and pentachlorophcnol, the more highly chlorinated dioxins can be formed. It has also been proposed that the dioxins may be formed after the manu facture of PC P, for example when wood is treated with this substance under highly alkaline conditions. Wood may also be pressure-impregnated with pcp, with the combination of high pressure and alkalinity leading to the formation o f dioxins. Once in the environment there is the chance that p c p and 2,4,5-T (which may have chlorophenol impurities) can become exposed to very high tempera tures. This might occur when brush sprayed with either ol these chemicals ts burned or when wood products impreg nated with PCP arc incinerated after dis posal. Dioxins might in this way be re leased into the environment. (One way o f producing dioxins, for which there is a Canadian patent, is to heat 5 grams a gram is equivalent to 0.03S ounce o f pcp at 300.degrees for twelve hours. This vieids 1.5 grams of the ucta-dioxin r Toxicity neern about the reieasr of the dioxins -*to the environment stems from the 36 fact that thrt a*r extreme!' toxu sub stances For example, tin- iIiktoI 7('t>l> -needed to kill 50 percent ot an experi mental group of guinea pip. is ().<- microgram nine microgram e- one-milluinth of a gramI per kilogram of body weight." In an experiment in which rab bits were fed 1 Ci>l>, a Jose of 10 micrograms per kilogram killed* all the ani mals. while at a lower dosage of I mi crogram per kilogram, liver damagr and cUoracnc (a skin diseawi resulted. PCP on the other hand, is toxic to ex perimental animats in the milligram per kilogram range." (A milligram ts onrthousandth of a gram.) One of the higher chionnaled dioxins fthc hex-dioxw> found in pcp has abc hecn shown to hr toxic. This informa tion was gathered after the accidental poisoning of millions of commcrcialfy raised chickens in the I'-S. in l u5~. in some flocks, therr was as high as 50 percent mortality. Affected chickens had droopy and ruffled feathers as wel! as difficulty m breathing Autopsies showed fluid accumulation in the mem brane surrounding the heart 'hvdroperi cardium). beneath tht skin <subrutaneous edema l. and in tltr abdominal cavi ty, and ii*cr and kidnry damage. Un:i: the substancr responsible lo* :lu- ep>- demir was later idcnttfied. _it was callet the "duck edema factor.*'1* The cause of `.hr discasi was at first traced to par. ot the birds' diet - to tin fats added to commercial poulry feed Later, a toxic fraction was isolated Iron: these fats and found to contain cm: tine. It was suggested at fust that the substance could hr a chlorinated naptiwlcnc.13 The chlorinated nap'.lialenc:. have been known to cause a condition called hyperkeratosis in cattle, fHyper keratosis involves thickening and hard ening of the skin end proliferation o) crib of the mucous membranes i How ever. this substance was ruled nut when samples of letmchlorunapthaiem and hexachloronapthaieac failed to producc tbe symptom of edema disease seen in chickens. Finally, in 1906, x-ray crystallngraphers at Proctor and Gambit- de termined that the toxi; substance was hexacfaiurdihenzo-p-dinxir."* This teaconfirmed when a chemist at Proctor and Gamble synthesized the hcxa-di.-xtii end showed that it prnduerd chick eiirBn disease. During tfar search lor :ht- nientitt ot the unknown chemical substance whicti had caused the disease sampler ot u.xi. lat were tested in a variety iahoratt>*\ ammris. Adverse eftect: we*t- roiuid it. munkrvs. guinea pip. tiop rat-, and Ptf1 Became thr toxic lacto; c.-uid fir recovered front Uw tissw ->! chid enled thr substance ttu cucral Food and Drug Administration u ;.-. i ruled in l i ( I that eoRimercu! fats bad to hr screened lor the -chick etlema factor hr fore being placed .us thr market. Thr chick edrma (actor hid atv> hem found in fat samples which wen lo hr used in food tin human hemp.1* After identifying thr factor as a di oxin. the sourer ol the dioxin was still a puzzle One suggestitm was that thr di oxin fin contaminated samples of 2.4.5-T or P i n was originally sprayrd onto crops (such as enrni from which vegetable oil was later processed. Anoth er suggestion' was that since heat is needrd during thr preparation ol thr fats, tfar dioxins could have hern formed as condensation products ot p( P or other cfalorophenob which might have been present in thr onpnal vegetable material. LI 1 OCooO CCOO The chlorodilienzoiurans. which as wr mentioned earlier were found as con taminants in re samples along with thr dinxins are ais<> toxic Researchers have noted a possible relationship between tin- toxicity ol ;hr Pi h:. ilfar potvefaiorraa'ed biplicp-'L-.i and their level ol eontammatior with tetra- and pentachlonlibenzoturai. in a recent study, three samples o! pi b-PlM.-nocior iproduced in Francri. Clophen (produced in (ermanyt and Arocior tproduced by Monsan to ir thr l -S I - were fed to chickens lo: od days, after which thr surviving animals wife killed and autopsaed. The animals receiving Phenoctor and Ck phen all died during thr course of th r experiment; then symptoms included ruffled leathers, loss of feathers, and kiss ot (macular coordination. awl thry * wen- found to have hydrupcncardium, alulomuial and subcutaneous edema, and liver necrosis (death ol liver crilsi. On thr other hand, only 15 percent of thr group receiving Arocior died during the experiment, and only a few (tfarer of twenty) developed hydrnpericardium: Five Japanese quail and eight white rats were also fed Phenodnr. The quail all died within 13 days; necrosis ol tin- livet was revealed upon autopsy All eight rat: died wuhtii days and showed' enlarged Inert, iwith local ne crose.) and disappearance of part ol the structure of thr spleen."' A subsequent investigation turned ut> tin- tact that Phenocior awl Oopfaer b.<;n contained tetra- and pentacfalord*henzoturan The authors at'.ribu'rd Ibr greater toxicity o; ;im r cr-mpound th r iactot aw! -aiggr'.'rd that tin *u-ar. could *n , lo'nird wtien c*wi p h r dis'.ibeu O ccupation! Hazard Tin cfalortna'.rd pvdr-i-arbvins have king (mmanw. d it. * V 00i FKiMiit: I NOTICE: POOR COPYHUETO ORIGINAL , DOW 090040 Ma-ac S tru c tu re o l -/in (I .m o o r) Ui:<iMii(> may Lj in v jn o u s combina- tton-.. J t th e 1 . 2 . 3. 4, ii. L . / . d . or '} poM ti-jnv ( ] ) Tetrachlorobcn/crie (ij-ac S tru ttu re of U itic ru o lu id fi (f uran). (?) Ifexathlotobsn/cnc fncjh temperature (3 ) Chlorine * Phenols -- ... PCP (1`oxm <ontjrnifijritS K-i-n a k n o w n h a /a id In w orkers. Ilic chcm icals w hich wc have m entioned m > I j r m its. chlorinated napthalencs. thlorophenolsi cause a condition know n as chloracm.-. o n e ol the m ost frequently incurring toim s ol occupational derma- litis I uit lam in j l ion o f IIn- sk in I.1* ) lie iliH-jM- in n o l cau sed by c h lo rin e a lo n e , hul hy chlorine m cotnhinalion with certain organic eultstancc-s. micIi as p h e nol. In laic I 'M n . several i j m -n o l c r- acne were rc-poMed ill a (e rin an ci. ...t- cal p lant in v o lv ed ill th e p ro d u c tio n o f i-i I*, usiiip lie x a c lilo io h e n /e iie in th e process Som e of the sym ptom s, which the jltc c lc d m en began to sllow neaiiy live m o n th s a lte r th e s ta r t >>t i-i l* p ro duction. included severe acne, in v.rne >ases w ith u ile c tio iis. hrow u skin pig- ilicntaliori. and neuralgic i nerve I pain in the low er eW rcm ilies. It-w.is rc-cogm/cd th a t c ilh e r I'l l' ilsc-ll o r an in le ru ie d ia iy p io d n d in its p ro d u c tio n could have lieeu tlie- c a u s jtiv e .i|!ent in a d d ih o n . come ot the m en llad also been exposed to letiachK -rohen/ol and tinhlorophc- nol 1 ' \ lalei re p o rt ae.m i trorn t 'iiu.iiiv . in I*'- s chi-wed th at :t.e .<*iilal:u:iatil- in . l.li 'ii 't'h e t.o ! pit -d tic 11*ill in d ee d ..ill .aasc . In .|_c lie In iliic .jc .- -I w. :se : - w li-- | - : . | . i ; . d I n . 10 I.--I .u..i 2.4.5-I developed ci.lol.icne. U 'lU ) was .solateli Iro m th e lu n ch ed p ro d u c t jn d w as lo u n d t o Ih; a p o te n t a c n e -p ro d u c ing atteri I in ra b b its. I r i- a tid te trac h lo rodilH-ii/.oluran w ere also tested on ex perim ental anim als and found In be ac tive a cu e-p io d u cin t! atteri Is In tac t. I ( t il l p ro v e d to be su c h a slro n tt at-'e n t l a **.415 to O.tlfll p e rc e n t s o lu tio n was needed In produce acne on the rabbit e a rl d ial ill a late r a tte m p i lo dev elo p a stan d aidi/ed procedure lor i|iiantilyiut! the e tle cls ol chloracue producili)! sub stances I r 'lili was chosell as the lesi s u b s ta n c e ." 1 < llloraene has also appealed js a p ro b lem m l-.S . w o rk e rs. In I'M*# an acc id e n t ill w in ch iiiteiliie d ia rv . Ilciuicals were leleased al a 2 .4 .5 -1 plant I M o n san to t led to I I ' cases o l c h lo taciie In a later in cid en t hi th e I .Y . th is lim e at How t heiiuc.il in l'r.4. n n n was singled out as the causative aeenl a .ti l ll a d l e o u lb ie .il. Ill o ld er to in crease the- p ro d u c tio n rale <>i J .f .5 - 1 . llocc had .h a u tte d l- m-.i. I iou .o n d ilio n s le s itllni)* in a n h i. tea se in l< l ' l> colllelll '* ler.iliieeiucilv Itile ! -I I!.. ,1.-1.Is ..r Ills .1,....:. I .-t* J 5868 produce b u lli d elects beeau willi a stu d y carried o u l h y llio n e iic s Ke scare li la b e lH-lweett l' its aliti I -*-'< u n d e r eoulraet lo th N ational I aiicei Insti- tu te : 2 .4 .4 -1 was aiuoli)! ihe 4* pesti- eides w liicli w e re slu d iv d II vvas lo u n d thal pre)!ii.inl lince, tre a te d willi 2.4 5-1 al a Ic-vcl ol I I .* n iiliig ra m s per k ilo e ia m ol body wei)!ht per day prodiiccd a grcalcr pcrccnlape ot abnorm al litteis ol il|s|iriri|! as w ell as ari ilici case in th peicenla|!c- ->l a b n o iiii.il Iclli't-s per li! - ter. I Ile- iii.iioi ly p e >d abuoim .ibli-.-s intlc-tl w ere d e l l p a la le am i sy slic kid* lice . .Ahiiotm allllc-e. as well as ilii 'cV'C-s Iti te la i n io iia lily . veere al'-* piotili- e d in la te al v.uioiis dosc-s Al all Jusi-s ili Ibe la i. h e n io rth a p e ol ilu- ; islio iiile -liii.il tr a ile o| th le tu s veere o h e e tc -.l I ln-s-.- re su lis. Iioccever. c o n iti n-d be ilvttrtrli le linke .1 1- .4.> 1, h k!**>!c* 1 teas ll.M er ed lll.it (lu* N jinplc II>1 il iin rli,L Nl'ItlV tornam eli ;o p 'Il l( I t i ) l> * ' II tl.f Njfi.r r. .li tu .1 li. V.CVl'f Iti il 1 HI * c ir. . . *' - *li 1(| ili'!Vat n whenI !..l l-t l'fl'L'Jl.lM ' Idi .1if 1 li. ile 1ri .1 *;;ulv ni i.l.- le 1* !I*. i! <--tnpai.v d '.i.-e '.-..o e ' -e : ' I '> ' . :m . r -gl !:. - - ; ; - v-r >: : dv .e : :-d , c:: .' O O a iO > *1 m j . i c 1 v u m K i ' i ' ' a t I I ** N ) *; lu ll' ! I r u n . 'n m i lil.il III alili S.ii-ii. . > I l I I Il j s 11' , | i'il m i l l l l f c M i .i i i i - *1 m in ' .nut in m u ' s t u m -I 1 .1I > I' 1 p ii>ilHifil i ll'll p a la le III .ill llili'i* "I'.iilii ni unii- lull uni in tlif M l. fnl ncy .il' lu iii'ljlilii'i iii'ii; seen ill b o th IIn- m in ' uni tin* u t v * Liivinnim i'iitul (.'tm iam iiuiim i llu- ilinxiiiN. 2 .4 .5 -7 . jm l f i r iim i c u ter ilic I'liv iim iu ii'iii lim n .i n u m b c i ul sm itccs. O n e so u rc e n l M iilm ni-fi r is dii' i'll Iiia i l u l p a p er p u lp la c to n e s which use I'l l' i.im p o u n iK in sliJil ol nii'ii'iiry as slim c-cm iliolling agents. Many experim ents have show n dial I'l l', as n o il a s iiic io u ry , is to x ic to t'lsii in m u' ex p erim e n t one-hall o f dio guppies k 'sli'il at a li'Vol u l ppm I'll* were killed in sox on d a y s. T h e p u p p ie s itiulil survive a lelha! dose ni S ppm only aller tirsi having licon aciliiiialcil at lower doses (th at is. placed in w ater with a low I'lineontratim i ol I 'l l' toi a few d jy st. T he a u th o rs ol this stu d y compared the toxicity ol I 'd ' to that nl m ercuri' com pounds (such as phcnylmercuric acetate and inercuiic ch lo n d el and found d ial i'l l' is as toxic in 25 species o f fish as m ercu ry is In pup p ies, rainlxin tro u t, and stickleback. lu rlh c rm ore, th e a m o u n t o f I'l'l* needed for edicicnt slime rem oval was above the lethal ranpe lor m any fish, and m ore I'l'l* th an m e rc u ry is n e e d e d .2 '* The authors noted that the concentrai ion o f I 'l'l' needed to kill slim e was above dial w hich could kill aq u atic in vertebrates (such as insect larvae and snails). In fa ct. IV I' has been used to 'k ill schistosom e-(a type o f parasite I carrying snails dial live in Ig y p tia n irrigation ditches. Levels o f 10 ppm o f copperI'ci* are leth al to so m e snails, a n d 15 lo 20 ppm w ere used on the l-.pyptian snails. These c o n ce iilia lim is. m oreover, also affected th e local lish p o p u la tion.* While u e k n o w (h a t Ihe d io x in s a re extrem ely toxic substances, ano th er im porla!!! q u e st im i is w h e th e r <>r n o t th e y persist in die en v iro n m en t. A lthough Ihe d io x in s a re heal stab le i i i n n is stab le up In HOIJ d e g ree s c e n tig ra d e , o r 1.472 degrees Talirenheil I they can he decom posed by su n lig h t'u n d er eerlain circiim slum es. T \perim i-iils have show n lh.it m alcohol solili unis, um ici naturai siinliglil. i r m i tlccm nposi-s rapidly I u ni n i Immus W hen l i UH III m ilh a uoi was pl.iii-d m i sm l Ira lh e r iliau in he.ikeis m ijosi'.l lu b i'si. h o u e ie i. so Hk till-1fi J I,i .| Wiilklii cv.ip* >1 ali*. >1 li.iii tu 7 vi i \ * ::iip* iN(.J .ilU 'l ! tur* l e m il.tV * 1 (u* .itili h . t ' . . | rh i^ ' 1Ull \ y' l u i . li 1ll.ll Ih w .1 . k l t l u h ..1 II'.Ai ' ! . 1. ' 1 1 . tile i . > l i . :.i Ih u 'lt l ; i v f IIn v< ;; 111:1. (; r: I . Jn*`:<u j llv t .1' ( h i :... Hi <M i i .J ; ,.<I.J <i 'I.. I li Mi ` 'I l In- ill-111'i . Il I I .1 * *1 In ; i i . u . l *11 I1.Hi' *u l ' i . i ' . ou1.1 .ii i liluul.iie .:( ..| V ..|li` t 1 i* i * A III* *' r is'. " I l l . i t . h i f i i t i i - i l i * ii.i< Il l l l l l l l l l . l l l" M 11 <" 1111 1 h i - . .h l l.l ll o l l' o l I I II > I I I H . .11.>1 M in p p m III h c u /c i ic l n e i e ap t - l i . i l i. r . \ . . l*. PV*. >| i o ||. ,i vindy '.oil alul a . lay lo.im . Soil sam ples w ere th e n te s te d j l t c i .''l. 4 0 fill. |d ll. a n d 5 0 d a is . A llc l .5u il.i'. s. 54 p c ric n l ol d ie I p pm H l>l> -a m p le was ic c o ie r c d lo i b o ili ty p e s oi s m l.. pern-ill o | the Kilt ppm sam ple was rc c o ic ic il ill th e sandy soil, an d " I pet- eenl nl die Km ppm sam ple was ic in i- ered hi th e clay sm l * ' Ihe possihihly dial die dioxins couM be lo im ed hv bacterial a i l n m in die sml was also te s te d , b e ea u se il lias been show n, lor exam ple, dial m ic ol Ihe breakdown products of propam l (an herb icid e). .t.4 ili-clilm o an ilin e. is c o n v e n e d ill th e soil to 5 .5 '4 .4 '-te tr;i.i.'o - ih lm b e n /e n e. a subslaiice closely re lated In chem icals which have produced cancer hi experim ental anim als. Dichlo- rophenol and trichlm ophcnol were both incubaled in tw o ty p es ol soil hi order to determ ine w hether or not a baiterial condensation reaction might co m eri them lo d ie dioxins. T hey were iiicu- baled at levels o f It). 100. and I .m m ppm lor 70 days no dioxins, howevei. were reeoveied alter this tim e period. While th e I'hlttrophcnols an d their de- rival lies represent only a sm all Iractim i o f the agricultural and industrial ill- lin eals e n te rin g th e e n v iro n m e n t, il is clear dial Ihcy c a n y w ith llicm problem s lar beyond their ow n di-siruilivc proper ties. Sin c alm ost all organic ih em ical reactions produce com pounds other than the one specifically sought (the p o p u la r insect icid c `lo x a p h e n e w as u - cently found to be com posed of 4* d o le rent I'lim poiientsl. Ihe diseoiery ol these to x ic e o n ta m in a n is m ay be a sig nal lhal a th o ro u g h analysis o f widely used chem icals is long over d ue. Ii NOTES 1. Revenue, A rth u r, and H e r r u n H e rb m d d , A D is cu ssi on it it s f'r u p c r tic s n d Its O c c u r ie n e v as 1 M osid u c m H u m a n a m i / t i m i d i lissnus,** R esidue Re* views. J 9 H .1 B 4 . 1 9 0 /. 2. S y n th etic O rganic C hem icals. U .S P ro d u c t io n a n d Sales, w .5 I .in L u i m -.iv*,i .h . J. P lim n ic r . J a r * M .. J t.h u f.V h * ! ., a n d t U w in A , **r/,ss 'i i i u f i K 'n i p t n i iigr.-ut- l(ijctatilciMiit/iKo-of dtrti'eu*dMixentaandmdIhhcn. r. * *ir d ijt r r i 1 r t c n 'u c j i lV " ! jy d (i>.i't" j Aqr a n d P o o d C h e m .. 2 U I i * 9 j, Jj -> .*0 i*W 4. W.m.e...... i-|, i* . jio . Tn ..i*.. a n d Pet' 1 j | flilfi'tt.'.i j ili td (.d ilr I. f 1 ... 1 1 ... 1 ufl*'!." J. Agi 1 l*i/2 *. J*!.\ *. . ..< a.jU . . .1 '. ir. l *U '. l * / .4 s t . t * . . .. *,... i,.- . . .1 . ............ ............. /la . ............... * . i*.' .* . 4.* . * '*. 1 . V . 19 / 1, li 1`. / >., tr*'t` .a*.. .i**! *. *. *.p 1 *51 ; H * t, f *:i". * , " / * .i**.| / i *. C n a iio n r t tr n t , J .* |h | .*1 , J**t . 1 .*, r i t u x 9 R e p o rt un 2 .4 .5 T . */ ? . 0 4 9 |M f , M l . f ,, * ? I I . , " I * t* .|f|............I . . J , / . f J* tll ll 'i f M . i M I / 'i f ] l 'i l l l t i / lti***fl.|| M ir i li *.f ` finii*' .* .4 .5 > *. h l-.r i O(im ii N ature. ? v , jn| 1, 1 9 / 1 . Q 0 12. f *'l*'it:, I f i i t i f l l / , bVilii.i* | rftr lid i. f Hii'ti'*.**, > K.ul* V . K t/f* !ii* * *i, tei 1% *U " (' MdtfltS if K 'itlit* t*(-. j^l*-***! . S*!<lMir<t lV M tiK ld , fK i|titi'ii.tl< :l " J in d u c i. 1 tien e and T o *.. ? i( 5 | to n I /? . f/a y 1 9 (9 . 1?. " I ite (tue* >.<|i*ini t d i 't i , " N u t r i i R t r i r e t , ><<r..<lH, 1 9 0 I J / i / n t l , A u d i :* . ( j.iv i*- I iri!st*m:. D a fiani*,. V iliijrn M ' f f w i t / , 1 #?* I ii.*uuian, S l a n l e / N v U i m i i i i , * * 'it u d iv `< n i th e t.iu c t* t ini t a.tor, i l . is o la tim i u t 1 T o ic * s U n ie ," J. o t A m . O tl C h e m is t' & 3H CO 0 2 . &. i O O l. $ coo o OAi 14. '*^ea n ti tu C ln c ^ t dcna I mcI * , " C he cal and C n tm eerm t News, Jan. 30. 1 9 0 / 10. 15. Um igiass, t . j r i l i . , and O n n aid H " C 'tlU U td J lifU b i'U S M y fir C Ii k i * f d e n ta f to#,** J. Assoc. O ffre . A g r. Chem ists. 44 I9 t l. li. Vo s. J .ii., and J.M . Kr*e>tian( " C o m p . ture 7 d u (o lu i|it. U *udy w ith P d ly c h lirfiiu & iD h e n y ts m C lu c k c n s w it h Soc*.Mi M e le r tu P u rp h yii.t. t dom a l orn*ti*m , Li^cr N et sis. a n d Tissue M esidues.** T o * . A p p i. Rhar l/:tiS O *46. 197 0. 1 / . V o i . J.Ca., J .H . K if iu a ri. H . l . van W.ij s , M .C . le u N 'ttv v r d e U<iue, and M .H V o s . '*td e n lilic a tu n a n d u*ic>l</<jicai b v. atm n ut inatvd r)tb e fi/u fu M n and O rin ate ci N a D tlia ic n c n T w n C n in itu .'ic ia i, P< chiurin atcd H io h e n y ts /* Fd- Cosm et. T o e ic B .025- 3J. 1970. 16. S chw at/, L ou is. L o u is T tih p in , D o n B irm in g h a m . O c e u p a tio n a l Otscases o f SkiA . Lea A F c P tte r. Ptoiadeipnia, 1 9 5 7 , 33C. 19. B a a lc t. L .W ., an d H .J . B auer, " InduSt lntuBi.tfiitm f>uc to P e n ta cm ^ ru p h en o i.'* d u s tn a i M e d ic in e an d S u rf < 2 0 (0 | ?46 ?90. June 1951. 2 0 . K n n rm g . V o i , J ,, a n d K .M . S c h u i/. ** ru flich e A n n e (soq. C h lo tan n e) durch et iene arom attsche /y*lische A ttiv i,'* sumr ry. O erm atologica. 1 1 5 .5 4 o . 1 9 5 /. 2 1 . J*nes, f . L t n n , a n d H e le n K r /e e . T erm ite fu i Testm g Acne>tenic i*ntcncv RabDtts, A p plied tu lite Poteot A tn cg 2 .3 , / , 7 etractiinii!dt>cn,'o p d io * m ." v e it. O crm ., 3 9 :5 1 1 *5 1 /. 1902. J. 2 2 . R e p o rt o n 2 ,e ,S * T , ut c it ., pu. 10 . 4 6 . 2 L C o u rtn e y , K . D ia n e , (J.W . C*ayiOi. M H n g an . H .l . f a lk , M .R . BatcS, and I. f / i l t h " TeratiOcn>r t-vauatio n o l 2 .4 .5 1 Scicn I0 6 604 660. 1 9 /0 . 2 4 . S o arsth u . C.L., f .L . In in n , and V M o w e . ** M u d y t>7 Ih Teal'fqc*city 2 .3 . / . 6 - T e o j t h i * ii n d i o e o . < u < ! > '" M a t," F o o d C s m l. T o * . . 91 J i 4 0 5 * 4 1 ? . J. 1 9 /1 . m * 2 5 . C.f.HjrtnCy. K . P U M C , a n d John A . M<* M1 e ra t 'iiori S tu d ic s w it h 2 . 4 .5 7 n i.n io r o n - yarctic A c id and 2 .3 ./.e fe tia rtiin id ih p d i> t n tM T e e . A p p i. Fharm . 2 0 % - 4 19/1 2i Nurup. BiaMie. " To i m ! t 1 M!l Jl P a p e r r j i t o r y r f flu c r * fs .* * W a te r Resear* Pe*4J***.n Press, O J 5 6 5 l i n e . 1 . / b . r a<ur a**d B e . :.* 9. 91 no 2 8 ( u s o , l i . - . . M S % ;>. * a n d k M /nis>i, " M ti .? i t it .i* ia :i- i :(!*' i d i i.'* : / > / : * / a , a ?i, ; * t , Sere* cd 0c0o IO .1 .il;, ( , t f.'. I!.. li x r.i 1 .1 ni*ieniii Sw ir'K f 1 1. 1 i 1 / 1 ! 9 . f4 | Tr hi. >1*1 000 C2 ec Or > co O 00 er DOW 119368 Chlorinated Dibenzodioxins and Pentachlorophenol by R.L. lobasia,' P.l. Gehrinc/ R.l. Kacika/ aid B.A. Schwatz* Introduction Pentachlorophenol is a registered anti microbial agent whose principal use is for the preservation of wood. Typical commer cial pentachlorophenol contains a variety of substances which are considered to be "in active" from the aspect of antimicrobial ef ficacy. Consequently, pentachlorophenol is sold as an antimicrobial agent with 95% active ingredients and 5% "inert" ingredi ents. Analysis of acceptable commercial pentachlorophenol is shown in Table 1. The "caustic insolubles," sometimes re ferred to as the "nonphenolic or neutral im purities," include chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans (I). Recently developed analytical technology has allowed quantitation of hexachlorodibenzop-dioxins and octachlorodibenzo-p-dioxin in pentachlorophenol. Portrayed in Table 2 are concentration ranges for these two chlorodibenzo-p-dioxins in samples of currently available commercial grade pentachlorophe nol. Techniques capable of detecting 0.05 ppm showed no 2,3,7,8-tetrachlorodibenzo-p-dioxin in any sample of pentachlorophenol examined by us. The absence of this compound in pen tachlorophenol is not surprising, because the appropriate precursors for its formation are not present. 'Designed Products Department, The Dow Chemi cal Company, Midland, Michigan' 48640. tChemical Biology Research Laboratory, The Dow Chemical Company, Midland, Michigan 48640. Table 1. Commercial pentachlorophenol composition. Pentachlorophenol Tetrachlorophenol Trichlorophenol Higher chlorophenols Caustic insolubles (maximum) Content % 85-90 4-8 <0.1 2-6 1 Table 2. Concentration ranges of some chlorinated dioxins in commereiai pentachlorophenol. .Chlorinated dibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzop-dioxin Hexachlorodibenzo-p-dioxins Octachlorodibenzo-p-dioxin Concentration range, ppm None 9-27 575-2510 Heptachlorodibenzo-p-dioxin and hexa-, hepta- and octachlorodibenzofurans have been qualitatively detected in commercial pentachlorophenol However, the lack of ap propriate standards for these materials does not allow their quantitation. Severe toxicological responses have been attributed to certain chlorodibenzo-p-dioxins (2 ). For example, the LD50 of 2,3,7,8tetrachlorodibenzo-p-dioxin ranges from 0.6 fig/kg in male guinea pigs to 115 pg/kgr in rabbits of mixed sexes. A benzene solution of this agent containing as little as 0.04 /xg/ml produces acne in the rabbit ear bio assay. Very high embryotoxicity and the production of edema in chicks are other properties of this material. The no-effect September 1973 171 0001357 5871 2 DOW 119370 commercial pentachlorophenol. A positive re sponse was noted in both chick edema and rabbit ear bioassays. In the 90-day rat feed ing study, untoward effects were noted in a majority of the parameters monitored. Hem atological examination revealed a depres sion of erythrocytes, hemoglobin, and packed cell volumes at a dose level of 30 mg/kg-day pentachlorophenol. Clinical chemistry alterations included an elevation of serum alkaline phosphatase at 30, 10, or 3 mg/kg-day and a depression of serum al bumin at 30 or 10 mg/kg-day. The weights of liver and kidneys were increased at 30, 10, or 3 mg/kg-day. Pathologic examination revealed minimal focal hepatocellular de generation and necrosis at 30 mg/kg-day. Thus, it is evident that commercial penta chlorophenol induced untoward effects in each of the three toxicological tests. Table 3. Concentrations of chlorinated dioxin "indi cators" in commercial pentachlorophenol utilized in toxicological evaluations. Chlorinated dioxin Octachlorodibenzo-p-dioxin Hexachlorodibenzo-p-dioxins Concentration, ppm 1980 19 A chemically pure pentachlorophenol hav ing no detectable concentrations of any chlorinated dioxins was subjected to the same toxicological tests. The toxicological data on this chemically pure pentachloro phenol, summarized in Table 5, include neg ative responses in both the chick edema and rabbit ear bioassays. In the 90-day rat feeding study, the only changes noted were increased liver weights at 30 or 10 mg/kgday and increased kidney weights at 30 mg/ kg-day. However, in contrast to commercial pentachlorophenol, gross and histopathological alterations did not accompany these in creases in organ weights. Thus, by utilizing the results of these three tests, it may be concluded that the presence of the contami nants in commercial pentachlorophenol may be detected by toxicological evaluation. An analysis of a sample of pentachloro- Table 4. Toxicological data on sample of commercial pentachlorophenol. Study Chick edema bioassay Rabbit ear bioassay Rat feeding study Food consumption Body weight Hematology 30 m g/kg-day 10 m g/kg-day 3 mgAg-day Urinalysis Clinical chemistry 30 m gA g-day 10 m gA g-day 3 mgAg-day Liver weight 30 m gAg-day 10 m gA g-day 3 mg/kg-day Kidney weight 30 mg/kg-day 10 m gA g-day 3 mgAg-day Pathology 30 m gA g-day 10 m gA g-day 3 mg/kg-day * -I- denotes effect; -- denotes no effect. Result * + + -- -- + -- -- -- + + + + + + + + + -1- --: --. phenol representative of that which we are capable of producing is shown in Table 6. The toxicological data on this sample of pentachlorophenol are summarized in Table 7. Both the chick edema and rabbit ear bio assays gave negative responses. In the 90-day rat feeding study, the only unequivocal changes were increased liver weights at 30 or 10 mg/kg-day and increased kidney weights at 30 mgAg-day. There were no gross or histopathological alterations noted. To reiterate the toxicological findings on these three samples of pentachlorophenol, a comparison is provided in Table 8. Commercial pentachlorophenol gave posi tive responses in both the chick edema and rabbit ear bioassays; in contrast, the chem ically pure pentachlorophenol and the im proved pentachlorophenol both gave nega tive responses in these bioassays. In the rat feeding studies, commercial pen tachlorophenol was associated with hema- September 1973 173 0001359 5872 DOW 119372 Rodenticide Act. The composition specifica tions for this new commercial pentachlorophenol are cited in Table 9. In conclusion, it is feasible to produce a pentachlorophenol in commercial quantities which by comparative evaluations mimics pure pentachlorophenol in toxicological re sponses. Table 9. Composition and specifications of improved pentachlorophenol. Content Pentachlorophenol 88-93% Tetrachlorophenol 12- 7% Trichlorophenol <0.1% Higher chlorophenols 0.1% Chlorinated dioxins Octachlorodibenzo-p-dioxin 30 ppm (max.) Hexachlorodibenzo-p-dioxins 1.0 ppm (max.) REFERENCES 1. Plimmer, J. R., Ruth, J. M., and Woolson, E. A. Mass spectrometric identification of the heptaand octachlorinated dibenzo-p-dioxins and dibenzofurans in technical pentachlorophenol. J. Agr. Food Chem. 21: 90 (1973). 2. Schwetz, B. A., et al. Toxicology of chlorinated dibenzo-p-dioxins. Adv. in Chemistry, in press. 3. The Dow Chemical Company. Antimicrobial agents product literature, Section IV-7, Dow Chemical Co., Midland, Mich., 1969. 4. Adams, E. M., et aL The response of rabbit skin to compounds reported to have caused Acneform Dermatitis. Ind. Med. Ind. Hyg. Sec. 10: (2)1 (1941). 5. Official Methods of Analysis, 10th ed. Associa tion of Official Agriculture Chemists, Washing ton, D. C., 1965, Sections 26.087-26.091. September 1973 003Q t 175 5873 Il  DOW275Z82 TOXICOLOGY AND APPLIED PHARMACOLOGY 46. 279-303 (1978) Results of a Two-Year Chronic Toxicity and Oncogenicity Study of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Rats R. J.K ociba, D. G. K eyes, J.E. Beyer, R. M. C arreon, C. E. W ade, D. A. D ittenber, R. P. K alnins, L. E. F rauson, C. N. Park, S. D. Barnard, R. A. Hummel, and C. G. H umiston Toxicology Research Laboratory, Health and EnvironmentalResearch, Dow Chemical, U S A . Midland, Michigan 48640 X ro o o si 03 Received November l , 1977; accepted February 22,1978 Results of a Two-Year Chronic Toxicity and Oncogenicity Study of 2J.7,8-Tetrachlorodibenzo-p-Dioxin in Rats. Kociba, R. J., Keyes. D. G - Beyer. J. E , Carreon. R. M,, Wade. C. E,, Dittenber. D. A,, Kalnins, R. P,, Frauson. L. E , Park. C. N.. Barnard. S. D.. Hummel. R. A - and Humiston. C. G. (1978). Toxicol.Appi. Pharmacol. 46.279-303. Rats were maintained for 2 years on diets supplying 0.1.0.01, and 0.001 pg of 2.3.7,8-tetrachJorodibenzop-dioxin (TCDD)/kg/day. Analysis of these diets indicated 2200, 210, and 22 parts per trillion (ppt) of TCDD. Ingestion of 0.1 pg/kg/day caused an increased incidence of hepatocellular carcinomas and squamous cell carcinomas of the lung, hard palate/nasal turbinates, or tongue, whereas a reduced incidence of tumors of the pituitary, uterus, mammary glands, pancreas, and adrenal gland was noted. Other indications of toxicity at this dose level included increased mortality, decreased weight gain, slight depression of erythroid parameters, increased urinary excretion of porphyrins and -aminolevulinic acid, along with increased serum activities of alkaline phosphatase, y-glutamy! transferase and glutamic-pyruvic transaminase. Gross and histopathologic changes were noted in the hepatic, lymphoid, respiratory, and vascular tissues. The primary hepatic ultrastructural change at this high dose level w u proliferation of the rough endoplasmic reticulum. Terminal liver and fat samples from rats at this high dose level contained 24.000 and 8100 ppt of TCDD, respectively. Rats given 0.01 pg/kg/day for 2 years had a lesser degree of toxicity than that seen at the highest dose level. This included increased urinary excretion of porphyrins in females, liver lesions (including hepatocellular nodules), and lung lesions (including focal alveolar hyperplasia). Terminal liver and fat samples from rats of this dose level contained 3100 and 1700 ppt of TCDD, respectively. Ingestion of 0.001 pg of TCDD/kg/day (-2 2 ppt in the diet) caused no effects considered to be of any toxicologic significance. At this lower dose level, terminal liver and fat samples each contained 340 ppt of TCDD. These data indicate that continuous doses of TCDD sufficient to induce severe toxicity increased the incidence of some types of tumors, while reducing other types. During the 2-year study in rats, no increase in tumors occurred in those ran receiving sufficient TCDD to induce slight or no manifestations of toxicity. . The compound 23,7,8-tetrachlorodibcnzo-/>-dioxin (TCDD) is a highly toxic impurity that may be formed under certain conditions during the production of 2,4,5-trichloro- phenol. T C D D has been considered one of the causes of chloracne, which has been 2 7 9 <l-00SX/Tg/04M-0279J02.00/0 Copyright 1973 by Academic Press. Inc. AU rights of reproduction in tny form reserved. Primed m Greet Britain 5b i'o 0001969 I 280 KOCIBA ET At. associated with the industrial production of 2,4,5-trichlorophenol and other products made from 2.4.5-trichIorophenoI. Most of the earlier toxicologic studies with T C D D were concerned with the assessment of its short-term toxicity and teratogenic potential. Results of these earlier V studies have been summarized in a previous publication by Kociba et at. (1976). This same publication also reported the results of a subchronic study in which rats were given 1.0,0.1,0.01,0.001, otOftg ofTCDD/kg 5 days/week for 13 weeks. Doses of 1.0 ro Mg/kg/day caused multiple toxicologic effects, including mortality and morphologic 00 changes inliver,thymus, and reproductive organs. A dose levelof0.1 //g/kg/day caused lesser degrees of toxicity, and rats given 0.01 or 0.001 //g/kg/day had no alterations considered ofany toxicologic significance. More recently, toxicologic studies of T C D D have also been conducted in the monkey. McConnell et at. (1978) reported a single oral LD50 in monkeys of <70 ftg TCDD/kg. These monkeys died with loss of hair or nails, keradnization ofMeibomian glands and hair follicles, and hyperplasia of the epithelium ofthe renal pelvis,stomach, and bileduct. Allen et at. (1977) reported on a subchronic study inwhich monkeys consumed a diet containing 500 ppt of T C D D for 9 months. It was calculated that these monkeys ingested 2 to 3 fig TCDD/kg over the course of the 9-month study. Clinically, these monkeys showed changes similar to those described by McConnell et at. (1978) as well as hematologic depression and hemorrhages in various tissues. Hypertrophy, hyper plasia, and/or metaplasia were noted in the epithelium ofthe bileducts, salivary glands, bronchi,pancreatic ducts,sebaceous glands, skin, gastric lining,and urinary tract In regard to long-term or carcinogenic studies conducted in rodents with TCDD, Innes et at. (1969) reported no increase in tumors in mice given 2,4,5-Trichloro- phenoxyacetic acid contaminated with a level ofT C D D sufficient to supply 0.27 fig of TCDD/kg/day. DiGiovanni et at. (1977) conducted a study in which T C D D was reported to be only a weak tumor initiator in the two-stage system of mouse skin carcinogenesis with 7,12-dimethylbenz(a]anthracene (DMBA). Van Miller and Allen (1977) issued a preliminary report on a study ofsmall groups of male rats fed diets containing T C D D for 65 weeks. All 10 ratsofeach group receiving 1.0, 0.5, or 0.05 ppm ofT C D D in the diet died within 4 weeks, with acute toxic effects, including severe liver necrosis, bile duct hyperplasia and edema, atrophy of spleen and thymus, gastrointestinal hemorrhages, and decreased spermatogenesis. Groups of male rats on diets containing 5000 or 1000 ppt experienced increased mortality, decreased weight gain, and liver toxicity. Dietary levels of 500, 50, 5, and 1 ppt ofT C D D were also studied. Various neoplasms were found in some rats at aildose levelsof 5 ppt and higher, with only the lowest dose levelof 1 ppt reportedly free ofany neoplasms. These same results on tumorigenesis were included in an updated report by Van Miller et at. (1977), which included the data generated through the end of their 95-week study. No tumors were reported in the group given 1 ppt ofT C D D or in a totalof 50 control rats. In view of the need for an evaluation of the chronic toxicity and potential for carcinogenicity of TCDD, the study reported herein was conducted. In this study, groups of male and female rats were maintained for 2 years on diets supplying J 5876 0001970 r s z w moo CHRONIC TOXICITY OF TCDD IN RATS 281 various dose levels of TCDD, and numerous parameters were evaluated in order to assess the potentialchronic toxicity associated with long-term ingestionofthe material. METHODS Experimental design. Male and female Sprague-Dawley rats, Spartan substrain,16 to 7 weeks old, were randomly placed (two/cage) into suspended wire-bottomed cages forthisstudy. Food1and water were availablead libitum. Croups of 100 rats (50 males, 50 females) were maintained for up to 2 years on diets supplying 0.1, 0.01, or 0.001 pg of TCDD/kg/day. The diet of the control group of 172 rats (86 males, 86 females) contained the vehicle. Test material. The T C D D sample used for this 2-year study was prepared by the D o w Chemical Company. Purification of the crude T C D D was followed by gas chromatography and mass spectrometry. The final product had a purity exceeding 99%, as determined by electron-capture gas chromatography. This sample was used to prepare the premixes and test diets according to the following general procedure: Approximately 1 mg ofT C D D was weighed on a microbalance and dissolved in 40 ml of reagent-grade acetone. This solution was then added to 1000 g of control feed and mixed for 30 min toprepare a stock premix. A sufficientsample ofthestock premix was then mixed with control feed to produce a working premix to be used in preparing the testdiets.The stock premix was prepared six times during the course ofthe study. The test diets were prepared by diluting the working premix with sufficient control feed to provide dose levels of T C D D as required by body weight and food consumption determinations in order to maintain the designated dosages on a microgram per kilo gram per day basis. Samples of the working premix and the test diets were analyzed periodically to ascertain thatthe dietary levelswere being maintained as scheduled. The concentration of T C D D in the premix samples was determined using electron-capture gas chromatography and gas chromatography-mass spectrometry. The concentration of T C D D in the test diets was determined by gas chromatography-mass spectrometry afterextraction and suitablecleanup. Clinical observations. All rats were palpated on a monthly basis,with a recording of the number of rats bearing palpable masses. Body weights and food consumption of 20 rats/sex/treatment leveland controls were routinely recorded foreach week ofthefirst3 months of the study and at approximately monthly intervals thereafter. All remaining rats were weighed monthly throughout thestudy. Blood samples for hematological determinations were collected from eight rats/sex/ group at 3, 12, and 23 months of treatment The total erythrocyte count (RBC), total and differential leukocyte counts (WBC), thrombocyte and reticulocytecounts, packed cell volume (PCV), and hemoglobin (Hgb) concentration were determined using automated3 or manual procedures. Urine samples were collected from seven to eight rats/sex/group atthese same time intervals. Urine specificgravity,pH, and thepresence 1Spartan Research Animals, Haslett, Michigan. 1 Purina Laboratory Chow, Ralston-Purina Co.. St. Louis. Missouri. 1Coulter Counter Model ZB-1. Coulter Electronics. Hialeah, Florida. A 5877 0001971 4 ro 00 ~ 4. ' 282 KOCIBA ET At. or absence of sugar, protein, ketones, bilirubin, and occult blood were determined45at each ofthese times, and urinary urobilinogen was alsoevaluated at Month 23. Urinary excretion of creatinine, coproporphyrin, uroporphyrin, and -amino levulinic acid (-ALA) was determined by a consulting laboratory* on samples collected from four to fiverats/sex/group at Months 3-4, 12, and 23. Serum samples were collected from seven rats/sex/group by orbital puncture at Month 22 for determination of urea nitrogen (BUN), glutamic pyruvic transaminase (SGPT), bilirubin (total, direct and indirect), cholesterol, and triglycerides. Automated procedures were used for these determinations.4Serum samples were similarly collected from seven rats/sex/group at Month 23 for determination ofalkaline phosphatase (AP) activity,totalprotein, albumin and globulin.6 At terminal necropsy, serum samples were collected from allsurvivors or a maximum of 10 rats/sex/group for determination of BUN, SGPT, AP, and total bilirubin.6 A consulting laboratory* also made determinations of serum y-glutamyi transferase activity (GGT). All rats dying or culled during the course of the study were subjected to a gross pathologic examination. Representative portions of the major organs and any gross lesion suggestive of a significant pathologic process or tumor formation were collected from each ratand preserved inbuffered 10% formalin. Terminal necropsy examination was conducted at the end of 2 years of treatment (105th week). All rats were deprived of food overnight prior to killing by decapitation. The eyes of all rats were examined by gently pressing a glass slide against the cornea under bright fluorescent illumination. Any observations on the eyes were recorded as part of the gross necropsy observation records. The eyes for a maximum of five rats/sex/group were preserved inZenker's fixative. Eyes from remaining rats were fixed informalin fixative. The weights of the liver, kidney, brain, heart, thymus, spleen, testes, and ovaries/uterus were recorded for a maximum of 10 rats/sex/group. A bone marrow smear was prepared from most rats, and filedfor future reference, ifindicated. Portions of fat,liver,and kidney from a maximum offiverats/sex/group were frozen forpossible T C O D analysis, with subsequent analysis of liver and fat samples from three females/dose level, using gas chromatography-low-resolution mass spectrometry. Portions of esophagus, salivary glands, stomach, small intestine, large intestine, pancreas, liver, kidneys, urinary bladder, prostate, accessory sex glands, epididymis, testes, ovaries, uterus, brain (cerebrum, cerebellum, brain stem), pituitary gland, spinal cord, peripheral (sciatic) nerve, trachea, lungs, spleen, thymus, lymph nodes, heart, aorta, skeletal muscle, mammary tissue (females), adrenal glands, thyroid, parathyroid, tongue, lower jaw, and skull (including nasal turbinates, ear canal), together with any additional gross lesions were preserved informalin fixative. Histologic examination of tissues was conducted on paraffin-embedded sections of tissues which were stained with hematoxylin and eosin. All ratsfrom the control and top dose level, regardless of whether they died during the study or were killed at the 4 Ames Bililabstix or Multistix. Ames Co.. Elkhart. Indiana and TS Meter. AO Optical. Buffalo. New York. 5 Bioscience Laboratories. Van Nuys. California. ` Technicon AutoAnalyzer. Technicon Corp.. Rye. New York. j 5878 0001972 CHRONIC TOXICITY OF TCDD IN RATS 283 termination, were subjected to histologic examination of an extensive list of tissues, intended to include a majority ofthose tissues listedabove as those collected atthe time of terminal necropsy. All rats from the two lower dose levels were subjected to histo logic examination of those selected tissues identified as possible target organs and all gross lesions suggestive of tumor formation. The actual number of dssues specimens from each group examined histologically is on file and available from the authors. Additional sections of liver from selected females from the terminal necropsy were stained forlipidcontent using Oil Red O or Sudan IV stains. Liver tissue collected at the terminal necropsy was examined using an electron microscope1 to characterize qualitatively the ultrastructure of hepatocytes from three females/group. The liver tissue was fixed in 2.5% phosphate-buffered glutaraldehyde and then postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated through graded ethanol solutions, washed in propylene oxide, infiltrated with Epon 812, and embedded inpolyethene capsules. Sections of 1ftm thicknesswere stained with toluidine blue and examined using light microscopy. Thin sections were stained with uranyl acetate and lead citratepriortoexamination by electron microscopy. Statistical evaluation o f data. The significance of differences between control and test values for hematology, urinary and clinical chemistry parameters, body weights, organ weights, and organ/body weight ratios was statistically determined by one-way analyses of variance followed by the Dunnett test (Steel and Torrie, 1960). A significance level of p < 0.05 was used. Data on mortality, palpable masses, gross pathology, histopathology, and tumor incidences were analyzed using the Fischer exact probability test, p < 0.05, one-sided test (Siegel, 1956). Mortality data were also analyzed using the Mantel-Haenszel Test Repeated measures analyses across time were not appropriate because ofmortality and because the assumptions ofthe statistical testswere not valid. Statistical evaluation of gross pathology data collated for the entire study compared the data of each ofthe treatment groups with the data ofthe control group ofthat sex. Statistical evaluation of histopathologic observations and tumor incidences compared the data ofthe high-dose group with the data ofthe control group ofthatsex.The same evaluation was conducted on the lower dose levels in instances in which comparable numbers of tissues were subjected to microscopic examination (apparent target organs). RESULTS Dietary Content o f TCDD Analyses of feed samples indicated the dosage levels of 0.1, 0.01, and 0.001 p% of TCDD/kg/day equated with approximately 2193,208, and 22 ppt ofT C D D in the diet. Six repeated analyses of the feed samples indicated good agreement between the intended contentofT C D D and resultsofanalysis forT C D D content. Clinical observations Females given 0.1 pg/kg/day had statistically increased cumulative mortality dining the latter halfof the study, whereas those given 0.01 or 0.001 tg/kg/day had mortality 1Carl Zeiss. Inc., New York. o O ro TO 03O'i 5879 0001973 t)82CLZ it&a 284 KOCIBA E T A L . rates comparable to that ofthe controls. In males, there were some isolated instances of statisticaldifferences between the treated and control groups. However, as the mortality ofonly the group ofmales given 0.01 //g/kg/day was significantlydifferentfrom control using the Mantel-Haenszel test, these deviations in the male rats were considered of questionable toxicologic significance. Mean body weights of males and females given 0.1 //g/kg/day were statistically decreased from control values throughout the major portion of the study, from Month 6 to the end of the 2-year test period. Mean body weights of females given 0.01 //g/kg/day were decreased to a lesser degree during this same time interval.The mean body weights ofmales given 0.01 or0.001 //g/kg/day and females given 0.001 //g/kg/day were sometimes lower than controls during the middle of the study, but only occasionally were the differences statistically significant. During the last quarter ofthe study body weights of these groups were comparable to those of controls. There were no consistent deviations in food consumption of males or females at any dose. The few sporadic cases in which there was a statistical increase or decrease between the control and treatment groups followed no consistent trend, and were considered of no toxicologic significance. The firstpalpable mass was noted at Month 5 in a male ofthe control group. There were no statisticallysignificantdifferences between the control and treated groups except during Months 15 and 16, when the males given 0.01 //g/kg/day had an increased incidence of palpable masses. This was considered of no toxicologic significance because ofitsisolated occurrence and lack ofdose response. During the last 12 months of the study, females ingesting 0.1 //g/kg/day had a consistent trend toward a decrease in the number of rats with palpable masses. This observation was not noted atlower dosage levelsinthefemales. Hematology The hematology data collected after23 months oftreatment are listed inTable 1and are similar to the patterns observed during the study. In rats given 0.1 //g/kg/day, there were statistically significant decreases in the P C V and Hgb values for males after 3 months as well as decreases in the Hgb values for males and decreases in PCV, total RBC, and W B C counts and Hgb values for females after 1 year. At the preterminal examination, this high dose group again had statistically significant decreases in RB C and Hgb values (males) and P C V and Hgb values (females); reticulocyte counts also appeared to be slightly increased. Thrombocyte and W B C differential counts appeared to be unaffected at alldose levelsofTCDD. Rats given 0.01 or 0.001 //g/kg/day had no hematologic changes considered relatedto treatment Urinalyses Repeated examination of urinary parameters revealed no consistent alterations that could be attributedto any ofthedose levelsofTCDD. Urinary porphyrins and 5-ALA Porphyrin data collected after 23 months of treatment are listed in Table 2 and are representative ofthe patterns observed during the study. Urinary excretion ofcopropor phyrin was statistically increased in female rats at a dose level of 0.1 //g/kg/day after each evaluation at 3-4, 12, and 23 months. Coproporphyrin excretion was also 5880 0001974 r TABLE I _M_e_a_n_H_e_m_a_to_l_og..i.c__Va_lu_e_s o_f_M--ale Rats (D-ay 6--81) and Female Rats (D ay 682) on D iets Containing TCDD Dose of Number Reticulo Thrombo WBC dilferential count (%) TCDD of PCV RBC Hgb cytes cytes WBC (MsAgAJay) Sex rats/group (%) (x IO*/mmJ) (g/100 ml) (%) (x lOVmm1) (x lOVmm1) Neut Lymph Mono Eosin Buso 0 0.100 0.010 0.001 0 0.100 0.010 0.001 M 8 46.9 4.7" 7.99 0.64 15.6 1.6 1.0 1.195 0.350 14.9 5.4 27 65 7 1 0 M 8 43.4 t 4.J 7.19 0.65 13.9 1.5 2.2 1.214 0.358 12.1 5.2 33 62 4 | 0 M 8 47.6 2.8 7.65 t 0.46 16.0 0.8 1.0 1.482 0.342 15.8 4.0 34 60 5 | 0 M 8 47.4 f 1.8 7.73 0.27 15.7 0.6 0.5 1.042 0.143 18.7 8.0 30 65 4 1 0 F 8 43.6 t 1.4 6.84 0.66 14.1 l.l 0.8 1.046 0.210 9.1 1.5 34 60 4 2 0 F 8 38.9 t 3.8* 6.38 0.90 12.5 1.2* 1.2 1.176 0.358 7.1 2.1 30 67 2 1 0 F 8 45.1 I I 7.35 0.32 15.1 0.4 0.5 0.962 F 0.161 9.4 2.2 36 60 3 1 0 F 8 46.9 4.7 7.56 0.69 15.7 1.5* 0.5 0.857 + 0.239 9.5 1.9 23 72 3 2 0 "Mean i SD. * Statistically significant fromcontrol mean usinganalysis of variance and Dunncil's lesl, p < 0.03. 250 o z n OH n3 3 o n o o 2 000197 or co oo c.n K> OO la moa r K 0OO\ TABLE 2 U rinary Excretion of C reatinine, Coproporphyrin, U roporphyrin, and -Amino Levuunic A cid for Male and Femai.e Rai s (D ays 678-680) on D iets Containing TCDD Q~ O o3 Sex Number of rats/group Total urine 48 hr (ml; o> C3 OO 3 'b <j *C>C* .g 8. S3 u5> > O & G 'S uaoIoom. se a u U 00 B G c >C* IT o8 *0^0 2% D5 G J>lSa 4) G & 1fr Ou(4 Ot* D U 60 se S o go Sf S C " iS < 00 E u(*> E 0 0.100 0.010 0.001 0 0.100 0 .010 0.001 M M M M F F F F 4 51-0 + 21.6- 27.6 3.2 18.0 + 3.7 0.69 +0.19 5.4 2.2 0.200 + 0.092 0.27 + 0.38 0.010*0.015 5 63.4 40.5 19.6 9.1 19.6 11.7 1.22 + 1.08 7.3 4.0 0.418 + 0.295 0.08 0.02 0.005 + 0.003 5 61.4 26.1 24.4 5.7 16.5 8.0 0.64 0.25 5.7 2.1 0.228 0.056 0.26 0.42 0.009 0.013 5 41.0 6.4 30.8 3.2 23.8 4.8 0.78 0.19 5.3 1.6 0.174 0.053 0.06 0.01 0.002 0.001 5 60.0 34.9 23.3 6.2 9.8 1.3 0.43 0.49 3.8 1.7 0.157 0.050 0.07 0.03 0.003 0.001 5 51.2 22.8 18.6 4.3 17.4 4.0* 0.98 + 0.41 5.7 2.3 0.296 0.074* 0.12 0.05 0.006 * 0.002 5 54.2 t 20.5 19.4 2.3 16.4 4.7 0.83 0.18 3.5 1.1 0.181 0.053 0.08 0.03 0.004 i 0.002 5 57.2 + 20.0 20.8 4.7 8.6 2.0 0.42 0.06 3.0 1.1 0.143 0.037 0.08 + 0.02 0.004 + 0.001 * Mean t SU. * Slalislically significant fromcontrol mean by analysisof variance and Ounnett'stcsl.p < .0S. KOCIBA ET AL. 000197?! cn GO O to j DOW 75209 CHRONIC TOXICITY OF TCDD IN RATS 287 statistically increased in female rats at a dose level of 0.01 /g/kg/day after 3 and 23 months. Urinary excretion of uroporphyrin was statistically increased in females after 3 and 23 months of receiving 0.1 /g/kg/day and after 3 months of receiving 0.01 /ig/kg/day. Urinary excretion of -ALA was statistically increased in females after 3 and 23 months of receiving 0.1 /ig/kg/day. Total urine volume or creatinine excretion was not affected by any of these dose levels in the females. Males had no alterations considered treatment-related in any of these parameters at any of the dose levels of TCDD. Clinical Chemistry For sake of brevity, only the results obtained at terminal necropsy after 2 years of treatment are presented in Table 3. Analyses of serum samples collected by orbital <1"i 'j 'j j TABLE 3 Mean Terminal (2-Year) Clinical Chemistry Values for Male and Female Rats G iven D iets Containing TCDD Dose Number Total y-glutamyl TCDD of BUN SGPT AP bilirubin transferase 0/g/kg/day) Sex rats/group (mg/100 ml) (mU/ml) (mU/ml) (mg/100 ml) (mU/ml) 0 0.100 0.010 0.001 0 0.100 0.010 0.001 M 10 M5 M4 M 10 F 10 F4 F 10 F 10 33 34" 28 11 36 19 20 9 21 9 20 3 17 3 18 5 49 17 87 3 0 42 6 105 17 4 7 + 1 0 88 2 6 4 3 + 9 86 2 5 39 11 60 2 9 54 + 12* 205 146* 49 + 7 61 2 0 42 5 54 2 8 0.2+0 0.2 0 0.2 + 0 0.2 0 0.2 0 0.2 0 0.3 0.1 0.4 0.2 0+0 10 10 00 00 14+ 10* 10 1+0 * Mean SD. ` Statistically significant from control mean using analysis of variance and the Dunneu's test. p < 0.05. puncture at 22 to 23 months oftreatment revealed no alterations considered related to treatment in regard to BUN, SGPT, total, direct, or indirect bilirubin, cholesterol, triglycerides, total protein, albumin, and globulin. Serum A P was statisticallyincreased in females given 0.1 /ig/kg/day. A statisticallysignificantincreasein serum triglycerides noted in males given 0.01 ^g/kg/day was considered of no toxicologic significance based on the lack of a dose-response relationship. Analyses ofserum samples collected at terminal necropsy after 2 years indicated a statistical increase in SGPT, AP, and G G T activities for females given 0.1 ^g/kg/day. Females at the lower dose levels and males at aildose levelswere unaffected inthese parameters. The B U N and totalbilirubin values ofeithersex were unaffected by any leveloftreatment with TCDD. Gross and Microscopic Observations on Tissues Detailed descriptions of all gross and microscopic observations made on all rats killed or dying during the course of the 2-year study are on fileand available from the authors. On account of the voluminous nature of the data, the results are summarized below. Tumor and tumor-like lesions arelistedinTables 4 and 5. 5883 0001977 r ai GO GO TABLE 4 Tumor Incidence in Mai.i; Rats Maintained on D iets Containing TCDDu Time iniervals during study: Months 13-24 Terminal kill Dose level in /rg/kg/day: 0 0.1 0.01 0.001 0 0.1 0.01 0.001 0 Number ol'rals examined: 65 41 46 38 15 5 4 . 85 Rais with luniors/lum or like lesions Hepaiocellulur hyperplastic 2 12 0 41 1 0 6 nodule(s) Hepatocellular carcinoma(s) 1 0 0 0 110 0 2 Bile duel adenoma 0 10 0 00 0 0 0 Strulilied squamous cell 0 4 0 0 00 0 00 carcinoma ol' hurd palate or nasal turbinales Paravertebral or sub cutaneous malignant 0 1 ' 0 00 0 00 schwannoma Carcinoma of renal tubules 1 0 00 10 0 12 pelvis, or bladder Adenoma of renal tubules 0 0 0 1 0 0 0 10 or pelvis Keratinizing squam ous cell 0 10 0 00 0 00 carcinoma of lung Pulmonary adenoma 1 10 0 00 0 0 | Pulmonury adenocarcinoma I 0 0 0 00 0 0 1 Oligodcndroglioma/astro- 2 0 0 1 0 0 0 0 2 cylom a of brain, or glioma ol'spinal cord Interstitial cell adenoma of 2 0 0 testes 100 0 12 Adenoma ol'prostate 0 0 0 0 00 0 10 Subcutaneous fibroadenoma/ 8 6 4 1 10 1 0 10 libroma/lipoma .* . ' ' ' n U o G iZ m o o 0000 Total 0.1 0.01 50 50 0.001 50 KO CIBA ST AL. 23 10 10 4h 0 1' 00 00 10 10 00 00 00 00 65 0 0 0 0 0 1 2 0 0 0 1 2 1 1" 0001379 r Ol GO 00 or Subcutaneous librosareoma 0 1 1 Benign mammary neoplasms 0 10 Mammary gland adeno- 200 carcinoma Cutaneous papilloma/basal J 0 0 cell tum or Squamous cell carcinoma of 0 0 0 integument Zymbal gland carcinoma 00 1 Pituitary adenoma 19 10 11 Pituitary adenocarcinoma 10 0 Stralilied squam ous cell 031 carcinoma ol' longue Fibrosarcoma of tongue 10 0 Squamous papilloma/polyp 0 1 1 of gastric mucosa Mucocysladcnocarcinoma of small intestine Leiomyosarcoma of cecum 0 0 0 Intraabdominal 020 schwannoma/sarcoma Acinar adenoma of pancreas Acinar adenocarcinoma of 24 00 pancreas Islet adenoma of pancreas 6 3 2 Islet adenocarcinoma of pancreas I 00 Adenoma ol' adrenal cortex 0 5 2 Pheochromocytoma of 19 3 10 adrenal Inlerfullieular C ecil 240 adenoma ol'thyroid Intcrfollicular C ecil 2 10 adenocarcinoma of thyroid Follicular adenoma of 0 I1 11 thyroid Follicular adenocarcinoma 0 0 0 of thyroid CHRONIC TOXICITY OF TCDD IN RATS 0 00 0 0 00 0 0 00 0 0 00 0 100 0 , 00 0 5 72 0 0 10 0 ' 00 0 0 00 100 0 0 2 00 0 0 0 10 0 00 0 3 30 1 0 00 0 1 60 1 0 10 0 0 00 0 3 9 10 0 60 0 0 10 0 0 00 0 0 0 10 001 1 0010 0 20 0 0 30 0 0 00 0 0 00 1 26 13 0 30 0 0 3* 1 II 0 1 0 10 0 00 11 0 10 0 00 10 002 0 4 14 2h 5 0 10 0 2 12 3 3 02 00 3 28 00 Sh 2 4* 10 0 `s 4 0 0 310 001 1 0010 0 0 0 0 1 1 6 0 1 0 1 2 0 0 7 0 3 0 0 6 0 0 0 0 M oa i KOCIBA ETAL. 0001980 TABLE 4 -- continued Time intervals (luring study: Months 13-24 Terminal kill Total Dose level in pg /k g /d ay : 0 0.1 0.01 0.001 0 0.1 0.01 0.001 0 0.1 0.01 0.001 Number o f rats examined: 65 41 46 38 15 5 4 II 85 50 50 50 Malignant lymphoreticular 5 0 3 2 00 0 neoplasm Hemangioma of lymph node 1 0 0 0 00 0 F ib rosarcom a/osleosarcom a 1 0 0 0 00 0 of musculoskeletal system Infraorbital malignant 0 0 10 00 0 schwannoma Mediastinal fibrosarcoma 000 1 00 0 Not available for 0 0 0 0 00 0 pathological examination 1 50 0 10 0 20 0 00 0 00 0 10 33 00 00 10 01 00 * No tumors occurred during Months I through 6. Tumors occurring during Months 7 to 12 included I subcutaneous fibroadenoma (control), I pituitary adenoma (0.1 ig/kg/day). I pituitary adenocarcinoma (control), I osteosarcoma (control). These four tumors, which were present in the 10 males dying prior to Month 13, are included in the above total tabulation. * Statistically diiTerenl from control data when analyzed using the Fischer exact probability test, p < 0.0S. Appropriate tumor data have been combined Tor the sake of brevity. CA CO CO oe CHRONIC TOXICITY OF TCDD IN RATS 0001081 or oo oo vi- TABLE 5 Tumor Incidence in F emale Rats Maintained on D iets Containing T C D D ' Time intervals during study: Months 13-24 Terminal kill Dose level in ftg/kg/day: 0 0.1 0.01 0.001 0 0.1 0.01 0.001 0 Number of rats examined: 60 36 34 32 23 4 14 16 86 R ats with tumors/tum or-like lesions: Hepatocellular hyperplastic 2 20 8 1 6 3 10 2 8 nodules Hepatocellular carcinoma(s) 0 10 10 11 10 1 Bile duct adenoma 0 2 0 0 00 0 10 Stratified squam ous cell 04 10 00 000 carcinoma of hard palate or nasal turbinates Keratinizing squam ous cell 0 70 0 00 000 carcinoma of lung Pulmonary adenocarcinoma 0 0 10 00 000 Astrocytoma of cerebrum 0000 10 00 1 Malignant schwannoma of 1 0 0 0 00 00 1 pelvic canal Nephroblastoma of kidney 0 1 10 00 000 Adenoma of renal tubules Carcinom a o f renal pelvis Granulosal cell neoplasm 0 0 0 1 00 0 0 0 0 00 0 0 1 1 30 00 10 00 0 0 3 of ovary Benign tum or o f uterus 16 3 7 5 12 2 Malignant schwannoma of 2 0 3 100 4 7 28 002 uterus Adenocarcinoma of uterus 6 4 0 1 00 Fibroma o f cervix/vagina 1 0 0 0 10 Subcutaneous fibroma/ 1 0 0 1 00 00 10 00 6 2 1 fibrolipoma Total 0.1 0.01 49 30 0.001 50 23* 18* II* 2 20 4* 1 3 0 1 0 7* 0 0 0 10 00 0 00 0 110 00 1 0 10 011 7* II 03 12 1 40 1 0 10 00 1 Kvo* MOO J * ' . iV*i \ * V p > ,o i ^ v b o -! ^ o a r K VtoO KOC1BA 7" AL. 0001982 OJ GO QO GO Time intervals during study: Dose level in //g/kg/day: Number of rats examined: Subcutaneous fibrosarcoma Benign neoplasm of mammary gland Carcinoma of mammary gland Stratified squam ous cell carcinoma o f digit Cystadenoma of Zymbal gland Pituitary adenoma Pituitary adenocarcinoma Stratified squam ous cell carcinoma of tongue Papilloma of esophagus Squamous papilloma/polyp of gastric mucosa L eiom yosarcom a/sarcom a of small intestine 0 60 0 50 5 0 0 26 4 1 0 0 1 TABLE 5-- continued Months 13-24 Terminal kill 0.1 0.01 0.001 0 0.1 0.01 36 34 32 25 00 10 22 23 24 23 4 0 2 0 1 3 60 10 0 00 0 0 0 00 12 8 12 17 0 2 0 0 20 2 0 0 00 14 0 12 5 0 0 5 1 0 0 0 0 10 2 0 0 10 0 0 0 00 0 0 , 0.001 16 0 It 1 0 1 6 0 0 0 1 0 0 86 0 73 8 0 0 43 6 1 1 1 1 Total 0.1 0.01 49 50 00 24 36 0 4 10 00 12 13 21 20 00 20 01 0.001 50 1 35 4 0 1 18 0 0 0 1 0 CHRONIC TOXICITY OF TCDD IN RATS 0001983 Polypoid adenoma o f large 1 0 0 0 00 intestine Acinar adenoma of pancreas 0 10 0 00 Islet cell adenoma o f 1 0 0 1 20 pancreas Islet cell adenocarcinoma 1 0 0 0 10 of pancreas Adenoma of adrenal cortex 7 5 12 20 Pheochromocytoma of 430 1 30 adrenal Intcrfollicular adenoma of 9 4 1 1 5 0 thyroid Interfollicular adeno 3 20 1 10 carcinoma of thyroid Follicular adenoma of 0 10 0 00 thyroid Malignant lymphoreticular 1 1 10 10 neoplasm Hemangioma of abdominal 1 0 0 0 0 0 muscle Not available for pathologic 0 0 0 0 00 examination 00 10 0 0 0 10 10 1 12 30 13 00 20 0 0 14 95 2 6 1 173 12 1 0 14 4 2 1 0 142 0 2 000 10 0 00 2110 0 0 10 0 0 000 10 0 * No tumors occurred during Months I through 6. Tumors occurring during Months ? to 12 included two benign tumors oT the uterus (0.1 pg/kg/day) and one benign neoplasm of the mammary gland (0.01 jg/kg/day). These three tumors, which were present in the 14 females dying prior to Month 13, are included in lire above total tabulation. * Statistically different from control data when analyzed using the Fischer exact probability lest, p < 0.05. Appropriate tumor data have been combined for (lie sake of brevity UtvoOi 6 MOO 0u 0i CO DOV* 2752 0&,- 294 K0C1BA ETAL. Gross necropsy examination of the rats of the top dose level indicated the grossly visible target organs to include the liver, vascular system, respiratory system, and lymphoid organs; the general body condition was alsoconsistently affected. Microscopic examination of tissues from rats dying during the study or killed after 2 years revealed the following treatment-related affects: Liver. The liver was the organ most consistently affected, and rats given 0.1 or 0.01 /ig/kg/day had multiple hepatocellular degenerative, inflammatory, and necrotic changes noted upon light microscopy. These hepatic changes, which were more extensive in females than in males, were characterized by cytomegaly, distortion of F ig. 1. Lesion classified morphologically as hepatocellular carcinoma in liver of rat given 0.1 n of TCDD/kg/day. Note adjacent fibrosis, inflammation, and fatty infiltration on left. H & E stain, x 200. lobular pattern, and resultant atrophy ofhepatic cords, cytoplasmic vacuolization, fatty metamorphosis, altered tinctorial properties with increased basophilia, hepatic necrosis and inflammation, multinucleated hepatocytes, and foci or areas of hepatocellular alterations. They were accompanied by increased aggregates of pigment, bile duct hyperplasia, and some increase infibrosisand periportal inflammation. During the latter phase of the study and at the terminal necropsy the females given 0.1 ig/kg/day also had hepatocellular proliferative lesions classified morphologically as hepatocellular carcinomas (Fig. 1) and hyperplastic (neoplastic) nodules. There was no evidence of metastasis of any liver neoplasms. Female rats given 0.01 ,ug/kg/day also had an increased incidence of these hepatocellular hyperplastic nodules. Upon examination using light microscopy, livers of female rats given 0.001 tg/kg/day had a statistical increase above the background incidence of foci or larger area of slight hepatocellular alteration (swollen hepatocytes). However, in male rats given 0.001 //g/kg/day, there was a statistically significant decrease in the number of livers with an area of hepato cellular alteration ofthistype. 0001384 moa CHRONIC TOXICITY OF TCDD IN RATS 295 As part ofthe ultrastructural evaluation of hepatocytes, light microscopy oftoluidine blue-stained sections of liver from females given 0.1 /ig/kg/day revealed an increased number of individual hepatocytes containing large accumulations of lipid droplets. Ultrastructural evaluation by electron microscopy of liver sections from this high dose 5, W ro CD Fig. 2. Hepatocyte from female rat given 0.1 n of TCDD/kg/day for 2 years. Note disorientation of RER and focal cytoplasmic vacuolization. Uranyl acetate-lead citrate stain. x3350. level revealed the most consistent change to be in the rough endoplasmic reticulum (RER), which appeared to be undergoing proliferation with some distortion and fragmentation (Fig. 2). Smooth endoplasmic reticulum (SER) and mitochondrial structures were within the range of variation observed in the control sections. Other changes noted at this high dose levelincluded focal areas ofcytoplasmic vacuolization, increased lysosomal activity with residual body formation, and an occasional multinucleated hepatocyte (Fig. 3). Upon ultrastructural examination of hepatocytes from ' 5891 0001985 296 KOCIBA E T AL. rats of the 0.01-ig/kg/day dose level, the most notable change was limited to a lesser degree of proliferation and disorientation of the R E R and some proliferation of SER cn fO (Fig. 4). There was some slight increase in the number of individual hepatocytes with lipiddroplet accumulations. CO 00 The hepatocytes of female rats given 0.001 /g/kg/day were ultrastructurally within the limits of variation seen in the controls (Fig. 5).There was no general increase inthe lipid droplet content, but an occasional cell contained increased numbers of lipid droplets. Lymphoreticular tissues. Treatment-related effects, noted only in females ofthe 0.1//g/kg/day dose level, included isolated occurrences of thymic atrophy and/or splenic atrophy. 5892 0001986 CHRONIC TOXICITY OF TCDD IN RATS 297 Respiratory system. Treatment-related effects were noted in both males and females at the O.I-/tg/kg/day dose level but were much more extensive in the female rats and included an increased incidence of focal alveolar hyperplasia (Fig. 6), aggregates of hematogenous pigment in lung and thoracic lymph nodes, focal ' * 10 \ Cl iv to t o J Fig. 4. Hepatocyte from female rat given 0.01 n of TCDD/kg/day for 2 years. Note proliferation of SER and disorientation of RER. Uranyl acetate-lead citrate stain, x 2070. accumulation of alveolar macrophages and cholesterol clefts,pulmonary edema, focal interstitial inflammation and fibrosis, keratinizing squamous metaplasia, or squamous cell carcinoma formation (Fig. 7) within the lung. Focal alveolar hyperplasia was also increased in females given 0.01 /tg/kg/day. The lower dose level of 0.001 /tg/kg/day had no discernibleeffecton the tissuesofthe respiratory system. Cardiovascular system. Effects probably related to the ingestion of 0.1 /tg/kg/day included an apparent increase in the incidence of hemorrhage in the brain and possibly 5893 0001987 DOW 2 / 5 3 0 0 298 kociba e t a l . spinal cord of females, an increase above the background incidence rate of mesenteric/thoracic periarteritis with accompanying changes, such as thrombosis and hematoma formations in both males and females, and an increase above the background incidence of myocardial degenerative changes (females only). At the 0.01- Fio. 5. Hepatocyte from female rat given 0.001 ftg of TCDD/kg/day for 2 year*. Morphology within normal limits of variation seen in controls. Uranyl acetate-lead citrate x4100. jug/kg/day dose level, probable treatment-related lesions were limited to an increase above background incidence of periarteritis and thrombosis of testicular or thoracic/mediastinal vessels of male rats. There were no alterations considered related to treatment with 0.001 ig/kg/day. Reproductive system and mammary gland. Female rats given 0.1 ig/kg/day had a statistically decreased incidence of uterine changes, including endometrial hyperplasia. 5894 0001388 CHRONIC TOXICITY OF TCDD IN RATS 299 cyst formation, and adenomatous polyp formation. This same group of high dose level female rats also had a significantly decreased incidence of subcutaneous mammary tumors. These observations correlated well with a decreased incidence of pituitary TO Ol CoO Fig. 6. Focal alveolar hyperplasia near terminal bronchiole within lung of rat given 0.1 Mg of TCDD/kg/day. H & E stain, x 100. Fig. 7. Lesion within lung of rat given 0.1 Mgof TCDD/kg/day classified morphologically as squamous cell carcinoma. Note accumulation of keratinized material within lesion. U A E stain, x 100. 5895 0001389 o 0 -S ro -i Cl o, C O : j to 300 KOCIBA. FT / L. adenomas noted in this same high dose level of female rats. There were no discernible effects in female rats given 0.01 or 0.001 ftgt kg/day. The reproductive organs of male rats appeared to be unaffected by these dose levels, with similar degenerative, inflammatory, and proliferative lesions in all treated and control groups. Endocrine organs. Female but not male rats given 0.1 //g/kg/day had a significantly decreased incidence of pituitary changes, including hemangiectasis and adenoma formation. Adrenal changes noted atthis high dose level included a decreased incidence of medullary hyperplastic nodule formation (males and females), a decreased incidence of pheochromocytoma formation (males), an increased incidence of cortical necrosis and hemorrhage (females), and an increased incidence ofadrenal hematocyst formation (males). A statistical increase in the incidence of adrenal cortical adenomas noted for males given 0.1 ftg/kg/day may have been the result of normal biological variation of the incidence of this tumor, which does occur spontaneously in this strain of rat (approximate 10% incidence inthe control group offemale rats used in thisstudy). The pancreas of male rats given 0.1 ftg/kg/day had a statistical decrease in the incidence of acinar adenoma formation. A statistically increased incidence of fibrosis of atrophic pancreatic tissue noted in the group of females given 0.1 ftg/kg/day may or may not have been associated with the increased incidence of periarteritis noted in this group. The thyroid glands of the high dose group of male rats appeared to have a low incidence of various follicular changes that may or may not have been related to treatment: this included isolated cases of follicular cyst or microcyst formation, follicular adenoma, or follicular adenocarcinoma formation. The parathyroid gland was unaffected by treatment, except for a decrease in secondary parathyroid hyperplasia as a result of the decrease in severity of chronic renal disease of the males given 0.1 //g/kg/day. Gastrointestinal system. A wide variety of degenerative or inflammatory lesions occurred in all control and treated groups, with no indications of a direct treatmentrelated effect in the salivary glands, esophagus, stomach, small intestine, or large intestine. However, the group of male rats given 0.1 //g/kg/day had a statisticalincrease above background incidence of stratified squamous cell carcinomas of the tongue, which were considered to be probably relatedto treatment. There was also a statistically significant increase in the incidence of squamous ceil carcinomas of the hard palate/nasal turbinate region of male and female rats given 0.1 /tg/kg/day. Historically, squamous cellcarcinomas of the tongue and hard palate/turbinates have occurred at a spontaneous incidence rate of 1 to 3 % in this strain of rat It appears as iftreatment with 0.1 //g/kg/day increased the incidence ofthistype ofneoplasm. A secondary effect of treatment was noted in the stomach only of the high dose group of males, in which there was a decrease inthe incidencerate ofmineralization ofthe gastricmuscularis and mucosa. This was secondary to the decreased incidence and severity of chronic renal disease and uremia inthishigh dose group ofmale rats. Nervous system. The only observation considered as probably related to treatment was the increased incidence of focal hemorrhage in the brain (and possibly spinal cord) of female rats given 0.1 //g/kg/day. This was described previously in the description of the cardiovascular system. All groups of rats had the expected spectrum of degenerative, inflammatory, and proliferativelesionsconsidered spontaneous inorigin. / 5896 0001390 CHRONIC TOXICITY OF TCDD IN RATS 301 Urinary system. There appeared to be a decrease in the severity of the chronic nephropathy affecting the kidneys of the male rats given 0.1 /ig/kg/day. Other degenerative, inflammatory, and proliferative changes occurred in the kidneys or urogenital tract of control or treated groups, with no observations considered related to treatment. Musculoskeletal system, eye. and miscellaneous tissues. Various degenerative, inflammatory, or proliferative lesions occurred in a scattered pattern in all treated and control groups, with no indication of a treatment-related effect. No toxicologic significance was attached to the statistical decrease in the incidence rate of subcutaneous benign tumors noted in males given 0.001 ig/kg/day. Organ Weights Statistically significant differences in terminal organ weights considered related to treatment included (1) an increase in liverweight calculated on an absolute basis (males given 0.1 or 0.01 //g/'kg/day, females given 0.1 ig/kg/day) and on a relative basis of liver/body ratio (females given 0.1 or 0.01 /ig/kg/day) and (2)a decrease inthe absolute weight of the thymus of females given 0.1 /g/kg/day. Additional changes in organ weights were considered to be secondary to decreased body weights due to treatment with 0.1 jUg/kg/day. TCDD Content o f Tissues Results ofanalysis ofsamples offatand livercollected atterminal necropsy offemale rats after 2 years of treatment indicated that rats given 0.1 //g/kg/day had an average T C D D content of 8100 ppt in the fat and 24,000 ppt in the liver. Rats given 0.01 /ig/kg/day had an average T C D D content of 1700 ppt in the fat and 5100 ppt in the liver. Rats given 0.001 ig/kg/day had an average of 540 ppt ofT C D D in the fat and alsoin the liver. rw tO o - a o- w w DISCUSSION The findings of this chronic toxicity study on T C D D in rats are an extension of the studies of shorter duration reported previously from this laboratory (Kociba et al., 1976). Continuous ingestion of diets containing approximately 2200 ppt ofT C D D (0.1 fig of TCDD/kg/day) for 2 years caused multiple toxicologic effects, including increased mortality, decreased body weight gain, slight depression of certain hematologic parameters, increased urinary excretion of porphyrins and -ALA, increased serum activities of AP, GGT, and SGPT, and morphological changes primarily of the hepatic, lymphoid, respiratory, and vascular tissues of the body. This high dose level of 0.1 ig/kg/day also caused an increase in the incidence of hepato cellular carcinomas of the liver (females only) and squamous cell carcinomas of the lung, hard palate/nasal turbinates, or tongue. The occurrence of numerous age-related lesions usually encountered inthis strain ofrat,includingtumors ofthe pituitary, uterus, mammary gland, pancreas, and adrenal gland was reduced at the high dose level. Also reduced was the incidence and severity of chronic renal disease in the aged male rats. Female rats given this high dose level for 2 years had 24,000 ppt present in the liver. This compares with 34,600 ppt of T C D D present in the liver of female rats given this 5897 0001991 DOW 275304 302 KOCIBA ETAL. same dose level for 13 weeks (Kociba trations were achieved during the early pehtaasle.,o1f 9th7i6s) and indicates 2-year study. steady state concen Ingestion of the intermediate dose level of 0.01 //g/kg/day (-210 ppt in the diet) caused a lesser degree of toxicity. The primary effects noted at this dose level included (1) increased urinary excretion of porphyrins (females), (2) liver toxicity, including an increased incidence of hepatocellular nodules, and (3) increased incidence of focal alveolar hyperplasia in the lungs. Terminal liver and fat content of TCDD averaged 5100, and 1700 ppt, respectively. This compares with 3700 ppt present in the liver aftLerife1t3imweeeiknsgoesfttiroenatmofen0t.0w0i1th /th/gis/kdgo/sdealyev(e-l2(K2ocpipbta eintalt.h,e19d7ie6t)). caused no effects considered to be of any toxicological significance. Light microscopy of livers from females of this group indicated a statistical increase above the background incidence of swollen hepatocytes; conversely, the livers of the males of the group had a decreased imncicidroesnccoepyo,fthethhisepoatbosceyrtveastiforno.m Wthehefnemlailveesrwteirsesuweithwinastheexliammitisneodf vaursiiantgionelseecetnroinn the controls with an occasional hepatocyte containing increased lipid droplets. The liver and fat each contained 540 ppt of TCDD at the end of the lifetime ingestion of 0.0I0f1th//eg/rkegsu/dltasyo. f this lifetime study in rats are compared to the preliminary results of tnheeopsltausdtyicinresraptosnsbeys VinanthMe illulenrgeatnadl.li(v1e9r77o)f, riattws imll abientnaionteedd tfhoartebxotetnhdsetdudpieesrioredpsoortf time 5000 on high doses ppt produced of TC both DD. liver In the preliminary report by and lung neoplasms, while VinanthMisilsletur deyt,al2.2(01097p7p)t, produced both liver and lung neoplasms. Thus, there is agreement between the results obtained in both studies at higher dose levels of 2200 to 5000 ppt of TCDD in the diet. However, at lower dose levels, there are odifffneereonpclaessminsthine rtwatos sgtiuvdeinesa,swilothwVaasn 5Mpilpletroeft aTlC. (D19D7,7b) aresepdorotinngaazdeirvoerisnecsidpeencctreumof neoplasms in a total of 50 control rats examined in their study. Conversely, in this study, there was no carcinogenic response in rats given 210 or 22 ppt of TCDD for 2 ysteuadrise.sDoifGmioovuasennsikient caal.rc(i1n9o7g7en) erseips owrtiethd TCDD DMBA. to be only a weak tumor initiator in In summary, data collected in the study reported herein indicate that doses sufficient to induce severe toxicity increased the incidence of some types of neoplasms in rats, while reducing the incidence of other types. No increase in neoplasms occurred in rats receiving sufficient TCDD during the 2-year study to induce slight or no manifestations of toxicity. REFERENCES Allen, J. R., Barsottl D. A.. Van Miller, J. P., Abrahamson, L. J., and Lauch, J. J. (1977). Morphological change in monkeys consuming a diet containing five hundred parts per trillion of 2,3,7,8 tetrachlorodibenzo-p-dioxin. Food Cosmet. Toxicol. 15,401-410. DiG iovannl J., Viaje, A.. Berry, D. L-. Slaga, T. J., and Juchau, M. R. (1977). Tumor initiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Arochlor 1254 in the two-stage system of mouse skin carcinogenesis. Bull. Environ. Contam. Toxicol. 18, 552557. 5898 0001992 CHRONIC TOXICITY OF TCDD IN RATS 303 Innes. J. R. M.. Ulland. B. M.. Valerio. M. G.. Petrlcelli. L.. F ishbein. L.. Hart. E. R.. Pallotta. A. J.. Bates. R. R.. F alk. H. L.. G art. J. J.. Klein. M.. Mitchell. I., and Peters. J. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Sat. Cancer Inst. 4Z 1101-1114. Kociba. R. J.. Keeler. P. A.. Park. C. N.. and G ehring. P. J. (1976). 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCCD): Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35. 553-574. McConnell. E. E.. Moore. J. A., and Dalgard. D. W. (1978). Toxicity of 2,3,7.8-tetrachlorodibenzo-p-dioxin in rhesus monkeys (Macaca mulatto) following a single oral dose. Toxicol. Appl. Pharmacol. 43. 175-187., Siegel. (1956). Son-parametric Statistics for the Behavioral Sciences. McGraw-Hill. New York. Steel, R. G.. and Torrie, H. H. (1960). Principles and Procedures o f Statistics. McGrawHill. New York. Van Miller. J. P., and Allen, J. R. (1977). Chronic toxicity of 2.3,7.8-tetrachlorodibenzop-dioxin in rats. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 36, 396. Van Miller. J. P.. LaLich. J. J., and Allen, J. R.. (1977). Increased incidence of neoplasms in rats exposed to low levels of 2.3.7.8-tetrachlorodibenzo-o-dioxin. Chemosphere 9. 537-544. 275303 ) 5893 0001993 5900 --art I t r C f ! - (/< r d ) ^ . D u r i n g t h e y e a r s .f r o m . 1 9 6 5 t h r o u g h 1 9 6 9 , a c h e m i c a l p l a n t producing herbicides - pentachlorophenol and 2,4,5-trichlorophenoxyacetic acid derivatives - reported a mass outbreak of acne chlorina. The number of persons stricken, 78, is one of the largest, e v e r recorded. Furthermore, some of ou r p a ti en ts were, also stricken by a complete intoxication manifested by liver lesions, by a.deficiency in porphyrin, fat and protein metabolism and by nervous and physical changes. In two cases, the intoxi cation was fatal. This part of the research will deal primarily with skin symptoms and the internal and neurological changes will b e d i s c u s s e d i n a- s u b s e q u e n t w o r k (23] a n d i n m o r e d e t a i l i n an ot he r w o r k [32]. The acne forming dermatoses are caused in most cass by crude oil derivatives - known as acne oleosa - and less often by tar and its derivatives - in these cases it is acne picea. In contrast to those acnes, acne chlorina is relatively very rare because it is only caused by a small number of certain chlorinated hydrocarbons which are produced and used only on a limited scale. Acne chlorina also differs from other acne forming dermatoses by its localization, clinical picture and course. Mass occurrences of acne chlorina have been recorded during s e v e r a l t i m e p e r i o d s w h i c h a r e s e p a r a t e d b y d e c a d e s , .in a c c o r d with industrial and chemical development and with the gradual i n t r o d u c t i o n i n t o p r o d u c t i o n o f new- chlorinated- h y d r o c a r b o n s . T h e f i r s t ca se s w e r e d e s c r i b e d b y He r x h e i m e r [18] in 899 i n the electrolytic production of sodium chloride and sodium hydroxidfr om brine.. Herx he im er assumed that the illness wa s ca u s e d b y t h e i n h a l a t i o n o f c h l o r i n e a n d b y its e x c r e t i o n t h r o u g h m e m b r a n e s and. he therefore named the disease acne chlorina; i.e., chloracne. The. F r e n c h a u t h o r s T h i b i e r g e [43,44] a n d H a l l o p e a u [16] w e r e of. the same opinion as to the incidence of this disease. Further more, Hallopeau thought that the black coloration of the comedones .5901 0011890 09 cn  I \ Page 3 -- -- ---- w a s c a u s e d b y s o m e l e a d c o m p o u n d t h a t f o r m e d on- "the c h a m b e r 4s lead w a l l s as a r e s u l t of a r e a c t i o n w i t h ch lo ri ne . R e n o n [35] a l so co nn ec te d chloracne -with lung tu be rc ul os is in one of h i s patients and ascribed both diseases to gaseous chlorine. H o w e v e r , H i r s c h [20] c o n c l u d e d i n t h a t s a m e y e a r t h a t t h e cause must be a solid substance which formed on the anodes during electrolysis because exposure to chlorine gas alone did not cause . t h e d i s e a s e . T h i s f i n d i n g w a s s u p p o r t e d b y B e t t m a n n [2] w h o o b served 21 patients, who were engaged in the production of hy dr o- - chloric acid and who were in contact-with chlorinated tar com pounds but not with chlorine gas. Lehmann [26], in 1903, co nc lu de d th at the actual c a u s e s o f th e disease were chlorinated products such as hexachlorobenzene, penta- chlorobenzoic acid and hexachloroethane which formed on the car- bon anode during electrolysis. Because similar cases of acne - chlorina were also observed in the production and processing of p- n i t r o c h l o r o b e n z e n e , W. L e h m a n n [27] c o n c l u d e d t h a t a c n e c h l o r i n a was caused primarily by chlorobenzenes. As soon as carbon anodes were replaced by magnetite anodes, the incidence of acne during - electrolysis ceased. B e f o r e W o r l d W a r I, W a h l e [46] o b s e r v e d a c n e c h l o r i n a i n t w o chemists which was probably caused by a chlorine compound - phenylei dioxide - not identified or known in more detail at that time. T h e n e x t w a v e o f t h e d i s e a s e 's i n c i d e n c e o c c u r r e d a t t h e e n d of World War Z during the production of chlorinated naphthalenes or perchloronaphthalenes. In this period the disease was given an additional new name - Peraakrankheit (Wauer [421). Chlorinated naphthalenes and diphenyls with a waxy consistency, also known as halowaxes, have some very valuable properties such as waterproof ness, flame resistance and acid resistance. These properties earmarked these materials for the production of protective gas masks and other military equipment. A new increase in the incidence of the disease occurred in the nineteen-twenties during the use of chlorinated naphthalenes '5902 MllSSi 16909 IZMOQ \: : ~ ~-- ' ; : : rage a-----:---- -- ~ i . as a n i n s u l a t i n g ' m a t e r i a l , in th e m i n i n g i n d u s t r y {Teleicy {423 ) Acne chlorina also appeared in the electrolytic production of chlorine when insulating coatings of tar were used. This fact b r o u g h t a b o u t n u m e r o u s d i s a g r e e m e n t s a n d q u e s t i o n s a s t o w he t h*e r th disease, in those production processes was not actually acne picea. On the other hand, Teleky supported the contention that ' the tar acne could be caused by chlorinated hydrocarbons contam inating the tar. The excellent dielectric properties of the chlorinated naphthalenes ("haftax", "halowax", "nibrene") led to their wide utilization in the production of cables and condensers and mother mass occurrence of the disease was produced in the nineteen-thirtie when the U.S.A. and other countries recorded hundreds of stricken i n d i v i d u a l s . B o l t z m a n n [22] a l s o n o t e d th e o c c u r r e n c e o f a c n e a m o n g t h e f a m i l y m e m b e r s o f w o r k e r s e m p l o y e d i n t h e p r o d u c t i o n of. condensers. These family members handled and washed work clothes' contaminated with the chlorinated naphthalenes. Based on these observations, Boltzmann corrected his previous original' conclusions and stated that the chloracne was actually tar acne and judged the effect of halowaxes to be that of an external acnegenic cause. . O z m s b y [31] o b s e r v e d a c n e c h l o r i n a w i t h t h e u s e o f a f u n g i c i d e used in the impregnation of lumber* (sodium tetrachloro-c-phenylp h en ol at e) . J o n e s a n d A l d e n [24], Sc hw ar tz [39] a n d la te r m a n y others also observed total disorders in their patients and Flinn a n d J a r v i k [8] n o t e d a d e a t h d u e t o y e l l o w l i v e r a t r o p h y . I n 1 9 3 8 , M a y e r s a n d h i s c o w o r k e r s [28] w e r e u n s u c c e s s f u l i n c l a r i f y i n g t h e . m e c h a n i s m of o c c u r r e n c e of d e r m a t i t i d e s and- a c n e c h l o r i n a i n t h e i r patients with the aid of epicutaneous tests - the tests were nega tive. Du ri ng Wo rl d Wa r ZI, c h lo ri na te d n a p h t h a l e n e s we re u s e d for the impregnation of ships' hulls when it was discovered that these substances could protect ships from damage of magnetic mines. The use of halowaxes increased significantly, especially in-the electi.cal-industry. American authors gave acne chlorina another name,' 00li89^903 C/1  "cable rash", because of the occupation (cable production) of the p a t i e n t s ( G o o d a n d c o w o r k e r s (14], S c h w a r t z -140]., .Morris ( 2 9 ] ) . At that time, massive outbreaks of acne and several' deaths were . a recorded. After World War'll, the incidence of acne did not sub side because the undcsirab-le experiences with the chlorinated naphthalenes did not prevent their use in th electrical .industry for some time. An interesting observation was recorded b y Hrzberg [19] i n 1947. H e o b s e r v e d s e v e n c a s e s o f a c n e c h l o r i n a i n p a t i e n t s who had ingested chlorinated paraffins in the form of a food s h o r t e n i n g . T h i s p r o m p t e d H e r z b e r g to. c o n c l u d e t h a t a c n e c h l o r i n a m u s t o c c u r a f t e r t h e i n t e r n a l i n g e s t i o n o f c h l o r i n a t e d -h y d r o c a r b o n s . S i m i l a r l y , F u c h s [9] a l s o n o t e d t h e a f f l i c t i o n . o f t h r e e children and their mothers after they had eaten potatoes fried in t h e "paraffin". W i n k l e r [48] r e c o r d e d 10 c a s e s o f d e r m a t i t i s a n d 13 ca se s of a c n e c h l o r i n a a m o n g 23 w o r k e r s in a c o n d e n s e r factory., t h a t u s e d n i b r e n e i n .t h e p r o d u c t i o n s e q u e n c e . F r o m t h e s e c a s e s Winkler concluded that "chlorinated hydrocarbons cause the forma tion of reagents that in turn cause an increased secretion from l u b r i c a t i n g glands". In 1949 G a v r i l o v a [10} w a r n e d o f the photo-; s e n s i t i z a t i o n e f f e c t o f p o l y c h l o r o n a p h t h a l e n e s .' I n r e c e n t y e a r s , plastics have replaced chlorinated naphthalens in industrial, use. Even today the production of sodium hydroxide may be a source of acne chlorina. We have observed more cases between 1950 and 1965. The main causes of acne chlorina in these cases have been the combination of chlorine gas with the hydrocarbons in the tar gaskets and fittings on the electrolytic containers and tubs and . with the mineral oils which were used to protect the electrode c o n t a c t s . T h e a u t h o r s n o t e d s e v e r a l c a s e s o f a c n e 'c h l o r i n a a m o n g l a b o r a t o r y w o r k e r s a f t e r t h e u s e o f n i b r e n e i n 't h e e l e c t r i c a l industry in the 1950's. Furthermore, the acnegenic effects of the mineral oil based coolant mixtures used in metal working increase when chlorinated . hydrocarbons are added. Therefore* some cases of acne oleosa were observed which exhibited a partial acne chlorina character (more 0011803 z Z \ i Ii I I i i r f a j j C s Iff distinct follicular hyperkeratosis, less inflammation, cyst forma tion and atypical localization). The last wave of the disease's occurrence was recorded in the 1950's. This outbreak was noted among persons engaged in. the production of chlorinated phenol type insecticides and herbicides. From 1950 through'1951, Baader and Baer [1] and. Brinkiaann 171' published information on the occurrence of acne chlorina which involved the complete internal,, nervous and psychic symptoms among 17 workers engaged in the production of .pentachlorophenol.; Several years iater, Hergt [17],. Oettel [30] and Hoffmann [21] observed a total of .80 afflicted workers who were employed in the production of 2,4,5-trichlorophenol by the alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene. From 1954 through 1956, Kiramig and Schultz [25] recorded 31 cases of acne chlorina in one Hamburg (Germany) plant that produced 2,4,5-trichlorophenoxyacetic acid . and its esters. By very detailed research and tests, these authors have shown that'acne chlorina and total intoxication with damage tc the internal organs, mainly to the liver, with psychovegetative dis orders were not caused by the basic chlorinated hydrocarbons, i.e., tetrachlorobenzene, trichlorophenol and trichlorophenoxyacetic acid, but rather were caused by a ballast substance concentrated in these compounds in small quantities, namely, 2,3,6,7-tetrachlorodibenzodioxine. These authors contributed significantly with their other experimental works to the question of what sub stances cause ache chlorina.and how these substances act. .In 1964, Bleiberg and coworkers [3] observed 29;cases of acne chlorina among persons engaged in the production of 2,4,5-tri- .* chlorophenol and 2,4-dichlorophenol. In eleven of these cases they also diagnosed symptoms of porphyria cutanea .tarda. In 1957 Bowen and coworkers [4] observed acne.chlorina among workers em ployed in the production of DDT from trichlorobenzene and hexa-- chlorobenzene. The fates.of 53 patients Who in 1953 had worked .' In th alkaline hydrolysis of tetrachlorobenzene. to trichlorophenol verc described only last year by Goldmann [11]. In addition to acno, Goldmann also diagnosed in his patients bronchitides and' O - 0011894 .tsv* *< '5305 disorders of the liver, kidney, myocardium and nervous system. The author described the death of a patient -from "necrosis of -the pancreas as a special case. Furthermore, he also observed . dermatitis in a nurse who had had contact only with experimental animals. Finally, he diagnosed acne chlorina in the fourteen year old son of an employee whose disease was contracted only from the household environment - by wearing his father's con- taminated scarf and by usingr his father's towel. . Actual observations The process called-PCP (pentachlorophenol) which caused mass damage in Czechoslovakia was, as far as production is con cerned, identical to the process from which Kimmig and Schulz drew their patients for observation. The final products of this process were sodium pentachlorophenolate, 2,4,5-tricholorphenoxyacetic acid and the latter's sodium salt and butyl ester. The primary raw material was technical grade trichlorobenzene which was produced in a neighboring building for.the production of hexachlorocyclohexane (HCH) and lindane. .The table shows the entire production scheme (Table I). The chlorination of tri chlorobenzene produced tetrachlorobenzene and hexachlorobenzene. Sodium pentachlorophenolate was formed by the alkaline hydrolysis of hexachlorobenzene. Sodium trichlorophenolate was produced by the alkaline hydrolysis of tetrachlorobenzene. The hydrolysis of tetrachlorobenzene with sodium hydroxide in the presence of . methanol took, place in an autoclave at 190C and at 45 atm of pressure for one hour. After cooling, the methanol was distilled from the hydrolysate, the hydrolysate was diluted with water to a 25% concentration and it was then syphoned into a storage tank. The. condensation of sodium trichlorophenolate with monochlo'roacetic acid produced sodium trichlorophenoxyacetate. FIollowing cooling, centrifugation and flushing with water, the dried sodium trichlorophcnoxyacetate was picked out by hand and placed in small barreOls.. Some of this product was used to produce the butyl ester of- o ro . 03. Cff 5906 P a^cf trichlorophenoxyacetic acid. This production tok place in . another building. The production of both the primary products took place in a four story building with grated floors (WEMA grates) which was one reason why the entire_building was contaminated with the high risk substances.. The building was not sufficiently airtight, the local exhausts were in most cass improperly installed and . hermetization and mechanization'were insufficient.' Initially, the sodium tetrachlorophenolate [sic] was escaping .from the centri fuge into the work space in the form, of a mist. A series of tasks such as the removal of solid substances and the pumping of liquids were performed by hand. The workers were dressed in linen work clothes. The thorough cleaning and frequent changing of the work clothing was impossible to guarantee over the.entire interval of production. Rubber gloves and respirators were used at some work sites. The conditions surrounding the production of the butyl ester of trichlorophenoxyacetic acid were the same as those described above. Kimmig and Schulz [25] found that the main source of acnegenic hydrocarbons was that segment of the production process in -which sodium trichiorophenolate is formed by the alkaline hydro- ' lysis of tetrachlorobenzene in the presence of methanol. A co worker of. these authors. Doctor (of Chemistry) Sorge, had de duced on theoretical grounds that if this reaction were conducted at 190 *C and 45 atm of pressure, chlorinated hydrocarbon by-pro ducts such as polycblorodibenzo-p-dioxines, polychlorodibenzo-; furans, polychlorodiphenyl ethers and polychlorodiphenl oxides may be formed in small amounts (Schulz [36]). Sorge also pre pared some of these substances by his synthetic process. Animal tests carried out by the authors (Schulz and Kimmig). w i t h t e t r a c h l o r o d i b e n z o f u r a n a n d 2 , 3 , 6 , 7 - t e t r a c hIl o r o. d.i b e n z o d i o x i n e have proved an extraordinarily high toxicity for these substances. A simple, smear of a 0.1% solution of tri- and tetrachlorodibenzo furan on a small area of a rabbit's auricle caused heavy liver .5 9 0 ' 7 DOM2I6059 A j c, f damage and death of the test animal within 2 to 3 weeks {Schulz (36]).. 2,3,6,7-Tetrachlorodibenzodioxine, which also has very strong acnegenic effects, was proved to be far more toxic. Only then was Dr. Sorge successful in the identification of this enormously toxic substance-ih the production process and in the final product. He proved that this toxic substance is formed only at certain temperatures and pressures. If during the alkaline hydrolysis of tetrachlorobenzene to sodium trichlorophenolate the temperature is reduced below 153*C, the toxic substances do . not form. However, at this low temperature, the reaction time is extended from one to five hours and the productivity is ob viously lower. Furthermore, it was proved that the condensation of two molecules of trichlorophenolate to give tetrachlorodibenzodioxine is an exothermic reaction. Several explosions of auto claves with injuries and the lethal intoxication of workers were reported to be the result of this reaction in the German Demo cratic Republic (East Germany) (Schul2 [38], Goldmann [11,12]). .Tetrachlorodibenzodioxine was also identified in the Czechoslo vakian production process and in the products as well. Th first two cases of acne chlorin were noted in 1965 in two technicians who operated the production machinery. It was as sumed at that time that these tw o cases were caused by careless work and an undeveloped production technology under semiproduction conditions. After one year without a reported case and during which production was temporarily halted, a mass outbreak of 78 cases was recorded within the following three years. Finally, after a detailed investigation and study of the entire problem, the process was terminated and production ceased in 1968. Workers from all stages of production were stricken. At that time, it was impossible to determine which production stage was the most risky because most of the workers were involved in several stages at various locations in the plant within a relatively short time. Workers directly involved in production as well- as maintenance per* sonne1 and workers digging sewage ditches were stricken. Later a research scientist working to isolate the acnegenic ballast : , 5 9 0 8 '. DJ>H2 16059 substances, from the products of the individual production phases was even stricken, ilis research work vas carried out outside the production plant in a research institute's laboratory. In addi- z/ 4 tion to the 78 patients with dangerous'symptoms of acne ;chlorina. or porphyria, other workers in this plant had symptoms of acne which were, however, symptoms of juvenile acne or of a similar character and could not therefore be counted as an-occupational disease. With the exception of two female laboratory technicians> the patients consisted of males from 18 to 57 years of age (4 persons below 20, 46 persons from 21 to 30, 11 persons from 31' to 40; . 10 persons from 41-50 -and 7 persons from 51 to 57). Therefore, . the acne chlorina patients were mostly young people. On the other hand, more than half the patients with porphyria cutanea tarda - were older than 40 years. Generally, the disease followed a normal course as described by other authors. In only six patients did the disease begin sud- ' *denly as acute solar dermatitis,\ i.e., by.erythrema and edema . in regions exposed to solar radiation and continued by acne sym-' ptoms (Photographs 1 and 2), as observed by Goldmann [12], Grimmer [15], Braun [6] and before these authors, also by Touraine and co workers [45]. Therefore, chlorinated phenols in some cases have similar photodynamic effects as the chloronaphthalenes described by Gavrilova [10]. Erythema and edema in these patients were obvious mainly on their auricles. Sometimes, small blisters in thick groupings were observed there. In all the other patients the acne developed slowly. Among the less severe cases the disease was limited to single or densely grouped comedones, conditoned by follicular hyperkeratosis, which were so small and sometimes so numerous that they appeared to be. a slate grayish-brown color, like dirty spots on the skin. These symptoms were noted mainly on the face, with max ilium occurrence above the cheek bones (Photograph 3). In the more severe cases; . the comedones gradually developed into small white cysts similar rs> 5909 to nilia, although with -black centers, and into large cysts ranging from the size of a pea to that of a nut (Photographs 4, S and 6). As opposed to other acne forming dermatoses, the described symptoms and manifestations mainly lacked signs of an infection. Only in the more severe cases and rather in the vici nity of large cysts were some reactive inflammations, infections . and abscesses noted. The emptied cysts and abscesses left behind ' large atrophied scars with raised edges and with bridges of. intact skin under which a probe could be inserted (Photograph 7). This basic clinical picture was supplemented by diffused hyperpig mentation in the face and by papulopustules on the torso and ex tremities. Patients who were found to suffer from a disorder in - porphyrin metabolism .had' symptoms of porphyria cutanea tarda in regions exposed to the sun (hyperpigmentation and hypertrichosis,-' blisters,'excoriation and small scars). However, in this group there were two patients with prophyria but almost without any acne at all. , . -, * -. Histological investigation is not much, help in diagnosis.. The symptoms begin with hyperkeratosis at the mouth, of the follicle After one to three weeks, the follicle changes into a cystic for mation filled with a h o m y substance `[keratin?] (Photograph 11) and to a small extent with sebum. However, the epidermal changes are small. After the rupture of the full follicle, the contents of the cyst penetrate into the surrounding tissue and a granuloma of foreign bodies forms around the area (Photograph 12).. The disease normally starts on the face above the cheek bones. However,- in 17 patients the disease started in an unconventional manner by papulopustlar eruptions on the extremities, primarily on the lower extremities. This characteristic, resembling.more ' acne oleosa, remained in some of the patients for the entire dura tion of their illness. The most common localizations of acne chlorina were.the cheeks temples and'to a lesser extent, the entire face." Eyebrows, eye lids, the perioral region and the scalp and hair area remained r*o cn 5910 cn 0011897 POW2I6068 Page 12 ~ ` Other predilected areas vere the auricles, th back, of the neck, the back and the chest in .a seborrheic localization and the genitals. The auricles and genitals and especially the scrotum were stricken by the formation of large cysts (Photographs 8 and 9) Among the five most seriously ill patients, the disease spread . all. over the body and had a discouraging.effect that was heightened by the. disease's usual persistence, the long term healing and by medical helplessness (Photographs 10 and 7).` The.contents of the cysts produced a repulsive and rancid odor. For some of the patients the disease disrupted their social and family relations and caused severe depressions.- Some authors, such as Braun [6], emphasize itching as the initial symptom, but we noted this symptom in only three cases. In 20 patients with extensive symptoms,, the occurrence of pustules, cysts and abscesses was also noted in axillae although this location was not mentioned by other authors. Because of the. great number of follicles in the axillae and the similarity of the axillary skin to that of the genitals and for several other reasons, this finding appears to be just as valid as localization on the genitals and the genital area. On the other hand, it is surprising that acne chlorina does not strike at the hairline or scalp. In the case of one patient, the ache symptoms disappeared in the facial areas when he grew a full beard while in other areas, the symptoms continued without a reduction in intensity. So far. there is.no explanation for this phenomenon. Hypertrichosis or hyperpigmentation or both were, determined in the faces of 19 patients without a laboratory diagnosis of a deficiency in porphyrin metabolism. Bleiberg [3] made similar ob servations. These symptoms receded simultaneously with the healinc of acne chlorina. Mbm younger pogoone-ouffared from aeae e^lerina-than"eldeg persene.* This study group contained SO persons (85%) below 30 TraitelabegTa. ate i Beginning-od-this-sentance illegible-- Many authors believe that younger, persons, have a greater tendency to develop acne .chlorina than older individuals. ' (Correction by NIH Translation'Unii I Page 13 years of age. However, this fact cannot be used as a hard and fast rule or to support the findings because the production plant under study employed mostly young workers. The preillness medi cal history among the patients did not indicate any significant predisposing factors and no incidence of serious illnesses, intoxi cation or etiolation*. Furthermore, no relation to hair color, eye or complexion color, to the degree of.body hair, to the per spiration of skin oils or to hereditary follicular hyperkeratosis * and other skin diseases was noted. Only one patient reported a more severe prior case of acne juvenilis, 12 patients reported mild cases during puberty and 14 patients reported only insignificant acne juvenilis symptoms. 18 patients also had some infrequent Con tact with mineral oils either during the appearance of the symptoms or at some time in the past. ' These patients were mostly involved in maintenance work as mechanics. However none of them had ever had an oil acne in the past. Their illnesses following 'contact with tetrachlorodibenzodioxine were of the same character as the illnesses of the other patients. Their diseases were not similar . to acne oleosa either in appearance or in localization. The latent period from the initial contact with the risk substance to the appearnace of symptoms was quite varied.. Same workers became ill after exposure of from several months to several years and the disease in their cases.was rather mild although the indications pointed to a massive exposure. In other cases, severe damage occurred after a very short time of several weeks and after an apparently small exposure. For example, one of the most serious: ill patients worked under the semiproduction conditions for only, two and a half weeks before the outbreak of the disease (acne and porphyria cutanea tarda}. After that he had no contact with the risk materials. It is not clear whether individual predispbsition or the level of personal and work hygiene is more responsible for the intensity *Sdtsea note:-- This may be a dubious sheiso The Caoch "atylismu fcgensliterates to eteiliam^r ._ alcoholism (Correction by NIH Translation Unit) : O. Page 14 i of'damage or latency of the disease. Most authors discount individual predispositon. However it is believed that the deciding factor must be the degree of actual exposure which is ' undoubtedly dependent on a series of other factors such as work discipline, personal discipline, adherence to preventive measures, frequency of changing work clothes and undergarments, the degree and regularity of hygienic decontamination, work site order and cleanliness, dust in the environment, etc: These patients also had a series of subjective difficulties: tiredness, weakness in the lower extremities, muscle pains, sleepi ness and insomnia, increased perspiration, lack of apetite, head aches and other disorders in the mental and sexual spheres. These complaints were more recurrent and more intense in patients with the more extensive skin symptoms. In more serious acne cases, a . significant weight loss was also noted. It is our opinion that all these difficulties and symptoms are manifestations of a rather overall intoxication than of only the acne chlorina. . All the patients were admitted to a dispensary. Regular examinations of their dermatological, internal and neurological conditons were arranged. The patients who were still working were transferred to prevent further contact with any acnegenic factor. All contact with chlorinated hydrocarbons of any type was prevented even with those substances whose acengenic effects have not been thoroughly proved. Furthermore, all contact with, mineral oils and tar and its products were prevented. The application of mineral based ointments was discounted for external therapy. The plant's medical doctors were cautioned against prescription of any internal medication with an acnegenic effect (iodine and bromium preparation corticoids and others). The patients were given detailed medical directions concerning their daily regimen, personal and work hy giene and secondary disease prevention and treatment. All available medications for acne were utilized in the treat ment. The expressions of comedones and the pressure relief of the contents of cysts and abscesses necessitated many periods of 09IZHOO ; 5913 0011902 O) ( yage'xa-- hospitalization and special ambulatory treatment hours attended by a specially trained nurse. No special results were, observed as a result of repeated hospitalization. The best results were noted after the expression of.comedones by the use of a sauna constructed for the patients by the factory on the author's re commendations. It was not possible to test the effects of vitamin A acid (retinoic acid) which has been praised by some authors in . the medical literature. However, in agreement with the findings of other authors, it was determined that no local' or internal . medication exists which has an effective and permanent healing effect on acne chlorine. Some of the patients have been under .. observation for eight years and their disease still persists. Dr. Schulz's [38] patients have manifested continuous symptoms for more than ten years. Goldmann [11] has now. been treating one patient with comedones and cysts on his penis and scrotum for 18 . years.! In most of the patients discussed by these authors, the . symptoms and manifestations of the disease progressed, and reached ' a maximum after about one year, even after transfer of the patient from the high risk work area. . A regression takes place after two or three years at the earliest. In some-isolated cases, the pro gress of a mild course of the disease was observed even after, five years. However, the regression of the disease in all the patients was very slow and, it may be said, even lasts a number of years. Of the 78 patients treated by the authors, 76 suffered from acne chlorina. Of these,.11 simultaneously had hepatic lesions with a deficiency in porphyrin metabolism. Two patients did not have acne chlorina. One of these two had porphyria and the second one died in 2.966 from acute intoxication. Pentachlorophenol was considered to be the cause of death in that instance. When the toxicological analysis was carried out at that time, the investigators were unaware of tetrachlorodibenzodioxine and, therefore#. * it is our contention now that in that case, this highly toxic substance was primarily responsible for the acute intoxication^ ' ; ro 5914 , S 0011903 S , f nagre"-xu Gombos 1131 mentioned one patient with acne chlorina and porphyria in connection with this production process. The authors analyzed '55 patients internally and neurologic cally in detail.' About.half, of these patients had a deficiency in lipid metabolism and in more than one-third, some minor.biochemi cal deviations.and mild hepatic lesions were noted in the first stages.of the disease. In 17 patients, the authors found symptoms of central nervous system disorder, the majority with lesions of. the peripheral neurons of the lower .'extremities' (verified by EMG . examination). In most of the patients, physical disorders, pri marily an acute neurasthenic syndrome, were diagnosed. In addi tion to the death due. to acute intoxication (pentachlorophenol? tetrachlorodiberizodioxine?), three more patients died.' In one patient, the.porphyria developed'unusually rapidly into an .- ' arteriosclerosis of the brain with an atypical morphological diag nosis and demention. Two patients died from bronchogenic carcinoma All these patients with their complete symptoms 'ire discussed in other papers [32,23]. Discussion In agreement with Kixnmig and Schulz, we cite the tetrachlorodibenzodioxine which.is formed during the production process as .. the main reason for the skin symptoms as well as for all the other deficiencies mentioned and diagnosed in the authors1-patients. This toxic compound was confirmed in considerable quantities in. the final product (Arboricide E50)., in the butyl trichlorophen- oxyacetate and also in the mortar and wall paint of the factory building. 'Even adjacent rooms such as worker dressing and locker rooms were contaminated with this compound. Test rabbits placed into these rooms soon died. In his monograph, Braun [5] cited the following chlorinated hydrocarbons as the then known causes of acne chlorina: chloro-. . benzenes with various degrees, of chlorination, chlorinated phenols (sodium tetrachlorophenolate, pentachlorophenol), p a ra -nitrochlorobchzene, sodium tetrachloro-o-phenylphenolate, chlorinated .- O '5915 0011904 naphthalenes, various chlorin ated diphenyls,, trichloroxydiphenyl, chlorinated diphenyl oxides, sodium o-(2-chlorophenyl)phenolate and othrs. However, Kimmit and Schulz [25] ascertained that pure pentachlorophenol and pure 2,4,5-trichlorophenoxyacetic acid do not have any acnegenic effects. Therefore, they checked the presumed effects of the other substances that were blamed for causing acne and found that neither pure nor chlorinated benzenes '.(from 1 to 8 ch lorinated positions) nor chlorinated diphenyl- * ethers have any acnegenic effects. A definite acnegenic effect was proved only for the following substances: chlorinated naph thalenes (5 and 6 chlorinated positions):in agreement with Shelley and Klingman [41], higher chlorinated dlbenzofurans (diphenyl oxide: and 2,3,6,7-tetrachlorodibenzodioxine. Therefore, it is possible and even probable that some chlor inated hydrocarbons that have been previously considered to have an acnegenic effect do not have this effect and the actual causes . were other ballast chlorinated hydrocarbons contained in.those hydrocarbons. Tetrachlorodiphenylene oxide (tetrachlorodibenzofuran) 2,3,6,7-tetrachlorodibenzodioxine (tetrachlorodipheny! dioxide) In an animal experiment, Kimmig and Schulz found an ex ceptionally high toxicity for tetrachlorodibenzodioxine. In a local application on rabbits* auricles (keratosis test), this comr pound produced a strong reaction even at a concentration of 0.001%. Also; a skin application of this substance at the same concentra tion produced acne chlorina (Schulz [38]). Tetrachlorodibenzofuran was proved to be 10 to 20 times maker. Chlorinated naphthalenes had a thousand times weaker effect. Generally, acne chlorina is caused by the direct contact of tetrachlorobenzodioxine with t3 skin. Absorption through the skin is not ruled out and ah e f f g p _ IS) ' CO- 001190$ t'aytf-- xo through the ingestive or respiration tracts is also conceded.,. However, in a pure ingestive experiment with a test animal, acne chlorina did not occur. However, the cases of acne chlorina con tracted after the ingestion of chlorinated naphthalenes as food fat substitutes should not be forgotten (19,9]. Tetrachlorodibenzodioxine used internally has a strong he- patotoxic effect. A dose of 20 to 50 mg per kg of rabbit's body weight causes death in all the test, animals by yellow liver atrophy. Because of this enormous toxicity, all the'preventive measures taken at the high risk processes in East Germany and Czechoslo vakia were proved to be completely ineffective. The only de pendable solution to that problem was the reduction of the re action temperature and pressure during the alkaline hydrolysis of tetrachlorophenolate to sodium trichlorophenolate. In East Germany, the decontamination cost of the high risk areas and buildings was extremely high. One chemical plant attempted to use for other purposes a contaminated building which* had not been decontaminated after the building had remained unused for two . years, but. new cases of acne chlorina were shortly reported. With normal decontamination procedures new outbreaks of the disease occurred and therefore, decontamination had to be carried out using special pressurized chemical protective suits with pressurized breathing apparatus because gas.masks were not sufficient. Doors, windows, the wooden trim and other wooden building components had to be burned; the steel construction materials in the building had to be sandblasted; the plaster, facade and floors had to be torn out and cast into cement blocks together with the tubing, plumbing and other equipment. These blocks were then transported to the, Atlantic Ocean and sunk. The disposition of those non- , flammable materials by burying would have run the danger of con taminating the subterranean water supply. Only the most expensive. components, autoclaves and some valves, were successfully decon taminated by repeated washing with special detergents. The Czechoslovakian plants are still closed and. their fate^a' still undecided. The patients are still under a continuous o b - O servetion. ^ 001190$ 5917 O) i* ___ References 1. Surfer, E. VA, Basar. II. I Industriai . intoiiealMin Una io pcnueblurplicooL lad. ihul. Sur*. 23. ISjI. . 233. -- 2. BoUiuaun: Cblcr-Akuc. clan besandere Fera too pr* Icssiuaellcr ll>lcrkr<mka>t DiscIl ocd. W tch r, 27. 1*131. x 427. -- 3. B U ilu rr. ) , IC alles, K , B rodaio, I-* In d o tti JaU raciiair* ed purpiirr. Ardi. Dora, U , USI. s. 733--797. -- 4. Sansa, S. S , Mmimiod, M. Tu Oliarcene (a llu oanutactoro oi DOT. . AreS. Ocra. Syph. (Cblcagol, 75. 1SS7. x 743--743. -- 5. Brasa, I7d X^tlaratoo. Mo* , nogupitiM zor Zolische. Oenilsdcraotesca. t Dead I , Auletidar! L, WUrtt, Lili'loa Cantor; ,,1355. . 73. -- 9. 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FiaaafcraskbsIL Arslu IItf. 117. 1333. 1. x. su ved eas -- dC Uraaa S. . -- 23. Jlrdisk, U. Z slu ci:/. ) . Asbix, IC. . HttliU, Z . I*axJerari. J. al nU Vilduky tslUatd imasikaca a alino chiodila pii vj* ahi hcrbhdd -- piip.uvorduo do Usha. -- Page 19 i. 2 L Jonca, J. VA. Aldui. IL Sa An aconiarm * dcnuatorcosti. Arch. Dtan. S/ph. (Chini* . co) 13. 1233, x. 2L22--liUX -- 23. KbciLC L Sdisia, K. IL: OeruUldio Akaa (or. ' Coloratoci durcb chlorlene orooalUdio zykllidie Uier. Dcrsatolostea, 115. 1557, . x 540--549. -- 20. Lohoiasa, X. IL: Experi* . .menIella SUuilon aber dea ELnlluts tedi- . . aitch a. fafciealzdi widillscr Cara u. Oao* ' io o si dea Organimi!. XL Siudlou Otar. .Chlorakne*. Arch. n r r . 40, 1003. . 322-- 333. -- ClL Urani 5 . - 2 7 . Lrbiaaan, W: .*Otar Chloratoa. Arch. D ora, 77, 1905. . 205--203. Uditesi 323--344. -- 23. Marasx. 7L K . SOrerhers. K. &s Stia condition .Temiuse from exposure to certain chiarina* - led hydrocarbons. ). ludusir. llyg. Toxicol,' .20, 1933, x. 244--253. cU. Uraua 5. -- 23. . Harrix, `C. L , Zatasshow, L. JL_*- .Cabla . rash* -- A nolo on s now deenxiar mix* ' lata. J. Amcr. acd . Axx, 121.1913, x. 132-- { 333. -- d L Brami i -- 30. DdloL U s JOl-. r a u dio a . tlcnujialam tcH a Erlshnagaa Jialt liochloxlxchca Chlorkohleatsassemol* * ,i lea; ala Bcltras zna Penu-Prohlam. Wetk* - AXnio Koalernax, Bed DarMiclm,19S5, ctt.*. ...................... . , V . . _! .Baaor 40.'*--* S t,- Oimihy,' Oj^DUkussf y ,. p oxointto'k drfnku Jon-- ?f. -- .32.^. T o sin o ti. U tokiL S , Spiritati, I I , ]t- } . risale, L . XiOsBsk/. J, F itto ti, J, IlxircV. * A . K tei l II, JUis, J- I'uiLosjal ztlravl- pwcMoBU pii vfrobA Z.4.S-1!IchlOrltsiaxy. ocuna tedadia. fratta. U*v, (v lis ta i.-- 3X FlottU. IL: Zur Klsdlk net Ccatsdaacn* BOdtiar UJ Chiaraluto (llalow sxauo). Arch. klla. sxp. Dorai, 239. 1970. X 223-- 24L -- 3L FLnrir, LobalLehsndlui.r dcr Cbloracae (llalowaxacaal aur vuotala A* Ware. Haitiani, 21. '35170. a. 405--470. -- * 35. Hfacrt. i t i Inioxlcatloa praiesslsndl par lux repours de cblorc; a<to chiurlotto ot lultsrclatc putuuaalre. Scia. m iti. 1305, x.423. -- ctt firata 5 . - 3 . SelutU, IL Zs JQlolecite a. cxpcrloieaudlc Usicraschua* pea sur Atiolofia dcr Cbloracns. Arch.- * kiln. cxp. P era, 223. 1357. 589--C30. -- 37. Setola, IL IL: Zar KUnik s . AUologlo dcr Chluraltnc. ArboUsuulizbi (ASA), 3. 1933, X. 25-23. -- 33. Sshulx, li. IL: Osotoi sdiicol. -- 33. SdnrarU, t s DerstutlUx i n a syathciic resus osti v tu c x Amur. J. paid, ltllh , 20, 1920, S. 53d--592. -- <40L Schararix, t i Aa ohlbrcuk al llaluiax acne {.cablo rash*) aaump clckiricanL J. Amor. ned. Axs, 122, 3313. t. 2S3. -- 4JL ** Shxllc/, 57. A , Xllasfea, A. t s The epo* rinculai predaevea ul cetw lt p&tu* and JicxachlBttwplitalbae. Ardi. Ucna. Syplu (C higol. 75. 1357. X. 6SX -- 42. Tahikor, Id Dio I'ltnukraakhd: (Chlorshae). Klla. VAlChr, 1 2 . 1327. x 45-443, iU iU a 097-- . SOL -- 4L Tbiblcrra, Cd Acni coBrfdaa. ptocralisCs dite 3 Htihslallod pralcsstoael* le de vapours chloriqucs. Seas vn/.d, 1300, x. 24, ClL Brau &. -- 5918.0011907 0909 I.ZM0Q .. Pag~2'CT UThiWco;. C., Feg. Miei, ' >tu L`ze*e c^kji. ui*. a.u iierm. _ Ji-U. W X1, tm *15--`523. -- cl. ` ,, B.-jsa 5. -- 4S. Tojrutt'o, A, Soleste, B,, J/ --alluri, B . A etna: Si o de ilcnaitltei ' per ir:ihIurae|tUMllae. tlulL Voc, 'detta,' MjiB,, i l . 1931 s. 3.5-270. -- i 3. WaUlsT- " i\: Vu.-i 2 Felle tut .CIietfM'. Iiuvt* . Ml DIU. Leipzig. 2914. -- ctL litt. S. -- . 47. vreuet: tidoeblirhe ftkrenVang curdi tiitU u u KiAieiwturtuili ll'e n u b u l . liciti. ZK. Ccvcrbciiyt, 9, 1911. . lO-- ' 201. -- U WlniJitf. Zar CUckI m (Per- R.".rssUicj. 2. lU st. CjssLIVt^ U . ; iU z. 415--491. -- 43. Hattet. Sclial. X . u , S,>U ltu . De U ent3lriie y*7jUla* jtc* Itti der HunlcUuas cs CLierjUiesel--*' - W itisutnagen, Arck. C airnb^PataoL Ca'** 4txb. lly s, 18, 2931, i. S52-5W . -V [10] [13] Pathogenesis of photodermatides from chlorinated naphthalenes. .. . 4. Gombosr et al., Porphyris cutanea tarda and acne chlorina ..occurring during the production of cyclic chlorinated hydro- . .carbons. _ [23] Symptoms of total intoxication in acne chlorina during the production of herbicides in final preparation for.printing. [31] Discussion on paper by Jones [24] [32] Health hazards'to workers in production of sodium 2,4,5-txd; chlorophenoxyacetate- I. ; OOH90S 5919 '7 TABLE I . P roduction Sehea*. Plane fo r production of . HC8 (htxachiorocydohexane) Page 21 1*0 1,2,3-erichlorobanzaaa and its' isooers (1,3,5- and 1,2,40) .. V * -> . pastachlaropbanolaca .i..1f*,(.v.*.k >. v 1 1. f l f l J. *. v i * . y*+ R *V M * : t . * *. ** 0011909 5920. X i 1 9 0 9 IZMOQ ' fl ras rematad thnt Um lila and general tactirma tr e n ca a d by 2JA7-l*tre- ttlorodltoeozadlaxln wblch la tonned dunng alkallcw bydrolyso o inTirlilniiraciiijji. uua eodiua irtriUargphanoUiA Um rractloo w at perfonncd by a a anaritabla pepeada ra ai a im p e m a n o t IW C and p m n n of 49 a in . From tfw -sor* o Sllala and KUaarig tt la kamra Uul liria hlgiriy lale labrtincat la aoc. tem ad whea iba a b a n n iriin n la earrMd oat at a lo t r (amparaten o< 1S3*C KM onJy rock*re angagod la prodac Iloa af feerWcldaB wara alaciad bol also raraataaaaca ararfear cartn diggta ti am aga cnaaaai. la ala iba dtaanaa basta aa acata aotar enastilo. tn all tbo nratlndra II by a atoar arapdoa ot cewtdonaa aad thatr dera lopraeat lata eyata. iMflimraanoaa aad acam as arara tomad eoly la iba leoei ta r a n a n d e * tanatee atractioaa. Tba aba aad tan-al auatfestaiioea am tpoodad lo d a u taponad la tba 11ten tara, la 17 harn ear. acaa eblortaa-ttartad la a n a ta tl cay by pa'paloparaulam anpU oaa aa stnrattMA la 20 panano tria oleara tia m a d loeaUaatioa la tba axtllaa trie* r oot taponad p n n o a tly. Tba diarara doaa a et atlact tba balry p a n al tba aba i la oaa patlaat tt duappaana aran Ireot ibera site* la tba laca and aa tba ba (tara a baard, wbila tba cooditloa pactaod ai b a r m oa Pailoats wtm aa uapaind porpbyn atctabollaat tu lc n d (rom typwal byprrpigmanianaa and brpanrieboaO aad wriy naa o( iba froai bolloait aclnica aractuaici. Hypmncbociaaad byparptg u aa atas obrarcad olmo la U tablacta triUt acaa vriinoat labeniar d y n t al la tba pattod prior ta tba dlaatar aa goUicant pndtapeatns factara tan i Tba Utancy d tflm d . la tora# Insuacaa ta r a n drmatniogtcal , n lo i t d altar io n asparon, la b a r lotiaacrt appanm ly tong a a d .: c a n oaly arild tyatptooo. Oatplta th o tba a atb c n a n caa riactd tbat tba lataaaHp ot Iba dlaaaro dapaada a t a oo dxpoaan tora pradOporiUoa. Ib a potleao bad ranoaa corapirtno-tMDtlie. sa n ta l aad aotaal aad roara o rirara bad canridanblr araight lanar. Trra dlod froai genaml liuorlc rilnw. uro (ro a hmg cancar ta i ao la r tt la a ppealbla to a ta nlOMy whatbar lt na relatad te tba oerspaltana! asparan, p w rtfect o t cblortaatad bydmcarbom prodacad ht tba patttralar p a aras a : lar. Tba raedle! aad aaanlpsicsl tira arar la i iitai'YMOV sanarsi latoxtcarton llttla fa e ttn . A cartata bai rad anear, la an paltana cooda ry la srrnrdtnca i 4A 0 7 3 , . J, A JOS--317. Hidratara a, E. W, ia a n , . btdestroi das to pnm tilorpbanol. bid. Mad. Sor. 20. USL a U A -- 2. Satlraaaat Chler-Afeon. n aa baroadara Fona roa pro- Waebr, 27. 1S0L a. 4 . -- 2. tartars . J , W a n t. K , A iadtla. Id tadasuM Iy acaalo ad porpbyn. Arca. D m . *3,' U H a. 739-7*7. - 4. la m a . A A, M aarnri; M. Fd O r ia m o bt tba aaafactan al DOT. Aten. Dana. Sypb. (C blcaiai, 73. 1337. a 749--743. -- 1 S n aa. W j O rio n i Ma> ! L, AaOaOorf L W trtt, Cdhton Caator , a 73. -- A I m a , d KlbrircMt leo - worn aar t n oob ng dar Chloracoo. H aotrnt, M, USE a 123--123. -- 7. Irlab- Vanrag. 0nr*ranm . Tagg. Hata L W. ISSO -- cri. Baoer--- 43. -- A Fibra, V. 3 . Jarrflb D. Ed Aerina crtala cblartaaiad aaphm aaaa rat tba livor, Frac. Soci aspra-. Slot. Mod. 33. 1393. a 123--1281 -- A Fosb Fd eli. Brrara -- A -- 1A l . Md O p a la s i ara fctodarranlimr t n ratraaaycb aatotbra*. Sara. Darra. Va* aar, 134A A 3. a 3A -- IL Dtlla a t a. L Fd SeHo m a r afean QUorabaa darcb TH* ChlorpbanM-Tai eem ingepiudubta. Arbori* (ASA|. 7. 1372. A 12--1A -- 12. F. IJ Scbtrrrao abata China* Mararairaoaifeani darcb A X A 7-Tttfracftlerdlhaaio dlon a . Haotarct, SA 1373. a 143-132. -- 13. t r a t t i, B , IO* SI' [JLJiiura.sa'm a* 'mg-ip rtt THIS IS A SIN G LE PH O TO CO PY MADE BY THE NATIONAL LIBRARY OP MEP< o o im i "F922 >c :- Ob . * :v I\ iV4..^*l;i-.s*1 *. \ .,>. .I*4 *J v ;vJT~^-iVf#ir I ta lv ec with V. C. hove. a f Sot, " I t nam in g. .s c : t : t : :ne fa llo v in * i So haa 20 casea or enloracno. No Id not t o l l so another or not th e ir plant a town. They analyted tfta trlchloropnenoxyacotle acid ve one then and found 3 - 10 ppn o f 3 ,* .7 .8 - c n c n lo r o dlbenxodlozaae. Thla, ho atatoo, ho la auro la tno chloracno acont, although tnoro nay bo other l a p u r it ie s . a t any r a te , no la ao eoneomad that tnoy ara flavin* a aanaxoaont aootlnx to docldo notnor to uao tU M flT o earloada of a a to r ia l vo t*Mc then. 3 . Bo had d o fln lto ly cauaod onloroeno on rabbit oari v lth th la a a t o r ia l. When 1 t r lo t to aay that aoao o f our poopla nave not boon afclo to cauao I t vitr. other aato ria l ho juat aeoffod and aald theta vaa no doubt la tua alnd that ho can t e l l enloracno by ta la rabbit oar t e a t and that ho naa run about 500 toota on rabbit ea ra . b . Sow haa boon ro eo lv ln c eoaplalnta froa th e ir euatonora (vnothor fom ulatora or u ltlaato uaore, 1 don't imam). Bo vaa not too elo a r on vnoao a a to r ia l I t vaa and ho auch enloracno developed. 3 Ho had hoard th a t "the ?HS la looking Into th la problta and haa eontaetod aono o f Sow'a cuatcnort concam ln* the oocurrar.ee o f enlsracn e." <*Oam v l U have a nanacosont so o tlh s tetay to dec Ite whether or not tnoy v i l l a tto ct thla *".. Be ttfcei I f t ' one o aeni o ;raar.:t : t.-.* L ve d lt r.;;. lit wanted ste a so r t o f era an Eaetlr4*-*ith a l l the produaora to try to roach tone o p a cifica tio n le v e l in order to aliaHnate thla particular contaaina before the PUS pot Into the a c t. 1 . *** . .* #J , . A r5*" I fooruar 2, 1? : , t e - user, H :::r.f. ;.rvar. s.* : . a l l or tno aeovo m a naaaac over acalr.. Tm only tnln e ^ A gricu ltu ral D ivision pooplo could add as th a t they had no hlatory o f chloracno froai any o f th o ir eu atoaors. Thay aro so ln c to c a ll AaChoa, no haa boucht about 1 .2 d l l l o n pounds o f 2 ,u .o ,t o find out I f they had any tr o u b le . Ruaor la to chock Thoapaon Coapany te too I f tho res haa boon a fto r ho*. Thoy alao atatod that I f auch a a a otlag occurs Dr. Jaeobs, no la a acobinatloo q u a lity aentrol/prooosa aan, would bo th o ir roprasontatlvo. aloe with aoaoono froa tho Modloal Depart- aont. fX K /la l . W t S a lly . X. V * 005H73 * J i . ?>?. * * *; *-;! 1.-5. -i t rv.^r\ '> K l./ a y ft* ** ' *iW "SiTj - ?vj.' * 4-, #.P "O L t ST r K e .\ S T 2.R - V I T S it I' V' I 5926 5927 DEBORAH A. BARSOTTI, Ph.D. I . QUALIFICATIONS Dr. Barsotti is an Assistant Professor of Tocicology in the Department of Pharmacology and Toxicology of the Philadelphia College of Pharmacy and Science in Philadelphia, Pennsylvania. Her curriculum vitae is attached. II. SUBJECT MATTER Dr. Barsotti is a toxicologist who has done substantial personal investigative work with halogenated aromatic hydrocarbons. This category of chemicals includes various biphenyls and dioxins, and specifically includes tetrachlorodibenzo-p-dioxin (TCDD). She will testify as to the relative chemical stability and lipophilicity of these chemicals as well as their resistance to degradation, and will testify that these qualities are what account for the perceived and documented bioaccumulation of the chemicals in the human system. Dr. Barsotti will emphasize the difference in results between chronic and acute exposures to these chemicals, testifying as to the varying types of responses to acute and chronic exposures. Dr. Barsotti will discuss her own work as well as review the literature in these areas, noting differences in routes of 5928 administration, and in the species and genetic strain of the animals involved. She will explain these variations and where they permit extrapolation of results from animals to humans. In particular, Dr. Barsotti will also testify as to the similarities in response to these chemicals by rhesus monkeys and by humans, and will explain how the similarities permit extrapolation in this instance. Dr. Barsotti will also discuss reproductive toxicity, teratology and post-natal toxicity, including instances where no gross or clinical signs of maternal toxicity could be detected- III. SCIENTIFIC PRINCIPLES See General Statement of Scientific Principles and Assumed Facts attached. IV. SUBSTANCE OF OPINIONS A. That dioxins are toxic, and in particular TCDD is exceptionally toxic. B. That dioxins bioaccumulate because of their chemical stability, lepophilicity, and resistance to degradation. C. That chronic low level exposure to dioxins is significantly ^toxic. 5929 D. That TCDD accumulates and persists in the lipid stores. E. That TCDD, in chronic doses, is toxic, is parti cularly toxic to reproductive system, is teratogenic, and is 'f e t o t o x i c . F. There are ample scientific bases, for extrapolat ing animal experimental data to man, in the case of TCDD, for example, as demonstrated by Dr. Barsotti's work on rhesus monkeys. V. GROUNDS FOR EACH OPINION*1 A. General Review of applicable medical and scientific litera ture; education and training; personal research. The work on the chronic toxicity of HAHs in rhesus monkeys has culminated in numerous publications as presented in Dr. Barsotti's curriculum vitae. In addition, she has been invited to speak or present her work in various atmospheres including community meetings and scientific symposia. B. Specific 1. With respect to opinion IV A, see generally references attached. 2. With respect to Opinion IV B, note the following: I In the course of Dr. Barsotti's graduate and post graduate studies, she has had the opportunity to conduct research and evaluate others' work on members of the class of chemicals called halogenated aromatic hydrocarbons (HAHs). She will discuss the fact that this class of chemicals includes the chlorinated *dibenzo-p-dioxins and the halogenated biphenyls (polychlorinated and polybrominated biphenyls) and that HAHs are similar in chemical structure, in the pattern of toxic responses that they produce and in the mechanism by which they are believed to act (Allen t a l . , 1979; Poland and Knutson, 1982) . Although individual HAHs vary in potency, their chemical stability and lipophilicity, as well as their resistance to degradation results in bioaccumulation (Isensee and Jones, 1975; Meselson et a_l. , 1978; Marinucci and Bartha, 1982; Mes et a l . , 1982). 3. With respect to opinion IV C, note the follow ing: Thesis work performed by Dr. Barsotti at the Univer sity of Wisconsin with polychlorinated biphenyls indicated that evaluation of the toxic effects of HAHs in the Rhesus monkeys was valuable to the understanding of the potential hazards of HAHs in the environment (i.e. chronic exposure). In addition, this work indicated, unlike rodent models, that there were similarities in the response of humans and rhesus monkeys to these chemical toxi cants (Bar-sotti, 1980) . Like man, the rhesus monkey is genetically diverse and responded to PCB exposure on an individual basis. / Dr. Barsotti will contrast the acute toxicity that one observes in accidental or intentional ingestion of toxic or lethal doses of chemicals with the effects of chronic or lower levels of exposure to chemicals such as HAHs. 4. With respect to Opin i o n IV D, note the following: Dr. Barsotti will comment on the state of the literature on the fate of TCDD once in the body, i.e. TCDD metabolism (Vinopal and Cassida, 1973; Ramsey ert a l ., 1979; Schlatter, 1979) . Efforts to identify metabolites in tissues and excreta from various studies have been unsuccessful. Recent advances in detection methodology indicate elevated levels of TCDD in Vietnam veterans and other exposed persons. Generally, however, the data indicate that if metabolism does occur the rate is slow (Guenthner et al., 1979) The ramification of this finding is that the body would be incapable of or slow in ridding itself of TCDD and TCDD would accumulate and persist in the lipid stores. In Dr. B a r s o t t i 's work, as well as others', this appears to be the case with rhesus monkeys and man (Montagna et a l ., 1979; Van Miller, 1981; McNu l t y et a J . , 1983). 5. With respect to opinion IV E, note the following: Not only did the rhesus monkey appear a good model for HAH toxicity but established, as had been in other animal species, that the reproductive system was extremely sensitive to PCBs at intake levels below that required for the manifestation of toxic signs-in the adult (Barsotti et .al. , 1976) . "In addition to PCB induced reproductive toxicity, PCBs were found to be fetotoxic three months in animals receiving TCDD in the diets at levels of 500 parts per trillion (ppt) and in animals consuming diets c o n t a i n ing 50 ppt TCDD these same symptoms occurred after consuming the diets for 30 months (Allen et _al. , 1977; Schantz el: a_l. , 1979) . This indicates cumulative toxicity associated with ingestion of TCDD. Thus, there is evidence for bioaccumulation and associated chronic toxicity at lower doses than that required for acute toxi city in rhesus monkeys and p resumably man (Pazderova et _al, 1981; Ideo et al., 1982.) 6 . With respect to Opinion IV F, see generall references attached and V A above. I 5933 D. A. Barsotti, L.J. Abrahamson and J.R. Allen, Bull. Environ. Contain. Toxicol., 21, 463, 1979- R.E. Bowman, M.P. Heironimus and D.A. Barsotti, Neurotoxicol,m 2, 251, 1981. K.D. Courtney and J.A. Moore, Toxicol. Appl. Pharmacol., 20, 396, 1971. T.M. Guenthner, J.M. Pysh and D.W. Nebert, Pharmacol., 19 12, 1979- G. Ideo, G. Bellati, Al Bellobuono, P. Mocarelli, A. Marochhi and P. Brambilla, Clinica Chimica Acta, 120, 273, 1982. A.R. Isensee and G.E. Jones, Environ. Sei. Technol., 9, 668, 1975- E. E. McConnell, J.A. Moore, J.K. Haseman and M.W. Harris, Toxicol. Appl. Pharmacol., 44, 335, 1978 a. E.E. McConnell, J.A. Moore, and D.W. Dalgard, Toxicol. Appl. Pharmacol., 175, 1978 b. W.P. McNulty,K.A. Nielsen-Smith, J.O. Lay, D.L. Lippstreu, N.L. Kangas,^P.A. Lyon and M.L. Gross,4E d . Chem. Toxic., 20, 371, 19 8 2 . I ' 5934 I. Manara, P. Coccia and T. Croci, Drug Met. Rev., 13, 423, 1982. A.C. Marinucci and R. Bartha, Bull. Environm. Contain. Toxicol., 29, 326, 1982. J. Mes, D.J. Davies and D. Turton, Bull. Environm. Contain. Toxicol., 28 97, 1982. M. Meselson, P. O'Keefe and R. Baughman, In: Symposium on the use of herbicides in forestry, 91, February 21-22, 1978. M.-Montagna, A. Fornari and S. Facchetti, In: Forensic Toxicology, 78, 1979- J.R. Olson, M.A. Holscher, and R.A. Neal, Toxicol., Appl. Pharmacol., 55, 67, 1980. J. Pazderova, M. Nemcova, J. Pickova, L. Jirasek and E. Lucas, Arch. Environ. Health, 36, 5, 1981. A. Poland and E. Glover, Molec. Pharmacol., 17, 86, 1980. A. Poland and J.C. Knutson, Ann. Rev. Pharmacol. Toxicol.,22, 517, 1982. H. Poger, H. Buser, H. Weber, U. Zweifel and C. Schlatter, Experimentia, 4 484, 1982. I s R. C. Ramsey, J.G. Hefner, R.J. Karbowski, W.H. Braun and P.J. Gehring, Toxicol. Appl. Pharmacol., 47, A 162, 1979* S. L. Schantz, D.A. Barsotti and J.R. Allen, Toxicol. Appl. Pharmacol., 46, 180. 1979- S.L. Schantz and R.E. Bowman, The Toxicologist, Vo l . 4, Abstract number 333 March, 1984. J.P. Van Miller, Doctoral Thesis, University of Wisconsin, 1981. J.H. Vinopal and J.E. Cassida, Arch. Environ. Contam. Toxicol. 1, 122, 1973- I J K.J r> jmm aw I UNITED STATES PATENT OFFICE 2,509,245 PREPARATION OF 2,4,5-TRICHLOROPHENOL EdJGGweluravsmraednpyJd, oaUsnpeppCheorNrMpiokoranawttciloltzan,i,rP.aaNs.csJao.ir,cp,aosarsnaidtginoWonrisloltifoamNTphSwe. No Drawing.SeAripapl lNicoa.ti7o3n6,1MXa8rch 20,1947, 5 CUlnu. (CL 260--623) cpotdliodlrocedtse2bapdigcststtasshdppmw1carcaelbihosauohehuoiuoioatvf.nshitietiflei.rcihcmreiehnde4dh2dyhssqgmtoaeuaeihT2apsOIrhyfeolnerldtitlaTtenosd.ae.uea2cfoeneduuienemtlpeyoo.ro5ei4cezhotfindo-d4ersuzeorr.tsh.brsrriibrl2lecr-te.lerrrsaf14alateifreeo.or5to,.qnvhlmrpbiileaigivjdon,dhsi5hi,,bea,,tmcaadesnse-iye42hmyluraeecw5iwepieseru-rdetoiypliobnslrcecenI,y)nddapthmrtmmfee-n.nrap5rcfprntlpit.iihcreatdtetogrdtttrbuemreeernoticwee.eirol-feaoecvnrupleidoe.iaoalharicesraenitnisorlepddnwextosocgTtamzecsrrdoahxtyeephnaen.derptarrcsrillaeifhtiTmmeihneni.xaiiinhlzhrhlooecoantdnurs-ieerpnbslcdt,onlsoeltleaedishcefhtwcelaoerh:ncgasopsrioiklheFiedaedreywnhttbqexoTeoovenleoiuchsaialorriia,lwheyooarSIosenliupiontyneweorokgutrn.haliaalseplcouseoiofrrehrtsekrsihvieianddyaeprtdoebo,eitarafhinphfrnrsr,eepdaoeneelilsupfnbehletexlontSaiemaedosipmd.roaaplnpbo;hennrepndihxecgepmltitnfb2ppeaunthnofrysotrrtimtarenretiaeneterthooaplti.behn,eeoorainefabdatelthnnottTo4melnhooddnonss'ictehlececoetlsehheyvn1ceenrpne,.hutfo,nyaiustlhigetriisnd5tephiinihr,teeolaswdhipyageckalnb2ednlnsniueaco-u-ylthzdetelo1lattitotoslieliseol,ettoetlohthselphoehmedtf4dnenasarvpipmucft,,satrschrfinopxdnoinnrareomre.sd,eceghepiochdmccfoiatt5eooyaederrttenaepgfndcoalerirharnehheechrsremh-alndbtsacacuologgaeanfdrayntettriprdlbstereypyrcosreptaecmeldclhoirraiibddcensoseearouetylefsogertdlmdstetnttraiaeroiytyemwuotaialoconlnrexhlacncrlaasfbeordeaocel.ddacebyesreserecmdrzpotwobtkimceheondditr36neec.vseiesecduomseae1prleixhltanpeevtic.s0-hooqsdioaooeeviscpncs,hncrlywinmt4tasthencTeewa02lsoltrmf,ucihndeoethetlhoteodecTeqaetit-eoh.erisuiphpe,feswhmihoTaeifn46dtrrmmatnlrsurshotteouootitnaehoomlmoae0w(ne.edoehhysehohohipiyar5ueeafnrptruceldeds0m.tpur'xfpuosoleyeyrneynaeihtn-sohbtterrceppmermabomxrwtacbthmrhdzeltepooidtddutesrsheeobrheqneekrtecpleehtMiodyydataovpsuneeietasnueyenfcggruneyptanlybfexerclcslcuedesn.aziklldiapgtltrorsctezaodneolpemrthatttiiiyohnenhneottpeituaeoiocsuirnretatairrrrwlrhlhncnncnobdhennheemoeooeleanahabirbooeunellrlitIetioygnegeeyy-dnnsgesnrorfeeyfeshlefsTloer132132B4H46o0060550O5ba'esihmoygt4meaatcavibptregNbnemoiwsedbrlwtdacd1tdethhhenxu5hyeaxfosbnraqrxeaeolr8cyfoeerretaaefovyieoWeaiT0gcoeaATtctlndncn06yodouTatoacrnstltgirOvryacmmariueshhuauhne0zmrtoogahipluoehatsolegheosranabilophillcelxeynHsmefb2enecetbsmnlie.sCdntdultorliegbspeexnmyphheeac.h0rettnemergsh,agt)rtrestec.loho1odneitdae0aelnimitimcereholibtdmeeaoemoen7aeanfurlyca.taionraarc(theorswaaenedemHnn0rsurneolimipsgtmhetnvriqmitonebclrrneaos1tCeasdarsgxehodwgaTleoaeebepe0ulidlide3orspepdboiscarca.diettemCsr1iawenlnii,n0ehufbesniubeaowtennmrlraoyaatnvduat5rt2tbi.useziihecetrtueperfsgotrthytdogdinoealdeemmiiodaetehoteualotosventtalcielbfnreloltvrfhwdnoteetlsomonyainluyteeeernunzthefsdeeiiesgwtu.duta1erlcelyezecgecntdlrnhemasotndsl,skoaiew5tnet2,orolercmeroahmontuuupoteibstaatocnlraepb0e0amriltwnstdlihvparHleshpentrdaritooae1slttxctEovlcupiieefoseepsehieeohno.npeeaiaf8dudpxgrmoutitxheiifsatdupeffoCeaedrahgwuen0lnegmiosevtwend.aeetdatluaea,rlrmnlrrsamsruon.hrr.rtmoeulueirtomteae2e-mesout.eoereweososbhrbegoer2cia1rrnvur0se2pCtes2ecpdbeieyntfo1weleutolry1touoafu6emTtiagdna1etyohlruea.l-4bliswddrsr6prro0lnuuer.tsiuar6alhsdnalh4eatiyCsrta5epeaemo.bew*faieadennhancrmepemtedsge0htidntdlplrh4*eupHg)nremstenhewgenidtdiitreaca0a,oIyrre,AlaCneziAmsnreeoonddes.hsyrdla,otkinnn2fbsd,swaerhzeaaohrgast,,cltscugmgmoxaogesiggiyeiiirephddwuensmeerhecemwlhtncnegabarwssolduilnneioLyatroviaoesrscydsdaueadlqnidssythahnxorshe.hsmfpbscoltechsyhuscamerprtsutdoetcigia,.fboo(aaeylrroibhmobieeicnr.dselostpwoedae1seegnltebrrlotwsgTeceafnlrwoohtecetehefyslfdrslnlitat.siftxaons2osteedmnenfylhniesheemlhbs1soeto.whetiksatsacidf-zyrue1vocuganta0ehteysetee3oweakoeelleeiitafpteoo,etbhirefytpssnm2larnsiefngeaaunpstmeceaolherp)sbsntaoy1aadms.mchsfd.e1rsior,tsoitt4lanrprefretu7dtcdudfrhphrlnsaoh6yetwoahoheei,omnel0otnuooohsodnrtq05lliy,dvogewdfetsA(tqflceoatwolnttxhncu*luim9tushc-ylsieideierhtaouatd7ytioexvtdiseinidtgfnh5eurooiCaeisnrahrldcnen5draoieeatoineneo-eCdoe%ehottdraeylnloreshintoeet0ddnsys.ofIltgdseenrs.dtt.-t.-tf;rt y UNITED STATES PATENT OFFICE 2,509.245 PREPARATION OF 2.4.5-TRICHLOROPHENOL EdJGGweiuravsmraedopyd.JoaUsnpeppCehorNrMplokoranawttciiotlxan,i,rP,aaNs.csJao,,ircp,aosarsnaigdtinoWonrisloltfioamNTphSwe. No Drawing.SeAripapl lNicoa.ti7o3n6,M11a8rch 20,1947, 5 Claims. (CL 260--623) ctedpooceddaltio2lrdcsdatsspbptsigtpmschaw1rcaleibhuushoahueauoosiotiinhttvifch.cieiilrremfe.ihnee4ddhdsh2aymustoeqagaeypOIoireThf2slhniletdtTlraedntso.auefede.noece2inatmpyeeouu5leicoro,ez4fothnioedszu-dh4eorrtrb,ssrir,rrl-rbicrreetle2lr.saf1ta4lafeoreeo.ionrmhqbtr.5lv,iieapliigvhdndoijis,,5,,htecbmaaaedn,ssyeIhlm-2yeeecu4arwwe5iieepurersdi-toipybllneeroscncpydt)dn,Ieammrmf.hnnte-aprrcfi5iptrhdpie.lttancretogrtdbttmrrutoeeicneetee.rwolelrcfno-eoi.ualerodlieepavahraaeerlcissnnlriwednotptoxdeTosgctzadasmpoyxetchrneren.aehdrtaarrrplecsitTifmeimelhiin.ehixohnzhriilehnadooaulnsntcnrelep-ibtrdc,sneosleodlllirhefetsheaacetcw:ohngarpsciioeFslohkyddeiaeewtnrhTtqxnebevoeoeoisocualhiliaroi.rwlyaeoaShrIooepsnoiiwutglyentrkehauoonnra.ilallsioocpuefrsoheetsrrhndevkairseyiIedtaerodeipbt,faaoihhrpnrrfneesrp,oedsineleaflbuneplxethelontmadsaieSieo.rdmppnabpaoonhn;reledpxneichgttep2fmlnioebautppnyohsnrritfrttermrieaantteetenlhr,aoeptioo,nfenbrihoaealebteahdTnl4nmotoddhtootennn'osschectietolelecesyecvehhnren1,eftnhpaui.y,unsohittgielrd5ieptsinrhtnilhiaseohe.pdwikgcebayanellnnsnudea2i--cluotdzyeethitltoeoia1ostlltlielttleostetoe.hsohhhtelpmeondednrfapasv4ipmt,fuca,stfschrxrilnondnooepeandrrmreg,cesihhe.pcimcoaotdtcfoyeentoa5tdeepgrflrnaedeciorahreahcrerhnhrehtsmnseaadaclcb-guoldoaagearfnnribtlytertdpreteyysscrrotpapeedrolcmhaclriIiecebdodeensoetsarlueytledfsosgermdtnaarttitrtemyteyaooiilnwulnaocoxelrhaacrdacenbolerfscleao.ddbeseceyzasreertkdowocrmpibtcmeoeidhdntr3nu.vseo6dseeeeeimcacseie1lhxarptplevietnc.-oi0qsavscdnhoeoipceoossnhlc,tyi4awrhtnwTetmcensaemto20ihn,esouedlftlclrhhetotedceTeaoiettqeos-p.rhhe,iiuewhipsmoenifahTedtf4rn8alsmrtrrmthusottooiueemlonothoaawemneeohodhey0.o(hoshiyuapheeiean5furctlsrmreddpxtuefou.'0prlposnenyeseriatyyheno-rhtebctmerabrtaxewbcoppmmerrmtzhteodhdltirtpdodsushboheqreeeelnetkceepdthoeridaMyvaysteioueupnengtanyfgnersuyectpaenyblxeccefrsledls.icuenlllzktdaargosctizpedranmtpooelehrttytitihiaeonehnuetotnpteiooiiunestcrrtarirarawrrlhchlnnoehnbnnnhecandobomeaoboeeoeaeunihllretrlIloeitIgngyyennyeodegyeeenes-rsrffhefsslTloer1331K242<go000550055ba`eimmsyhov4icnteaagamtebobpeNgtdsrctbdirwedlwa1tdhtenhuehheaox5xosnbxqayfr8lareecreferoyraeevyoatfieoigeactoWTtela00ocATyonnc6TnaatudndcortlstivrOrgaycmiar*mhsuheuhnue0gmaolzatortihaesuohopheeloginrsaahbplolinlleexHcmeyesn2fncsbeiC.mndetebltsuoldtelprsIgbeeymhnepxahrt.e0mnceteghhg,esra)trtrse.clteho1odd0teoeneiainlmaiirtecelmohibmtaeedena7ooame*nareufo.rcarylaacni(tthesoawraenned0rHsmelneonuirgpimmhttsrnevqiicltmbenone*r1asroCetdasgaoderxswhaTleago0bieeueepidel3lrdsopedsocpairbdcet,,itaire1Cmweansinlin0heebiwufunsantmbreoaanenratltdoavy5ut2rbitzs.iuiehtetecueotrrsetthpfgrnodyideogedammleoidietoeauotehoealvtstneactfinllrboroelnldvhefwttelnanoitslymouuryteeeeetnhnzeifsidsgeewut1daul.etlerezegnccytercndalmnhesslsidokaw,te5oten2t,ermrooltmcoaoeuuohpbrntutetiasran0ocaempelib0rwntailsvtahdrlplthpHadeenrsot*Iroatlcesx1Eottvpliieuescseefpeoohieenoneh.uipadeaxpfuo8dgrmtxfteisiieuhaptfdCofhreedwauagnigs0eelemwnotvnte.daeaedeaurtamrrls,lsnlumra.rrno.rerhtmeueaoilu2mtroet-mueosetee.weoerrossohobbgerc1ri2e0unartvrepssCe22cbpdeeiefyn1teeolwulaoyuotT*f6t1roueagmittdeano1ylhel-il4ra.uwbdsdrrsprn60reolsuruiurht.nasl6hadCr4lyaet5aaitaeemspe.ewobi*afadeeamnnnhrtcedepsee0mhdldptngit4lr,,)hpHreugnsemeitegwteinhntecaidd0oar,a,yIralerCzAAniemsennresooddhsdtyorkela.,ninnfbs2sdweh,eaarhza.rlaogststcgcguomxsmoe.agiggiyiiredpmwehednsemueemhretcwceglhaarubonidswsllineatnariyoLvroeoddscysaudsaneldqhaihsynsrxots.hbsmhoteplcsfechsuyhmcesrsatrptdouetaigicbfo,y.o(eiaiamrbriheoob.iencdsroelspdwoetlaen1seeetgrwtelsbrToragcolnfewohttechefefedslrynlstiat.txlsiossoeaf2tneemndlhnnifheehesy1lomwbssoeett.ihkactdafsIsy-urzeov10angucteaeetyefhswkoeeoe3eallaieieopttefteo,ihbrtfepnysmsselfgra2neasiuaptnnceaemohrplets)ba1syosnaasdmcm.h,fridos1retoti,tsalA7puterdnrre4tdcdufrahhnsohplrte6yhwooaeeh0mnnleotouodn.osoohrmqt0lylv,deowe5A(*ffstgdqetcitowlotactnhix*tcu9ulmuihldyesseei-itrtahudiaoosy7xdetetnifihgvdio5eutnirCiserolnrahancdneito5idenaoeraneo-eeCd%eooehyeantrdotlresltihnoetedin.0dylodgssefnsted.srt.-tft-.t;rt 5938 oboCctgmmIoszuo6dtmtemwaecpwTiieneoelcybn0ffdfxprhll.hyaomrmia.lTnpliTntedtedteeaecpscevaitreohasotgneht.h.ndcdiohdtraeio,uorrlfiedron.geeeli.dceuooadaco.wna2tblcleObut6tthfeyn1,ita0fltgeeiyideec0easiifgno2rce0pnnTodbntmdoe.nsl0omdhiemidoyoInghhdmsztttraltcenyaCweofcohaieheeeeeclrnrlboCdpsatdp.nsfefiryi.ecaettorndwliritrcinore.hlhnunnheshd.odaoogfueidTbtcameeeerstaiuicmanpfwnirghylo4odtdlhtaetnyytteoeolthr,0idiagmadrwaee.dlthollfrvo0aswligtitfecdbtysabennheaftohaa1vledhe.ftaeoclcsyitgae01irnniatcenlneos6copnbcBlhett1o0cohdzl3.rermioloAartmay6iadruetnshe-yadanloleo3lmlitcis.neuaotlfgitdCceseg*fsmyhnugtanemlotnrtneap8.b.gceeilTrsbni-odCpptlwr0ctfehlalea1dteileiullhyridae.mone0knae6.nboi(lxrcstgelirntl5s3zned0gaehze(lpolCthalsk*eyo0uusmmereyeaeylt.,anoa,Iwmlndtnurcn8tlocnCeooecedatoeb0cbtrtwcaafaamcvhtulii.,eloeeod.as,alsnurlocaaneciteeirtiw.p2omdacnvlhltbohl)ortwxpbi,efrwu.n,geeymatef4nheeortoreataorcwdsisloreeg,nosef2etnemi5stafutrrfsstllxh3edahngteeto-chasulftwohb2tpetdtwecfeehutrymsaurreaptesebooedcs)ilaereveherrgtnt,chengyvreamelrheriylllahzttoarrwenwewwybaa,hloeatrwldfdkrwgecyyeu1oiefnr8eeieymrfiieeon0atine9ereerrrardsse0llhndelao0seeee04rrss...' E x a m p l e II woirhI2pmbovttascsooosat5n(tenhfyhhee5youtbneeo0cfffgaiieW1dTmneegAlnis70hlttancrtmbi.grfairtns3hr2trtopdhydttoehueofpseiiihaceiro0Tefrmtbedounbnonxerlemectmedolgeihlnrcwezcys,n.iregfrepc.oforadietdlaea,oc)ataoreowadeyafusbnh,eiptobaratrmnlrmdfinuluteewe4sely1uhtpmceemnepdoehigrr5m.ooil,iifwvstenrnthiew2dnecseso:oeoplnanihegioioehwn,geraiTnsrociocled4tene(rovcdnwet(hethf9auaha6,egebwctgcufasagi5msseo3mrutdelrooyobllgsh-eyea1i%samahmn.patfollbsdsdodcpabo,coreylfo2dtrfuoteuuniredlpttfectoowohs,HtauerleItsnhrsid4worrof,atcpnriusescuritateziie,mlcoser.ha22ltoem5tSlvetfn1ploaehennf5tpahil,rei-Oesne9hgs4atdrnlttcns.r2edreefoeii0fth,eh1aete,4idoob5hrestr-)rul0ytlCypsor2ounertu-slehgt0onaoideayt.mw0npahessan>0racerwlfdrbrr0:nzrdlutoaedeeh.oairdceeioeeandmtfnrcdttnletxhinrpi1pnarheocgtsCooragielzrssyeTsa.redaaottntn.aageloohrlnaneoerhcbaodc2enderwlbgpoewdfoavtearntftd,eabyrronIih4olosiemecaeaocetpnietwlwtai,aldmmoshedl6se5unhgeddnkfgabsnn4a0d2-pzedhiiomieialdatluset5lst5cttennyyorrtfnheleir0t0-tenieocioscirviaereod2ettcanrlleoteool2l.hadtidlhh0egegst0mllnwmascbbidc,rur0lrelro0swtWscwuoaoeaepeuTa.`iteiilitouitroimatoiitremeelshctihoneihnoenCanaemstlnmtrrtcedyghndsegh-restl.., 8 10 18 SO 28 30 35 10 48 80 68 80 85 tip2ptpIpIipitptpppIprscpvgw2aw2duawtwgegtonnhnihnninhe,uoohhthhhhrtnrlelhrrnram4eeemeyhgeaeeoeeeaa24r5gm3ureeeeeedled.ttiiiictetttcssytr5slx..mms..hnennrhnnngngggreohhhheyaiosrpsptua-plieooooeooohdhheTThTfTiersuemeeuedeslrmlarbtocLo,nlltlllpstteauetmnmreehrhhuhhn,,ta,,.ropsnretocseffhfrffeosapeeeeeptemdtmrooiofowwwleowoootiefamefhnpbnceiiaenwnllllllffncarntcoophhhllhrshgegellee1t1prpppoocefooiooocentoetilniclliiiclite,e,rotrrrehltienwwyecccws1weosc212nnhiduouoooenha2rrcqmsthgcehhhE,u2br,W,,vcoelcoc0cciicellieiti2424uuooaunnoannlep,eneDeueeefcafaef,4,y,t,ccccrtllprrggIgagfippr4s54sss5ooolc,aheconooooLW,7sthasrssstor,5-,-rtnrwstwohei5hm5mmgtmef75erpLtppitpesocwoutu-idenoeA05-swae-humtnreefrfocrarffInanfppppittutcwtre0vionooooAoootiibofeeonRrmertiegtdrrrr2onggceeriapcpaphrrrrptinitiesiilcioc0MrtsDagcnrthnrhlssrsscp.hcgootoiiiaovheibearsfaneeeeeamnrgdththtslelhppppprrmredeircasescoeaossssgpnuhnSulottltttrrtraraJreihsohtoeiimoithrsofietueeensettsnc.eoOoooobnchhhrhtool2ifflsrreipupppntgoaoiffGtnonsoeneodeopeeo.nte4ossScinaaaaa4lrdorafasassoatnhouf5dqafiisUslsoorEbrrdrbth,ore:e:t:fntcytft0oefo:tnu5iiiiaaeiiioieistenMrfmsfenPonnnutlinndndnn-anhtninslnoi1cerug1ltbbnagtghgHgcngoiigrtgogopgPzroabazprbuueetoediycaadligdtggeamrsgeeemse.tnn.emmcsouoallaaoae2p2n22mi2r2nntrriirtkherzNztuncononatppraatoatat.e,h.,,eit.eneal444444tms4vfmsgmmmshcmnhIfyloeesn1nl5e11ae,1aKu,,ap,,e,thylyitr5rq55do5565tete0oe67tt7eninrbheohdmhedgAc-u0-a-mf--h-s00mm0mnmaddtpestttesltorlrgilaayrtrreausWretrryanotoorulob-e-o-ioino-orilrhlaiityuadaitnp2cn2cn1dnsccax1fcxceecatlchldllnerIofbgt0atp0ho8edheh8dhedtnhehimiTioyuttmdcdlp0l0lcf0slcfhc0ccllllleolloic,iyoixlcoooooeeZoeoteusuaruniuernehloct2aote2trieaarrrmr.rrllllr.hclen2dhotoyCnCoonooCn-aloIaCsoa-Isassio0hdaye3aenle3c-nerfr--t-rtt-r.-..,. REFERENCES CITED filTe hoef ftohlilsowpiantgenrte:ferences are of record In the FOREIGN PATENTS Nu3m49b,e7r94 G erCmoaunnytr_y_________ JulyD2a9t,e1914 OTHER REFERENCES LppahJHSpgi.aupeasbiCre(lr1il2hisms93eho52maen1nd.a)ne,nb,Stdyp"oaaRcPl2g..,.3iec"B6s(hP1ltao99aer8lk4rye,l3'shs)9tpa.Oo9leonrprg(g'tas2iaegnSnnepeoiosacnn--gt2C)efD3t.sh5C)ee-.rom7i.v,ias(P3ttirhvyipel,"aasd.gv"eeolsl,. I ? r-' f >. U n ited States Patent Office Pate'nte:d Jul2y,71969, ,1791537 * ; .| 1 2,799,713 METHFORDOOMFTMETARKAINCGHLTORRICOHBLEONRZOENPHESENOLS A kHMamxeiumdlfLdlaoe,ncarnkc,Hotoar.pnoTdWrhahGetliioogDrnedoroo,wfnJDrCF,,ebl.aeMwmDaiuidcrgealaainnC,d,oBmMeapivakeneryt,oLnM., iAMdalakrnohdn.,,, No Drawing.SeAripapllNicoat.io4n83J,a7n9u8ary 24, 1955, 5 Claim*. (CL 260--623) uCpcitcgitrzrwaoatiicbamiffttdssctfoavpppcfstmchtPhwndennirehrwnioirooohoeuelhnfrohrenneoirhrheorn.niyiatkoagescigsoeateenp;rrplcrlncslernldTTdaWIMhexeeUanteedpsaovtrmraoomseamstdasfheahpteeozlsnneeeeooleeaa(hdrrhoalrsoeercriosnureilntpnllronhirloooadbisrlnollloouhlvoadoelhnnipiaho,rongppnnvuitcdkept,sreelsllpgmm)tbbrtrasatreftttedrlrwyaesraehsee2rgteetcrosnweaoeiebeaooo1hsooneer.o.iiontzn2.yssadiolttmNnebaesetnp7rdnhnceptiiiinrphcioeni5tbdn1ewgnoatnlrmbnanmsa0dynageehcntuyzzrhhmooees*tffyr,ipghrobnvSe*elIerilscipgeTeehicod2nootaceirs.paerbeenaetetsleexweelhhsnpttmnenrteee,erlunhlnxetmhtvilahedachg.r4onptnslholtaeaqiehoaeoordvldichedqr2noeityohatfprmeersi,sedtqzadnledufe,yeyelbafoais5alti.,eetuwdeydryrt,itinsehcruu,5doeoselitiAlinratecioncoor-mpcfovearladslirnsenaxye0cheditnhtaeelotaenyoabbapmlinetoin3relhdloifgcdeotPtacd9yasopnaln.yenvspcrln1rlgerurtti0glnoxaialycerul,tsdgaoplrironabeddeaoyh9angseoc2k0aoferotectgovrateymsflrsot,lpet0rstch,ielmostozo4rmn*oacoleo.ocifaebgddeoetmohot*moieyytaosoeerhthd5sludnohhnumltrsyhsleurwlhgleetmiditdnpsirnyyyofyyevCooloblcetcinemtonbec1ttyrroiaCaioeodsxepciilsireerysbmooltjsoumde.ygyf.nmtldpieorfoarfarseoreu8fitNeeed,rhhk.isemhfulaoisuoamrlrecncniflentrtrmaaeaeaynsacaatoproatspeoaicetbrtraelethas^ptipanhmtlutdeteokooaoldzddpiestlrnpahtruhd.atpeqhilttonahapydaardrrCkeeueenhrrelmorlh,ltnca,oeytlueaeeoronro4ceztanosaiofsaemmoorco2rtsaryrzbttpflccasiahbeoyte-fseliricistoenraiiaeHc,lvyfcateiydx5nehhlorcahfac6rypceodysimttsronbpitktomaonui2aantimaesr,tttcolllnsuahutei1ltidb,nhnoaueriooctaolinapoesbo,opnCngrmholp.l5eloieeroseaea4nnlwuoenoerxrrrltamtaygynldoehsritrln,clnnntneaod,ooevyatzshismr9ysrr,rlhlepe5aaahxhtyosxtziadtpmdlpbaaycutlasee,epTho2ladfnnt-hepywytyiedanoeedeeailcfolnrncohecsietdrobe3ltpksihurerennodrrdnrpsrledhiuofonegereniefreeeecepoofliirrottaaiectvd.rgaeriawtllldsnzcrcytesganooibh.mlroamtbootlfoorwtgebosecatmrrtsheyuimoieldshvdoyghngrxiterrlresmepodluultwnsselsnchilieibieyfteoeur.yatmteehepimog.ctostnpTricupisoithertmo2adceerchpcnobmnittsneyshlc.huirorhrfseesthrepr4ohoaefpopveeot1,oeseiefohtdo:rslapmrgldhtrposcyc0cvillieoterrofrl,notaepe2atnrdoac.coomiara2veorhoeial*aroneh(nsofarcztrt0hopshinotsmltyannl,edbtucraorero.epyrtstgxeret4uie2dupvelrnaydmihteoirphrqlnriap)tprdotnhlc,ibmincapetucerlehbecoesehi5edfrhertuah2ohrihryeemheneomoaohmasito0hcoyseeco-lydaeoeettieswnoeesfndlicccltceuledrdfrdl2alltrldpoq.obfhnesenro,ontetmikcrtyohtneai8aruaiibptufonruteoatorontrobtterasarlo1iortbnhd0hocooooocarhtoooeiaanSouovtinxnylewiaal0rell4oind*nydsnseersee-s-sreffrfil-.lr-tt,,, 6 10 15 20 25 30 . 40 4- 50 5,-, 00 05 70 ppjpcecirmmocduaphh4lscmp4b2aipeobttaaopatclaapaltthaCduhmfmosibiireeneihxharoioriheebornsmsyynner4cq0rheretctfteefoeneyraseeifenaeeerog.hrlpetreolnTelnrTTt0idddegTnouSrtc0arefdrtlinorntatdsIurfcetaitinprxiarfwheentimagamtazata*urreamauncaoltyhneoue,osohtherheatzehnrthaldurhniyosyoslvotetpsletcmteilrrtscioorutoonerihocnsdddmneerswfeueuholeoceoeoqmtstearnsednxdexatdeloarhtapoburrx.lihyneeihdehapisctirdreiresnootsnubdescriigetiuyihnebhvalsoresseteer.iyeel3teiydeoddr,snisenhhoaomasphpa.ialdemxesdyr,coerlnesarnstdeedh0mryedeenvutyylltcyolt2rr.etoteeoomPpiest,reoo.dtecksnlyzitpdih,osa,ry0seoartoteutddh8aah,rtaotfThaqrrrronetrttooeisfuriosaobrihlae0fllrindbpnaafnrretircyre0tcnehorwntaonebeliuseeiotnuehxrcltixgceoreeroaoeetimrtrinoasteTefrt*dsdiu.ynnooroexesscgtotahaopdiennpiteiietolitmceieadllfscrnihndoirasdowfoeotiddudmnhyifoynscmsog,erceirniahnrltutrwahontdoummzc-leneCsilaoeunyiirnoelatasesettohf.tuteingnofplraiwoloaigocereomfrhossuisnin;iomsnooatbdsanleybawore.ienredadrsurctsorh2ntnn,oa2mx,iexrvn.ceasufmrlmluhfsoteontsanpaaaonsdtesmcsneakehyeflhopmisartttiteadteucftmpenrapildinprsuitohstetshetotaitihaitcaoleabrxnsromeoumecooeosromidcihnaxrnrtsdoIp2r,ecaerranoelleehttfmecrtefpeeuttyeihssonrhfen,irimeu.eedt2erenrrnrcoiucrhdpplitcaanieaemnueatfaeestt8ctooese8ratxerotu5eoaa(ptdanlhnfirrrtiibymaec.odaasbilriczrirdtneifseta,onsmcho,iotuae*ruitliroTknrlaeprhfealueo,errtsl.rtdorctieseonucsaicae1gotcsn)iseoefinspentraionnloarcgtteenplnuihiorandofashvisocsrutuo,..tiihcrq,prtguettnsrlehndton8rmiaaeetheeorfetatosanseauoenfooligccuntrtnioo.eouaetteira2gaesolxacnendar.onhcnbedehliunfh2lfarpeoc.rtemtspqntralctc5lhaotnvrr6ineadeaihruinaatssorekpinsotehthlrdrrnorgestti0heynaozyeqtTerssi,dcisiltawrmohomopotomueaandoaoTeareenpm.npynoaa*pyebsddosterutftitoahsuihcb4pelurr3mtnitnuorisdnteesiahhirgrdtcasioireai1neyhehneoyertisur0.npehewtposotoanotrrtciiitehttoehcbert5ouet6f,iueasiibd0elnnsttdeimhrgyoeprc2lbocablooemnmegmohl1syoyoehe,sagfutrroeapayew*ermryreszrodeehr,xtfeerrnse2oyhmlfldtoeircnls4cisrufapsoeempoi3eepeowfroprrspotarnli0iihw4raasolieulistlroioeoi,wdomwitydrtnend0delgsCusexmflretrm.oasysm5zlnety0rustashrhenoonydsrnarlrepeao,ne0esseaotcuabmettses9eeha-llyaddr0p.o.ehdyoimuisdofdriuihi*epb(pumd,,liatucliemesncehg0otxsnrfrifleemoamaseuiuaigretaAomseiielerlgcpthheeuaornltiooeineynhiunclrtetegtotps)fetndpetcihesor,ntCxtnreelayyuemyrTaxoormmighoduottlmihaio,kids.tesrtaor,cmoiytdtcntte2obdsarettio..leiprnstvhefnehtouea.tfrc,,icachdeiesatnoatmlie2ooadbrohrnhorluofarrnfhmeeoeel(seliero1fnohtyceeltoeosriv5iellOhenelaoeerirtefea,ralbultataeetfp5oeeyno.ua,lmsaouoxaemtys*daTeeahrnaeetulliot)cldgtliicu0lhtzhunhfhlcabtfssqmknoneirisfkdiettonrnmmapdeohhttrahhpuirdslt0hiaeulrieybtyettaetusracboraataftwnhntoTyriageoaraiell2reuesniofareh.itt6epdldaoltnobeelgrbenebrpigaaeaacermarnnccyhgheohliteihtmotetcep,qeefrreroytmihatledemrodentcshihrtdhdeqteheeoaioirrmetteehmfoourhnatmuaulnqorsnodhha,crowilenmllreaueallf,mfumllmsxuoefteop2eftkoszhnryyl3nftogiolrrokloattipi(m1ieeraupiaothecsuonuteokeroooeuralviras0sid2eertvscvahrt1t1udaiutmnrirvirtgohunomacuyaobnmiiedrternhtaltmmaneon002rna0sretatee)ealoaeessseraiodsomneinhyn-seeedeleit,sl*etd0.-rrlde-,til,-, A V 5941 I i M,7VV,713 *'*. ptfcrrvhwaafobotfasaatoeeohhirroudeiqebdufloicaageeTrsotcmnTnulbtdtoectmshylhuzicehereheritvaiteetiinibirosxeleaseqrpnont,anacguuittuhsriulngchcoueshnaeflwesceehtacaenrtoyldhiizahokelhonbtrolsdofoohlnleuastnedeianrofarrnnwdeotlsornrttouwhisaeieedtltlztnipeevduuaeiyil,haiitesmmnidubrsneethssceqnuao.pscunsoeigteeueofsltghairontaoulfhisttr,oolseianouelaoreesnTueiqdnrravrltxdnrLfetlwguhoiousiioeammitcwmoioselpauncnhmunarupanrytasarhtensigarelTiopttsuntutieciooonoripew.tc,lspstnnhvaiienetetfrrhhcduohlgheeiitiesa,behot,raatrereshhtahwltilluohsrisrooihatstcel3tsuorobsaerwslhyrrhialuuyrlnyoascayldseieanctssuohsrpdblhcrrryh,ktedwdiryi,oebleofarttsclooaeeeeaedalaeonpoao,halybserrn.tbnrfetidrorgecldzeeoetaol.oogeprmipee.nircabbatilnr.r-rwdbrazbhpogyeteiegeteembieplpnapt.seamatsonnadeyrylsezhrdcmibtreloeux,imseihseeitrsyxldrcitnehidainacesorepstiiipeynnaaouufnssddnIaeladcbnddstlrrterbrdirteeetraduazlcwpolaibodtltrhetototaartrhfeexuh,eerannnowtrcdelaireiidoaccetesddopcoerirsiitoohma,saeneetuhrsnvieshrfslliseaegc.devoorrttaaffn.itorttatelretnnehhaehvooaeinomngriddndndeeeessrrtt.t EXAMPLE 1 mttothwaTbwt1twot4ctarhiaeehcyfe.oicp2hioh2ieractientd.nnpe6Ardeeh.ilhsazr4cnsee3edoc.gloacea.chd)reoo.afr5ul5hngicoctdarym-oTto3w*oeilhwnomoo1stod8hmp0aavapigafraCrsts.srntoee0ebso3eettbegoeadcrn2ntebldiaewsg(rcrfroiae,nnoe1ia,cqt4ctrvnnaoaeinouhic.a2.u,onwnd0steddn5zslhme0sohpgfrid5.e-a.lcwreo1tpospcnts,rmhmdh.r1rormamreio8eTetpoTcewo1snaiiwfasilhbono3ghdhhttxaaust6ueellu.einreeuaecus4toent6nnarntudvhercrsierrmtorzeesaardiot)pseseganeoewfcl,apsear.twngoilriiocarhbwcdasuneoaetfctmatfeethimtaeafTeefeistnnat8eicdossednrhrosheonzn0onfmoetodeveeealr.,n11sb(d5xenienpci0,,swbrmthwfied22tedduaoeou.iraza,,p,5a.loiramioin4m4stivnsxtade2arcrnel,,hieta5ee.rtr55ntdTbgnuudciedtdd--hdeogedhrertitd.oTeydseepmem,ine5ittntltd.hotnahtiiruor6boh.o,larmeiaTetlt.fn.olrcf3eechetTfeehbyxifht2drhb.ex)aehmeeai5iehgTleeldottseoe0toyunhrttlaheeeozfrriobard*rdzretmdeotieeofmeeoernrbo1Caabarsoanemwgmsc3einwfoeecq1sc.eat8npnbd,hanh,uii2ula(p2*zoxzassslele0,sn,eooooer4ntwa49oo.tduannfiClrr,2f.hc,0un5uotbaoieir5ee8hlece.0es-strr-.,, EXAMPLE 2 6 10 15 20 25 SO 35 40 45 00s If1 M1l 4 TabU I ReacttiioonnsCondi Products Run No. TMtmins., rrp . siCooenen.nvPteerr- BC1TeO.bn3rmlx,to4rer,sa6no.--e-, 3,4,6p-bTerlncoblloroOms. PYeriecledn,t I.............. 130 Ss............................................. to to 10 S*4I0 3X00 6ns4i I1770t...4666 I1S0..33 1I0S.84 8763J, e7Ss 6s EXAMPLE 3 TcotEtwtcoitttt2tboahuhhrieeoooqfef,fhoxoemtereenvnurIaenienst2aosangezehmpcampunc,rtnehoeeoaie4breeemhdpdnteumairsn,odarlcsl5easticecocemoephpnst-sgdeehruadrto1lsltr2robnnroioaronoidt,aai.ob.eu2ithtytrcdldminlreouef,muiehsauco4dnnTptmlttTaoc,itfgowaizh5foahhmnothiqroes,2e-bnneyovnopeetnuhrno7ledbeprryeee5oolrgttoiorramhaeoehrde*lsofrscfIasceausrieaxiIeweicounpoctCxtsmmdidhohhnloxiteadtfa.diiueulseor,ilisoeoorledtimrurle,aaonrenrntessleonxrhooerp.dsoooatbdtpibcwernwddtlitifteheooneseonioadiii*aericruuwnvepgn'imfissunmmzetogsmtpehhlsueerit1r1iraalehoaenefdtg0o,taghrihfd2lnaetehorpiuyiayb,tc,etaten4omtsefuddailtqinrm,e,xretotrotir5ouihtodneno,poaonnip-miedtgoxtexutvf.lwhectenrowigitahdowitdhaentasyrtlrmhaeTessehenaaosaee,eternmedcecuhdiegiipgpthnosnertnrsdeehhllthreitpaoesptnrertetpaeocoocrecsarrtncaeaoooecrfseeifbEttnnrafppnrdsbneelcoxci1cdsteoocetddbehorau,hgernni2emnmeonggrtyrlnmzd,daietoaiil4ioteipvvteemirrr,nabannox5edileaolenngneyadesssf-s. T a b U II *9 Ban No. HSyodlruotliyosnis Products CHInSPoytoniedWordcrncoeiauenxotmnetitdrtra,e CPseoirnocvnee,nrt BC1TeOhAnelmzt4ore.rsa6no.--e-, 3,4,5p-bTernleohlloroOms. PYeirecledn,t tSo--.-.-.---.-.-.-.-,,-.-.-.-.-,-.-. sSO 10 07M08 4sS0oO.0 33IS0S.O04 81 nSS7 tptswbfIatt1haTbTcmftaCg3rahruhrnaqoh.yeibaor0hhi2oecmsiiednulndra)sldxm.0Ieedohe4ezemer,neetbdrluatcoea.odumodnoh5ecrsacnucaur1etrtbee-ncortrahiileethioso4a,ar.feexsoznnslteiipcocnfaetotigeelwthouomhtbgrcniztestredeqe1hhr2feairtaeetgnedscesiauro,esen,snitooo2eia4mevwhosfdioewunpa,uc,rnodeamtre4.5ulestaheasda.sgla,.e-auttrtmal5pdistieerooacpt(ttrts-mTaraarxieT0tihirttnferTooencicfeoathripe.eerohetldhh3niuwthnbieseteatrndeet5lluetsehdearsrahlowoes.truisctcnfesposemerraadtirtehtmzoooladrnethitiwftleeepporbsbfdgorodoeoin1ndttlahdeleihocbrioarlees,ettonelruehat2tsfehctxlewinabzi)cacl,sgenelpa4oiioeeitettldwahianndneihr,rnnvLpono5itteerowiadaezceeehacifdn-midgpnseshmdtarneernhdasmtsoomsaTtajettgtaeteohahhfrvafmeacnainrnhisoadteevreeinxett(edtcodeltsies'tdehfctlipuhhawpi,lhurrosnoefiameieloteypwrigrepaoavhfreeterodftrbirlaerreuidechzgsooroonnemtmcrhenie,oheetdmenbgdnhnyhwrxyltapduzeguaeybttdeittttheeincterarhscooeertabcngraptetlzichotremmfhdbausocyneeeoitxfteanlewnt.npbiuarretbieldnorhhovdeoec.qe.raedetbfeaeoeefemrTuunsatacaoroenezcwc1asTst3ttTma2laoteoo3utdiiibate7rieonnoh,hnnhu1nbirteladb4.ddhnngdeeeeed*8esr-.,.5-776666606050 EXAMPLE 4 Tpcbpbtrbhthrbaegc3hheehornynyoeeeer0hmseoealnAnnoarddm4eiownnicdtzszduronreebohiouceeeusovsscdenc.lncnericetpepyhxggo.tiaeetodnbrsathaot,t.ooAoerwio.geferntlsarsfgefd1niaocTsoudcel8ocpdwsutifiaTh1.enqhlrirlo,ptaa,2sedoraheua72intete,ydaraeoi5,ae3Toaarwgl4neu4rqrlc,clfthe-pr6dcyi3eeuwrttwtettee-trezwmn.hehohti2adra1eerotc2efceabspcir7dhbu2cesgoeotmetl1mes.ehnerpbofrdam8n6adaltataaeenraiorzmdstotb1nnrgdgabdereutdcb,zdmcoriersynr2releaweeafhipele,oanadoamnnmoe4wlh.altorct.duzoe,fseesgs5seire..nforndoia-fcTnidTgoootfoion2bareechThpffulhr5lTiet2t,sectrehdwo0newatsh,an1r3o*eezolcbeiiawap8cme,retfcdhreyshoedetCnheeipIenitenru1xoerrlp.etwhizroes,miarsortrslw2eycuiauor.aancdsn,hodcwnrbsca3tdeeheehprloesdexn.eooooyT4lbdiefttrstowbircfd-rehhoerzoo2torahtrnaeieeb6eoal0tpocfslnustonh5tiobetxrban2tmecleeaarepaiiaseie,poidcodqnng4a.ncinadaehnnneuzrei,uonr5tlatuaeedwaioce1tTdlat-wnrtodo.teenrt6enoieheruodgdaatn.ts7dindh4ses-sss-t,., EXAMPLE 5 In each of a aeries of experiments, a charge of 1,2,4,5- 584 i 9,700,718 .pihbttpahgttcbcIprneoheeeIayonyieeortgvImgearsodrodrdmcecceeaipulirrpmershtededtcobooroihendneyheox.xarntrento.dtslhntiitaortiddnerce,tbiosorsrfeueowToooniiiaTltnrematylmhhnbeiseiihusvltsmnemede,hitbeeiltonisoahertp.opts1zhfsprehfrlnuwr,ieoeera2eo2ofTetonayanf,o5ponxii4ychdesefd,0co,fptr,2ideuti*5eeecrbehe(.ctttd-le4afrminahoiedttnCrizros,estemsme5riimetn.ewnxtoier-dhepsewmtgztfafaoeddauneeoitoetctsdnerter2perrnhsrheoasigr,aoresuyl4cituaactoiatuwmtrd,phnatnhor5lir.rprsotaebotdlee-oeeosfbiethcodlntrrogrwTdeyyoecaiifotrincosotdvghevmibrhziitdnrrneiee1ansheeteolrsd3caoennaeorxtt1ihtEthrenzeadrtefih*otliaedeexbdrooeapccm2neialCrnecqoehtf,0gmeaoimooshuneo.rnbwv)allpamnesnpdloeulaeumoolao.slrnaeindnmsowulhononzddysemupiTe2edopefginathdn.5rnblhroeitgoaescvgetselwcoanmoeiwe,dTtnesidocsdatentthdaapiubaliihauebt.tugbitrltlrhhcehdmoamheillthnneeeeea,r,t TabU IU KNiom. BC1eM-h2nl.oot4rlr.ne9o-e-, HSoyddiruomsMtriis ReadcittiioonnCs on TMtmme., TeOm.p., CscPvieooeenrrnnt, 2,4ro,0p-tTvrnlcohlloYcPieeenlrdt, PPortaeci*en.st-, 42a1................................ t11 1 42S2...009 20 20 20 20 22229900 S6SSO4I 8S77O00 99990929....1S7S mceacoba.ponlhefrkermnto23lWoa1wtaosutt..ap.ielhrieeslxiorsoIAIenettrhnntcbsunpetoyliearrpaohdianaele2firnnkyrszmoo2osadsopepcr5hxlu:rmnrfrei*ooyorioesdelfodccms,ayCeneerfnosaaoess.reiinsfoslstssaayamgqcstlonzffarholuoiodfaalnhedekr1rmiagyoima.3u8mmdmlut0mieemarsti0adtaooxrm*kok2xtahte4iiuliicenynentdtC.rhtro5dcggeeaac.lru,.olgcl1oulaaarh0raaxhoinalmrtiyobmtdwtdrrdprcreeiieoercncr1.rmioboahhzg,xeapcoellhwoonittnoltdierrzheorlpoeocetee,nieuppnraotarelhhhesntacnieeeetrnednnmbontseoohyttftqpheslleuusepeabobfrbiaevwfalyaytiuuataeclhutttklitoiittierernaohhcegnanlhgneesitt 3 10 13 20 25 80 35 w h2e26oohw2aw1qyl2.ffe,a-kht2tu45adotttoiat.,h*.tirech54ivloerh1nie-,4Aanax25tgtslcrne0i-oeomidogptadcndeemnrrfhithtiamoxosr3uliyimicoatprs0nmsiudmecrxr0turorseoht*etimosuhmplpenaolyrhsooyaCnoperh,edsrslofl.yentrioatobicshodcoqumoyfelxeurlaunneononaiolieddfsdnzalmtrfoyeeecereooiueznrro,nmfipgaidseaoaanrrs,tllata2goioitsftmnnhrmpredlet6og1eeftooicoa,rouamm2rlacuthsmarth,1ctint4yio0ioeh1maotd,lt.hn5leh8nmwrocys-rcoe3otreettdiule,oeoxraipyraltgrmbwctoarzeue4uhaterxertsh.pirtcno5doippeeehdgzgpnorrgrmeleraoeeeanorannnirpttadiramnicuexodmoomnier,ntubrtdenurhetgsmemseitrteonsehcpoobtsounz.oesoorflefleoveeefltonidwusceffcscferrsriututieoouiuietellonhbemmmparanngneyerrst equivalent proportion of the tetrachlorobenzene starting mfpetmwppr3doacmureqlho0iuorokeisiaraou0nesnr5xeismacttpln*gis.uoteyiltviuohooiwirtlsnaIoverritlnCtfsnaileetsneeatseipl.,fidheo.ihnonercazddnoyutcfroooiiscndomnhnnrnpmrperrnydgeoereosenroatdeafuoslitatwtcarrcyhnpfeptoenstfsuehhtorioiasloieoiocoeyenssratltthinruztrudelhoseiaysilsftinooefenicinoldzntonyrfdagahgrroonsiwolatntutorhtwaelmoteooamyrteaft,itorftzahttefaebehkmerhrreatctmetdeodihhynrhnaatnrmaepedoadltgcazmcoeerutwsehorrirtft1ioasooniaoelxoixa.eodntbne8tgirtluprrdtigumeserauotraatenrinoceimbrancemztohsapaerhhd.4etuelnneelehib.onossn5zryerehoorageotrd,ogepgfdqwnebrtarprriuaefhotaeueaethrhceeexmnsemmoetoedbsinzesmnduuyetdhmmoieers2tnnyerl,p62ceooim2dsoc5,iallbtorneeh*nwdioyotxfcctoltmxphoiotuua1nuttiirrilln2hhdc1uxoraao00dhaeeee-rr References Cited in the file of this patent *tV UNITED STATES,PATENTS 2,059,245 Nikawitz et aL ---------------May 30,1950 FOREIGN PATENTS (C1,o0r4r9e,s0p2o3ndingFrGarnecaet_B__ri_t_ai_n_,__7_1_8_,7_7_9_,__NAouvg. .1172,,11995534) OTHER REFERENCES ( 1 Hpiangaeji):. Chem. Abstracts, voL 48 (1954), Col 2774 / i 594" 3 I 11 3 SRCU L E S P OW D E R C O M P A N Y August 2, 1962 JACKSONVILLEj ARKANSAS OPERATIONS BOOKLET t This booklet gives information on the chemistry, properties, equipment, method of manufacture etc. for the various items produced at the Jacksonville, Arkansas plant. It is a sussaxy of the operations as they currently exist. It should be noted that Jacksonville personnel helped considerably in compiling the data presented in the booklet. A 11 2,4-DICHLOROPHENOXYACETlC ACID (2.4-D) 2,4-dichlorophenoxyacetic acid is prepared by coupling monochloracetic acid with dichlorophenol as follows: A' CHgCIC-OH + NaOH --^ sodium hydroxide MW 1+0 CHgCIC + HgO + CHgOHC-ONa + NaCl ^N a 11 NaMCA + sodium glycolate, etc. + NaOH ONa Cl OC l + %0 2,4 DCP MW 163 Caustic MW 1+0 o -c h 2c -o h sodium 2,4-dichlorophenate /r OCHgC? NaCl + 0 " < - s - Cl 2,4-D acid MW 221 MP H+1C Gf Cl + NaCl 6odium chloride MW 58.5 sodium 6alt of 2,l+-dichlorophenoxyacetic acid MW 243 This is essentially a. Williamson synthesis. The coupling process produces the sodium salt of 2,4-D acid. The 2,4-D i6 released by acidifying with HC1. Final product is a white to tan granular solid and contains about 1^ impurities including NaCl. EQUIPMENT: One 1,500 gallon stainless steel, agitated and jacketed coupling reactor. One 1,200 gallon steel, agitated and Jacketed coupling reactor which is generally used with 'the recovered DCP and TCP. v MCA, DCP and HC1 are stored in tanks as mentioned in previous sections. / t - 12 - 50$ Caustic storage tank, steel - 12,000 gallons. Weigh tank to measure DCP, glassed steel on platform scales-300 gallons. Caustic drop tank, steel - 350 gallons Rotary vacuum drum filter. Auxiliary items such as vacuum pump, etc. Rotary drum filter feed tank, wood, with agitator - 5,000 gallons. Slurry tank to hold cake from rotary drum filter, heated and agitated. Filtrate tank for liquor from rotary drum filter - 7,500 gallon. Acidification equipment including: Small stainless steel mix tank with agitator - 75 gallons. Cooling tank, steel - 350 gallons. t Large vacuum filter, Cypress wood, similar to a large Buchner funnel. Carts to convey wet 2,U-D acid to drying area. Atmospheric tray dryers. (Screen bottom trays) Miscellaneous pumps, etc. METHOD OF MANUFACTURE: DCP, water, and half of the caustic pumped to coupling actor. Initial temperature held at less than 55*0. MCA and the remainder of the caustic then added with coding, pH of 10.0 to 10.5 important. Temperature allowed to go to 75-85*0. Mixture now contains various products including sodium chloride, sodium glycolate, sodium dichlorophenate, sodium salt of dichlorophenoxyacetic acid. The coupling reaction takes approximately 8 hours. 13- - Material pumped, to rotary vacuum drum filter feed tank along with more vater. Slurry filtered on rotary vacuum drum filter. Filtrate containing unreacted DCP, 6odium chloride, degraded monochloroacetic acid and some dissolved sodium salt of 2,4-D acid pumped to a storage tank. The 2,4-D and DCP are recovered by pri adjustment and toluene extraction. Cake from the rotary drum filter washed and dumped into a slurry tank. This contains the sodium salt of 2,4-D acid. The cake slurry is pumped to a mix-tank and adjusted with HC1 on a continuous basis to maintain a pH of less than 1. This releases the 2,4-D acid. The adjusted slurry fed by gravity to a large Buchner type filter. Filtrate goes to wash tank. pH adjusted. Secondary filtrate to sewer. t The cake is washed to remove most of the NaCI and the wet 2.4-D acid shoveled into carts. The carts are moved to the drying areas and the 2,4-D acid dryed for use later in the esterification operation. Wet product is used when formulating amines. Obtain about 4,000 pounds 2,4-D acid per batch. CAPACITY; Currently about 200,000 lbs./month. UNIT QUANTITIES (per 100 lbs. 2,4-D) DCP MCA NaOH (50$) Theory 73-8 42-7 ^ 73 Actual *105 64 *125 Ai'6. 67 14. ^ $0t 0 HC1 as 32/0 (by-product from DCP & MCA operations^ Toluene (gal) f' 1 This should drop to 82 assuming 9 0 $ receovery of DCP. Excess caustic used. 165 4 .3 ( <$1 * ^ V,, v - 5948 - lU 2 , k ,5-TRICHLOROPHEHOXYACEnC ACID (2,U,5-T) CHEMISTRY: 2 iU,5-Trichlorophenoxyacetic acid is prepared by coupling oonochloroacetic acid with trichlorophenol (in a sodium salt solution). The reaction is summarized below: CH2C1C-0H + NaOH MCA MW 9U . 5 ChgCICf-ONa sodium MCA + HgO NaCl O-CHpC-OH \ D H rP HC1 ONa Cl Cl Cl sodim 2,k 5-trichlorophenate MW 219.5 (TCP MW 197.5) & 0 OCCHHJ 4 C l 'ONa = ,C0l + NaCl - 2,Uy5-T acid MW 255.5 MP 153SC sodium salt of 2,U,5-Trichlorophenoxyacetic acid i . Mt .WJ2-7T7'.5' j. r*>- 7' ^ As with the 2jk-D reaction this is essentially a Williamson synthesis for amyl-alkyl ethers. The coupling process produces the sodium salt of 2,4,5-T acid. The 2 , h 5-T is released by acidifying with HC1. Final product is a tan granular 2%solid and contains about impurities such as tetrachlorobenzene and sodium chloride. EQUIPMENT: I -------- - -- One 750 gallon stainless steel agitated and Jacketed coupling reactor. 5949 - 15 - MCA, HC1 and caustic are stored in tanks as mentioned in 2,^-D section. Rotary vacuum drum filter with auxiliary equipment such as vacuum pump, feed tank, filtrate tank, and cake slurry tank. Acidification equipment including: Cypress wood mix tank with agitator - 100 gallons. Cypress wood cooling tank - 200 gallons. Large vacuum filter, cypress wood - similar to a large Buchner funnel. Carts to convey wet 2,U,5-T acid to drying area. Same atmospheric tray dryers as used with 2,U-D. Miscellaneous pumps, etc. METHOD OF MANUFACTURE: f Essentially the same as 2,L-D except less caustic required due to excess amount already in the TCP slurry. w Cycle time for coupling about 8 hours. Separation made as with 2,L-D. Obtain about 1,100 pounds 2,L,5-T acid per batch. CAPACITY: Currently about 70,000 lbs./month. UNIT QUANTITIES (per 100 lbs. 2,U,5-T) TCP MCA HC1 (as 32$) Theory 77-3 37 - Actual A'jo y' *111 i* t,n 60 44, ez 150 4Z.1-! y Toluene (gals) - 6 .0 NaOH (50$) (, 50 c 2_. 7 This should drop to 86 assuming 90$ recovery of :TCP. 5950 I 5951 : 2.2 s T U X K ' o m C . Y (II' IIICIM lIl'IDKS /)cfKirlnn`tit of 'inirnmrottnf!/ nini To.rirolnft;/, Ixoijnl V oh iiin tnj nini .1f// iritffimil ('olirti!-, ( fij/ir/f/ffff/rr, t h i i i / n u k T a III.U o r ClINTUNTH I. I ni rullili') imi 11. Inorganic herbicides A. A I'H C lli I I'M . M. Sodium chlorate. C. Sulphuric acid . . III. Organic herbicides ......................................................................... A. C hloriiialed phcrioxy-aeids................................................. II. Chlorinated aliphatic acids and their sodium salts C. Carbam ates and allyl alcoh ol............................................ D . Substituted u rea s..................................................................... E. T riazin os....................................................................................... F. S ubstituted p h en ols................................................................ G. M iscellaneous organic herbicides..................................... 1. Tributyl phosphorotrithioate (DEF) 2. M aleic hydrazide 3. Endothal sodium 4. Diquat (Kcglonc) 5. Aminotriazole IV. Conclusions 231 234 230 235 240 241 241 243 243 244 244 215 I. INTRODUCTION In their endeavours to raise productivity, agriculturists have, made use of ahciifrnnoihmasrshskeeoimwcbbninteeiigstceev,oandorwlflssuwvch,teonirohdcngihtaatitb,rveysoentwtlhe.uaeeaasMebtdidtnoiiranslguay,nstcteyhietchnetteecocidmc.rrf,ibedtcaiiahencossieni,gdpn'srertgioeadse,acxesetptiixri.ccaeetoneecb.l,,uinloaeocmgtlroliiybtmcFgoaeoipxrlntriohacdtuoetohaefnlteodfoapgrprsoeiugucwmsnaratphnildccoioeicosidprnhenleh,tyfeshooiirdnsafehepgvscnahteo.asta.nibsattsbReerlee,eosenenclsllnhamoifnntooeigdul-vnipenneituvnedlsyebsjultoluoeirflsspriiehtoateftehusudldeeesl, reports placed at the disposal of the authorities concerned. Such data have not been included in the present survey. Of a number of compounds, the toxicology of which has been elucidated in relation to their use for purposes other than as herbicides, only a brief account will be given with references to relevant literature. Regarding the practical use of herbicides, reference, is made to handbook dlgckirriteloeolsprutarasplotlswuy,vriineestgehgl(eoep7tuca0ottt,tiiavdo8teano0m.,ahSna1augd1ubi9lnms,ngtoa1antn2hn-c0esde).e.slcfcWuoocrfltteitidvevheedaes.ktielciTaldcltheatretpesirloasnugensrltoeuosfc,uatglpwilrvyehmeeeanarsryeuep.abtaschsrlletatahrssneesocfifoefnirsepoedliclab-ainsennettlsuoebscweettdhiwviueacoshlestmdoympafoaoeinnyrs unduly delay harvesting, in which case they are often called desiccants or de foliants. The herbicides arc grouped, according to their mode of distribution in 225 5952 i;ui.. : Ini-i n--. m- u ** *.i.-. i.<*. . from >wpo?*cd parts of foliage or rool>, ami residual herbicides, which arc spri-ad on or in the soil and arc effective mainly against germinating seeds. accFoorrditnhge tporecsheenmt iscuarlvecyonofifgutrhaetiotonxihcoaslogbyeeonf cthhoesehne.rbIincaidsems,ucah calassscifoimcamtioonn names, abbreviations, and registered trade marks are used indiscriminately in tmheendlietedraftourrpe,eswticcihdaesveb,yasthfearBarsitipsohssSitbalne,dainrcdlsudIendstitthuetioconm, mmaornkendam(*e)s, rneacmomes approved by the British Weed Control Council (f), and those approved by the Weed Society of America (J). ir. .in o r g a n ic h e r b ic id e s Prior to the Second World War, mainly inorganic compounds were used for chemical weed control. However, as their actions are not very selective and they arc often very persistent in the soil, so that damage to cultivated crops has been difficult to avoid, they have been replaced by organic compounds to a steadily increasing extent. Yet, various familiar inorganic compounds arc still used as dchhaelrtoabriicodinde,etsph, oeets.ages.,saicruacmlcwiuceymllankcunyotacwn,anamnfidrdocsm,odctuihupemrihctasenutrdlpabbhooaortkea,tleiftee(rrbraooturuasrxes)u.. lTpThhhaeetesta,omxmiceeorcliosugrtiorcuuaesl of arsenites, sodium chlorate, and sulphuric acid, but these compounds will nevertheless by described briefly, because their use as herbicides has introduced special problems of toxicity. A , Arsenites Arsenites are used as non-selcctive herbicides, especially for destruction of potato haulm, as aqueous solutions of potassium-ortho-arsenite (Kj.VsjOs) and sodium ortho-arsenite (Xa*Asj03). The preparations may also contain metaarsenitc and pyro-arsenite alkali salts. In rats, the LU50 for alkali arsenites has been found to be 70 mg 'kg when administered by mouth and 150 ing/kg when applied dennally (32). In domestic animals (40) and man (103) the toxicity is considerably higher, fatalities having occurred in the horse, cow, and man after oral ingestion of 2 lo 10 mg/kg. The solubility, preparation, and purity have a considerable influence on the toxicity of arsenic compounds (48); the toxic properties, actions on the animal organism, (m10aSn)if.estations of poisoning, etc., have been excellently summarized recently The use of arsenites as weedkillers has occasioned an extensive series of acute, often fatal cases of poisoning in domestic animals, especially cattle, which have eaten contaminated crops or residues of sprays (40). Concentrations toxic to innii hCaav-eesboefendifroeucnt dhuinmmanilkpofirsoomnincogwhsafveedaolsnocbroeepns coobnsetarmveidn.aAtend ibnystaarnsceenihteass bMeieln). described where one woman died and four others fell ill after drinking water from a reservoir which had been contaminated through a leaking pump valve in the iii 1iii-iit Britain resulted in a voluntary agreement between iudu-try .aid 'in; authorities for discontinuing the use of arsenite herbicides (32 i. 5953 B. Sodium chlorate Sodium chlorate and other chlorates are extensively used for killing all vegeta ptiootnatoof hfaarumlmy.arCdhs,lorraaitlewsapyostrseascsksn,orpoaardtsiicduelsa,rleytcg.,reaastwaeclulteaslofxorieidtevs,trtuhceti1o.n1Xo0f for sodium chlorate administered by mouth to rats having been stated to be 12r0 mg/kg (32). Nevertheless, oral ingestion of both sodium chlorate and potassium chlorate, used in oral hygiene, has caused numerous cases of poisoning 2i man (21, 33, 105). In domestic animals, too, cases of acute poisoning have been ob served after oral ingestion of chlorates (10). The mechanism of the poisoning-- methemoglobin production and its consequences--has been well studied '21. 33. 10A6).special problem is that of the high inflammability of sodium chlorate. This can be reduced in commercial preparations by admixture with calcium chloride or sodium chloride, but after dissolution in water and spraying, dried residues may become ignited. In Denmark, a tractor driver whose clothes had ' come timhepyrewgenraeteidgnwiteitdh bsyodaiurmed-chholtoreaxtheaudustripnigpes.praying, died of severe bums after C. Sulphuric acid useSdulpfohrurpicotaactiod,hwauhlimch dinesctreurtcatiionnc, oiusntorfiteesn, ea.gp.p. lGiedreaitn Bhriigthaine,nnisc.c-momrm. ol nirl.-y Accidcnls with severe skin burns and eye burns halatiop of sulphuric acid may also exert a toxic emffaeyct,thaesreifnodriecaotcecdurbyi:',s2tu.dIiens- on guinea pigs (3, 4) and on volunteer human subjects (5). ti t. ORl'SAMC HF.nBICIOIfs A. Chlorinated phcnoxy-acids Chlorinated compounds of phenoxy-acetic acid, propionic acid, and butyric acid, have been very extensively used within the past 15 years as herbicides under the common name of "hormone weedkillers.'' Tln v stimulate pars of susceptible plants, particularly within the group of dicotyledon*, to excessive, uconmcopnoturonldlesd, wgrhoicwhthi,nwthhiechfocramussesofthweaptelar-nstoslutoblediesaGltO- Po)r. Tlihpeoimd--osothiihmlcpoenratenr:s constitute the active components of commercial preparation-, are listed in Table 1, where the LD50 values iu rats are also given, in addition n> ill" mdpiocuhnlodrsolpishtecdnoixny)tchtehytlabsulel,pph-acthelo(2ro,4p-hDcKno.Sx-ysaocdeintimc ,acSiedso(n1e-,CSPMASi.d-oadnidumy-f22-.42..34-torficthhilsortoypphec.noxy)butyric acid (2,4,o-TI5, 4-(2,4,5-TB'i> ire usi d as heihi. ites 225 DALGAARD'MIKKKLSEN AND rOULSEN TAUU5 1 LO CO LO TOXICOLOGY OF HERBICIDES 229 asakunnilsdTiocmgher2apeal,tsm4aibc,i5lusfe-ot,mTerwottohhlduxeecirisncse.ipitbeteyhe.ceeifnTeosLlhlfDoeeoxw5uaLi0nmnDdgfi5ono0treordbanlosb(ta8ehnd9)m1(r,20uie,0ln4exi-csmditesirpegaoht/tikflinooggtnrhod(ept2ooh7oger)as.dn, neowEruxxhmyopi)fcbaeh.rec'1ric0mst0eoieecftmnoeatxsc7tpio0dwe0rbi(itm2emh,gm4eno-poDrtreaaer)ll. administration of various salts and esters of 2,4-D as pure chemicals and as commercial preparat ions showed no significant difference in toxicity to a number of small animals fnm that of the free 2,4-D acid (53, 89). Short-term studies on rats revealed no signs of reduced intake of food or inhibition of growth in response to admixture of 2,4-D at 400 p.p.m. in the fodder for 30 days or 1000 p.p.m. for 14 days. Subcutaneous injection of 50 to 100 mg 2 .4-D per kg to mice daily for 90 days had no demonstrable-effect on the general condition, fertility, or tissue histology (53). In Table 2 are recorded the results agosoblfyolfcieenoetldtooibxniutgcotypleelhfrfeaeehcttheot exporynr-oecslsoohtmenergepsepo,duotnihfndeg2ssee,t4sote,i5xola-pnrTegorPeifrm,paaehnndeitmnmsoiansxlhiysso.-tcweEorexmedcdpeptoihnutanfttodhrseth.tthfeooerltrmaharegthoesfearhmpecreroobnpeisxvyioldteerennerest as rats. In dogs, on the other hai\d, these compounds were found to have a pro pnooiusnonceindg.toxic action: 20 mg/kg given for 2 to 3 weeks produced severe, fatal dapwniihscPiptimcualharraeyelsnhaotbaefsrdriimansblgiyensaeocnnatloibdnnosaitouaturtidoaadinleecdavthodealmpmoraapironcstvitiseecwtru.riilaTtsahtthriicolirysncigopioimnadfsipstdtliyoevoxxcgoinsofc.cfstdAshsioegfistsneessgsrkraeoaanlfeddtuf2aesal,wy4llmmy-Dhpuaotsguoctgmolrresassevtx(aha1pnet8eedr,da2ia,mn7tai,aemnxn5dia3tual)as.l, The condition may improve transitorily with movement. In severe cases the animals show progressive apathy, depression, and muscular weakness, especially of the hindlegs, with periodic, clonic spusms, and, finally, coma. The muscular signs ure accompanied by marked anorexia; frequent!}', irritation of the nose and eyes is indicated by scratching reactions. Further, bleeding from the nose and mouth may occur, as well us diarrhea with blood-stained stools. Local irritation eovfetnheafatleirmoernatlaardymtriancisttoraftteionnctaousdeosgvso(m27it)i.nAgu. tHoopwsyevmear,ytrheivsesaigl nnemcraoytibceualcbesresnotf, the oral mucosa and signs of irritation with histologicully demonstrable inflam matory changes and necrosis of the small intestinal mucosa, as well as focal necrosis in the liver (27, 53) and degeneration of the renal tubules. When given by mouth to dogs, even in fatal cases 2,4,5-T has produced only weak signs in the forms of ataxia and stiff movements of the hindlegs (27). As foi; the human response, n report is available (9, 75) of a man who in a srlf-ex-pf.nrnont ronsumed .7H) imr of 2 .-1-D daily for 3 neck wih to n* r* TAIJLU 2 Chlorinated phenoxy-acids; short-term studies in doQ, sheep, and cow Compound SApneicmieal BDoadmiylyW/kDeaoigteh,t Retult RKeof.. MCI'A. Con- 30 To2l1erdaatyeds without detectable symptoms during (24) 2,4-0 -- -- 2,4,5-T -- -- 2,4,5-TP Fbeep Dog D or Sheep Dog Dog Sheep 100 2-10 20 3TTooo03llf05ee4rrddaaaaattyyeenddssimwwaiilttshhooduuiettddd,ee1ttee8ccthttaabbtollee4ss9yytmmh ppdttaooymmss during during (85) (27) (27) 100 2-10 20 . TT4 ooo93llf05eer4rddaaaaattyeyenddssimwwaiilttshhooduuiettdddee1tt1eetcchttaatbbollee75ssytyhmmdppattoyommss during during (85) (27) (271 100 Lethal after 11 doses (85) The action of ingested phcnoxy-acid herbicides on muscular function, which is reminiscent of that following administration of halogenatcd acetic acid com pounds (22. 23), suggests an interference with carbohydrate metabolism. Two cases of a transitory diabeliform condition observed in spraying personnel following work with chlorinated phenoxy-acid herbicides point in the same direction. However, hyperglycemia and glycosuria could not be reproduced with certainty in rabbits; only one out of five animals responded in this way in ex ploratory experiments, where the doses ranged from 125 to 500 mg/kg and the period of administration from Gto 50 days (08). The results of a long series of investigations into the toxicity to domestic animals and game of hcrbicidal preparations and treated crops suggested that acute poisoning caused by consumption of crops from sprayed fields is unlikely to occur (lt>, 24, 4t>, 75, 87), excepting that "grass-eating" dogs may be poisoned by newly sprayed lawns. Human beings and domestic animats arc presumably liable to poisoning only by oral ingestion of highly concentrated preparation? or spray solutions. tcroamtiposonpusen--cdiatsol isipmrtohpbealreltemntodminwiracjtoenro,nfmetchitlieko,nparnwedpitaohrtahtteihorennsuu--stereiveoenfntscihnaloverxeirtnryaetmpeederlsypishltoeewnnotcxcoyhn-laocceriond- phonoMike odor and taste. Spilling of highly concentrated preparations close to gtwiioevnlelsnoofar2dw,i4as-at1eg)rrecteooaudbralseierstyahscatasetittnloesedthvoeeerswanlaoitnetrsstefaoenmrcelostno, gbryepsepurelirtocdionslaaetxfitocenrrewtthiaorrnoduso.gfhAbdtihomleoisgntiircsaatirlnlay, demonstrable amounts in the milk (75). aciVdse,ryinfterwodtuocxeidcoalolognicealordaintacaormcbaivnaaitliaobnleasrepgraer-dcimngerogtehneerr-cahnldori.nateeledctivaenhmerabdie ctridicehslofroorbaegnrziociucltuarcaidl c(r2o,p3s,.62-T,3B,6A-Tfrti,chTlBarAo.phTcCnyBl)-acaontdic .'^avaimdir.ho--nSa.oc-id)i.cL2J.3o.r0o-abfetnezronicsiancgidle (oarmalibdeonsje.) taorerautssewdafsorstathteedsetopubrepowsietsh.inThthee LrDanogO- ooff 720.03,8to-TIB'OAO mg/kg (32). 5955 B. Chlorinated aliphatic acids and their sodium sa'.'-. sSpooldaTmniuhotmesopcfthrrtiilhcoorhersilnoetoraaotre-mcadceuccatshlaeiaptdcnh, ia(ctTthaicClerAhaefca-oifrdrJveses,soptfdioonirsugsdm.eseT)sf,ohsmloieadotrimiueomongsteamnnioedmrnaopdlcochrspltiohcarcnyoatt-oiaiv'f..r.x-*m'icoaprfocprur'unSoldMptsie-Crata'tiAreeesd):., and sodium-dichloropropionate (Du!apon-sodium*fi), as weil as 'he com': ined compound, bonff). 2-(2,4,5-triehlorophcnoxy)-ethyl-r2',2'-dichloropp. r.:onate Er- Studies substance on the acute toxicity of is not very toxic (118), tshoedLiuDmoOtriarfhtelwr nor-nalataadtcmhinaiv-era.T-hoonw1-U-hnag* 'bis 320 mg/kg for thesia-Iike rats and 4970 mg.'kg state, which lasts for for 30 mice. hours, The animals quickly to lx* succeeded by <sgo-,r>r.iantaor.dand-a2.r*hs-. or by awakening and survival. Sodium trichloro-acetare ha* a :`ar less lo.-al- irritating action than the free acid, which can corrode the ski:. and mucous membranes. baSctoedriiuamanmdoinnorthheloaron-iamccatlaotergfaSnMisCmAi)s is considerably more reminiscent of that of other It* a-?-,.*:, on x.'inohalogen- substituted acetic acid compounds, such as monoiodo- and monobromac^tic acid, the enzyme-inhibiting and bacteriostatic proponies of *.\h: h have K-en thoroughly to be 70 m gs/tkugdiefodr(rvaidtes,2a2n).dTShOe LD5G mg kg after oral admini.-tra'y'n bis r.-Q for guinea pigs (US . and of the I :::d same order for geese (20.), whereas for mice the toxicity is somewhat !<v.v*r, the LD50 being 2o5 mg/kg (118). In the small experimental animals apir.hy and loss of weight were noticed, and in the fatal cases death occurred wi*h:r. 3 days. The fatal oral dose for young cattle was found to lie within the ra:.ir- : 10Q to '.30 mg/kg. Colicky-like restlessness and incoordination were nb-*rv::i. developing in the course of 4 to 5 hours into universal fascicular twitching?-. gr.i-hinc of -he teeth, anxiety, dyspnea, and tachycardia. Coma and death (23). This picture, which is probably referable to blocking offolol-x.v:.vl-a.'ivdeioiur 0*ahle:hrvs processes, is reminiscent of that scon in pigs 22) and dogs p .-oned wvh njonobromacetic acid. The gross and microscopic findings . .-op-y w-re likewise identical with those described for monobromacotic a-id-r honed pig o(2w2Si)on.dgiutomit2s,2h-cdribcihcliodraolparcotpioinnnoantem(oDnaolacpontynl'et dhoanss,bseuccohmae*ogfnpi-;.'r-*v:. small quantities, therefore being useful as a selective herbicide ce r:mnj`miAnrrecalrauoteipvrs^elosy!f cultivated plants. Dalapon is absorbed by plants and then trandoci*-*d. On tki* toxici-trylUui prohensive the su-busdtance has been stIuAdielldi^.from .sovrrul series of experiments (81). Administration of n.-|><vis brumdi single doses to a m.1imrub;:or of small experimental animals has given LD50 values for rats, mice, guinea pigs, rabbits, and chickens within the range of 4000 to 9000 mg/kg. Two young cattle survived 1000 mg/kg given by mouth daily for 10 days. Beyond transitory symptoms in one of these (anorexia, diarrhea, indisposition), no signs of a toxic action ere found by clinical or pathological examination. After oral administra tion of 15, 50, and 100 mg/ kg daily to dogs for one year, blood and urine analyses, as well as liver function tests and histological examination of tissues revealed no dsiognse.*ooff 1a00toxmicg/akcgti.oEn,xabmeyionnadtioann oinf ctriessauseedspweeciigmhetnosf ftrhoemkitdhneedyosgfsolslhowowinegd tuhpe to "S p.p.m. of Dalapon in kidney and liver tissues (81). Feeding of rats with Dalapon for 2 years in doses of 100, 300, and 1000 p.p.m. in the fodder, corresponding to about 5, 15, and .50 mg/kg daily, was tolerated with no signs of a toxic action, apart from a minor increase in kidney weight on the largest dose, fn these experiments with 1000 p.p.m., 10 to 30 p.p.m. of Dala pon was fourni on chemical analysis of the liver and kidney, and 20 p.p.m. in milk. Thus, cumulation docs not take place. In rats, reproduction and lactation proved to be uninfluenced through three generations with daily administration of 300 to 3000 p.p.m. of Dalapon in the fodder (81). The local-irritating action of Dalapon has been studied on rabbits, the skin of which was exposed daily for 10 days to a 10% aqueous solution. No more than signs of a mild, transitory irritation was seen (81). C. ('nrhnmairs and aUtjl alrohol Within this group of herbicides, which, as shown in Table 3, comprises enrhu amPrreotpeuhss,aemdthieoascnpadercbeiaaMmlloyarptaorsso,ppahrnuedm-edmhiteahrvigoeecnaaclresboaombreaestenelesu,cstieisvdaetnhoueprmbrbeicveirednoetsf pcooonmtacpteooreutsanidinnstecwnrhodipecsdh. fwoirthcopnrsoulomnpgteidoninfgreosmtiosnprisouthtienrgefworheiloefsptoarretidc.ulTahreinttoexriecsitt.y of these compound haPverosphhoawmn (O-Awpropyl N-phonyl carbamate, JFC). that carbamate esters possess a carcinogenic Various investigations action. Thus intraperi toneal strain oinf jmecictieonfroomf is1o7ptroop9y0l%ca(r0b0a).mTahtee raised the frequency of idea of inquiring into a lung tumors in a possible cam* gonic action of Propham, which splits off aniline by acid hydrolysis, therefore suggested itself. However, with prolonged oral, intramuscular, ami intrapleural administration of Propham to rats and mice, no signs of a carcinogenic action were found (50). This is in agreement with the results of experiments on group* of four rats each, given 400, 800, and 1000 p.p.m., respectively, daily in the fodder fcoornd3itmioonn,tghrso. wNtoh,soigrnfserwtielriteyd, eamndonnsotrpaabtlheohloegriecaolf cahatonxgiecs action on were seen the. general at autop.\V or on histological examination of the tissues (101). 5956 i..vd'ii t tii a n d i.iln- >, J . J '-*0 l>\ i.>r;ti miiiiiii. t..i. . ^ ....... to be of the order of oOUO mg per kg for both species (I l'. Ir- feeding experi ments on male rats given 310 to 20,000 p.p.m. for 00 days, no efT-6*?? was cr a n ed on growth. Doses of 12o0 p.p.m. and higher produced an increase in w*.-:,*ht o: theIinr ltihveerlisg,hwtitohfotuhtehaibstoovleogiincvaelscthigaantgioenssb,etihnegodbesmerovnasttiroanbloefit'hll**'l.cve!opn.*-nT of skin tumors following painting of the back skin of Prophan.- an-: Chlorpropaam- fed rats with croton oil is of considerable theoretical mere4' 34). However, cthormoupgrhe*he2nsyiveaersfeetodinrgatsexapnedrim1eynetsar(Gto7) dwogitshreavdemaliendistnraoticoanr.imnoc2eCn0i0c pcr.pT.cmct.. Feeding at 20,000 p.p.m. provoked in both species signs of a to:.;'* action. which was manifested in rats by retarded growth, increased m ortify of the male animals, and increased liver and kidney weights, though witho ;; deir.onsirahle histological changes. In dogs, retarded grotvth, increased weights of liver, kiiney. and spleen, and splenic congestion were noted (fi7). wadhBmaatirngbisratenraeattei(ro4n-acchhulatoevriont-og2x-ibbcueitetyynntyfhola-uXnn-d(t3h-tecohPbloreoropGphOhnOcmnmycgloJpmcaeprrbokuagmndf3ost,re:t'rhhedeii-.rDpa"ltayUa*ncdiaor.ar^boobmriaet],- and 240 mg/kg for the guinea pig. Dermal application of 1*300 me kg ov-r a panerdio3d7omf g24/khgoduarsilycafuosred22ndoadyesapthrsodaumceodngnorattos.xiOcrraelaacdtimonin, iwsthraertieoans o7:-30n. .j1:*k.5g. . over the same period efTocted loss of weight. Feeding esporin.-mts with rats showed no toxic action of loO p.p.m. for IS months (36). Barbane is a potent akin-sensitizing agent in man, in whom allergic reaction with r^h dev-dor? at subsequent contact. Protection against cutaneous contact i- 'her- ."re :rv during its use. Plastic (polyvinyl chloride) seems to be a more suitable protr-.-ive . materiaf than rubber (36). ' ^. as.pneSdcMitahlDesCplihk(eseoredbiyoufmsoa-pNcpa-lmlilcecadtthisoyonli,ldshittoehirnioiglcizauarstbeiodanmf.oarWtckhdielilnhinvagdprwpalteieee.ddmsineeerd:hhsae.n-;~-.-;oillduiit*u?.nI'.i&ib.e>Lra;i.steesas. gaseousmefAi//iso//itnc>/ano/e, the active substance, which is also available commer cially as an aqueous solution (Trapex) for similar purposes. The I.D-30 o: SMDC bTOIryhfremiotraemattilhnaaygidnliamscotoittnihxoiiisnoctocroylaoaftgimotihclneaesltieosinrscuatoetbnrsseshtsiadatnescarbeattesbaelocnynhshettsiahg,tehehedsorkw,tionethvbaeeonr8,dL2Dt0mo.3mu0tcghobeuok;szp::rmg1o2'on.!>ro7..u.imnTrchage~nd*ktsozl.ox.;-->-p.i2itle)yy-. cially those of toxic amounts the respiratory* organs, as well as the by these routes. The LD.30 of SMDC apfotsesribdielirtmy acl>:..phpaoi:r.p-.:i;::-:. of to rabbits has been found to !> 800 mg kg (20). whereas daily ru: hi*_ -.f u.. *V\ - v- iWheloakcyraenaactteionin(8120)`.7? ethanol for 0 weeks into rabbit ears g:iv<- r. nor-* ?h..\ a ..S. MAlDlyCl aanlcdohmole. tThyhliissouthnsioactuyraantaetde;ailtcoishoalpphlaisedthine same rang* of solution to the ~ '.I. -vher as ,;s # fi 5 i 5 %y p, >, j .' 5960 5961 MIDLAND, 'Michigan March 2y, 1965 c CHEM ICAL ' t / / > O*. 019193 K . Rowe Biochemical Research Laboratory 1701- Building Riley R. N. Smiley L. B. Grant C. 0. Hutchenreuther F. C. Amstutz W. P. Falsey G. E. Lynn ' W. M. Gill M. G. Wiltse D. E. Pletcher W. L. Corbin D. D. Irish J. E. Peterson J. C. Tucker J. W. Harris H. W. Feinauer E. C- Staehling C. E. Otis K. Y..Hansen R. C. Hoff W. J.' M c C o y J. D. Doedens K. C . Barrons H. R. Hoyle B. B. Holder, M.D. S. E. Sadek REPORT ON THE CHLGRACNE"PROBLEM MEETING ON 3 /2 ^ / 5 Present: Dr. J. Wilkenfeld and Mr. Raymond Verhoeze, Hooker Chemical Corporation Mr. Francis Kennedy and Dr. Ed Chandler, Diamond Alleali C o m p a n y Mr. C. L. Dunn and Dr. John ?. Frawley, Hercules Povrder Company* V. K. recaoped the Dov? situation in terms of the p r o b l e m and the initial studies by Toxicology and Environmental Research Laboratory regarding the in-plant situation. He expanded this in general terms to the study of end products, ours and other peoples. He made reference to symmetrical tetrachloro-p-dlbenzodloxln- He referred to the evidence for unknown acnegens. There were some questions from the group about the unknowns- We (Dow) were not able to a n s w e r these questions except to review the evidence for their exiatence In the process samples and end product's. CONFIDENTIAL - ELGJCT TO INJUNCTION JXC, E.D. /.U. 4-4-73; COW/EPA AGREEMENT 9-79 PLAINTIFF'S g EXHIBIT 5962 V- K. Rowe j 1-iarch 2g r 1965 Dr. Holder reviewed the medical side of the Dov; experience; he said that we now have approximately 60 to 7 0 cases of individuals w i t h chloracne ranging from tvjo severe cases to some very mild cases that were difficult to diagnose. He showed slides of the more dramatic cases. The slides were., exclusively views of the faces of the individuals afflicted. He described in fair detail the appearance of the indivi duals mentioning the blackheads specifically. He then reviewed the clinical studies that are being made on these - people with emphasis on the liver function tests. He men tioned the single liver biopsy that has been taken and studied in w h i c h the liver was normal although the man. had a rther pronounced case of chloracne. Dr. Holder also .mentioned the incidence of fatigue among the afflicted~"D^oole as" being the o n l y other significant .finding in these folks. ~~ He touched briefly on treatment indicating that various topical treatments were not particularly effective. He described the cycling of this disorder in individuals who had been completely removed from exposure. He mentioned that some fellows are approaching the end of their trouble two cr two and one-half years after onset of the skin dis order. He also described "acute chloracne" which is an acute inflammatory condition that appears considerably sooner than the normal chloracne in individuals and appear? after pro- * nounced single exposure. The acute chl'oracne shows up within, a few days of exposure. Dr. Holder mentioned five to eight days specifically.. There w a s 'considerable discussion b y t h e group on the skin disorder Itself. The Hooker representatives related experience of skin condition thirty years after expo- surel Their cases were more similar to the Dowiclde Dumps vw Hl~dh D ow has experienced in that there were large Polls o F . hnmns 'than one multitude or small blackheads and eruptions wnich~~D5w is seeing IrT the" current, cases-. Dr. Sadek showed slides of ears and livers of rabbits that . had been exposed 00 zne symmetrical terrachloro-p-dlbenzodioxl He discussed the pathology in detail which I will not attempt to summarize. : ' *-- V. K. mentioned the studies in which the rabbit ears have been treated with TCBD in benzene or corn oil and then washed w i t h soap .and water at various time intervals l a t e r - If exposure occurs for very long, washing does little good. He also briefly mentioned the oral studies but without detail. Silver3tein described the plant study on washing of contamination from tools and surfaces. This study Indicated that benzene, acetone and Chlorothene NU were effective in removing the contaminant from tools and also that'detergent and water with, scrubbing action.could clean up tools and equipment. Some discussion ensued on the use 5963 V. K. Rowe - 3- March 29, I955 of detergent-and water and the point was made again that strong scrubbing action was necessary for this approach to be successful. . . Harold Gill then discussed the analysis for tetrachloro-p-- . dibenzodioxin by vapor phase chromatography. He listed the limit of sensitivity on various process materials. He mentioned the oil which he defined as a non-saponlfiable mixture of chloro anisoles, tetrachlorobenzene and trichlorobenzene; the limit of sensitivity for TCBD in this material is 10 ppm. The limit is 1 ppm for 2,4,5-trichlorophenol, and for 2,4,5-T Acid, either acetic or propionic. Gill then defined 1 ppm as a very discernible, peak. H e men tioned that he-might estimate 0.5 ppm in some instances but 'to be conservative the analyst reports <1 p p m i f the p e a k does hot measure up to the quite identifiable level of 1 ppm. The analytical problem has not yet been solved for the .T-Acld esters. The general procedure used f o r the T-Acias is to extract the sample (arbitrarily about 20 grams) w i t h chloroform (about 40 milliliters), filter the-chloroform to remove solids and wash with an equal volume of N/lO caustic to remove any acidic materials* The chloroform extract then is concentrated by evaporation to"'one-tenth the original volume; t h u s , 'the concentration of the d i o x i n in the chloroform will be ten times higher than in the original sample. When the analysis is conducted on t r i - . chlorophenol, the material is dissolved in N/l caustic to the extent of 10^, and this solution is then extracted with the chloroform and handled as Indicated above. A question was asked about the utilization of detectors other than the flame ionization which is specified in the Analytical Laboratory write-up for this analysis. Gill h a s `not tried the micro coulometrlc detector becausq h e is not set up to do so, but he has experimented w i t h electron capture. He stated that theoretically this uriit should not .provide any greater Increase in sensitivity. In actuality he found a slight increase in sensitivity but there are usually too many chlorinated species present which may saturate the electron capture cell whose recovery is too slow to be of practical use. He summarized b y s a y ing that the slight -increase in sensitivity' is not worth the effort -to switch from flame ionization t o e l e c t r o n capture. A question was asked about how' the e x t r a c t i o n is performed. Gill stated that it is performed in a wid e m o u t h bottle on a shaker for one hour. (It w a s "n ot m e n tioned, but it is the case that this is done at room temperature.) He mentioned that spiked samples have been V. K. Rowe - 4- r-Iarch 29> 1955 run this way-and the recovery ranged from SO to 100 per cent. The ratio of solvent to the material being extracted on this step is not critical according to Gill. -Their t standard .procedure Is 20 grains of sample'and 40 mil l i l i t e r s of chloroform. On trichlorophenol samples specifically, .20 grams of phenol Is converted to phenate -- about 10 p e r cent concentration in water. The phenate solution is extracted with 20 milliliters of chloroform in a single extraction. The chloroform Is then concentrated so that the concentration of the dioxin will be ten times that In the original sample. The question of volatility of dioxin came up and Harold Gill .stated that he*found he can distill o-dichlorobenzene away from tetrchlorobensodioxinJ 'He said that in his opinion the secret.was to avoid distilling to dryness. A member of the group asked if samples of standard TCBD were available. ' T h e answer w as yes and 100 mg samples trere p r o vided to one of the representatives from each company. (a sample had previouslj- been given to Dr. K e lly of konsanto.) A question of laboratory safety in the analytical work up and the basic precaution of wearing vinyl gloves was mentioned. Information relative to the gloves_.we used was provided to the group. "" . Disposal of contaminated, laboratory materials and plant materials was discussed. We mentioned that Dow burns some small amounts of waste. - Harold Gill stated that his lab oratory study of combustion showed that 99*9o p e r cent of.-' .the dioxin sample was burned at 800C. We described w h y w'e felt that our practice of burning small amounts of d ioxin was a. safe one. V. K. then outlined the project In which plant samples and products (not mentioned by name) were spiked wit h known., amounts of the TCBD. The spiked samples were split.for the purpose of checking our analytical procedures for recovery and correlating these results with the bio-assay method. The question of specification, quality control specification that is, was raised and we were asked if ve could give levels of dioxin contamination which were permissible limits. V. K. mentioned that at present w e are using zero w i t h a confidence of 1 ppm in process samples. There was some discussion on the problem of customers using finished pro- ' ducts under far less desirable conditions of health control than we can provide our workmen in our own plant. There seemed to be agreement among the group that we could not afford to sell contaminated products. I 5965 V. K. Rowe -5- l'arch 29 , 1955 \ Jack Peterson then discussed the data from animal experiments using pure symmetrical tetrachlorobenzodioxin. Doses ranging from 2 parts per billion to 1000 parts per million.. of tetrachlorobenzodioxin in benzene had ,been administered to the. rabbit ear. Dosage in most cases was 0.1 m l per day. Both single and multiple exposures have been studied and multiple exposures administered on a five days per week basis'. The significant factors in the study a r e dose,, t h e number of applications and. the days on exposure of the animals- The response which is reported in the gross ob servation of the condition of the r a b b i t 's ear b y the toxicologists. This does not include pathological findings -- there is not enough .data in this area to discuss. The .level. _ response ranges from none through very slight,, slight, slight to moderate, moderate, moderate to- severe, severe, and extremely severe. Jack Indicated 'to the group that there `is not a sharp definition between these categories o f response and indicated also that there is some difficulty in graphing this type of response. He .described the resoonse from single applications to the rabbit ear firstr at 100 parts per million there was a severe response in eight days; at 40 parts per million there was a slight response in eleven days; a t `20, 10, 7 and 4- parts per m i l l i o n there w a s no response. These tests were run on s i n g l e .rabbits and -without washing the material off. Jack then discussed the .multiple application data which he took from his major graph of this data. The. important points that h e m a d e f r o m " this data were first that at the limit of .VPC sensitivity. .a severe response m a y be produced. I n other words, e v e n if. the VPC does not detect TCBD, an animal response m a y still occur. His. second important point was that the Induction period for response averaged about ten days on the animals in the studies. There v:as a brief discussion then about the a i r samples''t h a t were taken in the plant. Silverstein mentioned that some air samples have shown activity on the animals. The degree of response is slight and the number of samples t h a t show activity is small out of the total number taken and the amount of air that must be sampled is very much larger than the amount a man normally breathes in an eight hour day. -The meeting was'adjourned. The group then proceeded to t h e Toxicology Laboratory to view some of the test animals. T h e y v/ere shewn responses of varying intensity and these w e r e described. This demonstration appeared to have considerable Biochemical Research Laboratory 1701 Building LGSrsjl 5966 C V. K. Rowe. - 6- rch 29, 19 6 5 Postscript All participants seemed to appreciate well the problem a n d all indicated that they would return home and attempt to convince their management to institute safety specifications (really - Equality control) for- their various products i n this area. All agreed that the industry should meet its own responsibility. All were very appreciative of Dow's effort to steer them away from a danger area. Time xfill tell whether w e a c c o mplished our mission, but as of now X feel satisfied with our effort and the reception it received. . . VKR 5967 r'..:' tEKv,- 5968 8 DEFENDANT'S EXHIBIT iu a .3 T JKtIoHJUC2 D4 . 1 9 6 5 Boss BulboHand lu ip r H oprud ucta | m C henleal o f C*aad* S a r n ia , Canada DOW CONFIDENTIAL i t,4,5TSZCSLOAOPSS(QL, TBS "T" ACIM , AXD ASSOCIATED ACBBODtS j g Z kftT* o o t boon n e g le c t in g your roq u oat f o r la f o n s a t io o t o uao ^ i a d is c u s s in g tb* su b jec t probi* w ith Naugatuck and tbo Co-Op. Z bar boon s ty u le d , however, b*causo tbo a n a ly t ic a l otboda ^ hare boon changed and aro la tbo procoa* o f b e la g cloarod and * raproducod. Z oxpoct tbo any day, but ratbor than a lt lo n g er, Z thought X abould a d v ise you o f tbo a ltu a tlo a . Z 111 aood you eoploa o f tboao otboda aa aoon aa tboy bocoao a v a ila b le . Za regard t o tb o o r o r a ll p r o b le a , *o aro a t t o a p t la c to do everyth ing poaalblo to avoid tbo poaalblo oeourroaco o f e h lo r aooe la any a p p lica tio n s involving tbo bandllac or so o f t r i abloroph onol, trlcb lorop b ooozyacotle a c id and I t s d e r iv a tiv e s . Aa you o i l t o o , 0 bad a s e r io u s a l t u a t l o a l a o u r o p e r a tin g p lan ts because o f oontaalnatlon o f 2 ,4 ,5 -trleh lo ro p h eo o l ltb la p u r ltlc s , tbo oat a c tiv e o f bleb la 2 , 3 , 7 , 8-te tr a c h lo r o d lb o a zo d lo zla . This a ttr la l la o x ecp tlo n a lly to x ic ; I t baa a tremendous p o te n tia l fo r producing ehloraene and sy a to a le In ju ry . I f I t la p resen t la tbo tr lc h lo r o p h en o l, I t 1 1 1 bo aarrlod through In to tbo T acid and la ta tbo o s to r s and bonce l a t o fo r m u la tio n s h lc h aro t o bo s o ld t o tb o p u b l i c . Qdo o f t b s th in g s b le b wo s a t t o a v o id l a tb o o c c u r r e n c e o f a ay sen* la ooosu sers. X aa p a rticu la rly concerned boro w ith per sons bo aro u sin g tbo s a ta r la l on a d a lly , repeated b a sis such aa ouato operator* aay use I t . I f th is abould occu r, tbo b ole In d u str y i l l be fc^^d h i t and X w ould e x p e c t r e s t r i c t i v e le g is la tio n , e ith e r barrlag the s a ta r la l or p u ttin g very r ig id e o a t r o ls upon i t . T h is la tbo a m rea so n by we a re s o e o o - aern ed t h a t v e e le a a up our own bouse f r o w it h in , r a th e r than h a v in g a o io n e f r o w ith o u t do i t f o r u s . Xa t h i s w ay, we can approach the problea la aa orderly aaoer. Zf the producer sad handlers o f t a ls a te r la l w ill coop erate, th ere la bo ro a so o why we ca n n o t g e t t h i s p r o b le un der s t r i c t c o n t r o l and thereby h op efu lly avoid r e s tr ic tiv e le g is la tio n ; la oth er words, l e t ua p r a c t ic e good o lt lx e n a h lp . At th e p r e s e n t t i n * , we a r e f the op in ion th a t a te r la l co n ta in in g no tetra o h lo ro d lb en x o d lo x ln w ith a c e r t a i n t y o f 1 ppw d o es n ot p r e s e n t aa a p p r e c i a b le hazard t o consum ers; li k e w i s e , we do o t b e l i e v e t h a t su ch a te r la l c o n s titu te s a s i g n i f i e s * hazard to person s working la 5969 0-A K DOU DOW CONFIDENTIAL 1 M u lh ollu d - 8- June 2, 1965 p'-ants handling such phenol, ? acid , or T acid e ste r * . Z s i g h t add th a t we t r t c o n tin u in g our resea rch * on t h i s A rticu la r prob Ira fro a tha standpoint of studying tha othar im p u r itie s which u r hara tha cap acity to produce th is typa o f r e a c t io n . A ls o , we a re s t t e n p t ln g to q u a n t it a t e th e a f f e c t s o f th a taxewn acnagena whan added t o b ase a a t a r l a l s . T h is work i s p r o g r e s s in g w a ll, but I t w i l l be s e v e r a l o a th s b e f o r e we hare a com p leted s t o r y . Z would urge a g a in th a t I f your b ig custom ers such as Co-Op i arv< la u g a tu c k hare p a r t ic u l a r q u e s tio n s about t h i s p r o b ls e t h a t you l s r l t e th ea t o cooe to Midland where we w i l l be g la d to d is c u s s th e n a t t e r in d e t a i l w ith than and show th ea srhat we h are le a r n e d . Ve a r e not l a any way a t t e a p t l o g to h id e our p r o b le n under a heap o f san d , but we c e r t a i n l y do n o t wane t o hare any s it u a t io n s a r i s e w hich w i l l c a u se th e cc r e g u la to r y a g e n c ie s t o becone r e s t r i c t i v e . Our prim ary o l - J a c tlr e i s to avoid t h is . Z tr u s t th a t you w ill be very Judicious la your u se o f th is In fo r e st io n . I t could be q u ite ew h a m s sin g I f i t wars m is interpreted or nlsused. % T O /jd a c t L. S ilr e r s te ln C. O tis fr a d y Holdeman F. Asstutx 0 . Ooergen 1 . Boyle V .. Fa 1. lfsteswr e (2) T 1 7 .* -1 2 -2 0 ---~ Correspondence F .3 flbder no circu m stan ces nay t h is l a t t e r be reproduced, show n, o r s e n t t o anyone o u t s id e o f Dow. TO 5970 1 3 i r* I 4 5971 / . r.-... \B. JOH.N%SON/ vV ; 0000005 ih DOW 75187A F eb ru ary 15, 1967 ' -v Vw* . . ; * * W * V 4- m l m i (W ' * , ;. , M r. U , S. Buckley - -- >c|-**4**** fI * -.Technical D irector .: Thompson Chemical Company k 5023 Locust S treet . *, - f t . L ouis, M issouri 4 3 1 0 1 ? / ' i *'*>:-v*>>:.*'.L>.)v. .. **4%^> . ^e ^^ # > % , f ** i #V - ; . a-- ;; tV .-rf.y - ' :*:-\L.e, * r--M...r,^ Bu.c. kl. sJyt* V . ; - - ^ V - r V' * j j j .,*7* . v * % / : '.* * * 2 ^ ef # _ * 4 v` ^ - V rl.r . - . ' " ' . * . . i - : " v<*.* . % . * , % * . ,*i '.^*T.*'T* ' *'Tv- *' Pp ruorbsul eamn t , tX,o*hoauvre dhiasdc ut>h*ssrieoen paubwliesehkedora rsto.iic_al*ge_os^ about repro dyuocuerdehaniodraaemn e *; i'`T _ _ - i enclosing them herew ith, Two ot them a re translations which we . /* : - have m ade. The tran slatio n s m ay not be p erfect, hut the im portant ~\? : *points a r e th e re . One ie entitled "C linical O bservations about the O rigin oi C hloracns," and the other la antitied "Industrial Poisoning 1'; '* ^ ; ^ in the M anufacture i Chloropbenol Compounds, *. In thie la tte r cj... ,'r . , 2,9* .** .t-----^-*-*-*----*,-T------* *. i; ' * S in c e re ly yours, %tr-fV " /< ; ^ ' ** , * lr*e/| . - -- - Y* K, Howe ............. . *< .``t* . f ^- . B iochem ical R esearch L aboratory . .. McCoy J*. * , *r T:v "; 1803 Building - - `` w - . W, R, Dixon ' ^-.T.5 v. -:, 1 B. B, H older, MD vr E n c l o s t t * ^ e o * r - ? ' i --:.r - * ; v*^ `vr* r*V#r*^*ri -^v^r-s'v->". * , iZ U Z * y + A P I; V  t or r JUCHNSON . iKR24'83 T ran sl. MID. no.3728 ~ / Hautarst Tel.10 so.3 p.126-129 (1939) C lin ica l observations about tha origin of ehloracne. \ Author: Braun, y . Translator: Trans. Chess. Inc. . Bata: - Hay 27, 1966 Requested by: Murray, J io c h a a ., 1701 Bldg. O riginal language: GasBtt~ Translation no.: 66-3-46 , J ' 'r .5 - 1 tl S' 4 / Tl _ i 5973 2 -f 10 ULES POWDER COMPANY ,4 ( 0 C * ' C 3 000034 M . A . `raves - R. C. J. J. Ford - ?.. C. A. I. Conner - R. C A. D. Sidvell - Jacksonville Wilmington, Delavare November 18, 1965 TO: C. L. Dunn - Synthetics Department FROM: E. E. Christofano - Medical Department The following comments will summarize the investigations ve 2,k,5-Tcontemplate on chloracne possibly associated with esters. 1. Badische uses a bioassay technique to check each batch of TCP purchased. Their test consists of feeding each of three rabbits with. 1 gm./kg. of TCP, with a determination of bromsulphalein retention after 5 days. More than 5$ of BSP retention is cause for rejection of the batch. Sensitivity is claimed at 3 ppm. [ fc 2. Dov bioassays by a rabbJLt ear painting technique. The internal surface of one rabbit ear is painted daily with 0 . 1 ml. of a chloroform solution of test material (10 or 50$) of an extract of TCP in chloroform. Unless severe irritation is noted 10 doses are applied. The ears are observed for two additional weeks for the appearance of folliculitis and cyst formation. They have noted fatalities from a single application and liver damage has accompanied the chloracne reactions. Sensitivity is claimed at about 1 ppm. While the Dow testing may be more sensitive, the Badische method is faster and less expensive. In order to evaluate both methods we recommend that Hercules supply material to Badische for their testing and simultaneously begin gathering confirming data in a commercial laboratory. Because of a lack of information on the toxicity of in-process materials, these latter studies should begin only after oral toxicity has been determined. For this first stage the acute oral LD50 studies would cost approximately $3 0 0 . Subsequent acnegen studies on these materials are expected to cost about $6,000, however, an accurate estimate is not possible until Stage 1 is complete. Preparation of the samples and subsequent analysis is estimated to cost $5 0 0 t ^DEFENDANTS t EXHIBIT t HERCULES POWDER COMPANY C. L. Dunn - Synthetics -2 - November iS, 1 9c 5 Dr. Oettel of Badische has agreed to bioassay several spiked samples to permit comparison of results with the gas chromatographic determination of dioxin and with the Dow ear painting procedure. To permit this study we request that 25 g. of each of the following spiked samples be prepared: a. Standard 2,k,5-T acid spiked with 0, 2, 5, 10, 20 ppm dioxin. b. Plant grade 2,k,5-T acid with a known dioxin content. c. Plant grade sodium trichlorophenate spiked with 0, dioxin (based on TCP equivalent). d. Dioxin standard approximately 2 mg. 3, 5, 10 ppm CP Plant grade pnapyl ester of 2,U,5-T acid as 6 lbs/gal. solution in xylene spiked wit^ 0, 2^ 5, 10, 20 ppm dioxin. Approximately 1 0 ^gms. of each sample should be prepared for ship ment to Dr. Oettel and the remainder retained for future confirming tests conducted by Bio-Test. Chemical analysis of the spiked samples would be required after the biological testing demonstrates physiological activity. U a, i -- -- -- ^.Acute oral toxicity data on sodium trichlophenate, 2,^,5-T acid and finished propyl ester formulation should be begun promptly. Approximately '50 gjns. ofeach material should be-sent to Dr. Joseph C. Calandra, Industrial Bio-Test Laboratories, 1810 Frontage Road, Northbrook, Illinois. Dr. Calandra has a supply of dioxin. After these preliminary tests are conducted we will establish a more comprehensive program for the evaluation of hazard during manufacture and in the product as it would be used by the customer. Without these pre liminary data a test program would be unnecessarily expensive. A U / ,~ EEC:w h JL IftC U U S * ,u _____ I) - - 7- ---j r^ .f>-- .1 ) 5975 t i*--'..;. / to RESSA.RCH 3 P 4 3 : m ENT HERCULES INCORPORATED RESEARCH CENTER ' . r . C. L . ~ nn - Syr.. rn-' i'Or i'ir. A . Z . Conner 0000347 k- V/ilmington, Delaware June 15, 1966 MEDICAL DEPARTMENT DIVI SION FOR 3I0ASSAY TESTS In accordance with your letter of November 18, 1965> the samples listed below are being sent to you for transmittal to Dr. Oettel of Badische, for use in the bioassay tests. Approximately 10 grams of each sample (except for the pure dioxin sample) is included. The samples are packed in four shipping containers, and the contents of each are listed on the outside of the container. The individual samples are also clearly identified with an appropriate label. I have- indicated which samples have dioxin added to them, but have no t 1shown the fortification-level on the label. If you wish to provide Dr. Oettel with *this information, it is included in the attached list. It would be useful to point out to Dr. Oettel'that the samples from Group 3 and Group 6 are more easily sampled if heated as indicated. ' \ i JJp/cbb Attachment minti im y . a . DEPOSITION f EXHIBIT lX tfvA -- )2- 5977 Mr. E. E. Chris toino P2--i~"9V*--' 10 *"t ^ jur.e 1 SAMPLES PREPARED FOR 3I0ASSAY TESTS (1) Standard 2,4/5 T Acid With Added Dioxin Sample Designation XI4867-41-1 X14867-41-2 XI4867-41-5 XI4867-41-4 XI4867-41-5 ppm. Dioxin Added O' 2 5 10 20 (2) Plant Grade 2,4,5 T acid with Known Dioxin Sample Designation ppm,. Dioxin X15280-56-8 XI5280-56-9 13 (5) Plant Grade Sodium Trichlorophenate with Added Dioxin Fortification is based on a 35$? NaTCP Content. Sample Designation ppm, Dioxin-*0 X15280-55-1 X15280-55-2 X15280-55-3 X15280-55-4 X15280-55-5 0 1.2 2.4 1 53 .. 07 Heating the above samples to 65C. aids in sampling. T t (4) Dioxin Sample - 2,3,7*8 tetrachlorodibenzo-p-dioxi.n - Designated XI528O-56-IO. (5) 2 Ethyl hexyl, ester of 2,4.5 T with added dioxin (Brush-Rhap LV-4-0) Fortification is based on 44.Iff? 2,4,5 T acid. Sample Designation ppm. Dioxin X15280-56-1 X15280-56-2 XX1I 55 2288 0O-- 55 6 -3 -4 X15280-56-5 0 2 5 10 20 (6 ) Non-saponifiable fraction from wash solvent still. Designated XI5280-56-7 . This sample contains unreacted tetrachlorobenzene and relatives, anisol of trichlorophenol and related phenols, toluene and a small amount of NaTCP. Sidwell believes this is where the dioxin would end up in our process. We are storing this material at the rate of 2-3 drums per day. To melt, heat this sample to 50C. 5978  II 0000004 DOW 75187ii ' . MIDLAND February 15, 1767 : W U llam B , D ixon ' '.Jr.''I * *' Byproduct* Department r.' f> CC1 ' - I f . U C o r b in , P C :>4* *' ... P . P iet ch a r, B P C ' * .i*. **' H* C o w ell, B P C ^ ~ i- W. J, B. B. M cCoy, H o ld e r BPC H P ., 407 . , ... j. v `tsV* > # % . ~ * . * * . * e .t TH O M PSO N C H E M IC A L C O M P A N Y , S T , L O U IS , M ISSO U RI Oc F ebruary 2, 1767 W. J . McCoy Informed me that a Mr, M, S, . . . V 1 :-: * V /'/.V Buckley of had had an Thompson Chemical had accident associated with called Pew indicating that they the manufacture e ftrlc h ln ro - 'v' . V/V< phenol, and that they had torn men with what they thought was ' chloracne, They wondered If w could advise them la regard to . /, medical practice, 1 contacted Dr, Holder, who then called Mr, Buckley and discussed with him medical aspects of the problem, ' <t- .. It is my hade rstaadlng from Dt, Holdr that M r, Buckley'then ''. started talking about the chemistry of this m aterial, and Dr, Holder < # i.W .suggested that he contact me In regard to anything along this lias, On o r about F ebruary 7 M r, Buckley called mo to seo what we knew about chloracne caused by contact with m aterials associated with the manufacture of trichloropbenol from te tra cklorobonxtne, 1. told him that under certain circum stances, which 1 did not describo, the cauitic Insoluble oils could contain considerable amounts of a cry highly toxic substance which we had Identified as 2 ,3 ,7 ,8 tetrachlorodibensodloxin 1 Indicated to him that this m aterial was act only extrem ely toxic systemically, but it was elao an extremely potent cbjoracnogea, lie Indicated that they had been distilling some ' e o ils or ta rs containing anisles when the' operation got out of control, and the m aterial either spilled or splashed onto a hot autoclave, .Ha / V_ '/ *# ! rf.1 -,c- v\ f*Vr"-- v * -A thought that tyo fumes and vapors of difficulty, ' 1 told him 1 did not think anislet but ra th e r by other m ateri 2V"t. V " O - -3 V leV . SJi-/J-Vi . .- V. - ' , . v y.,> .. . . . . . . j. - the the als anisles had caused the acne was caused by the present In the acicales, ir T -***. ;; e*i* a- \ r V ' WUlUra R. DUon 2- February 15, 19&7 OOW75187J "* Apparently Mr. Buckley had asked Or. Holder how te clean up tha . `equipment, etc. and Or. Holder Indicated to him that be meat taka *:* * , entrama precautions, Including tbo oao of rubber auita and glove a and .*m- > .* p iralo ro . Ha comed aurpriaed that wo would recommend uch Severa a a a o ro s. but Xreiterated that ouch precaution a ro h at .. . .Ora believed n*eeaary. Ha than wanted to know if Xthought they <!; could cXaaa up the contaminated equipment with steam . 1 felt at thia point an obligation to tall him Chat team tree tha w orst thing ha could use, imply bacana# it would volatilise tha m aterial causing it to . / . recryataU laa and deposit elsew here. Thia waa not tha way to get rid ... .of it. Ke suggested acrubbing down the equipment with detergent e r . * / 'solvents. ~X indicated that thia coold ha done if be made aura that a ll ' *. neccaaary precaution were taken to prevent contact with the people. .. I auggeated that ha aaaay o r take wipe ample of the equipment to ~ determine the degree of contamination. 2 auggeetad that thia aheuld be done before and after the clean-up. Ke eaked how auch aaaaya ; could be mado. 1 indicated to him that we had developed a method of | .* \ analyaU and alao uaed biological aaaay employing a rabbit e a r. He . -v.i did not aak me for i to him. *' v - -* 1**1 *. *v ' . -* detalla ef cheat p r oc e d u< r.e. t",. ' co 1 did ..*f'-*"r+ti-mm*'* m n .-o*,t. g--ive` ; V . them * *V* - 'm 4 lie then asked where he could find information about chloracne, and Z A.V volunteered to end him coplea of pertinent articles from tha published , ` .H>h*- VV. literatu ree*which .X....h...a..d..... had r-^My coevaraationa with Mr " *dIctaaion of the problem. thia.haa. breei<a'di-o, nc. ** *fj>d f* - Buckley were within the framework of The information on the phyaiologlcal eur .Vih:*i.#...** T-wtir;r activity waa perhapa even lea than waa given to tha other producers of trichloropbenol over a year ago. This waa because it waa quickly . . apparent that Mr. Buckley had little understanding of tto toxicological ' tap ecta of bis problem , liad he aaked for method, e tc ., Xwould have agreed,to tend them to him. However, linca he did not, Xsaw no reason to volunteer. -- * 5~ - I,IP0*mf lM^^> IM-* . . e** " . .0 *' v . ' *** Xdon't know whether this information la of any value to you. but It . will a t le a st, keep you Informed. If you have any question o r eug- . geatlona for further action, piece do not hcaitale to contact mo. ft >(^, Va4dt.f qr n.. *'* * *..* smc . * e *,i . * , * ^i' * _# ' ' m V*W'. :!**' *.>*.tw #i* f-*# '. *H i Oi V j i i ,H n S " ' - - ` - a ^ i O x. if V, It, Rows w * # * t * ; * ^ * V ^ | v < *' ** X ^ * * ,*^#* T T** .............. - ......b ccj^ V , K. Rowe (2) S iw k * Biochem icalR aaaarch X ah o rato ry - *V% t i 7.:4-23-20 * 1203 Building . Chlorcne fe T ... - - *"" T" "**. *--*. vC,'oeTrr*or',a`"p^ o*ndanca r' *!.w-i**.-- f-V *1r*"'V''-'-'..*';: .vr.v /* **`v 5982 INCORPORATED * 0000334 Dr. J . P. Prowl ey - Medical Dept. |Wl!mington, Delaware June 30, 1967 TO: ROM: Mr. H. E. Wilder - Jacksonville J . M . Eagan Dr. Frawley sent to me the attached copy of a report dealing with five cases of cloracne in Colombia which developed from the use of phenoxy herbicides. The inference in these reports is that some producers allowed contaminated 2,4,5-T to enter sales channels in 1963 and 1964. No prior cases of cloracne outside of manufacturing operations have come to-our attention but this report should remind ' all of us that we must be certain to continue to be on the alert for abnormal operating conditions which might contribute to the presence of the chemical which is reputed to be responsible for this type of cloracne. JME/abk Attachment i o h -a s 5983 >3 /3 t> T H E D O W C H E M I C A L . C O M P A N Y i*' :**** Septem ber 26, 1967 C o l o n e l C . C , She6 <SAO> D i r e c t o r a t e o f A i r F o r c e A e r o s p a c e Fue l* K elly A ir Force Base T c u i 7b24t |- *" . Dear C olonel Shcad: Uv h av e been a sk e d t o b id on tht- d o v i p ) , c o n v e r s io n and p c r itio n ( a n , 0 0 0 , 0 0 0 g a l l o n p e r > * a r " O r a n g e " p l a n t a ? l*. < V v c r n ^ r r . t o v u i c h e m i c a l p l a n t a t W# Id em S p r i n g , M i s s o u r i . T h is Is to in f o r t you t h a t we h a w a d v is e d V . C . K r a f t , C o lo n e l, C o r p s o f E n g i n e e r s s n K a r . u * C i t s . M i s s o u r i , t h a t we do n o t u i l e n d t o b i d o n t h e i r s o l i c i t * : ; : "*. rWCAN^-bB-R-WK, I , d a t e d S e p t e m b e r 1967. #\ " U i o b a s i i . r e a s o n f o r o u r c . . * below : due te r it n a l lu s te rs m il lined "flu* t i n e a l l o t t e d ir s u b r : * - ; ~ h id * , Sc>Ur?N-r , 967 to O c t o b e r 1 2 , 1 9 6 7 , i s n . i a --; % ;**r p r e p a r a t i o n cm h i d ? . a project of th is nagnitude. " H i e p r e t e n t a t ?n o t a l c e r r j f i v Q r i m a* **Sr t h a r l > d c n S p r i n g w e n p r e c l u d e d h e t r i ' i i ttu* t i:r v u l t c % ' c U r t ' e p a r a t i o n o f a r e s p o n s i v e b i d i n a n u v v r t- * .t *! i t i t a t i -m . **Uc w o u l d l i k e t o s u a u p * * ' v f u r r . . i * . i > t o r n o t f*r* s e r . t i n ; a b i d a t t h i s tin*, as fo llo w s : 1* T h e W e l d o n S p r i n g s i t e , I h j t v d a s i t i s h% t v c v n t h e U n i v e r s i t y . ( Mi . r i E p e n r x n t a l F a i ^ a n d t h e A ugust Busch R eserve, p re s e n ts too m u h of a h arard to t h e p l a n t l i f e i n in..** a r c u s , i n i u ` f f a n a c c i d e n t a l re le a s e o f p ro d u cts fru o the Orange r i a n t . 2 . I k e r e q u i r e m e n t o f 15 n i n t h s s t a r t up and f u l l y e r a - 3 , T h e l a b o r s i t u a t i o n i n t h e S t , L o u i a r e a w o u l J # r c > c a t . to o many p ro b le m a . 4 , I t w o u l d b e i l m > U i o g H * i h l e t o m ` I vl* t h e p o l l u t i o n problem s in the a re a . ' 5985 C o itftc t C. G. Shcad ^ 01 pi .-.nhef 2i*t t*#h7 S . D i s p o s a l o l v a s t . o n : ! . i : a r r i v e a t a p o s i t i v e s. .. .-n M f o r c o l l e t I*n o j tit. j i j n t . itt t i-w* t o :< i*u- t : ^ a l l o t t e d 0 . The I h l o r - A c n e pi h l a a hv\ u (<'<) w i t h tc* r a n u t a t t u r c *f T r i c h l r p h v n o l ioi: .t be s o l v v d w t t h ' m t ' r s t re c e iv in g p erm issio n i r*ir a ru ropcan C heaical M anufacturer. T h e C h l o r - A c n e p r o b l e m i s o n e o f Human H e a l t h v h i v h D.tv h a s h a d t o co m b at i n t h e d e s i g n o f lt% *~**n p a n t s at i t s Midland o p era tio n s. Several phases of the ra n u fa c tu rin g process for 2,4-D an d 2 , 4 , 3 * T : a v o * w Dra k n o w h o w t h a t wc d o n o t w i s h to lose to our c o a ^ c tite rs. 9 . The p e o p le t h a t a r e now e n g a g e d , the factory Ics^ l. in th e pr*duction of C.O'iO.i'iO , a l l o n s / w a r of Orange f o r th e G overnnent a r e fru arn- l u o p l v ( h a t would have t o b e a s s i g n e d t o t h e W iw* S p r i r g p r o j e c t . " T h e ih*w C h e m i c a l Compart. m n u * t o h a v . a h i c h d e c r e e o f i n t e r e s t i n t h e p r o j e c t a n d r n , u 4 1 > i h . a *.i/. t o r y *r b i d d e r s l i s t t o r a n ) f o l l o w i n g < o l ic H a l to n i - ****c t i.n w i t h t h e .apply raw or lnh*m(JIu< m a te ria ls . *Vhvn t h e TVH RFP i s r e l e a * - '1 - no;.* : * h e stx t* b i d d e r s l i s t , and f e e l t h a t we W i l l be m a h e t t . . * ; t * e t . * h i d *n t h t . p r o j e c t * MIf voi h a v e a n y q u e s t i o n o n e Ik*. ' ** b o s t o n w i t h r e g a r d t . : u - . DACA-i 1-68-R -O OO1. , p K a - e %**. * t n C la r.ir.ilv oivir O irector of Ovcrtunrnt A ffairs i' 5986 /V : /V - 5987 .T 0000343 October 16, 1963 Kr. V. K. Rowe Biochemical Research Laboratory The Dow Chemical Company 1701 Euilding Midland, Michigan Dear Mr. Rove: As you may recall, Hercules has been attempting to develop a bioassay technique to simplify detection of dioxin in herbicide formulations. Based principally upon work initiated by Dr. Oettel at BAS?, the tests measured liver function changes which result from feeding rabbits with i herbicide. We feel such a technique offers several advantages over rabbit *' ear painting tests. \ Unfortunately, ve have been unsuccessful. But to state it so bluntly is really unfair. First, ye attempted to determine the sensitivity of liver change by feeding test materials with knc*.m additions of dioxin. The toxicity of test materials prevented use of liver function change as a bioassay for dioxin even at 10 ppm. We then dosed animals vith 0.01 U 0.1 ng/kg of dioxin suspended in c o m oil. Carbon tetrachloride vas administered as a positive control at 100, 250 and 500 mg/kg. Clinical determinations including SCOT, SG?T and ESP retention were conducted prior to administration and every two to three days thereafter for at least lU days. Significant elevations in one or more of the clinical determinations vere found in scee but not all dioxin animals. The changes vere not veil correlated vith dose nor vere they consistent vith regard to onset or duration. Animals on the positive control test responded as would be expected with significant blood chemistry changes only two days after dosing vith 100 mg/kg. Although positive results vere obtained with dioxin, the usefulness of this technique as a bioassay for the presence of dioxin is questionable. At the Toxicology Roundtable I mentioned these results to Ken Olsen vfao suggested that you might comment upon them. But the other reason for writing is to solicit your opinion on the usefulness of presenting our data at the Society of Toxicology meeting next spring. Certainly such a presentation vould raise questions as to the reason for conducting an unsuccessful study. 5988 Mr. V. K. Rove - 2 - October 1 6 , 1968 On the other hand, someone night be spared useless effort if such negative information vcre offered to the scientific corununity. I'd like to have your consents before ve proceed. Sincerely, EEC :rba Bail E. Christofano Industrial Hygienist t k I 5989 Jr i i 1 5990 /5 Ilo 5993 I/