Document zdz3dnvwJVqqJao00EXrdpr26

51 5380 ottonserd when crom-feriiUzed. No oihnr effects of treatment were noted, other on the treated plants or in their sbrid progeny. 'Hie application of .ther pantothenic acid or o-ribose par>>ally reversed the .d ie of die c. tated acids. .susceptibility to dalapon may be tinder partial genetic control. Scott ir|xjrtrd father widely differing results in his gatnrtocide experiments with several varieties of cotton. Funderburk and Davis (/-A reported that hybrid varieties of corn dilfered in their susceptibility to dalapon, and Uuchholz at Wisconsin as well as Behrens (J) studied a number of inbrrd lines of corn which dilTcr widely in tolerance to dalapon. Although higher plant systems seem to have little effect on the various chlorinated aliphatic acids, these acids have varied and profound effects on higher plants. Obviously a number of plant processes are affected, and it is likely that more than a single pathway is inhibited. The evidence points to multiple pathways and to more than one ste of action. Utrahm Gtnd 0 ) Barrons, K. C., U. S. Patent 2,642,354 (1951). "*) Ibid^ 2^07,530 (1957). Barrons, K. C , Hummer, R. W., Her. Otttn. 6, No. 6, 46 (1951). (4) Behrens, R., "Nummary of 1962 weed routroi trials in field crops," Minn. Agr. Expc Sta., 1962. (5) Blackman, G. E., Tcmpieman, W. G., Holliday, D. J., Ann. Bra. Plant Phuiat. 2, 199 (1951). (6) Blanchard, F. A., Wrtds 3, 274 (1954). (7) BiNiupict. F.. W., U. S. Patent 2,393,0(6 (1944). (8) Corns, W. G., Can. Bat. 34, 154 (1956) . (9) Crafts, A. S., Ann. Bn. Plant Physiol. 4, 253 (1953). (10) Fawcett, C. H., Wain, R. L., Wighiman, F., Xalurt 17*. 972 (1958). (11) Foy, C. L., Ph. D. thesis, University of California, 1958. (12) Foy, C. L., Plant Pkjsial. 36, 688 (1961). (13) Foy, C. L., Wrrds 10, 97 (1962). (14) Funderburk, H. H., Jr., Davis, D. E.. Ibid., 8,6 (1960). (15) Hilton, J. L.. Jansen. L. L., Genincr. W. A., Plant physiol. 33, 43 (1958). (16) Hir*ch, P., Alexander, M ., Can. J . Microbiol. 6,241 (1960). (17) Holstun, J. T ., Loomis, W. E., Wrtds 4, 205 (1956). (18) Ingle, M., Rogers, B. J., bid., 9, 264 (1961). (19) Jensen, H. L., Can. J . Microbiol. 3, 151 (1957). (20) Jensen, H. L., Main* ISO, 1416 (1957) . (21) Juniper, B. R ., Xtw Phylologist 58, 1 (1959). End of Symposium K. ANIMAL METABOLISM OF HERBICIDAS The Fate of 2,4-Dichlorophenoxyacetic Acid in Sheep (22) Juniper, R. R., Bradley, D. IL, UlbostnaUrt Its. 2, 16 (1958). (23) Kutschinski, A. H., Down Earth 10, No. 3. 14 (1954). (24) Kutschinski, A. H., J. A cs. Food Ciii'.m. 9, 365 (1961). (25) Lousialoc A. J., Ferrer, R., Agraa. J. 42, 323 (1950). (26) Magee, L. A., Colmer, A. R., Cm. J. Microbial. 5, 255 (1959). (27) Miller, S. R., Corns, W. G ., ibid., 35, 5 (1957). (28) Norman, A. G., Minarik, C. E ., Wrintraub, R. L., Am. Bn. Plant Physiol. 1, 141 (1950). (29) Rrdcmann, C. T., Hamaker, J. W., H ie Dow Chemical Co., Internal Report, 1959. (30) Kcdcmann, C. Tn Mriklc, R. W., . Arch. Biachm. Biohhys. 59, 106 (1955). (31) Scott, R. A., Jr., Plant Physiol. 36, 529 (1961). (32) Thicgs, B. U A rm Earth 11, N a 2,2(1955). (33) Ibid., 18, No. 2, 7 (1962). (34) Tiblteus, T. W., Holm, L. G., IIW i 3, 146 (1954). (35) Toornmaft, B. Vn U. & Patent 2,880J)82 (1959). (36) Wilkiraoo, R. E^ Ph. D. thesis. University of California, 1956. (37) Woodford, E. X., Holly, 1C, McCready, G. C , Asas. B n. Plant Pbpiol. 9, 311 (1958). Rtttm d far m ini Angast 1, 1963. Amptrd Naatmbrr 22, 1963. Dimsim at Agriadtorol and Food Oamssry. 144th Muting, ACS, Las Angrlts, Cdif^ April 1963. DONALO L CLARK, JOEL E. YOUNG, L L YOUNGER, L M. HUNT, ltd J. K. McLARAN Animal DIsoom and Parasita Resaorch Division, Agricultural Rasoareh Sarvicas, U. S; Deportment of Agricultura, KarrviUa, Taxas W ( , I f ' / ' The ir f r c n ix v u i of 2,4<iichloro- can tolerate rather large quantities of phenoxyacetic acid (2,4-D)' and 2.4- D salts and esters for extended related compounds as plant growthperiods of time (7). However, whether regulators has been recognized tor a 2.4- D is metabolized, stored, or excreted number of yean (/, J). The herbkidal unchanged by the sheep has not been activity of 2,4-D has been attributed to established. its hormonelike activity rather than to direct dehydration or necrosis of plant Apparatus tissues (3). Although previous studies have shown that phenoxyacetic add is excreted unchanged by man and dogs (6, ) and almost quantitatively in urine by rats and rabbits within 24 t (2), no work has been done on u , metabolism of 2,4-D or related The instrument used for carbon-14 quantitation was a thin-window (mica) Gctger-Mfiller tube enclosed in a Tracerlab SC-59S Shielded Manual Sample Changer and attached to a Traceriab " Versamatic 11" Scaler. eor funds in ruminants. Previous ok. auons have shown that sheep ffoogmts Chromoiropic acid (4,5-dihydroxy- 2,7-naphthalcnedisulfonic acid, dira- > Present Address: Wadlcy Remareh In- dium salt, dihydrate) : 0.05% in concen aittue, Dallas, Texas. - trated sulfuric acid. 2-Pheaaxyethanol-silver nitrate re agent: 1.7 grams of silver nitrate in 5 ml. of water. Add 20 mL of 2-pbeoexyethanol and dilute to 200 mL with acetone. If the mlutioo darkens, 1 to 5 drops of 30% hydrogen peroxide may be added. Electrophoresis buffer: dimolve 5.4 grams of KHtPO and 0.93 gram of NaH,PO in 1 liter of water and adjust pH u>6.0 with NasPO* Administration of 2,4-D and Sampling P rocedan. A gelatin capsule containing 539.6 ac. in 106.3 mg. of 2.4-dkhlorophenoxyacetic acid-2-CM (Traceriab, Inc) in 95% ethanol was administered orally to a yearling ewe (weight 264 kg.). The dam of 4.0 tr q O i' DOO1 QQ QOg ^ " 5 linn Approxim ately 9 6 % of an orally administered dose of 2,4-D-CH to a sheep was excreted unchanged in the urine within 7 2 hours. Slightly less than V .4 % of the administered Tub. radioactivity was excreted in the feces over the same period. The nature of the urinary j C u w as established by paper chromatography and electrophoresis. In no case w as there | a test for chloride, carbon* 14, or phenoxyacetic a d d groups at any Rj or migration other than that of standard 2,4*D. From the results, it was concluded that 2,4*0 is excreted s unchanged by the sheep. Very little residual radioactivity was found in edible tissue. , Thereafter, aamplcs were taken at Whatmar Whaunai N*CC Glaw fib various intervals over 24 houn* duration. Levels of carbon-14 in ethanol ex tracts of these blood samples arc given in Figure 1. Activity rose rapidly during the first half-hour, reached a peak Whaunai Whatmar KatCC Glmrfibr ; at l 1/ , hours, and diminished rapidly thereafter. By 24 hours post-treatment, Whatmar the blood radioactivity had diminished to essentially background levels. Whatmar NatCO Claw fibe Excretion of 2,4-D-C1*. About 15% of the original dose of 2.4-D-C* was The i oceanic c found in die urine collected during the , first valu* . first 1*/ hours post-treatment. At phenoxya 8*/i hours post-treatment, 50% had S p o t. been recovered in the urine, and by the end of 28 hours, over 90% of the 2,4-D -C '4 mg. of 2,4-D per kg. of Keep weight was calculated as the approximate minimum daily dose a sheep would ingest from' grazing on a pasture treated with the herbicide. Continuous samples of urine were taken by means of an in-dwelling catheter which fed into a collection bottle attached to the side of the sheep. Fecal samples were obtained by a plastic collecting bag taped to the animaL Blood samples were withdrawn from the jugular vein at timed intervals following administration of the doae. The filtrate was made slightly alkaline, concentrated, and assayed. Recovery of a sample spiked with 2,4-D-C*4 and treated in this manner was 99.3%. Urinz. Samples of whole urine were diluted 1:100 with 95% ethanol. Onemilliliter aliquots were evaporated to drynea and aaaycd for C" without further treatment since it was determined that little 1ms due to seif-abrorptioa occurred. Blooo. High recoveries (above 90%) were obtained from blood by extraction dose had been excreted. By 60 hours, the total recovery exceeded 95% ;' thereafter, urine radioactivity was neg ligible. The total urinary excretion of Chramau oxyacetic modificat sulfuric a 2.4-D^T** by 70 houn was 95.8%. Extraction of total feces collected over fiber filt< essential the 70-hour period following administra tion of 2,4-D-C1* yielded approximately cause of t add. Ti 1-4% ofthe dose. Identification of Excreted 2,4-D-C1* dipped r inotropic by P ip er Chrom atography and Paper Electrophoresis. Urine from the sheep treated with 2,4-D-C1* was compared chromatographically and electro- and obre spots or b Electro 2,4-D wz phoreticaJiy with pure 2,4-D-C**. The Paper El urine sample was a pod representing the Durham e total 7(W>our collection following ad ministration tsf the herbicide. Com parisons of Rt values and electrophoretic migrations frisai pure 7,4-D-C** samples were made with pooled urine tarnpin glass fibc usual dec buffer w. Na'H tPO , gration v; and with pooled urine to which pure : current dz 2.4-D-C** war added. The data ob tained from the various chromatographie The id< system* and electrophoresis are given in On the fourth day poet-creaunent, w ith hot 70% ethanol (adjusted to Tables I and II. the animal was anesthetized and ex* sanguinaied. At necropsy, sample of various tissues and organs were removed, weighed, and frozen for asuy of carbon* 14. pH 1 with H Q ) followed by chilling and filtration. Tissue. The extraction method used was unsatisfactory for carbon-14 assay where infinitely thin plates are required. The papers used for ascending chro matography included Whatman No. 1, washed; Whatman No. 1, washed and impregnated with 2% sodium carbonate; and glass fiber strips (Hurlburt Paper C o Sample Preparation. Fanes. To 20 . grams uf fresh feces was added 120 ml.'nf solvent which consisted of a 50:50 mixture of 95% ethanol and diethyl ether (v./v.) plus 1% by volume of However, allowing for 50% absorption of carbon-14 by the residues in the planchets, a confidence level of 0.05 p.pjn . was established. Company No. 934-A4). For decor phoresis, Beckman Electrophoresis Paper No. 300-028 and glass fiber strips were used. C** was detected by radioautog- QO CT3 0 in (r concentrated H Q . The mixture was 'irred and allowed to stand 30 minutes ftmmdH raphy. The chromatograms were covered with a thin sheet of S an a Wrap c room temperature. The slurry was Appearance of 2,4*D-CH in Blood (Dow Chemical Co.) and then placed then power-filtered through Whatman Following Oral Administration. Sam in contact with nonscreen x-ray film for ce No. 42 filler paper and the residue washed twice with 50 mL of solvent and once with 50 ml. of chloroform. ples of blood were withdrawn {ram the sheep at 15-minute intervals for 2 hours following administration of 2,4-D-C*4. approximately 1 week. For identifica tion of organic chloto compounds, the method of Mitchell (S) was employed. V DoW 0 5 0 8 7 7 7 V| r * Y .. r ,l < U- . .1( U.S.S Table L N p r Chromatography of 2, 4-Oichlorophonoxyacotic Acid .. Stmlmmmrf htmm Whatman No. 1 WhatmanNo. 1and 2% Na.CO, Clam fiber Ueko/*Mmm AnimtiwRfi wwBm tmulM: etelk (40>0.St 40) mitwmfm ./*./*. tthmmol, 95% 4*wic mid, t% 2.4,-D-C" 0.86,0.85 0.31,0.30 0.63,0.63 0.84,0.83 0.65,0.64 0.41,0.81 0.83,0.83 0.76,0.78 0.90,0.68 Whatman No. 1 Whatman No. 1 and 2Co NitC.O, d au Jibrr Urine 0.79,0.80 0.24,0.25 0 .6 7 ,0 .M 0.83,0.83 0.80,0.80 0.62,0.60 0.90, 0.82,0.83 0.91, Urine and 2,4-D-C14 Whatman No. 1 Whatman No. 1 and I'Tc ha-COi Class fiber 0.82,0.82 0.68.0.64 0.83,0.83 0.24,0.25 0.75,0.75 0.62,0.58 0.90, 0.84,0.83 0.88, The tint Rt value with the papen is by radioaucography for C", and the second for | oceanic rhloridr with 2-phrmixyethanol-silver nitrate-acetone. With the glass fiber, the I first value is by radioautography and cite second by chromotroptc-iulfuric acid for the r pbrnoxyacrtic acid croup. * Spot near R 1.0 masked by impurities. Chromatographic identification of phen- the following criteria* -a single band or nyacetic acid was accomplished by a spot on each chromatogram as evalu modification of the chromotropic a d d - ated by mdioautography; coincidence sulfuric add reagent. The use of glass of a positive test for organic chloride ''her filter strips instead of paper is with the band or spot obtained by coential when using this technique be* radioautography; coincidence of a posi cause of the corrorive nature of w Ifuric tive ten for pheimxyacetic add with ^ . The glass strip chromatogram is the band or spot obtained by radio 4 sipped momentarily into the chro autography; a single band or spot in notropic acid solution at 140* to 150* C. electrophoretic patterns as evaluated r and observed immediately* for violet by radioautography, organic chloride, 4 ipots or bands indicative of2,4-D. and phenoxyacctic add determinations; 3 Electrophoresis of samples containing R / values and electrofshoretic migrations L4-D was carried out on a Spines of the urine CM coincident with thorn e Piper Electrophoresis apparatus with obtained from 2,4-D-CM alone and/or * Durham cells. The papers used included urine CM to which pure 2,4-D-CMhad glass fiber filter strips as well as the been added; and in no instance was usual electrophoresis paper strips. The there more than one spot identified by buffer was a solution of KH,PO/ either chromatography or electropho NaHjPO, pH 6.0. The time of mi* resis. By these criteria, the radioactive (ration varied from. 5 to 6 hours a t a material excreted in the .urine by the current time of 9.0 ma. sheep was identified as 2,4-D-C*4. The identity of 2,4-dichlMophcacny- Tissue Residues. Although the acctic ad d in the urine was judged by radiometric method for tissue carbon-14 Table II. Paper Electrophoresis of 2,4-0 chlorophenoxyacet< Acid Migrwlm*. Strip Cm.* 2 ,4 -D O Paper --3.8, --4. Ola --2.0 to 4-1.5 Urine Paper --4 .1 ,--4 .1 _ Class --1.Oto +2.0 Urine and 2,4-D- Paper --3.6, --3.5 C** Clam -2 .0 to +2.0 * For the paprr strips, the first value was olxained by ratlkiaulugraphy and the second by test for organic chloride with 2-phenoxyethanoi-tilver nitrate-acetone. For the glam strips, although the pattern is a smear, in all casta the same pattern was found by both the radioautography and by chromotnopsc-sulfuric acid detection methods left much to be desired, all the edible tissues assayed contained less than 0.05 p.pjn., and in most cases was fa r' below this level. Exceptions to this were the thyroid and the urinary bladder, which indicated 0.56 and 0.50 p.pja., respectively. No attempt was made to identify the nature of the residual rad io activity. Uorafuro O t (1) Hammer, C. L., Tukey, H . B-, Scirett 100,154 (1944). (2) Levcv, S., Lewis, H* B., J . Biel. Gfina. 10,213(1947). (3) McNew, C. L., Hoffman, O. L , /we Siete J . Sd. 24, 189 (1950). (4) M anh, P. C , Mitchell, J. W-, Boten. Gee. 106, 224 (1945). (5) Mitchell, L. C., J . Asm . Ojie. Age. Chemists 41,781 (1958). (6) Nendd, M ., Giacosa, P-, Nsppr- Seders Z. 4,337 (1880). (7) Xadcicff, R. D., Bushland, R. C., The Nature and Fate of Chemicals Applied to Soils, Piano and Animals, Symposium, ARS 20-9, Sept. I960, United Sutes Department of Agricul ture, p. 146. (8) Thierfelder, 1L, Schempp, IL, Ank. Get. Pkysiel. 167,280 (19(7). Batumi fee rteiem Jdy 36, 190. Amtpui Aegest 14, 1963. I i i * I I I \ 5383 n im io s 5n DOW 500343 9*i<iuiiuai C I O - T E S T Jlaii&uU&u&i, 9nc. 1810 F R O N T A G E R OAD NORTHBROOK, ILLINOIS Talaphona CRastwood 2*3030 REPORT TO THE NATIONAL AGRICULTURAL CHEMICALS ASSOCIATION REPEATED DERMAL TOXICITY STUDIES ON THREE FORMULATIONS OF 2 ,4 DICHLOROPHENOXYACETIC ACID r \ n n n c r* DOW 500344 'tuhLbUal B I O - T E S T lao*aive., Une. REPORT TO THE NATIONAL AGRICULTURAL CHEMICALS ASSOCIATION REPEATED DERMAL TOXICITY STUDIES ON THREE FORMULATIONS O F 2 .4 DICHLOROPKENOXYACETIC ACID I. Introduction and Outline of Investigation At the request of The National A gricultural C hem icals A ssociation (NACA), a rep eated d erm al toxicity study, using albino rab b its as te s t a n im a ls, w as conducted on the following th ree co m m ercially available form ulations of 2 ,4 dichlorophenoxyacetic acid (2,4-D ): 2 .4 - D D im ethylam ine Salt* 2 .4 - D Isooctyl E ster** 2 .4 - D Butyl E ster*** The p ro ced u re em ployed in the study was that supplied by NACA. * W eed-Rhap H erbicide A-4; H ercules Pow der Company, Inc. , W ilm ington, D elaw are ** M onsanto 2 ,4 -D Low V olatile E s te r Weed K iller; M onsanto Chem ical Company, St. Louis, M issouri *** E stro n 76 BE W eed K ille r; The Dow C hem ical C om pany, Mid land, M ichigan 5386 0002502 DOW 500345 '9ndu<iiual 3 I O * T E S T Jia&o'iaia'uei, 9*tc. 2 Skin application of the te st m a te ria ls, p rep a red as dilutions in w ater and/or oil, w ere made seven hours per day, five days per week for three weeks according to the outline presented in Table I. F ifteen m illiliter volum es of the appropriately p rep ared te st dilutions w ere used for the skin applications. 0002503 TABLE I R epeated D erm al T oxicity Study - Albino R abbits i Outline of E xperim ent t; w O m m Group Number Number of Rabbits Male F em ale Condition of ` Skin Test M aterial and C oncentration Employed (%. w /v ) P er Cent 2, 4-D Acid Equivalent C -I ' 44 44 C-II 4 4 44 Intact A braded Intact A braded w ater control w ater control oil control oil control - -- - T-I 2 2 Intact 1.53 dim ethylam ine salt in w ater 0.626 22 A braded 1.53 dim ethylam ine salt in w ater 0. 626 T-II 2 2 22 Intact A braded 7.65 dim ethylam ine salt in w ater 7.65 dim ethylam ine salt in w ater 3.13 3. 13 T-III 2 2 2 2 Intact A braded 1.36 isooctyl e ste r in w ater 1.36 isooctyl e s te r in w ater 0.626 0. 626 0002S T-IV 2 2 22 T-V u\ CO O 00 22 22 Intact A braded Intact A braded 6 .8 isooctyl e ste r in w ater 6 .8 isooctyl e s te r in w ater 1 .0 b u ty l e s te r in w ater 1.0 butyl e ste r in w ater 3.13 3. 13 0.626 0.626 Ul 9fc00S m o a TABLE I Continued R epeated D erm al Toxicity Study - A lbino R abbits __i________ G roup Number Number of Rabbits Male Fem ale Outline of E xperim ent Condition of Skin T est M aterial and C oncentration Employed (%, w / v ) T -V I 2 2 2 2 Intact A braded 5 .0 butyl e ste r in w ater 5.0 butyl ester in w ater T -V II 2 2 2 2 Intact A braded 1.36 isooctyl e s te r in oil 1.36 isooctyl e s te r in oil T -V III 2 2 2 2 Intact A braded 6. 8 isooctyl e s te r in oil 6. 8 isooctyl e s te r in oil T-IX 2 2 22 Intact A braded 1.0 butyl e ste r in oil 1.0 butyl e ste r in oil T-X 2 2 22 Intact A braded 5 .0 butyl e ste r in oil 5 .0 butyl e ste r in oil P er Cent 2, 4-D A cid Equivalent 3. 13 . 3. 13 0.626 0.626 3.13 3.13 0.626 0.626 3.13 3. 13 5389 * L P-429, 433 Furnace Oil #2; Texaco, I n c ., Lockport, Illinois P00 MOQ DOW 500348 ' SttdujJ/Ual 8 I O * T E S T Jlalxyud&u&i, )tic. 5 The rabbits in all groups w ere housed individually over the period of investigation and observations w ere m ade with respect to body weight effects, incidence of m o rtality and behavioral and local skin re a c tio n s. In addition, hem atologic studies and clinical blood c h e m istry determ inations w ere conducted. At the end of the test period, final m ortality tabulations w ere m ade and all surviving rabbits from each te st and control group w ere sa c ri ficed for pathologic studies. G ross autopsies w ere perform ed and a com plete set of representative tissu es and organs was taken from each rabbit and preserv ed in form alin. M icroscopic exam inations of selected tissu e s and organs w ere m ade to a sc e rta in the p resen ce o r absence of histopathologic change as a resu lt of derm al applications of the te st m aterial. A lso, a t the tim e of gross autopsy, h e a rt, liv e r, kidneys, spleen and testes w ere weighed and organ-to-body weight ratios calculated. 5390 O O O i L Q If ' l)tidu4/Ual 3 I O T E S T JlalwuiitvU&i, 9nc. 6 DOW 500349 II. Investigational P ro ced u re A . Experim ental Anim als The anim als em ployed in the repeated derm al toxicity study w ere adult, New Zealand stra in albino rab b its. The body w eights of the test anim als w ere in the tw o -to -th ree kilogram range. A total of 112 adult ra b b its w ere se le c te d fro m a la rg e r population, a fte r exam ination of each anim al for general physical w ell-being. Each anim al selected for the experim ent was housed individually in a w irebottom ed stainless steel rabbit cage for the duration of the te st. A ll rabbits w ere fed the standard laboratory rabbit ration* plus w ater ad libitum . B. .Method of A pplication In preparation for the te st, the back of each rabbit was shaved using electric clip p ers. The exposure site thus p rep ared on each anim al constituted approxim ately ten per cent of the total body surface a re a . The anim als w ere then retu rn ed to their stock cages and 24 h o u rs w ere allow ed to elap se b efo re the f ir s t d e rm a l a p p lic a tions w ere m ade. This p erio d p erm itted the skin to rec o v er from the slight disturbance of the stratu m corneum caused by the clipping p ro cedure and also perm itted the healing of any m icroscopic abrasions possibly produced during the shaving procedure. * Rockland R abbit Ration, Teklad, In c., Monmouth, Illinois 0002G HttduiifiiaL B I O - T E S T Jta&o'uxto'tiei., S*ic. 7 DOW 500350 At the end of the 24-hour reco v ery period, ju st p rio r to the firs t application of the te s t m a te ria l, the shaved skin of two m ales and two fem ales in each te s t group (four m ales and four fem ales in each control group) w ere abraded by making a se rie s of p arallel epiderm al incisions, ev ery two o r th ree cen tim e te rs longitudinally, over the a re a of exposure. T hese incisions w ere m ade sufficiently deep to pen etrate the stratum corneum , but not to disturb the derm a. The skin of the rem aining anim als in each group was left intact. Dosing w as accom plished in the following m anner: an 8-ply gauze patch (4" x 3") was affixed over the application site of each a n i m al. Fifteen m illilite rs of the respective te st dilution w as placed on this gauze patch and im m ediately covered with a piece of plastic sheeting (6" x 4"). The p lastic sheeting was held in place by thin strip s of adhesive tape. Finally, the entire trunk of the anim al w as covered by a sleeve of gauze elastic stockinette which helped to hold both gauze patch and p lastic sheeting in p lace. The te st m ateria l w as allow ed to contact the skin for a period of seven hours. At the end of the contact period the coverings w ere rem oved and all residual test m aterial was washed from the skin w ith soap and w ater. The anim als in the control groups w ere trea te d in the sam e m anner as those in the te st groups with the exception that either tap 5392 w ater o r oil w as applied to the skin. 0002638 ' 9*uhnibual 3 I O * T E S T Jialto'iaitvu&i., 9*tc. 8 DOW500351 The above schedule was followed 5 days per week for three w eeks o r a to ta l of 15 a p p lic a tio n s. C. Body W eight Effects Each anim al used in the study was weighed twice during the week p rio r to the inception of the experim ent. T h ereafter, the anim als w ere weighed three tim es during each week i .e ., Monday, W ednesday and F rid ay , the la s t weighing being im m ediately p rio r to sa c rific e and autopsy. D. M ortality and Reactions Checks for m ortality and abnorm al behavioral reactions w ere made daily during the three-w eek test period. Special em phasis was placed.on.observations for local skin reactio n s in o rd er to determ ine the degree of skin irrita tio n produced by the te s t m aterial. E. H em atologic and C linical Blood C hem istry Studies Blood studies including determ inations of hemoglobin concen tratio n , hem atocrit value, erythrocyte count and both total and dif feren tial leukocyte counts w ere made at the beginning of the study and a t the end of the three-w eek te st period. These determ inations w ere conducted upon each individual anim al. C linical blood chem istry studies including determ inations of u r e a p itro g e n c o n c e n tra tio n (BUN) and a lk a lin e p h o sp h atase a c tiv ity (SAP) w ere conducted on each an im al a t the end of the th re e -w e e k te s t __ 5393 period. 0002639 Dttdu4&Ual B I O - T E S T Xal/osiaivUei., Due. F. . G ross and M icroscopic Pathologic Studies A rrangem ents w ere made to subject any anim al which m ight die during the te s t to a g ro ss autopsy. A lso, in those instances w here UT CO ur to post-m ortem changes w ere not advanced, sections of representative tis s u e s and organs w ere scheduled to be taken for histopathologic study. At the conclusion of the investigational period, a ll surviving rabbits in each group w ere sacrificed and subjected to g ro ss path ologic exam ination. At this tim e, the h e art, liv er, kidneys, spleen and testes w ere rem oved, trim m ed and weighed. The organ weights w ere then tabulated and ex pressed as the percentage of the total body w eig h t. A lso a t the tim e of g ro s s path o lo g ic e x am in a tio n , the follow ing tissu e s and organs w ere fixed in form alin and p rep ared for histological exam ination: brain, spinal cord, peripheral nerves (sciatic and fem oral), thyroid, ad ren als, gonads, sk eletal m uscle (thigh) h e a rt, liv e r, spleen, pancreas, kidney, u rin ary bladder and m ediastinal and m esenteric lymph nodes. A ll sections w ere stained with H em atoxalin and E osin. In addition, sections of the brain, spinal cord and p erip h eral nerves w ere p rep ared and stained with L uxol-Fa3t Blue to evaluate possible m yelin sheath dam age. * 5394 * M a rg o lis, G e o rg e, M. D. , P ic k e t, John P h illip , H. T . ; New A p p lic a tions of the Luxol F a st Blue Myelin Stain; L ab. In v e st., 5, pp. 459- 474; 1956. ~ 0002700 DOW 500353 9*ui*i4&UaL B I O * T E S T JiaJvyiat& uei, Sttc. 10 III. R esults A . M ortality The m ortality data are presented separately for each test m a te ria l in T ables II, III and IV. A sum m ary of the m o rtality data for all m aterials is presen ted in Table V. Table VI p re s e n ts a sum m ary of individual m o rta lity data with re sp e c t to day of death and num ber of applications receiv ed . 5395 frseoosMoa jn d u ib a l S I O * T E S ? Jlalt&ia&yUeA., 9*tc. 11 Group CrI T -I T -II TABLE II TEST MATERIAL: D im ethylam ine Salt of 2, 4-D R epeated D erm al Toxicity Study - Albino Rabbits M ortality Data M ortality* Intact Skin M ale Fem ale A braded Skin M ale Fem ale 0/4 0/4 1/4 1/4 0/2 0/2 0/2 0/2 0/2 0/2 0/2 1/2 * Number D ead/N um ber T ested 5396 0002702 DOW 500355 .DticLu&al 3 I O - T E S T Jaiotiai&ed., *tc. 12 Group C-I C -II T-III T-IV T -V II T -VIII TABLE III TEST MATERIAL: Isooctyl E ster of 2, 4-D R epeated D erm al T oxicity Study - Albino R abbits - M ortality Data M ortality# Intact Skin M ale Fem ale A braded Skin M ale Fem ale 0/4 0/4 1/4 1/4 1/4 0/4 0/4 0/4 0/2 0/2 1/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 1/2 1/2 0/2 0/2 1/2 0/2 * Number Dead/Num ber T ested 5397 0002703 500356 D uduihial 3 I O * T E S 7 Jlalto'iaiosii&i, 9*tc. Group C -I C -II T-V T -V I T-IX T-X T A B L E IV 13 o O TEST MATERIAL: Butyl E ste r of 2, 4-D R epeated D erm al T oxicity Study - Albino Rabbits M ortality Data M ortality* Intact Skin Male Fem ale A braded Skin M ale Fem ale 0/4 0/4 1/4 1/4 1/4 0/4 0/4 0/4 0/2 0/2 0/2 0/2 0/2 0/2 1/2 0/2 1/2 0/2 0/2 1/2 0/2 1/2 0/2 0/2 * Number D ead/N um ber Tested 5398 0002704 9ndu6bual B 1O - T 5 S T JlaJto'iat&uei., !)*tc. 500357 TABLE V R epeated D erm al Toxicity Study - Albino Rabbits Sum m ary of M ortality Data M ortality# Number Dcad/Num ber T ested G roup___________________ M ale____________ F e m a le ___________ T otal C -I 1/8 1/8 2/16 C -II 1/8 0/8 1/16 T-I 0/4 0/4 0/8 T -n 0/4 1/4 1/8 T -III 1/4 0/4 1/8 T-IV 0/4 0/4 0/8 T-V - 0/4 0/4 0/8 T -V I 1/4 0/4 1/8 T -V II 1/4 1/4 2/8 T -VIII 1/4 0 /4 1/8 T-IX 1/4 1/4 2/8 T-X 0/4 . 1/4 1/8 o O * Figures rep resen t total num ber of anim als employed, i. e. intact and abraded skin com bined. K qQG* 0002705 9*uLtibual B I O - T E S T laMonaMyu&i, Due, 15 O O Group C-I C-II T -II T-III T -V I T -VII T -VIII T -IX T-X TABLE VI R epeated D erm al Toxicity Study - Albino Rabbits Sum m ary of Individual M ortality Data cn o o CO cn 00 Anim al Number and Sex Day of Death Number of Applications Received 7 -M ale 11 -F e m a le 19 13 15 10 20 -M ale 22 15 46-Fem ale 20 15 51-M ale 18 14 75-M ale 8 6 83-M ale 87-Fem ale 10 12 8 10 91 -M ale 7 5 97-M ale 103 -F e m a le 9 9 7 7 109-Fem ale 21 15 0002706 D w Lubual S I O - T E S T laJw akvu& i, Due. 16 B. Reactions 1. B ehavioral R eactions oo 3 500359 No significant untow ard b eh av io ral reactio n s w ere noted among rabbits receiving derm al applications of the respective te st m aterials in the form of aqueous solutions. Behavior of these anim als was the sam e as that of their corresponding w ater controls. H yperirritability becam e evident after the th ird or fourth day of testing among all anim als (test and control) receiving derm al applications of oil or oil dilutions of the respective test m ateria ls. These anim als cried upon application of the te s t m aterial after the fourth or fifth dosing. These reactions p e rsisted throughout the r e m ainder of the test period. 2. Local Skin Reactions L ocal skin reactions w ere noted after the fifth or sixth application among all anim als receiving the respective te st m aterials as aqueous dilutions. However, these reactions w ere identical to those seen in the w a ter co n tro l an im als and w ere c h a ra c te riz e d by m ild to m oderate erythem a, w rinkling and drying of the skin. R abbits receiving oil dilutions of the respective te st m a te ria ls as w ell as the oil control anim als exhibited the sam e type of local skin reactions after approxim ately three applications. The r e actions w ere characterized by severe erythem a, m oderate edem a, 5401 000270 U ndudhial 5 I O - 7 E S 7 JlaloAattvuei, 9*tc. 17 drying, cracking and bleeding of the skin. Loss of h air a t the applica tion site and surrounding a re a s was also noted. As the te st p rogressed, the skin at the application site becam e necrotic followed by the fo rm a tion of eschar tissu e. C. Body W eight Effects Salient individual body weight data for each control and te st group a re p re se n te d se p a ra te ly in T ables VII through XVIII. oaeoos MOQ 5402 !)rtdu4&ual 3 I O - T E S T Jalfo^aivuei, 9*c.. 18 T 9e00S M Q TABLE VII GROUP: C-I R epeated D erm al Toxicity - Albino R abbits Body Weight Data Anim al Num ber and Sex Skin Condition Individual Body W eights (kilogram s) Test Day Number: -5 1 8 15 F in a l 1-1 2.35 2.30 2.21 2.27 2.26 M ale 2-1 2.24 2.41 2.55 2.52 2.35 3-1 2.36 2.41 2.43 2.43 2.44 4-1 2.88 3.08 3.17 3.23 3 .3 4 5 -A 2.27 2.31 2.46 .2.41 2.66 - ' 6 -A 7 -A 2 .4 4 2.48 2.48 2.46 2.26 2.55 2.35 2.72 2.61 (2.72) 8 -A 2.69 2.70 2.72 2.83 2.89 Fem ale 9-1 10-I 11-1 12-1 13-A^ .. 14-A 1 5 -A 1 6 -A 2 .8 2 2.55 2.80 2.28 2.45 2.96 2.65 2.20 3.09 2.59 2.91 2.39 2.57 3.17 2.70 2.29 3 .2 6 2.72 2 .8 6 2.52 2.60 3 .3 7 2.89 2.15 3.37 2.83 (2.78) 2.55 2.66 3.49 2.95 1.87 3.48 3.01 2.61 2.81 3.00 3 .3 4 1.84 403 00027 Utidudiiial 3 1 0 * 1 5 S 7 JlaJurtaltyUei., 9*tc. 19 DOW 500362 TABLE VIII GROUP: C-II R epeated D erm al Toxicity - Albino Rabbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Number: -5 1 8 15 . F in a l M ale 17-1 18-1 19-1 20-1 2 1 -A 2 2 -A 2 3 -A 2.38 2.29 2.60 2.44 2.39 2.34 3.35 2.43 2.34 2.65 2.48 2.41 2.29 3.33 2.75 2.41 2.63 1.98 2.46 2 .1 8 3 .3 4 2.81 2.47 2.83 1.76 2.58 2.21 3.49 2.41 2.47 2.83 (1.47) 2.58 2.18 3.52 2 4 -A 2.22 2.27 2.41 2.61 2.84 Fem ale 25-1 26-1 27-1 28-1v. . 2 9 -A 3 0 -A 3 1 -A 32 -A 2.25 2.21 2.33 2. 51 2.33 2.33 2 .5 4 2.27 2.20 . 1.87 2. 20 1.8 9 2.35 2.26 2. 44 2.52 2.34 2.46 2. 22 2 .1 5 2.38 2.49 2.33 2.38 1.90 1.76 2.27 2.52 2.41 2.29 2.58 2.38 1.89 1.64 2.43 2.52 2.26 2.58 5404 2.61 00027 to 2.41 DOW 500363 S n d u it/u a l 3 I O - T E S T Jiaio'iaio'U ei., 9*tc. 20 T A B L E IX GROUP: T-I R epeated D erm al Toxicity - Albino Rabbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Number: -5 1 8 15 F in a l 33-1 2.66 2.64 2.75 2.75 2.83 M ale 34-1 2.58 2.59 2.80 2.92 2.98 35 -A 2.36 2.50 2.66 2.72 2.77 36 -A 2.29 2.40 2.49 2.58 2.63 F em ale 37-1 38-1 3 9 -A 4 0 -A 2.25 2 .6 6 2.58 2.38 2.25 2.74 2.78 2 .6 4 2.26 2.55 2.72 2.63 2.35 2.52 2.98 2.69 2.35 2.01 3.11 2.81 5405 0002711 DOW 500364 jrdusU ral 3 1O - T 2 S 7 2alt&ia&vei., Sue. 21 TABLE X GROUP: T-II R epeated D erm al Toxicity - Albino R abbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body W eights (kilogram s) T est Day Number: -5 1 8 15 F in a l 41-1 2.46 2.44 2.60 2.75 2.86 M ale 42-1 2.41 2.60 2.29 2.21 2.38 4 3 -A 2.25 2.23 2.24 2.32 2.52 4 4 -A 2.38 2.59 2.63 2.52 1.81 r Fem ale 45 -I 46-1 4 7 -A 4 8 -A 2.69 2.26 2.28 2.43 2.78 2.26 2.42 2.58 2 .9 4 2.26 2.46 2.75 3.06 2.29 2.52 2.89 3.12 (2.10) 2 .2 4 2.92 F ig u re in p a re n th esis re p re se n ts autopsy w eight 5406 0002712 DOW 500365 Dnduiinial 3 1O T c S 7 Jia&tyiai&iiei., 9*tc. 22 TABLE XI GROUP: T-III R epeated D erm al Toxicity - Albino Rabbits Body Weight Data A nim al Number and Sex Skin Condition Individual Body W eights (kilogram s) T est Day Number: -5 1 8 15 F in a l M ale 49-1 50-1 5 1 -A 2.53 2.43 2.27 2.68 2.37 2.31 3.00 2.46 2.38 3.06 2.35 2.35 3.20 2.41 (2.04) 5 2 -A 2.26 2.34 2.46 2.58 2.72 Fem ale 53-1 54-1 55 -A 5 6 -A 2.78 2.57 2. 38 2.31 2.77 2.66 2.41 2.55 2 .9 4 2.72 2.69 2.63 3.00 2.72 2.75 2.72 2.72 2 .8 4 2.75 2.63 Figure in parenthesis rep resen ts autopsy weight 5407 0002713 Dow 500366 Jnduii/Ual 3 I O - 7 E S 7 Jlao'iciio'ei, 9nc. 23 TABLE XII GROUP: T-IV R epeated D erm al T oxicity - Albino Rabbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Num ber: -5 1 8 15 F in a l M ale 57-1 58-1 5 9 -A 2.25 2.43 2.46 2.04 2.68 2.63 2.04 2.72 2.75 2 .2 4 2.89 2.78 2.41 2.97 2.86 60 -A 2 .9 0 3 .0 6 3. 17 3 .1 7 3.26 F em ale 61-1 62-1 6 3 -A 6 4 -A 2,53 2 .8 2 2 .3 2 2 .3 2 2.58 2.89 2.28 2.43 2.60 2.86 2.43 2.60 2.72 2.98 2.49 2,72 2.78 3.06 2.58 2.86 0002714 Doty 500367 Dncubal 3 I O * T 2 S T JlaLonaiotiiei., Dhc. 24 TABLE XIII GROUP: T-V R epeated D erm al Toxicity - Albino Rabbits Body iVeight Data Anim al Number and Sex Skin Condition Individual Body eights (kilogram s) T est Day Number: -5 1 8 15 F in a l M ale 65-1 66-1 2.57 2.45 2.73 2.48 2.69 2.72 2.78 2.81 2.86 2.89 67 -A 2.67 2.45 2.26 2.47 2.66 6 8 -A 2.26 2.36 2.49 2.58 2.66 Fem ale 69-1 70-1 7 1 -A 7 2 -A 2.46 2.33 2.32 2.41 2.61 2.14 2.41 2.19 2.75 2.04 2 .5 2 2.38 2.83 2.29 2.63 2.38 2.86 2.12 2.75 2.49 0002715 D ttdu ii al 3 1 0 - 7 1 5 7 Jtabo'iatve., D*tc. Oty 500368 25 TA BLE XIV GROUP: T-VI R epeated D erm al Toxicity - Albino R abbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Number: -5 1 8 15 F in a l 73-1 2.40 2.53 2.72 2.83 2.77 M ale 74-1 2.27 2.23 2.12 2.10 2.13 7 5 -A 7 6 -A 2.27 2.26 2.16 2.79 (1.58) 2.77 2.86 2.92 Fem ale 77-1 78-1 7 9 -A 8 0 -A 2.28 2.41 2 .5 2 2.58 2.23 2.57 2.48 2.71 2.32 2 .7 2 2.41 2.75 2.35 2.86 2.41 2.98 2 .4 4 2.89 2.38 3 .0 6 F igure in p a re n th esis re p re se n ts autopsy w eight DOW 500369 JkcuUUo 3 I O - 7 3 S 7 Jlaonatoni& i, Sue. 26 TA B L E XV GROUP: T-VII R epeated D erm al Toxicity - Albino Rabbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Number: -5 1 8 15 F in a l 81-1 2.37 2.35 2.32 2.47 2.64 M ale 82-1 2.48 2.53 2.43 2.61 2.69 8 3 -A 2.27 2.42 1.72 (1.72) m 8 4 -A 2.66. 2.82 2.86 2.98 3.00 Fem ale - 85-1 86-1 8 7 -A 8 8 -A 2.42 2.67 2.81 2 .4 6 2.62 2.62 2.82 2.46 2.58 2.60 2.09 2.38 2.66 2.69 (1.87) 2.38 2.86 2.86 2.44 F ig u res in p aren th esis re p re se n t autopsy w eights 5411 0002717 500370 !)rtdudUal 3 I O - 7 2 S T Ja&iaiaei, Jhc. 27 TABLE XVI GROUP: T-VIII O O $ R epeated D erm al Toxicity - Albino Rabbits Body Weight Data Anim al Number and Sex Skin Condition Individual Body Weights (kilogram s) T est Day Number: -5 1 8 15 F in a l 89-1 2.39 2.61 2.49 2 .5 2 2. 49 M ale 90-1 2.25 2.14 1.98 2.04 2.18 9 1 -A 2.24 2.02 (1.50) - - 9 2 -A 2.37 2.27 2.29 2.52 2.55 Fem ale 93-1 94-1 9 5 -A 96 -A 2.67 2.25 2 .4 2 2.48 2.60 2,42 2.64 2.53 2.60 2.32 2.52 2 .2 4 2.75 2.44 2.69 2.41 2.79 2.32 2.89 2.12 F igure in parenthesis rep re se n ts autopsy weight , 0002718 OW 500371 U ndiU tiial 2 3O - 7 2 S T 2a!m aivu& i., Joe. 28 TABLE XVII GROUP: T-IX R epeated D erm al Toxicity - Albino R abbits Body W eight Data Anim al Number and Sex Skin Condition Individual Body W eights (kilogram s) T est Day Num ber: -5 1 8 15 F in a l 97-1 2.61 2.67 2.12 (2.18) - M ale 98-1 2.29 2.35 2.35 2.41 2.55 9 9 -A 2.48 2.53 2.46 2.55 2.52 100-A 2.26 2.40 2.29 2.38 2.55 F em ale 101 -I 102-1 1 0 3 -A 104-A 2.47 2.81 2.30 2.23 2.52 2.86 2. 00 2.18 2 .4 6 2.55 1.45 2 .1 2 2.35 2.86 (1.36) 2.18 2.52 2.95 m 2.38 F ig u res in p a re n th esis re p re se n t autopsy w eights 5413 0002719 Dow 500372 9*tduiaL 3 ! O - 7 2 S 7 JlaoKii&&i, 9*c. 29 T A 3L E XVIII GROUP: T-X R epeated D erm al Toxicity - Albino Rabbits Body W eight Data Anim al Number and Sex Skin Condition Individual Body W eights (kilogram s) T est Day Number: -5 1 8 15 F in a l 105-A 2.41 2.24 2.12 2.35 2.49 M ale 106-1 2.36 2. 54 2.52 2.66 2.72 107-A 2.*66 2 .7 3 2 .6 6 2 .7 5 2.24 108-A 2.66 2.74 2.38 2.66 2.72 Fem ale 109-1 110-1 111-A 112-A 2,51 2,28 2.36 2.36 2.65 2.47 2.38 2.49 2,63 2.41 2 .2 6 2.41 2 .7 2 2.49 2.30 2.55 (2.04) 2.52 2.47 2.66 F igure in p a re n th esis re p re se n ts autopsy w eight 5414 0002720 D tid u iilu zl 3 I O - 3 S 7 JlaliO'uzt&u&i., Skc. 30 Doyy 500373 D. H em atologic and C lin ical Blood C h em istry Studies The re su lts of the blood studies perform ed at the beginning and again at the conclusion of the investigational period did not rev eal any significant differences betw een any of the test groups and th eir respective oil o r w ater control groups. A ll values w ere considered to be within the norm al range. D e te rm in a tio n s of u r e a n itro g e n c o n c e n tra tio n (BUN) and alkaline phosphatase activ ity (SAP) conducted a t the conclusion of the study did not rev e al any abnorm al findings. V alues for a ll te s t anim als w ere com parable to those of th eir corresponding controls. E. G ross Pathologic Findings 'W ith the exception of the skin at the application site, no gross pathologic alteratio n s w ere noted in the tissu es and organs of any of the te st or control group anim als. G ross findings w ith re sp e c t to skin w ere the sam e as those described in the section on local skin reactio n s (Sect. Ill, B2). F . M icroscopic Pathologic Findings M icroscopic exam ination of tissues and organs taken from anim als receiving derm al applications of the te st m aterials as aqueous dilutions as w ell as the w ater control anim als revealed histopathologic alteratio n s in the skin at the application site. These findings w ere ^ 5 ^ 5 ch aracterized by the presence of slight subepithelial fibrosis and mono* n u c le ar in filtra te . No sig n ifican t d ifferen ces could be o b se rv ed b e tw ^ g g o ^ DOw 500374 JkcLu4.oI 3 ! O - T 2 S T JjaMo'UitanieA, 9*tc. 31 either the incidence or the sev erity of these findings among te st a n i m als as com pared to c o n tro ls. No o ther histopathologic a lte ra tio n s w ere noted in the tissu e s and organs exam ined from these anim als. Again, with the exception of the skin a t the application site, no significant histopathologic alteratio n s w ere noted in the tissu e s and organs of anim als receiving derm al applications of oil dilutions of the te st m aterials. The skin of these anim als also revealed subepithelial fib ro sis with m ononuclear in filtrate. How ever, in this instance, the incidence and sev erity of the reactio n s found for te st anim als w as so m e what increased over that noted among controls. The histopathologic findings for the skin of these te st and control rabbits a re shown in T ab ler XIX. 00027 nk#rC DrMusiuaJ. 3 1 0 - 1 E S 7 JiaM&uzivu&i, 9nc. 32 OVV 500375 Group C -II T -V II T -VIII' ' T-IX T-X TA BLE XIX R epeated D erm al Toxicity - Albino Rabbits H istopathologic Changes - Skin N um ber of A nim als Exam ined Findings Incidence 15 S u b e p ith e lia l F ib ro sis 1 M ononuclear In filtra te 1 6 Subepithelial F ib ro sis 4 M ononuclear In filtra te 4 7 Subepithelial F ib ro sis 4 M ononuclear In filtra te 2 6 Subepithelial F ib ro sis 3 M ononuclear In filtra te 1 8 Subepithelial F ib ro sis 4 A verage G rade + + ++ ++ + ++ + +++ Grading System + = slight ++ = m ild +++ 1 m o d e ra te Dow 500376 jtu L u itia l 3 1O - 7 - S 7 JlaJiO 'iato'uei., S*ic. 33 G. Organ v/eights and O rgan/Body /eight R atios Individual organ w eights, viz. liv e r, kidneys, spleen, h eart, and testes, a re presented separately for each test and control group in T ab le s XX th ro u g h XXXI. Individual organ/body weight ratios for each test and control group a re p re se n te d se p a ra te ly in T ables XXXII through XLHI. r 5418 0002724 3 . O - 5 S 7 Jl<Ji&%GVU3<, 9rtc. 500377 Anim al Number and Sex 1-M 2-M 3-M 4-M 5-M 6-M 8-M . - 9-F 10-F 12-F 13-F 14-F 15-F 16-F TA BLE XX Group C -I R epeated D erm al Toxicity - Albino Rabbits Individual O rgan v/eights L iv e r Organ v/eights (gram s) O rgan: Kidney Spleen H eart 101.9 20.7 1.5 6.2 88.4 16.8 0 .8 6.0 94.9 17.8 0. 6 5 .6 148.2 19.3 1.7 7. 6 148.1 2 1 .6 1.0 5 .8 159.2 20.5 1.0 6 .6 113.8 24.7 1.2 6. 1 145.2 149.9 87.1 105.0 132.8 137.9 79.1 2 1 .4 23.8 18.2 15.9 19.2 25. I 12.8 1.7 2 .4 1. 1 1.2 1. 1 2 .0 1.0 9 .3 9 .6 6.7 7.1 6.8 8 .3 4 .9 34 o o ? T estes 1.4 3 .2 1.2 4 .9 4. 0 2 .2 4 .5 - m m m m m 9k m i1 3 JO - 7 3 S 7 2alxvtatoa, Une. 35 Anim al N um ber and Sex 17 -M 18-M 19-M 21-M 22-M 23 -M 2 4 -M 25-F 26-F 27-F 28-F 29-F 30-F 31-F 32-F TABLE XXI Group C-U R epeated D erm al Toxicity - Albino Rabbits Individual O rgan W eights L iv er O rgan Weights (gram s) O rgan: Kidney Spleen H eart 138.7 20.5 1.4 6.5 103.4 22.9 . 1.6 6.3 130. 6 19.5 1. 1 7.3 137.0 19.1 1.2 6.6 84.8 18.3 1.0 6. 1 151.8 4 179.8 27.2 20. 6 1.6 1.3 8.7 6 .6 104.2 68.3 141.4 96. 6 117.9 150. 3 99.7 144.2 18.5 17.0 17.9 15.3 22.5 18.6 18.3 19.5 0 .9 0.8 1.6 1.0 0 .9 1.2 1.2 1.2 5. 6 5 .6 5 .9 7 .0 7.5 7 .4 7. 1 6.4 T estes 2. 6 3 .4 5 .3 4 .9 2 .7 6. 1 4 .5 m m mt m m 5420 DOW 500378 0002726 500379 )rA it a l 3 1O - 7 2 5 T Jldiostai& ai, Sac. 36 Anim al N um ber and Sex 3 3 -M 34-M 3 5 -M 36-M 37-F 38-F 39-F 40-F TABLE XXII Group T -I R epeated D erm al Toxicity - Albino R abbits Individual Organ Weights L iv er O rgan v/eights (gram s) Organ: Kidney Spleen H eart 79. 1 136. 1 20. 0 2 3 .4 4. 1 2.2 9 .5 9 .6 110.0 19.4 2.3 9.5 127.9 23.3 1.9 7 .6 71.1 68.5 159.0 147. 1 13.5 22. 6 24.7 23.4 1.2 1.3 1.7 1.3 5 .4 6.5 7 .6 7 .8 D o T estes 7 .3 6.2 6.3 3 .2 - m m 0002727 500380 '9*uubal 3 3O - 7 5 S 7 2atw aiv& i, 9*tc. 37 Anim al Number and Sex 41-M 4 2 -M 4 3 -M 4 4 -M 45-F 47-F 48-F TABLE XXIII Group T -II R epeated D erm al Toxicity - Albino Rabbits Individual O rgan 'Weights .. L iv e r O rgan W eights (gram s) O rgan: Kidney Spleen H eart 170.3 33. 1 1.2 7 .3 139.4 2 7 .4 1.0 7. 0 158.9 175.9 2 0 .8 27. 1 2 .8 0.7 8.5 6.0 151.8 96.4 112.0 3 3 .0 26.2 25.4 2. 6 2. 0 4. 6 9. 6 7 .9 8 .5 oO T estes 5. 6 4. 1 6. 6 3.1 - - ?: ,. o ^ 0 0 0 2 i M j^daiU iC tl 3 ! O - i - 5 n JlaliOsicd&iL&i, Dkc. 38 Anim al N um ber and Sex 49 -M 50-M 52-M 53-F 54-F 55 -F 56-F TABLE XXIV Group T -IH R epeated D erm al Toxicity - Albino R abbits Individual O rgan v/eights L iv e r O rgan '/eights (g ram s) Organ: Kidney Spleen H eart 140.0 24. 1 1.7 8 .7 117.7 19.2 2 .1 6.7 192.0 19.3 1.0 5. 7 83.5 146.8 136.4 . 79.9 2 6 .8 16.3 21.7 18. 0 1.3 1.7 1.4 6.7 6.5 7 .0 6 .8 Oo i: ooor CO 00 T este s 4 .8 5 .7 2 .8 m m o 5423 0002729 DOW 500382 induifrual 3 1O - 7 E S 7 2aMonataAi&i, 9*tc. 39 A nim al N um ber and Sex 57-M 58-M 59-M 60-M 61-F 62-F 63-F 64-F T A B LE XXV Group T-IV R epeated D erm al Toxicity - Albino Rabbits Ind iv id u al O rg an v/ eights L iv er O rgan Weights (gram s) O rgan: Kidney Spleen H eart 146.9 22. 1 1.5 8.3 167. 1 108.1 22.9 15.5 1.7 0.8 9. 1 7.2 162.5 24.3 1.8 10.9 '36. 3 138.3 * 128.5 128.9 16.5 19.-8 17.9 19.1 1.6 1.7 1.2 2 .0 7. 6 9.3 7.9 8.3 T estes 3.3 5 .2 4 .7 7 .2 - - 0002730 DOW 500383 jriauM i/d. 3 I O * i : S i JlaJiOK+ye., Dkc. 40 A nim al N um ber and S ex 65 -M 66-M 67-M 6 8 -M 69-F 70-F 71-F 72-F TABLE XXVI Group T-V R epeated D erm al Toxicity - Albino Rabbits Individual O rgan W eights L iv e r O rg a n vVeights (g ra m s) Organ; Kidney Spleen H eart 143. 6 16.4 2.3 8.5 76.3 21.5 0.9 7.2 142.4 24. 3 1. 1 8 .0 118.3 22.5 1.5 9 .9 135.8 81.5 * 157.5 106.1 2 2 .7 20. 1 13.8 19. 1 2.5 1.1 1.6 1.0 7 .2 6. 1 7 .8 9. 1 T estes 5.0 5 .0 3.7 3 .4 - - - - 5425 0002731 500384 3 O - 7 3 3 7S n du i.t zl Jakyia& eS, Site. 41 Anim al N um ber and Sex 73-M 74-M 7-M 77-F 78-F 79-F 80-F TABLE XXVII Group T-VI R epeated D erm al Toxicity - Albino Rabbits Individual O rgan W eights L iv e r O rgan Weights (gram s) Organ: K idney Spleen H eart 103.0 18.7 1.2 8.5 78.7 23. 1 1.0 5 .8 120. 2 2 .8 1.3 8 .4 105.8 130.3 115.3 ` 159.0 16.6 2 2 .4 19-2 19.8 1.0 2. 1 1.4 0 .9 7. 1 8.2 5 .9 7 .7 oo 3 T estes 4 .2 3.7 6.0 - 54 2 6 0002732  3 O S 7 Jlai&uzicJUed., }m. 42 A nim al N um ber and Sex 81-M 82-M 8 4 -M TABLE XXVIII Group T-VII R epeated D erm al Toxicity - Albino Rabbits In d iv id u al O rg an vVeig h ts L iv e r O rg an vVeights (g ram s) Organ: K idney Spleen H eart 125.6 2 0 .8 0 .9 5 .8 109.9 128.5 18.3 20. 1 0.9 1.2 5 .2 6.1 85 - F 86-F 88-F ' 126.1 135.5 143.7 20.5 19.5 19.0 1.8 0. 6 0 .9 8 .4 7.2 7. 1 T e s te s JI 5 .2 4 .8 4 .7 - - DOW 500385 5427 0002733 9 8 8 0 0 5 ^ 0 (3 9kcuizI 3 O - 7 2 S 7 kc. 43 A nim al N um ber and Sex 89-M 90-M 92-M 93 -F 94-F 95-F 96-F TABLE XXIX Group T -V ni R epeated D erm al Toxicity - Albino R abbits Individual O rgan eights L iv e r O rgan Weights (gram s) O rgan: Kidney Spleen H eart 117.6 2 1 .4 1.0 7. 1 127.2 22. 1 0.8 6.6 137.4 21.5 1.3 6.2 186.5 136.7 178.3 * 100,7 19.9 2 0 .4 2 4 .7 19.0 2 .2 1. 1 1.6 0 .9 8.3 7. 6 6.7 6.0 T estes 1.8 1.4 3.5 - - 5428 0002734 Dow 500387 Dttdudi/ual B I O - T E S T a&Maio'Uei, Dhc. 44 A nim al N um ber and Sex 98-M 9 9 -M 100-M 101-F 102-F 104-F . TA BLE XXX G roup T -IX R epeated D erm al T oxicity - Albino R abbits Individual O rgan Weights L iver O rgan Weights (gram s) O rgan: K idney Spleen H eart 158.0 21.3 1.4 7 .9 97.3 18.2 1.9 7 .4 167.4 16.7 1.0 6.1 134.2 156.8 130.9 18.6 19.7 17.4 1.0 1.2 0 .8 6.8 8 .1 6.9 T estes 5 .6 3.9 2 .8 m - - 5429 DoW 500388 !t*tduii'ual B I O - T E S T 2 aJuyiayueA, 2*tc. 45 Anim al N um ber and Sex 105-M 106-M 107-M 108 -M TABLE XXXI Group T-X R epeated D erm al Toxicity - Albino Rabbits Individual O rgan W eights L iv e r Organ r/eights (gram s) O rgan: K idney Spleen H eart 128.8 19.3 0 .8 6.9 140.7 95.1 125.0 21.8 17.3 17.0 1.6 0 .9 1.3 7 .7 8 .6 6.9 110-F 111-F" ' 112-F 114.1 116.2 . 137.1 2 0 .6 19.8 17.4 1.9 1.0 1.3 7 .7 6 .6 7. 6 T estes 4 .5 4 .9 1.3 6.0 - ii_- 0002736 Stubui/iiai B 1O - T E S T ahyiatosii&L, 9*tc. 46 DOW 500389 TABLE XXXII Group C -I R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody Weight Ratios Anim al N um ber and Sex L iv e r O rg an /B o d y eight R atios (g/100 g) Organ; Kidney Spleen H eart T estes 1-M 4 .4 9 0.912 0.065 0.274 D.0%2 2-M 3 .7 6 0.715 .0.034 0.255 0.142 3-M 3.89 0.730 0.025 0.230 0. 049 4-M 4 .4 4 0.578 0.051 0.228 0. 146 5-M 5.57 0.813 0.038 0.218 0.150 6-M - 6. 10 0.785 8-M 3 .9 4 0.854 0.038 0.042 0.253 0.211 0.084 0.156 9-F 10-F 12-F 13-F 14-F 15-F 16-F 4. 17 4. 98 3 .3 4 3 .7 4 4.43 4 . 13 v* 4 .3 0 0.615 0.794 0. 669 0.567 0.640 0.755 0.695 0.049 0.080 0.042 0.043 0.060 0.038 0.054 0.267 0.320 0.25? 0.260 0.227 0.249 0.267 - - 5431 0002737 D tuLutai B I O - T E S T Jtalta'iat&U&i, 9hc. 47 DOW 500390 Anim al N um ber and S ex 17-M 18-M 19-M 21-M 22-M 23 -M _ 2 4 -M TABLE XXXIII Group C -II R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody W eight R atios O rg a n /B o d y W eight R a tio s (g /1 0 0 g) O rgan: L iv e r Kidney Spleen H eart 5.75 0.851 0.058 0.271 4 .2 0 0.927 0.065 0.256 4 . 62 0. 690 0. 040 0.259 5 .3 1 0.740 0.046 0.256 3 .9 0 0.841 0.046 0.281 4 .3 2 0.774 0.046 0.248 6.33 0.725 0.046 0.232 T estes 0. 108 0. 138 0. 188 0. 190 0. 124 0. 174 0. 159 2 5 -F 26-F 27-F 28-F 29-F 30-F 3 1-F 32-F 5 .5 0 4 . 17 5 .8 3 3 .8 4 5.21 5 .8 3 3.83 6.00 0.980 1.037' 0.738 0. 608 1. 000 0.721 0.703 0.810 0.048 0.049 0 .0 6 6 0.040 0. 040 0.046 0.046 0.050 0.296 0.342 0.243 0.278 0.332 0.287 0.272 0.266 m - m - 00027:8 DOW 500391 . 9*tdu ih ial B I O - T E S T JlaJto'iai&uei., 9*tc. 48 A nim al N um ber and Sex 3 3 -M 3 4 -M 35-M 36-M 37-F 38-F 39-F 40-F TABLE XXXIV Group T -I R epeated D erm al Toxicity - Albino Rabbits Ind iv id u al O rg a n -B o d y viTeight R a tio s O rgan/B ody v/eight R atios (g/lOOg) O rgan: L iv e r K idney Spleen H eart 2 .8 0 0.708 0.145 0.336 4 .5 7 0.785 ,0 .0 7 4 0.323 3.97 0.700 0.083 0.343 4 .8 6 0.885 0.072 0.289 T estes 0.258 0.208 0.228 0. 122 3.03 3.41 5 .1 1 5 .2 3 0.575 1.124 0.795 0.834 0.051 0.065 0.055 0.060 0.230 0.324 0.244 0.278 m ?! V-, 0002739 DOW 500392 U nduibuol B I O - T E S T 2aluviat<yuei., D*tc. 49 Anim al N um ber and Sex 41-M 4 2 -M 43 -M 4 4 -M 45-F 47-F ' 48-F TABLE XXXV Group T -II R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody eight Ratios O rg a n /B o d y Weig h t R a tio s (g/100 g) O rgan: L iv e r K idney Spleen H eart 5.95 1.16 0.042 0.356 5,87 1. 15 0.042 0. 294 6.31 0.825 0. I ll 0.338 9 .7 2 1.50 0.039 0.332 T estes 0. 196 0 . >1772 0.262 0. 171 4 .8 7 4 .3 0 3 .8 4 1.059 1.170 0.870 0.084 0.089 0.158 0.308 0.353 0.292 - 5434 0002740 DtvJLuifual B I O T E S T Jlahviaivuei, Unc. Anim al N um ber and Sex 49 -M 50-M 52-M 53-F 54-F 55 -F 56-F 50 TABLE XXXVI Group T -III R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody W eight R atios O rg a n /B o d y W eight R a tio s (g /1 0 0 g) O rgan: L iv e r K idney Spleen H eart 4 .3 9 0.753 0.053 0.272 4 .8 9 0.798 0.067 0.278 7 .0 6 0.710 0.037 0.210 OO coon CO CO CO T estes 0. 150 0.237 0.103 3. 07 5. 16 4 .9 6 3.05 0.985 0.575 0.790 0.685 0.048 0.060 0.051 0.038 0.246 0.229 0.255 0.259 m - - 5435 000274 1 500394 D rtduil'iicd B I O - T E S T Jtaio'iai&ue4; !)* 51 A nim al N um ber and Sex 57-M 58-M 59-M 60-M 61-F 62-F 63-F 64-F TABLE XXXVII Croup T-IV R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody W eight R atios O rgan/B ody Weight R atios (g/lO O g) O rgan: L iv e r K idn ey Spleen H eart 6.10 0.919 0.062 0.346 5 .6 4 0.772 0.057 0.306 3.68 0.542 0.028 0.252 4 .9 9 0.745 0; 055 0.334 oo 3 T estes 0.137 0. 175 0.164 0.221 3.13 0.594 4.52 * 0.648 4 .9 9 0. 695 4 .5 1 0. 669 0. 058 0.056 0.046 0.070 0.274 0.340 0. 306 0.290 m - m 000274Z DOW 500395 ' J n d tu b u o l B I O T E S T Jla A & u zityu ed ., Umc. 52 Anim al Number and Sex 6 5 -M 66-M 6 7 -M 68-M TABLE XXXVIII Group: T-V R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody Weight R atios L iv e r O rgan/B ody W eight R atio s (g/100 g) O rgan: K idney Spleen H eart T estes 5.03 0.574 0.080 0.297 0.175 2 .6 4 0.745 0.031 0.249 0.173 5.37 0.810 0.041 0.301 0.139 4.45 0.846 0.056 0.372 0.128 69-F 70 -F r ' 71 - F 72-F 4.75 3.85 . 5.73 4 .2 6 0.794 0.950 0.502 0.767 0.088 0.052 0.058 0.040 0.252 0.288 0.284 0.366 m - - - 3 4 J 8 00027i3 DOW 500396 ' HttcLidisual B I O - T E S T jaional&uei, 9*ic. 53 A nim al N um ber and Sex 73-M 7 4 -M 76-M TABLE XXXIX Group T-VI R epeated D erm al T oxicity * Albino Rabbits Individual O rgan/B ody Weight R atios O rg a n /B o d y W eight R a tio s (g/100 g) O rgan: L iv e r K id n ev Spleen H eart 3.72 0. 675 0.043 0.307 3. 69 1. 082 0. 047 0.272 4. 14 0. 781 0.044 0.288 i T estes 0. 152 0. 174 0.206 77-F 78-F 79 -F * 80-F 4 .3 4 4 .5 1 4.85 . 5 .2 0 0.681 . 0.041 0. 774 0. 073 0. 806 0. 059 0. 648 0.029 0. 292 0.284 0.248 0.252 - m - 543: 0002744 DOW 500397 r U nduihial B I O - T E S T Jao<2ve, nc. r r r TABLE XL . * Group T-VII 54 f R epeated D erm al Toxicity - Albino R abbits i f Individual O rgan/B ody W eight R atios . * A nim al O rg an /B o d y Weight R atios ( g / 100 g) N um ber O rgan; i and Sex L iv e r * H eart T estes 81-M 4 .7 6 0.788 0.034 0. 220 0. 197 L .* 82-M 4 .0 8 0. 680 0.033 0. 193 0. 178 8 4 -M 4 .2 9 0. 671 0.040 0.204 0.157 l 85-F 4.41 0. 683 0.063 0.294 - 86-F 4 .7 5 0.718 0.021 0. 252 - 88-F 5.89 ' 0.780 0. 037 0.291 m L L [ t L 4J '%K* O5 0002745 H*iduii/UaL B I O T E S T ^aluyiaiwieS, 9*tc. 55 DOW 500398 TABLE XLI Group T-VIII R epeated D erm al Toxicity - Albino Rabbits Indiviciai O rgan/B ody W eight R atios Anim al C cgan/B ody W eight R a tio s (g /1 0 0 g) N um ber Organ: and S ex _____L iv e r_____ Kidney_____ S p leen _____ H e a rt______T e s te s 89-M 4 .7 2 0.859 0.040 0.286 0.072 90-M 5 .8 4 1.015 0.037 0.303 0.064 9 2 -M 5 .4 0 0.845 0.051 0.244 0.141 93-F 94-F 95-F 96-F 6. 67 5 .8 9 6. 17 4.75 0.713 0. 880 0.855 0.896 0.079 0.048 0.056 0.042 0. 298 0.328 0.232 0.283 - m 500399 Dndudiruai B I O T E S T aio'iahvu&l, 9m . 56 Anim al N um ber and Sex 98-M 99-M 100-M TABLE XLII Croup T-IX R epeated D erm al Toxicity - Albino Rabbits Individual O rgan/B ody W eight R atios O rg an /B o d y W eight R atio s (g/100 g) O rgan: L iv e r K id n ey Spleen H eart 6.20 0.835 0.055 0.310 3.87 0.723 0.076 0.294 6.57 0. 655 0.039 0.240 o O T estes 0.220 0. 155 0. 110 101-F 102-F 104-F ' 5 .3 4 0.740 5 .3 1 0. 670 5 .5 0 * 0. 731 0.040 0.041 0.034 0.270 0.275 0.290 - 5441 00027 4*nI Oov/ 5 0 0 4 0 0 9*tdudJ/tial B I O * T E S T aluviat&Ue, Stic, 57 A nim al N um ber and Sex 105-M 106-M 107-M 108-M 110-F 111-F 112-F TABLE XLIU Group T-X R epeated D erm al T oxicity - Albino Rabbits Individual O rgan/B ody W eight Ratios O rg a n /B o d y W eight R a tio s (g/100 g) O rgan: L iver K idney Spleen H eart 5. 17 0.775 0.032 0.278 5. 17 0.803 0.060 0. 284 4 .2 4 0.773 0.040 0.268 4 . 60 0. 625 0.048 0.254 T estes 0.181 0. 180 0.058 0.221 4 .5 4 4 .7 1 5. 16 0.819 0.803 0. 655 0.068 0.040 0.049 0.306 0.268 0.286 - 5442 0002748 DOW 500401 *' 9*uLubuol B I O - T E S T ^aionaityueS, Smc. 58 IV. Sum m ary The following sum m arizes the re su lts of subacute d erm al ap p lica tion of w ater an d /o r oil dilutions of the dim ethylam ine salt, the butyl e ste r and the isooctyl e ste r of 2 ,4-dichlorophenoxyacetic acid (2,4-D ) to albino rab b its: A. M ortality Deaths occurring during the te st period w ere relativ ely few and w ere sc a tte re d among the v ario u s g ro u p s. No c o rre la tio n w as noted between the incidence of leth al responses and any given te s t m aterial or dose of test m aterial. B. Reactions H yperirritability and pain upon derm al applications w ere ob served among anim als treated with the oil dilutions of the butyl and isooctyl e ste rs of 2,4-D but w ere also noted among control rabbits receiving oil applications. These reactions w ere not seen among any anim als of those groups w here w ater was em ployed as the solvent. Local skin reactions w ere observed among all anim als includ ing controls. Among groups w here w ater w as em ployed as the solvent, only relatively m ild to m oderate inflam m atory reactions w ere seen and the reactio n s ^occurred to essen tially the sam e incidence and degree am ong both te st and control anim als. Inflam m atory reactions among the groups for which oil w as the solvent w ere sev ere and p ro g resse d to n ecro sis and eschar form ation. Again, how ever, no s ig n ifie d ^ q 7 4 g Utidudi Aio l B I O - T E S T aJwiat&u&i, Due. 59 differences w ere observed in eith er incidence or degree betw een test DOW 5p0402 and control anim als. C. Body W eight Effects Body weight data tabulated for anim als of the te s t groups di not differ m aterially from those recorded for their corresponding controls. D. H em atologic and C lin ical Blood C h e m istry Studies No re m a rk a b le d e v ia tio n s fro m n o rm a l ra n g e s w e re found for the hem atologic and clinical blood chem istry p a ra m eters studied in either test or control anim als. E. Pathologic Findings The only tissu e disclosing any significant pathologic altera* r tion among te st o r control anim als w as the skin. Evidence of the lo c il inflam m atory re a ctio n s a lread y d escrib ed (See IV. B above) w ere seen both grossly and m icroscopically among both te st and control anim als. C ro ssly , no differences could be discerned betw een te s t anim als and their corresponding controls, i . e . , those receiving the sam e solvent em ployed as a diluent, either oil or w ater. This was also the case m icroscopically for the w ater-treated groups. H ow ever, among the oil treated groups the incidence and degree of m icroscopic change, characterized by subepithelial fibrosis and m ononuclear infiltrate, appeared to be in c re a se d som ew hat in te s t an im als as com pared to 5 4 4 4 the corresponding controls. O O O ^T^i) DOW 500403 y+uludi/Lai B I O - T E S T -oJKyuiiosUeA, Uhc. 60 F. Organ V/eights and O rgan/B ody Weight R atios G ro ss in sp e c tio n o the o rg a n w eight and ra tio d a ta d is c lo s e d a num ber of inter group differences. Except for the observed effect of the dim ethylam ine salt of 1,4-D significantly increasing kidney w eights and ratios in both m ales and fem ales, none of the deviations observed w ere believed to be outside the norm al range of v ariation for the albino rabbit and w ere considered unassociated with derm al application of the te st m aterials. M oreover, in view of the lack of g ro ss or m icroscopic findings in the kidneys of the rabbits tre a te d with the dim ethylam ine salt, even the in creased kidney w eights and ratio s noted may have been coincidental. Respectfully subm itted, INDUSTRIAL B IO -TEST LABORATORIES, INC. R e p o rt p r e p a r e d b y . ______ . B. S. Departm ental D irector Acute Toxicity D epartm ent R e p o rt a p p ro v e d by: _ ,, __ , . ^ D. Associate D irector D irecto r M arch 13, 1964 5445 0002751 S 5446 DOW 500405 rtd u it a l B 10 -TEST a/uwatosU ei, Une. 1810 F R O N T A G E ROA D NORTHBROOK, ILLINOIS Talaphon CRuiwood 2-3030 ADDENDUM REPORT TO THE NATIONAL AGRICULTURAL CHEMICALS ASSOCIATION REPEA TED DERMAL TOXICITY STUDIES ON THREE FORMULATIONS OF 2 ,4 DICHLOROFHENOXYACETIC ACID i 5447 00027 DOW 500406 n d u iiw l B i O - T E S T JjaloAoliMiei, 9*i& ADDENDUM REPORT TO THE NATIONAL AGRICULTURAL CHEMICALS ASSOCIATION R E P E A T E D DERM A L TOXICITY STUDIES ON THREE FORMULATIONS OF 2 ,4 DICHLOROPHENOXYACETIC ACID I. Introduction This addendum re p o rt p resen ts the detailed resu lts of the h em a tologic and clinical blood ch em istry studies conducted on each anim al during a three-w eek repeated derm al toxicity study previously reported* on the three com m ercially available form ulations of 2,4 dichlorophenoxyacetic acid (2,4-D ) indicated below: 2 .4 - D D im ethylam ine Salt^ r 2 .4 - D Isooctyl E ster^ 2 .4 - D Butyl E ste r^ An outline of the investigation as a b strac te d fro m the p rio r re p o rt is presented in Table I. 1 See rep o rt to The N ational A gricu ltu ral C hem icals A ssociation "R e peated D erm al Toxicity Studies on T hree Form ulations of 2 ,4 D ichlorophenoxyacetic A cid ," M arch 13, 1964. 2 W eed-Rhap H erbicide A -4; H ercules Pow der Company, Inc. W ilm ington, D elaw are. 3 M onsanto 2,4.*0 Low V olatile E s te r Weed K iller; M onsanto C hem ical Company, St. Louis, M issouri. 4 E stro n 76 BE W eed K iller; The Dow C hem ical Com pany, M idland, M ichigan. \ 0002753 ^ ** e* Dtuiudfru a l 3 ! O - T E S 7 Jlaio'iaia'U & i., H*tc. 45 III. Sum m ary No re m a rk a b le deviations fro m n o rm al ran g es w ere found for the hem atologic and clinical blood ch em istry p a ra m e te rs studied in either test or control anim als. Respectfully subm itted, INDUSTRIAL BIO-TEST LABORATORIES, INC. 50045 Oo Report prepared by: Acute Toxicity D epartm ent fr{ j Report approved by: John H. Kay, Pn. D. A ssociate D irector J , C. C alandra, M. D. , Ph. D. D irector M arch 16, 1964 5443 0002797 SH l; Kirk-Othmer ENCYCLOPEDIA OF CHEMICAL J. TECHNOLOGY Second completely revised edition VOLUME 5 C h lo rin e to Colors for Foods, D rugs, and Cosm etics 5451 i EDITORIAL BOARD Chairman: HERMAN F. MARK Polytechnic Institute o f Brooklyn JOHN J* MftKKil'itA| JR* The University o f Texas DONALD F. OTHMER Polytechnic Institute o f Brooklyn EXECUTIVE EDITOR ANTHONY STANDEN Interscience Publishers a division of John Wiley & Sons, Inc. Neto York London Sydney 5452 Vol. 5 CHLOROPHENOLS 325 CHLOROPHENOLS This article discusses in detail the methods of manufacture and the physical and chemical properties, toxicology, and uses of the major chlorophenols while presenting briefer information on those compounds that are of no commercial importance. In general, chlorophenols containing other substituents on the ring, for example, chloronitrophenols, chloroalkylphenols, chlorobromophenols, etc, are not treated. Such compounds, when of commercial importance, are discussed in connection with the specific chlorophenol from which they are derived. An exception, however, is made in the case of some of the commercially important chlorinated cresols and xylenols, which are discussed a t the end of the article. Properties and General Reactions With the exception of o-chlorophenol, all of the chlorophenols are solids a t room temperature, and all have a pungent, medicinal odor. They are generally insoluble in methanol and acetone.' The chlorophenols are stronger acids than phenol itself: Unlike the latter, they can be converted to the sodium salts from sodium carbonate solutions. This property affords a method of separating phenol from chlorophenols. In addition, other metallic lialts, eg, salts of barium, potassium, calcium, zinc, etc, are also easily prepared. The chlorophenols and their salts show actual or potential utility in a wide range of ap plications. They exhibit outstanding germicidal and insecticidal properties and have demonstrated utility as flea repellents, fungicides, wood preservatives, mold inhibitors, etc. In general, effectiveness increases with the degree of chlorine substitution. References 1-4 typify the many studies which have been made on these materials (see Antiseptics and disinfectants; Fungicides). Generally, the chlorophenols react very much the same as phenol itself. The methyl, ethyl, propyl, and butyl ethers of all of the chlorophenols are well known and are readily available by the reaction of the sodium chlorophenoxides with the cor responding alkyl halides. An extension of the etherification reaction involving the reaction of the sodium chlorophenoxides with a-halo aliphatic acids has considerable commercial significance. Thus, the reaction of sodium 2,4-dichlorophenoxide with chloroacetic acid leads to 2,4-dichlorophenoxyacetic acid, Cl*CeH*OOCCHj, or 2,4-D; similarly, sodium 2,4,5-trichlorophenoxide leads to 2,4,5-T; these two compounds and other related compounds have gained a widespread reputation as weed killers (qv). Their evaluation and importance are discussed in several excellent review articles (5-7). Another important reaction is that of the sodium salts of chlorophenols with aromatic sulfonyl chlorides to form sulfonates: Cl . All of the commercially important chlorophenols have been converted to .the sulfonates and tested in various germicidal and insecticidal applications. The sul- 5453 326 CHLOROPHENOLS Table 1. Physical Properties of the Chlorophenols Material Bp at 760 mm, "C Fp, *C Dissociation constant at 25C, K . o-chlorophenol p-chlorophenol m-chlorophenol 2,4-dichlorophenol 2,6-dichlorophenol 2,3-dichlorophenol 2,5-dichlorophenol 3,4-dichlorophenol 3,5-dichlorophenol 2,4,6-trichlorophenoI 2,4,5-trichlorophenol 2,3,4-trichlorophenol 2,3,5-trichlorophenol 2,3,6-trichlorophenol 3,4,5-trichlorophenol 2,3,4,6-tetrachlorophenol 2,3,4,5-tetrochlorophenol 2,3,5,6-tetrachlorophenol pentachlorophenol 175-176 219 215-217 210-211 219-220 206 212-213 253 233 246 245-246 255 272 275 164/23 mm 309-310 8.7 40-41 32.S 43-44 67 58 58 65 68 6S 68 83.5 62 101 101 69-70 116-117 115 190 3.2 X 10-* 6.6 X 10-" 1.4 X 10-* 2.1 X 10- 1.6 X 10-'' 3.6 X 10" 4.5 X 10- 4.1 X 10"* 1.2 X 10- 3.8 X 10-* 3.7 X 10- 2.2 X 10-* 4.3 X 10-* 7.4 X 10-* 1.8 X 10-* 4.2 X 10-* 1.1 X 10-* 3.3 X Id" 1.2 X 10-* fonates qf the lower substituted chlorophenols show insecticidal properties equivalent to thoseftf phenylarsenous acid in apple sprays (8). The chlorophenols undergo substitution reactions, eg, nitration, alkylation, " acetylation, much the same as phenol itself except when positions occupied by the chlorines preclude further reaction. The lower substituted chlorophenols condense with formaldehyde to form phenolic resins which in turn serve as intermediates for the preparation of resoles and novolaks (see Phenoplasts). Generally, the resulting polymers have greater flame resistance than the usual resins prepared from phenol itself. The positions of the chlorine atoms on the phenol nucleus play a significant role in the utility of chlorophenols as starting materials for such resins. The resins, to be useful, must have a high degree of cross linkage which, in turn, is possible only if the starting phenol has open ortho or para positions. For this reason, o- and p-substituted chlorophenols have no real utility in this application. m-Chlorophenol and 3,5-dichlorophenol have shown promise, but high costs limit their utility to specialty applications. Small amounts of p- and o-chlorophenol are, however, used in phenolic resins where a greater degree of fire resistance is desired. The mono-, di-, and triphosphate esters are formed by the reaction of chloro phenols with phosphorus oxychloride. The products have utility as flameproofing agents, fungicides, wood preservatives, etc (9,10). Esterification proceeds by the general equation 3 ROH + POCh -- OP(OR)j + 3 HCl where R represents ClaCsHg-,,. Chlorophenols form salts with amines. The salts formed with diamines, such as ethylenediamine and propylenediamine, can be employed as bactericides, insecticides, fungicides, etc (11). 2 ROH + NHiCHiCHiNH, -- i$H,CHtCHiC$H, + 2RO- 5454 Vol. 5 CHLOROPHENOLS 327 In many cases these salts are very stable. Salts similarly formed with certain alkanolamines, such as N-cyclohexylmonoethanolamine, have similar applications (12). More specific reactions of the chlorophenols are discussed in the sections de scribing individual members of the family that have commercial significance. Toxicity The toxicity of the chloro-substituted phenols varies from isomer to isomer within a group. However, we may make the following generalizations: The monochlorophenols are likely to be moderate in acute oral toxicity. Most of them are likely to be corrosive to the skin and eyes, and are readily absorbed through the skin in toxic 4J amounts. Their vapors or dusts are very irritating and toxic. In handling these materials, precautions should be taken to prevent skin and eye contact and avoid inhalation of vapors or dusts. The dichlorophenols may be somewhat less toxic by skin and eye contact than the monochlorophenols and are less likely to be absorbed through the skin. Their dusts, however, are very irritating. Precautions for handling should include prevention of skin and eye contact and avoidance of dust inhalation. ^ The irichlorophenols are usually considered to be the. least toxic of the chloro phenols. Most of the isomers should cause only moderate skin irritation. Eye con tact may result in marked irritation, possibly some corneal injury. The dusts arc likely to be very irritating. Precautions should be taken to avoid skin and eye con tact and to avoid inhalation of dusts. While the telrachlorophcnols are perhaps somewhat less toxic than pcntachlorophenol, all of these highly chlorinated phenols are considered to present essentially the same degree of hazard on handling. They may be moderately to highly toxic when ingested, and highly toxic when absorbed by the skin. Strong solutions are especially hazardous in this respect. Eye contact is likely to cause marked irritation, or even a burn. Continuous daily skin contact has been known to cause acncform dermatitis in humans. Dusts of these materials are very irritating to the respiratory------tract and to the eyes. In handling, precautions should be directed toward avoiding eye or skin contact with the dusts. The inhalation of dusts should be avoided. None of the chloro-substituted phenols has been reported to cause skin sensitiza tion. The manufacturer should be contacted for specific toxicological information before any of these chlorophenols are handled in industrial quantities. General Methods of Preparation There are several general methods by which chlorophenols can be prepared. These include direct chlorination with various chlorinating agents, hydrolysis of chlorinated benzenes conversion of the diazonium salts of various chlorinated anilines, and chlo rination of phenolsulfonic acids and benzenesulfonic acids, followed by removal of the sulfonic acid group. The isomer desired determines the synthetic route. Direct Chlorination. One of the most widely used methods is the direct chlorina tion of phenols with molecular chlorine. The reaction proceeds stepwise and can lead to all possible products from monochlorophenol to pentachlorophenol. Generally, chlorination proceeds readily a t the lower levels of substitution by .. the direct addition of gaseous clilorine to molten phenol in the absence of catalysts. When greater degrees of chlorination arc desired, catalysts, such as FeCU. AlClj, and 5455 ? T 328 CHLOROPHENOLS SbCl, are employed in concentrations of 0.05-1.0%.. Usually, the reactions are car ried out in the absence of solvents, but carbon tetrachloride, acetic acid, and water have been used as diluents, ^ince the phenolic hydroxyl is ortho-para-directing, the product of monochlorination is a mixture of o- and p-chlorophenol, and the products of dichlorination are chiefly the 2,4- and the 2,6-isomer. The product of trichlorina tion is chiefly the 2,4,6-isomer and, of tetrachlorination, primarily the 2,3,4,6-isomer. Other chlorinating agents which have been used include hypochlorous acid, sodium hypochlorite solutions, and sulfuryl chloride. Substitution occurs in the same posi tions as with molecular chlorine; however, there is evidence for a greater degree of ortho substitution (13). Various mechanisms for the direct chlorination of phenols have been proposed (14-16), but none has as yet won general acceptance. Hydrolysis of Chlorinated Benzenes. The hydrolysis of polychlorobenzenes is another widely used method of preparing chlorophenols. This can be an attractive commercial route, since chlorinated benzenes are produced in large volumes in the United States. I t is used chiefly to obtain chlorophenol isomers not readily available by direct chlorination. Generally, hydrolysis is carried out in aqueous alkaline solu tions a t high temperatures and under pressure. Very often solvents such as methanol or ethanol are added to the reaction media to increase solubility and, thus, reaction rate; however, some methyl or ethyl ether of the chlorinated phenol is then obtained through side reactions. Reaction temperatures vary from 250 to 300C, reaction timfia-are from 15-90 min, pressures vary from atmospheric to 500 psi, and conversions are 85-98%. Some of the chlorinated phenols prepared by this method are 2,5-di-, 2,4,5-tri-, 2,3,5,6-tetra-, and pentachlorophenol. Other Methods of Preparation. Since both the direct chlorination of phenols and the hydrolysis of polychlorobenzenes result chiefly in similar o- and p-substituted products, other routes must be used to prepare substituted chlorophenols such as m-chlorophenol and 3,5-dichlorophenol. Two methods are commonly utilized. The first of these involves preparation of the diazonium salt of a chlorinated aniline, and its subsequent conversion to the desired product. The preparation of 3,5-dichlorophenol can be used as an example. 3 ,5-dichlorophenol The other method can be illustrated by the preparation of m-chlorophenol. It involves sulfonation, followed by removal of the sulfonic acid group, as illustrated below: Cl Cl Cl Cl m-chlorophenol Either method can be used for the preparation of m-substitutcd products, depending on the starting materials. These processes are, of course, more expensive than direct Vol.5 CHLOROPHENOLS 329 chlorination or the hydrolysis of polychlorobenzenes. For this reason, wi-substituted chlorophenols have never reached large-scale commercial volume. Analysis Several of the classic analytical methods used for phenol can be applied to the chlorophenols. When the ortho or para, or both, positions are open, the bromination method (17) may be used. The acetylation or phthalation of the hydroxyl group is an applicable method. The higher chlorophenols, ie, tri-, tetra-, and pentachlorophengl, have a pH sufficiently low to permit their being titrated potentiometrically with standardized sodium hydroxide solutions in the presence of appropriate indicators. For instance, 2,4,5-trichlorophenol is dissolved in an excess of aqueous standard NaOH solution and the excess NaOH is titrated with standard hydrochloric acid solution; thymolphthalein serves as the indicator. Tetra- and pentachlorophenol can be titrated directly by dissolving the sample in alcohol and adding water in the amount of 50% of the volume of alcohol used. Aqueous, standard NaOH solution is employed; metacresol purple is a suitable indicator. These methods, as well as other chemical .analyses, are well covered in the literature (18). ` Infrared spectroscopy is particularly important hi that it can be used to identify and determine the individual chlorophenols. An excellent review on the use of these spectrographic techniques on phenols in general has been published by Bellamy (19). These materials may also be assayed by determining the quantity of chlorine by one h of the standard chloride determinations after ah ignition procedure. Depending on 51 the degree of accuracy'required, the ignition can be carried out in an oxygen bomb, in the Parr sodium peroxide bomb, or by calcium hydroxide ignition. This last type of ignition is particularly appropriate for the determination of chlorophenols in wood, paper, pulp, etc. In this application, it should be realized that chlorides, as a rule, are naturally present, in these products, and a blank should be run on some of the same material that has not been treated with the chlorophenol. Other methods include ultraviolet absorption techniques; these are also applicable to both the chlorophenols or the derivatives formed from the chlorophenols. Various colorimetric procedures are available, such as the Gibbs method, inwhich 2,6-dibromoquinone chloroimide is used. The chlorophenols can often be separated from other materials by distillation or by absorption on suitable ion exchange resins. Individual Products o-ChlorophenoI is sparingly soluble in water at room temperature (<0.1 g/100 g); soluble in ethanol and ether (>200 g/100 g); and volatile with steam, -even from aqueous sodium hydroxide solutions. Melting point is not a good criterion of purity, since 20% phenol present as an impurity lowers the melting point only 1.7C. o-Chlorophenol is made most economically by the direct chlorination of phenol with molecular chlorine or by the hydrolysis of o-dichlorobenzene. Direct chlorination is carried out by passing gaseous chlorine into molten phenol a t temperatures of 50150C. This process has two disadvantages: (1) Chlorine ratios must be controlled to avoid the formation of di- and trisubstituted products; and (2) p-chlorophenol is also produced. Considerable work has been done to develop conditions favoring ortho substitution; however, no positive ways have been found. Chlorination temperature 5457 330 CHLOROPHENOLS or the presence of solvents has little effect. The chlorophenols can be separated from unreacted phenol by adding aqueous sodium carbonate to the reaction mixture, thus converting the chlorophenols to the sodium salts. The unreacted phenol is then re moved by extraction. After acidification of the sodium chlorophienoxides, washing, and drying, o-chlorophenol is separated from p-chlorophenol by fractional distillation, since the difference in boiling points is greater than 45C. Various methods have been proposed for the partial hydrolysis of o-dichlorobenzene to o-chlorophenol. The hydrolysis can be carried out in the vapor phase with steam and finely divided silicates, bauxite, magnesite, etc, a t 500-700 C, or in aqueous methanol or ethanol a t high pressures and temperatures in the presence of alkali, alkaline earths, or alkaline carbonates. Both of these procedures depend on distillation to recover pure products. Both likewise require strict control to avoid complete hydrolysis to o-dihydroxybenzene (catechol); however, the liquid-phase hydrolysis results in the formation, as by-products, of some methyl or ethyl ethers of th e chlorophenol. More exotic synthetic routes for the preparation of o-chlorophenol have also been studied. These include chlorination of phenol with ethyl hypochlorite (20) or N,N'dichlorourea (21), diazotization and subsequent decomposition of o-chloroaniline (22), reaction of 2-chlorophenol-4-sulfonic acid with water a t 180-200 C under pressure (23), and reaction of chlorine with p-phenolsulfonic acid in nitrobenzene at 55C (2&: o-Chlorophenol can be further chlorinated to 2,4- and 2,6-dichlorophcnol, 2,4,6trichlorophenol, and pentachlorophenol. I t can be brominated, iodinated, and nitrated to the mono- and disubstituted derivatives. Condensation of o-chlorophenol with formaldehyde under the proper conditions produces phenolic resin intermediates, or the reaction can be controlled to form 3,3'-dichloro-4,4'-dihydroxydiphenylmethanc. Treatment of o-chlorophenol with methyl chloride or methyl bromide in the RcimcrTiemann reaction gives 3-chloro-2-hydroxybcnzaldehyde and 3-chloro-4-hydroxybcnzaldehyde, and reaction with phthalic anhydride yields dichlorophenolphthalein. The ring hydroxyl of chlorophenols behaves very much the same as that of phenol in etherification reactions, Claisen condensations, allylic rearrangements, esterifications with phosphorus oxychloride, etc. In the United States most of the o-chlorophenol th a t is used commercially is recovered as a by-product from the manufacture of pchlorophenol by direct chlorination. Only small quantities are sold, and most of the production is used as an intermediate to higher chlorophenols. p-Chlorophenol has a disagreeable, persistent odor. I t is sparingly soluble in water (2.71 g/100 g water) a t room temperature, but is readily soluble in ethanol, ether, benzene, chloroform, and carbon disulfide. Like o-chlorophenol, it can be volatilized with steam even from aqueous sodium hydroxide solutions. jp-Chlorophenol can be prepared by the same general routes described above for o-chlorophenol, except th at proper positional isomers of halogenated benzenes are required when the latter serve as the starting materials. Commercial production is chiefly via the route of direct chlorination of phenol (see the procedure for o-chloro phenol). p-Chlorophenol undergoes the same reactions as described for o-chlorophcnol or, more generally, the same reactions as phenol itself. In the United States, the majority of p-chlorophenol produced is utilized as a raw material for the manufacture of other materials. Since further chlorination of p-chlorophenol results in a high con version to the 2,4-isomer (chlorination of o-chlorophenol yields a mixture of .2,4- Vol.5 CHLOROPHENOLS 331 and 2,6-isomcrs), large quantities of p-chlorophcnol are used to manufacture 2,4dichloropheuol. Reaction of p-chlorophcnol with phtlmlic anhydride and concen trated sulfuric acid results in the formation of 1,4-dihydroxyanthraquinone . (quinizarin). Reaction with 4-sulfophthalic anhydride in the presence of sulfuric acid results in l,4-dihydroxy-6-anthraquinonesuIfonic acid (6-suifoquinizarin). Likewise, p-chlorophenol reacts with a-alkylacetoacetates in the presence of phos phorus pentoxide to produce chromones. For example, p-chlorophenol and ethyl a-ethylacetoacetate give 6-chloro-3-ethyl-2-methylchromone. p-Chlorophenol couples with diazonium salts, such as.benzenediazonium chloride, to form products which, upon acidification, give chloro-hydroxy-azo derivatives such as 4-chloro-2-(benzeneazo)phenol. I t also reacts with indene hydrochloride, o-nitrobenzenesulfonic acids, etc, to produce indophenols. The above materials are useful in the manufacture of dyes and pigments. The reaction of p-chlorophenol and benzyl chloride gives 4-chloro-a-phenol-ocresol (Santophen I), a widely used germicide th a t is sold by the Monsanto Com pany. . p-Chlorophenol can be reacted with ethyl chloride to produce p-chlorophenetole, which can then be treated with ammonia to produce phenetidine. Phc; netidine can then be converted to the N-acetyl derivative, acetophenetidinc (phena cetin), a commercial analgesic (see Vol. 2, p. 390). p-Chlorophenol can be reacted with p-chlorobenzenesulfonic chloride to produce p-chlorophenyl-p-chlorobenzenesulfonate, a miticide sold under the trademark Ovex by The Dow Chemical'Company. In addition to its use as a chemical intermediate, p-chlorophenol is employed as a selective solvent in refining mineral oils and as a denaturant for ethanol. Certain salts, such as those of sodium, potassium or copper, are used as antigumming agents for gasoline, wash liquids for fuel gas purification, and germicides. p-Chlorophenol and its alkali metal salts are considered to be better germicides than p-cresol or phenol. United States production figures are not available for this material, but quoted prices approximate 38#f/lb in drum quantities. m-Chlorophenol has an odor similar to phenol and is almost insoluble in water (260 mg/100 g), but is soluble in ethanol, ether, benzene, and carbon disulfide. Likep- and o-chlorophenol, m-chlorophenol is soluble in sodium carbonate solutions a t room temperature and is reprecipitated by the addition of carbon dioxide. m-Chlorophenol can be synthesized by the diazotization of m-chloroaniline (prepared by the chlorination of nitrobenzene and subsequent reduction of the nitro group) followed by treatment with water. Yields of 60-65% have been reported (25). Hodgeson (26) has described a commercial method for the manufacture of m-chlorophenol by the diazotization of m-chloroaniline sulfate, followed by treatment with boiling water and sulfuric acid. During the latter step, steam is introduced into the reaction mixture, and the chlorophenol is steam-distilled out as soon as it is formed. m-Chlorophenol may also be prepared by treating m-chlorobenzene with water in the presence of methanol and dimethyl ether a t 180C for 30 hr. m-Chlorophenol on fusion with potassium hydroxide can be converted to mdihydroxybenzene (resorcinol). m-Chlorophenol will react with formaldehyde to form phenol-formaldehyde resins. Upon curing, the resulting products have a higher degree of fire retardancy than similar resins prepared from phenol. The high price of m-chlorophenol has prohibited its use in resorcinol manufacture or the preparation of any phenol-formaldehyde resins except specialty items. m-Chlorophenol reacts much the same as o- or p-chlorophenol, and phenol itself. Most of the derivatives described for these materials have been prepared from m-chlorophenol also. 332 CHLOROPHENOLS -2,4-Diehlorophenol has an unpleasant, persistent odor suggesting iodoform and is sparingly soluble in water but easily soluble in ethanol, ether, benzene, and methyl chloride. Generally, 2,4-dichlorophenol behaves as a weak acid and is soluble in aqueous alkaline solutions. It is volatile with steam but, unlike the raonochlorophenols, is not volatile from aqueous alkaline solutions. This difference affords a method for separating monochlorophenols from dichlorophenols. 2.4- Dichlorophenol may be prepared by the direct chlorination of phenol with two moles of chlorine. The resulting product usually contains some 2,6-dichlorophenol and 2,4,6-trichlorophenol as by-products. Foster and Bennett (27) have patented a manufacturing process whereby chlorine is passed into molten phenol at 80-100C until a product with a melting point of 34-36C is obtained. In this method, only very small amounts of the 2,6-isomer and trichlorophenol are formed; 2,4-dichloro phenol may be isolated in 80-90% yield. 2,4-Dichlorophenol of high purity may also be manufactured by the chlorination of p-chlorophenol; however, close control of the chlorine ratio is required to avoid the incidental preparation of trichlorophenols. The further chlorination of o-chlorophenol results in mixtures of the 2,4- and 2,6isomers; thus, o-chlorophenol is not normally used as a raw material for the commercial manufacture of pure 2,4-dichlorophenol. 2,4-Dichlorophenol can also be prepared from phenol and NjiV'-dichlorourea in HC1 solution (21) or from phenol and SOjClj (28). s 2.4- Dichlorophenol may be brominated, iodated, and nitrated, with substitution occurring in the 6 position. It yields condensation products with Formalin (37% J. formaldehyde solution in water) alone, Formalin in hydrochloric acid, etc. The product 2,2,-dihydroxy-3,5,3,,5'-tetrachlorodiphenylmethane may also be obtained by the reaction of 2,4-dichlorophenol with methylal, methylene diacetate, or methylene diiodide in the presence of sulfuric acid. I t has found application as a mothproofing compound, antiseptic, and seed disinfectant. 2,4-Dichlorophenol may be reacted with benzenesulfonyl chloride to prepare 2,4-dichlorophenyl benzenesulfonate. This compound is sold by Allied Chemical Corporation under the trademark Genite-EM923, as a miticide for fruit trees. The most important use for 2,4-dichlorophenol is in the manufacture of 2,4-dichlorophenoxyacetic acid (2,4-D) and its derivatives. Commercial synthesis of 2,4-D involves reacting the 2,4-dichlorophenol with chloroacetic acid in the presence of sodium hydroxide. The powerful plant-growth regulatory activity of 2,4-D was discovered at the Boyce Thompson Institute in the early 1940s and initial production occurred in 1946. In 1962, approximately 43,000,000 lb of 2.4- D was produced in the United States. While it is also possible to produce 2,4-D by the chlorination of phenoxyacetic acid, the yields are 70% or lower and it is difficult to purify the product. Reaction with alkali metal salts results in 2,4-dichlorophenates, which have found utility as germicides, antiseptics, etc. The germicidal activity of 2.4- dichlorophenol and its salts has been discussed in detail in the literature (3,29). Based on estimates from 2,4-D manufacture, United States production of 2,4-dichloro phenol amounted to 32,000,000 lb in 1962. Average quoted selling price was 32^/lb in drum quantities. 2,6-Dichlorophenol has a penetrating odor resembling o-chlorophenol or, if. dilute, iodoform. I t can be crystallized from petroleum ether, is miscible with ethanol and diethyl ether, and is volatile with steam. 2,6-Dichlorophenol can be prepared by the chlorination of p-hydroxybenzoic acid in acetic acid, followed by the elimination of carbon dioxide by heating in quinoline, or by the sulfonation of phenol, followed by Vol.5 CHLOROPHENOLS 333 chlorination in- nitrobenzene and final removal of the sulfonic acid by hydrolysis. Mixtures containing 2,6-dichlorophcnol can be obtained by the further chlorination of o-chlorophenol; however, 2,4-dichlorophenol and 2,4,6-trichlorophenol are also formed. Although 2,0-dichlorophcnol undergoes the same reactions as 2,4-dichloro phenol, it has found no commercial application. Material produced in the United States coincidentally with other chlorinated phenols is used primarily as starting material for the manufacture of trichlorophcnols, tetrachlorophenols, and penta- chlorophenol. ' *- 2,5-Dichlorophenol has a strong, persistent phenolic odor, is sparingly soluble in water, and is easily soluble in ethanol, diethyl ether, and benzene. I t is volatile with steam. 2,5-Dichlorophenol can be prepared in 90%.yield by the diazotization of 2,5- dichloroaniline followed by decomposition of the diazonium salt with boiling water and sulfuric acid. Agfa Chemical Company has a patent (30) for the manufacture of 2,5-dichlorophenol by the hydrolysis of 1,2,4-trichlorobenzene in the presence of sodium methylate a t 180C. The material has found no commercial utility. 3.4- Dichlorophenol is soluble in benzene, ethanol, diethyl ether, and petroleum ether, and is volatile with steam. 3,4-Dichlorophcnol can be prepared by the diazotiza- ^Jjon of 3,4-dichloroaniline followed by the hydrolysis of the diazonium salt, and by the hydrolysis of 4-fluoro-l,2-dichlorobenzene in the presence of sodium methylate (31). I t reacts with phthalic anhydride in the presence of sulfuric acid and boric acid to produce 2-chloro-l,4-dihydroxyanthraquinone (2-chloroquinizarin), which can be used as a dye intermediate (32). Further chlorination results in 2,3,4-trichlorophenol. Because of its expensive preparative route, 3,4-dichlorophenol has not enjoyed any commercial activity. 3.5- Dichlorophenol is slightly soluble in cold water and quite soluble in hot water. I t is also soluble in ethanol, diethyl ether, and benzene. 3,5-Dichlorophenol can be prepared by the diazotization of 3,5-dichloroaniline followed by hydrolysis of the diazonium salt. I t can be readily nitrated and brominated to the trinitro and tribromo derivatives. 3,5-Dichlorophenol reacts with formaldehyde to produce a typical phenol-formaldehyde resin composition having a high degree of fire retardancy. Although this product looks extremely interesting, the high cost of the starting chloro- phenol has precluded commercial development. 2,3-Dichlorophenol can be obtained as crystals from petroleum ether. I t is volatile with steam and its odor resembles o-chlorophenol and iodoform. I t can be prepared from 2,3-dichloroaniline via the diazo reaction and from 3-amino-2-chloro- phenol via the Sandmeyer method. This material has not achieved commercial importance. 2.4.6- Trichlorophenol is insoluble in water, but readily soluble in methanol (525 g/100 g), acetone (500 g/100 g), toluene (100 g/100 g), and petroleum solvents (16 g/100 g). I t is volatile with steam but, unlike o- and p-chlorophenol, it is not volatile from alkaline solutions. This offers a method of separating it from the latter mate rials. 2,4,6-Trichlorophenol behaves as an acid and can be titrated with O.liV aqueous sodium hydroxide. 2.4.6- Trichlorophenol is sold in commercial quantities by a number of producers. I t is available from The Dow Chemical Company under the trademark Dowicide 2S. Manufacture is readily accomplished by the direct chlorination of phenol. Mono- and dichlorophenols, obtained as coproducts, are easily removed by distillation be cause of the wide spread in boiling points. These mono- and dichlorinated compounds 334 CHLOROPHENOLS -are subsequently converted to th e desired product by recycling and further chlorina tion. In industry, manufacturers of p-chlorophenol and 2,4-dichlorophenol dispose of by-product o-chlorophenol and 2,6-dichlorophenol by chlorinating to 2,4,6-trichloro- phenol. 2,4,6-Trichlorophenol has also been prepared by the chlorination of phenol with sodium or potassium hypochlorite solutions in the presence of hydrochloric acid, and by the diazotization of 2,4,6-trichloroaniline followed by hydrolysis of the diazo nium salt. Considerable work has been done on the use of 2,4,6-trichlorophenol as a bac tericide and fungicide and in general antiseptic action. I t has been found that 2,4,6- trichlorophenol is a more effective germicide than o- or p-chlorophenol or 2,4-dichloro phenol. The literature provides detailed descriptions of the utility of 2,4,6-trichloro phenol as a wood preservative (9), glue preservative (33), insecticide ingredient (34), bactericide (35), and antimildew treatment for textiles (36). Oxidation of 2,4,6-trichlorophenol results in 2,6-dichlorohydroquinone and/or 2,G-dichloro-l,4-benzoquinone, _ OH 0 or2,(>-bis(2,,4',6/-trichlorophenoxy)-l,4-benzoquinone, depending on the reaction conditions, or the degree of oxidation achieved. Treat ment of 2,4,6-trichlorophenol with chlorine and fuming sulfuric acid results in the preparation of 2,3,5,6-tetrachloro-l,4-benzoquinone (chloranil). Chloranil is used as a seed protectant for a large number of food crops and ornamentals. Further chlorina tion of 2,4,6-trichlorophenol results in 2,3,4,6-tetrachlorophenol and pentachlorophenol. I t can also be brominated to the mono- and disubstituted derivatives. The direct nitration of 2,4,6-trichlorophenol has not been reported, presumably be cause nitration reagents readily oxidize the material to the benzoquinone derivative. 2,4,6-Trichlorophenol reacts with formaldehyde to form the bis(methylene) derivative, and with SCI* to give 3,3'-dihydroxy-2,4,6,2',4',6'-hexachlorodiphenyl sulfide, both soap germicides. The phenolic hydroxyl of 2,4,6-trichlorophenol reacts much the same as that of phenol; ethers, esters, amine adducts, etc. have been prepared. United States production quantities are not available. 2,4,Trichlorophenol can be recovered as colorless needles from ethanol and ligroin. I t is volatile with steam and sublimes. 2,4,5-Trichlorophenol behaves as a weak monobasic acid, but can be titrated with 0.\N aqueous sodium hydroxide. Like its 2,4,6-isomer, 2,4,5-trichlorophenol is available commercially under several trademark designations; Dow sells the product as Dowicide 2. 2,4,5-Trichlorophenol is manufactured by the hydrolysis of 1,2,4,5-tetrachlorobenzene. Hydrolysis is car ried out continuously in a coil reactor at 160C in the presence of methanol and sodium hydroxide. Contact time is approx 7 hr and yields of 80-85% are achieved. The 5462 Vol.5 CHLOROPHENOLS 335 chief impurity is 2,4,5-trichloroanisole, which resists from the reaction of 2,4,5- trichlorophenol with some of the methanol. After the hydrolysis is complete, the reaction mass is extracted with an organic solvent to remove the bulk of the anisole derivative, acidified with hydrochloric acid, and distilled to recover the trichloro- phenol. 2,4,5-Trichlorophenol may also be prepared by the diazotization of 2,4,5- trichloroaniline followed by decomposition of the diazonium salt with water and sul furic acid. 2,4,5-Trichlorophenol forms a sparingly soluble potassium salt, and in the pres ence of toluene and methanolic caustic forms the anhydrous sodium satt^which can be crystallized from water as the pentahydrate. The lithium, calcium, and barium salts have also been prepared. These salts have found utility as fungicides (37) and wood preservatives (9). 2,4,5-Trichlorophenol can be brominated and nitrated to the mono- and disubstituted derivatives. I t reacts with formaldehyde to form the bis- (methylene) derivative, and with sulfuryl chloride to yield the thiobis derivative. Both of these materials have found application as soap germicides. 2,4,5-Trichloro phenol reacts with thiophosphoryl chloride and then with sodium methylate in the presence of methanol to produce 2,4,6-trichlorophenol, 0,0-dimethylphosphorochloro- dithioate, which is used for the systemic control of grubs in cattle. The Dow name for this product is Ronnel. fS- The largest single use for 2,4,5-trichlorophenol is in the manufacture of 2,4,5- trichlorophenoxyacetic acid (2,4,5-T) and derivatives. Commercial preparation in volves reacting the 2,4,5-trichlorophenol with chloroacetic acid and caustic soda in a manner analogous to that used for 2,4-D. 2,4,5-T is used for brush control, being particularly effective against brambles, certain oak species, and woody vines. Total United States production of 2,4,5-T in 1962 was 8,400,000 lb. A closely related use for 2,4,5-trichlorophenol is in the manufacture of a-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP). 2,4,5-TP was introduced in 1953 by The Dow Chemical Company to control brush and bushes resistant to 2,4,5-T, and lias also found use in killing dande lions, plantains, and chickweed. Technical grades of 2,4,5-trichlorophenol and its sodium salt are sold in flake form for germicidal use. Total United States production in 1962 amounted to 12,000,000 lb. 2.3.4- Trichlorophenol is too weakly acid to be titrated, although it is soluble in alkaline solutions. 2,3,4-Trichlorophenol can be prepared by the direct chlorination of 3,4-dichlorophenol or by the nitration and reduction of 1,2,3-trichlorobenzene followed by diazotization and hydrolysis. I t has achieved no commercial importance. 2.3.5- Trichlorophenol is volatile with steam, and can be titrated with 0.17/ sodium hydroxide using phenolphthalein as an indicator. When 2,3,5-trichlorophcnol is dissolved in hot solvents and cooled, gels result. 2,3,5-Trichlorophenol can be prepared from 2,3,5-trichloroaniline by diazotization and subsequent hydrolysis of the diazonium salt. This material has no commercial utility. 3.4.5- TrichlorophenoI is too weakly acid to be titrated, although it is soluble in alkaline solutions. 3,4,5-Trichlorophcnol can he prepared by the diazotization of 3,4,5-trichloroaniline, followed by decomposition of the resulting salt, and also by the cleavage of 3,4,5-trichloroanisole. I t has not achieved any commercial importance. 2,3,4,6-Tetrachlorophenol is insoluble in cold water (100 ppm at 25C), partially soluble in hot water (500 ppm a t 70C), and soluble in methanol (319 g/100 g), acetone (570 g/100 g), and toluene (175 g/100 g). 2,3,4,6-Tetrachlorophenol bcliaves as an acid, and can be titrated with 0.11Vaqueous sodium hydroxide. 546 336 CHLOROPHENOLS 2,3,4,6-Tetrachlorophenol is sold by The Dow .Chemical Company under the . designation Dowicide 6. It-can be prepared by the direct chlorination of phenol; however, long reaction periods are required to achieve the desired product. Reaction time can be shortened by using catalysts such as iodine, antimony chloride, or iron chloride. Highest yields of relatively pure product are achieved through the chlo rination of 2,4,6-trichlorophenol. In industry, the starting material for the production of 2,3,4,6-tetrachlorophenol can be a mixture containing o-chlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol. These are materials available if the company is a primary producer of p-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol. 2.3.4.6- Tetrachlorophenol can also be prepared by the reduction of 1,2,4,4,5,6,6heptachlorocyclohexen-l-one-3 with stannous chloride/hydrochloric acid/acetic acid or with potassium iodide/acetic acid. Alkali salts are easily prepared in mixtures of organic solvents. The chief applications of 2,3,4,6-tetrachlorophenol and its salts include use as bactericides for latex preservation (38), insecticides, wood preservatives (34), and leather preservatives (4). In 1960, approximately 9,000,000 lb of 2,3,4,6tetrachlorophenol was produced and sold in the United States either as the phenol or as salts. 2,3,4,5-Tetrachlorophenol behaves as a weak acid with a neutral equivalent of 232. 2,3,4,5-Tetrachlorophenol can be prepared by the diazotization of 2,3,4,5-tetrachloroaniline, followed by hydrolysis of the diazonium salt. I t can also be prepared by the hydrolysis of pentachlorobenzene in the presence of methanol and sodium methylate. Ith a s found no commercial utility. 2^1,5,6-Tetrachlorophenol is a fairly strong acid and can be directly titrated with 0.11V aqueous sodium hydroxide having a neutral equivalent of 232. 2,3,5,6-Tetrachlorophenol can be prepared from 2,3,5,6-tetrachloroaniline via diazotization and subsequent hydrolysis of the diazonium salt. I t is also produced by the hydrolysis of the pentachlorobenzene as a coproduct with 2,3,4,5-tetrachlorophenol. I t has found no commercial utility. Pentachlorophenol is insoluble in water, but is soluble in acetone (53 g/100 g), diethyl ether (158 g/100 g), ethanol (143 g/100 g), methanol (202 g/100 g), and toluene (16 g/100 g). Pentachlorophenol has a low odor level when cold, but a strong pungent odor when hot. Pentachlorophenol is acidic, and in alcohol solution titrates quantitatively (bromothymol blue indicator). I t has a pK . of 4.86. I t is slightly volatile with steam, but not from alkaline solutions. Pentachlorophenol is sold by The Dow Chemical Company under the designation Dowicide 7. I t can be manufactured by the direct chlorination of phenol or polychlorophenols. One commercial process (39) uses a mixture of phenol, o-chlorophenol, 2.6- dichlorophenol, and 2,4,6-trichlorophenol as the stalling material. Chlorination is carried out in the absence of solvents and catalyst until tetrachlorination is achieved. At th at point, a catalyst such as FeClj, AlClj, SbClj, etc, is added to take the reaction to pentachlorophenol. The end point of the reaction is determined by the freezing point. When the proper freezing point is achieved, the chlorination is stopped, and the material is flaked and packaged. Pentachlorophenol is also manufactured by the hydrolysis of hexachlorobenzene with 5-15% sodium hydroxide in methanol a t ISOM O ^ (40). The hexachlorobenzene is easily prepared by the liquid-phase ironcatalyzed chlorination of benzene. Metallic salts are easily prepared by reacting pentachlorophenol with the appropriate base. 5464 Vol.5 CHLOEOPHENOLS 337 Pentachlorophenol is a broad-spectrum biotoxicant. I t is too phytotoxic to use on crops. As a fungicide and insecticide, its chief use is in wood preservation, where it . competes with creosote. Wood preservation with pentachlorophenol started in the late 1930s. An excellent detailed review of pentachlorophenol and its salts in wood treatment has been written by Sproule (41). Pentachlorophenol is quite dusty and exposure to the dust can lead to respiratory difficulties. To avoid this problem, several companies produce an oiled pentachlorophenol. United States production in 1962 was approximately 39,000,000 lb; the average selling price was 22ji/lb in drum quantities. 4-Chloro-2-methylphenol melts a t 48-49 C. I t can be manufactured by the direct chlorination of o-cresol a t low temperatures without catalyst or solvents. The resulting product is a mixture of 60-65% 4-chloro-o-cresol and 35-40% 6-chloro-ocresol. A purer product can be manufactured by using sulfuryl chloride as the chlo rinating agent. If this process is used along with catalysts such as ferric or aluminum chloride, the 4-chloro-2-methylphenol content can be raised to as high as 90% in the crude chlorinated product (42). The use of sulfuryl chloride can be made still more economical by mixing the off-gas from the chlorination with chlorine and passing the mixture through charcoal where the sulfur dioxide is reconverted to sulfuryl chloride '"X43). The chief use for 4-chloro-2-methylphenol is in the manufacture of 2-methyl-4chlorophenoxyacetic acid (MCPA) and its derivatives. MCPA was developed con currently with 2,4-D as a plant-growth regulator. MCPA is produced by a process analogous to th a t used for 2,4-D, eg, 4-chloro-2-methylphenol is reacted with chloroacetic acid in the presence of sodium hydroxide. The MCPA formulation is usually sold as the potassium salt and occasionally as the sodium salt. These salt formula tions have the advantage over 2,4-D amine salts that they are more water-soluble. MCPA and derivatives are widely used in Europe, but have not found wide acceptance in the United States. Agricultural methods and climatic conditions are probable reasons for this. 4-Chloro-3,5-dimethylphenoI. This product melts at 115-116C and boils a t 246C a t 760 mm Hg. I t is relatively insoluble in water, but'is soluble in ethanol (86 g/100 g) and isopropyl alcohol (50 g/100 g) and slightly soluble in benzene (6 g/100 g) and glycerine (1.5 g/100 g). 4-Chloro-3,5-dimethylphenol can be prepared by the chlorination of 3,5-dimethylphenol with SOiCl* in chloroform solution or with chlorine in acetic acid solution. Yields of 65-70% have been achieved. I t should be noted th at 2-chloro-3,5-dimethylphenol is also formed as a coproduct in this reaction and must be removed to prepare the pure 4-chloro derivative. 4-Chloro-3,5-dimethylphenol has received considerable attention as an antiseptic, disinfectant, pre servative, bactericide, etc. The material has found utility as a preservative in latex paints, adhesives, cosmetics, and pharmaceuticals. It has also found utility as an antiseptic in liniments, liquid soaps, waterless hand cleaners, etc (44). Its utility has been enhanced due to its relatively nontoxic nature. 4-Chloro-3,5-dimethylphenol is noncorrosive, nonirritating, and nonsensitizing; therefore, it can be used in many applications where other chlorinated phenols cannot be tolerated. Joseph (45) has carried out detailed toxicological studies on this material. 4-Chloro-3,5-dimethylphenol is manufactured and sold in the United States as well as in Europe. The product is commercially available and sold under the name Ottasept by Ottawa Chemical Company. 546 5 338 CHLOROPHENOLS Bibliography "Chlorophenole" in E O T le t ed. under "Phenol an d Phenols," Vol. 10, pp. 317-320, by Calvin Golumbic, Bureau of M ines, U B . D epartm ent of th e In terio r. 1. J . P . Landuska, J . H . Cochran, and F . A. M orton, J . Econ. Entomol. 39,767 (1946). 2. L . C. F elton and C. B . M cLaughlin, J . Org. Chem. 12, 298 (1947). 3. E . J . Ordal, Proc. Soc. Exp., Biol. Med. 47,387 (1924). 4. J. F . Richardson, Hide Leather Shoe* 99,28, 38 (1940). 5. E . J . K raus and J . W. M itchell, Botan. Gaz. 108,301-350 (1947). 6. A. J. N orm an, Botan* Gaz. 107,475 (1946). 7. H . E . Thompson, C. P . Swanson, and A. G. N orm an, Botan. Gaz. 107,470-507 (1946). S. E . H . Siegler and S. I. G ertler, J . Econ. Entomol. 39,662 (1946). 9. U B . P a t. 2,304,013 (D ec. 1942), F. W . N orton (to T he Dow Chem ical Co.). 10. F . L . Brensch and H . K . Eskin, Istanbul Uni. Fen Fek. Mecmuasi, Seri A 7 , 182-1S9 ( 1942). 11. U B . P a t. 2,362,464 (N ov. 1944), E . C. B ritto n and J . N . H anson (to T he Dow Chemical Co.). 12. TJB. P at. 2,382,976 (Aug. 1945), G. H . Coleman (to T he Dow Chem ical Co.). 13. D . Ginsberg, J . A m . Chem. Soe. 73,2723 (1951). 14. F . C. Soper and G. F . Sm ith, J . Chem. Soe. 1926,1582. 15. B. S. P ainter and F . C. Soper, J . Chem. Soc. 1947,342. 16. C. H . Li, J . A m . Chem. Soe. 64,1147 (1942). 17. W . F . K oppeschaar, Z. Anal. Chem. 15,223 (1876). 18. J . M itchell e t a l., Organic Analyses, 2 vola., Interscience Publishers, In c., New Y ork, 1954. 19. L . J . Bellam y, The Infra-Red Spectra o f Complex Molecules, John W iley & Sons, In c., New York, 1959. 20. S . G oldschm idt e t a l., Chem. Ber. 58,576 (1925). 21. rM . V. Likhoserstov, Zh. Russ. Fiz. K him . Obshcheslva 61,1019-1023 (1929). t f F . K . B eilstein, A nn. 176, 39 (1875). 23. Ger. P a t. 141,751 (M ay 1903), (to H azard-FIam m and). 24. R . C. H uston and A. H . Neeley, J . A m . Chem. Soe. 57, 2176 (1935). 25. H. Hollem an, Rec. Tras. Chim. 30, 81-82 (1911). 26. B rit. P at. 200,714 (Aug. 1923), H . H . H odgeson (to B ritish D yestuffs Corp.). 27. U B . P a t. 2,440,602 (1948), R . T . F o ster and N . B ennett (to Im perial Chemical Industries, L td.). 28. W . Steinkopf, W . M ieg, and J . H erold, Chem. Ber. 53,1145 (1920). 29. B rit. P a t. 356,192 (O ct. 1931) (to I . G. Farbenindustrie A .G .). 30. G er. P a t. 349,794 (M arch 1922) (to Agfa Chem icals Co.). 31. R . DeCram o, Rec. Trao. Chim. 48,1063 (1929). 32. U B . P a t. 1,655,462 (Jan . 1928), J . G ubelm ann and H . Lee (to N ew port Co.). 33. U B . P at. 1,946,057 (Feb. 1934), E . C. B ritto n and L. E . M ills (to T he Dow Chemical Co.). 34. U .S. P at. 2,161,654 (June 1939), G. H . E llis (to In su lite C o.). 35. F r. P a t. 709,788 (Aug. 1931), (to I. G. Farbenindustrie A .G .). 36. B rit. P a t. 259,690 (1926), (to B ritish D yestuffs C orp.). 37. U B . P a t. 1,955,080 (A pril 1934), J . F . M ills (to T he Dow Chem ical Co.). 38. B rit. P a t. 530,836 (D ec. 1940), (to M onsanto Chem ical Co.). 39. U B . P o t. 2,131,259 (Sept. 1938), W . C . Stoesscr (to T he Dow Chem ical Co.). 40. U B . P a t. 2,107,650 (Feb. 193S), F . B. Sm ith and J. E . L ivak (to T he Dow Chemical Co.). 41. J. Sproulc, Pest Teehnol. 41,3 (Nov. 1960). 42. B rit. P at. 755,676 (A pril 1954), (to M onsanto Chem ical Co.). 43. B rit. P at. 800,630 (Aug. 1958), H. F . Bandy and R . P . M arshall (to Coalite and Chcmie:d Fcitducts, L td.). 44. OUasrpt, T he O ttnw a Chemical Co., Toledo, Ohio. 45. J . M. Joseph, J . Amer. Pharm. Assoc., Sei. Ed. 41,595 (N ov. 1902). J. D . D o ed en s The Dow Chemical Company 5S 5467 a n u o V / y * l THE DOW C H E M I C A L CO M P AN Y '"MIDLAND, M ichigan M arch 15, 1965 H. L. Gordon, M.D. Medical Department 607 Building cc: EVDDKHDDLCT3......rrrr....6GMRKJP.BRCH2...... 5....RHSAOO-BDGH6do'oliaHswl6....yvmo6ealeKSHGens8,r\,Arr1taeoaBes-y,BlBmB4dw't,iBoeXiieioeaoociMirorcnrchc,,tchehhe,,hdeMMeemeBiMmmmceeimicaeoiddiicacclidciicaccaalhiDaaaclllReealllRRRmeplDDReeesaiReeceesssrteeeseaappm.asaaralacerrerrrRhcccLttanmmhhhaertcL,seebhLLL.anno6aaabttrLL0,,bbbao7aa..otr66,,obbarB00ra..t77y,,11out77,oriBB006y1lrd110y7,1uui70,7niiBB10ll1ddgB171uuBii07unniiB01llugigdd1ludiBiilnndiiBlnudggiungiilnidgldginingg Oo 4 wJ'J' BTSEHUNEBZCEFUONTAERNMEAOTUIOSNINOJFECFTOILOLNICOUFLISTYISMMINETRTIHCEALRTAEBTBRITACEHALRORFOODLLIBOEWNIZNOGDIAOXSININGINLE Problem Tdjeoiocpxtirinongd. ucvearifooulsl i ccuonl ict3ei sntriantiotnhes roaf bsbyimt meaertribcyalsutebtcrautcahnleooroudsliyb einnzo M a te ria ls Used 00db.e.i0b0n02ez0ne0Pnz1eoe.rdpicoeexr nincte, Ins0to.0ala1ntdepd0e.r0fr0co0em0n0t2c, a0pu.es0rt0ic1cepInnetsroscylumebnmlte,et0ori.c0ila0.l01teVptereahricchcleelonr-to, Experimental Procedure w0scuo.a0sbn1cciuenMtcnaotnirrleaploitoiluoirtsaneltyerodifnosifnryatemoabmcbhtehittesrieocsaalutrlustdit.oeynt.rAadccOehonsleconrrtirorbaodebldibbreiaatnbbwzobvoaiesdt iwofuosaxesrind.tinhoejne cestaoecldvhe n t A t o t a l o f seven anim als was used In the e v a lu a tio n . GG0430S JOW 749556 H. L. Gordon, M.D. 2 March 15 19^5 R esults and Conclusions - Ra0pnr.e0odsd1uuthlcmtesaldt ooaaff 0sdthe.i0esv2cesereptrunedfriobyclleeIlnincdftouicsllayliittmeci sdmu eldtithteriiavcsetalloaaptfeestdeturbraacceufhttleaelonvrreeoon2du1isdbdaeyaInnsyzjseoe. cxdptiioooxsnuinreo f p0dA.oil0sss0cou1e,rerpnt.heibarCltecoaennfncoteilnpnl ijrtceoradcutuitolicionet nidss aoleufssns0dli.eg0trh1hatthnmeflo0oc.l0olf0inc1adusiptlileiootrnwi sscaeaonctfftoeptnhrrcoeed2un6ectxredpadaetyirosinmnoeeaxnst.. BC17i.o0c1Ph.BeOmu'iiHlcdaairnlegResearch Laboratory BK17.io0cJ1h. BeOmuliislcdoaninl gResearch Laboratory s jl attachm en t 5469 GG04307 Table of R e su lts I F irs t Exposed 2/11/65 NAunmimbaerl 752 756 758 760 762 764 765 RCAnaadgcek SS toreluntgiotnh .. <w. 134-1 0.02 -2 0.01 -3 0.001 -4 0.0001 -5 0.00001 -6 0.000002 -7 Benzene FDaotellico uf lFitiirss t 2/22/65 2/25/65 /3 9/65 None None None None Appearance At Autopsy Severe f o llic u lit is Moderate f o llic u lit is S light fo llic u litis None None None None Autopsy Lung L ive r OK + OK OK O1 K 1 OK OK OK OK OK OK + OK OK Kidney OK OK OK OK OK OK OK 5470 0004308 LQ56fH, MOO . DOW 749558 Table of R esu lts F irs t exposed 2/11/65 Adunmimbaerl CRAnaadgcek SStor(el*un)tgiotnh DF aotellicOuf lFitiirss t Appearange At Autopsy 752 134-1 0.02 2/22/65 Severe f o llic u lit is 756 -2 0.01 2/25/65 Moderate f o llic u lit is 75 -3 0.001 3/9/65 S light fo llic u litis 760 -4 0.0001 None None 762 -5 0.00001 None None 764 -6 0.000002 None None 765 -7 Benzene None None Lung OK OK OK OK OK OK OK ^i LO Autopsy L iv e r + OK OK OK OK + OK Kidney OK OK OK OK OK OK OK 0004309 l 5k S te . MN069799 i `a _ \3&A6 -G<bei - ( THE DOW C H E M I C A L COMPANY MMIaDrLcAhN2D6, , M1i9c6h5igan B9SG KM O U HM60.e7dLBi.cauGlilodDridenopgna,rtmMe. nDt . cc: EVHHBRKCLSC........... EBDRHJGMKGP........... RAHHSKSGSOOdotaroaoli'eaasHwldyylwvmomd,eelaeeans,ekerMi*,r1reArrfBs,,c,.BBBB^Dt,ioeBMiMiiio.ooocM,ii..cnoccchDD.hhhhceM,D..eheee,m,.eBme,mmmdiMMmicoiiiiiMccccaceeciacaaaaleddhallllldiiReccliRRRDRmecaaReeeesaellieecssssplDDeeeeaasaDeaaaaerelrctaerrrrppmRhccccrpaahhhhceearrhLttsnrmmLLLLatetmL,aaaabeaeabbbbenrn.6,cb.n...tt0,,h,,,,.t,71,, 6761111B00770776111700000777u1111700iBBB11ldBBBBBuuuBBiuuuuuniiillluuiidiiiddglllllddddidiiiillnnnddiiiiinnnnngggiinnggggggg .T 3 6 .2 q -6 6 6 8 i-6 \ AFTOOHPILERPMLIIANCTEAIFOTFINOENCOSTFIVFOEOFNLE2LS,ISC3 U,O7LF,I8TW-ITSAESTIHNRINAGTCHHAELS0RRAA0BDPBRIIBTEEVENEAZNR0T-IpVF-OEDLILM0OXEWAINSINUGIRNERCEINOPERTANHTEED Problem wwadAtTsinroaoaiaoppssltvextrrhiheoexooiinenavpnfngmihotsiri.innuyanthlsetobaeeTofbcrhrthtiieeme2tilceps,am3eitonmet,area7tftreofwrt,aeeib8a(hscTrey-uliittc3mariehvefr6lrpeteo.rlp2oiaiinmnsety5cea-hs6srttitshhelse6iomdee6lraoe8oiaec1tlfsdaiaop-vsiw2rbrnpee)aetlwntlirseyncsohthcauizeliahnmsnotilngoool-myaipflqnuwap-usrfbIdpiineoztlilhesoesioledoxlfdibsiilcnnuovythuabainepabirllneiepiotaeb.npuincezfslsdnfyoeezinr.nnwepccegnor.taoenotdcoteihuaeIlrfce.tsneadsa M aterials Used d0.i0o1x inPeIrsocleantte dandfro0m.00c1aupsetirc ceInnsto2lu, 3b,le7 , 8o-itle.tra ch lo ro d ib e n zo -p - Solvent -- Corn o il Soap -- D ia l Tap water 0004311 5473 Dr. Gordon 2 March 26, 1965 J5W 74956 Washing Procedure wEanaadrtsesrwu, bearsefetqetuhreownrothluyicghrhinlytsheewdyaswhineedrecwobpitilohouttsreicdahmdosruyonatwps itolahfthpfreaepreeflryo-trfolow30welissne.gco' ntadps Exposure Times Selected For Study (1) Fscivreibesdecoanbodvse (toimn era tbabkeitnetaor)u. niform ly spread so lu tio n s de (2) Two hours. (3) Four hours. Experimental Procedure wmabvrtTtieiehmeleoelpeonrfaneoertsweser.usewiufrnefesdeegwneCercedwtasoettacsihndvsiirhnnetierbicnbsoneomygttlehudsr.psaedaosaftieeobTrecdarwohorvitvoeaafeeeimllndwaauittcnaacoohItiasthmnihloetrcraioeannchlmsg.lobeatmnabhbwwiciein,etceitrahnefbselotytarucusrradsorsoteeaycinodpp--.ocneneatatonnaA0rtdnteo.rd1dtdalolweeltyacitmaodactnoape/lenppraasaoeprnatrfrltrd/syiaiodc1itndae8aufogsylapaer oa--tnrrhotcifhmehepeatxahinpsmplydssoooelsaoliunuclrtteic Results And Conclusions WRabpineehecndersozniusercpclnceteoseonmrrtnahopata2afebs,rde3ttleehd,na7isssft,oo8s-ttlhhtatleuieectdpruydasreeoclvivilhnvtieolidelosouinrpcsotmaa,dstseeirbinmeatetshnirouleaazfltosrtsfu-oecplIoxt-lndlpnidoeiccoifrecuixmnasilnttieirxetanitiettsnitoeh.innnacsotwarnatphhsipecolhlioiculwabsepetianrooszondefusn0ce.0ei0ndw1as24 day weUvnietdhnerwsothhaeepn atchnoedndwdiitaoiotxenirns isiosfqatuhpietpelieeixndpeeffr-fime c-e-tnivt ei t aaspap--e--pa-rres vt-e-h-n-a-t-it-v-ew-am-sheian-sgurrme . BC17.io0c1Ph.BeOmu'iHilcdaairnlegResearch Laboratory B17io0c1hBemu iilcdainl gResearch Laboratory B jl attachment C004S1S Summary o f Results SS torelunt igotnh O00O...00.OO10OJ1l#6JI566 0OOO....0OOO1OOI5*6II5566 OO..OOII5566 OO..OOOOII5566 OOOO....OOOI5OOI656II5566 CCoornntroo li l CCoornnt roo li l ANpupmlibceartiOofn s 11114666 11111466 76 1146 16 1166 16 16 16 EDxpaoyssed 22224444 22210447 1113 2240 22224444 24 24 Washed NNNNoooo AAAAtttt oooonnnncccceeee 22 hhoouurrss 22 hhoouurrss 4444 hhhhoooouuuurrrrssss No 2 hours Ear Appearance MSSSelloiivggdeehhrerttatfffeooo-slllellliiivccceuuurellliiitttfiiiosssl l i c u l i t i s VSSSeeelivvrgyeehrreest lifffgooohlllllltiiicccf ouuu llllliiiitttciiisssu lit i s SSSS(Aeelliinvvggeeimhhrreettalffff ooood illllellll iiiidcccc) uu uu ll ll ii ii tt tt ii ii ss ss MMSS oolliiddggeehhrrttaatteeff oofflloolliicllclliiuucclluuiittlliiiisstt ii ss O.K. O.K. P ira t Appearance of P o lllc u litls A p plica tio ns Days Exposed 12 18 12 16 16 221844 9 13 12 16 16 221844 6 10 6 11 16 11 2147 1155 16 16 21 ' 222441 Ol 0004313 <en1 SH G0l6TtMOa DOW C H EM IC A L C O M P A N Y M IDLAND. M ICHIGAN 4 8 0 4 0 . June 22, 1965 ANALYTICAL METHOD MLW.Gp.ll THE DETERMINATION OP 2 ,3,7,8-TETRACHLORODIBENZO-p-DIOXlN IN 2,4,5-TRICHLOROPHENOXYACETIC ACID BY GAS-LIQUID CHROMATOGRAFHY 1. Scope TltiehmteraidtcTihoohlxfiosirn0om.d5ceiabtnphepomndbezpio'dso- pesa-tsedpibcipotlelexicdinaabatiltneopth2ttioem,4uot,mh5nee-torpdipcpeemhtrelaolrtermioovipnenhal cetiwononointdhxiyotiaafocn2leos,tw3.ice,7ra,8c-id. 2. Principle wscthahiemtrhopm2leTc,ah4hteiol,so5gr2-romat,rfp3eioch,ar7oems,dluo8..rer-otdepTTthhahreenaedcn2choch,lo3xolomy*r7raopo,acd8freoie-btidtmieecnttrozaeaocxcai-htdprlako-nbdcroyotiwodnmixsibiensaecntnasoisnzndoocas-feperndap-tnd.raairoeatextxeditndraanfcirndtoimottnhheen . 3- Safety Precautions t .opShwftismooraerahewlemnvplovmnedeepewrlrlleaese2cnvedst,oit,eod3dnurgassjeti7soabuallola,ysllc8vsygapt-eesasleTodswikcsve,ctith-noeethsirdscaeklaohpcitcnrpcoro(ohera.fnouflvrrooteseb'ecavrrsbsonmocpneinttdob,o-rtinawa,ibtlsaaiielneptnrlsyneyihon.zsa.(lgcpyokdhivo-niDCaplliotynnlie-esrycdyanpacoilcoonltonenisxcaftaeeaaihlnn)tlc.llyehioltnduiirsspcaiedoonwsqednunccuiocat,tiphamhmpImmneapiachnoebtincuhinalmeman.ltlo)dtaaaerwotnlodisafnoifchtteohnecoorr.aqaumnaauultlds,ciaslpWeicmnitttbteoio.ge.meaennreea.ts, 4. Apparatus .. ,' r? ""'Wofiouranllenlizquar((((tuebdactisi)))C)voparnoGSSRelneeayyesdsknrrceii,etonn.ctrggChetdiee-rmceVao,,trrmelo,.i~MHf.rao,-at0orum/nW.g0liirtla5aitilofpk,^inhittno-o,is;m5r--A+Iice1-ecn--c.rq0rso,ou5tgrBilruviamtenieplceehtriol,nnlnAittv'N.,-6aooD0n.ld0tic7,-Dk0R1ifen.uNwss,eliotlahnorscr:pahflnaea, dnmqI,uneCicoovmn.a.,eplea.-snn.eyt*c.,on.' d. . .) 4 C'J<^ O 547^ - V-. ^ DONWU9710 June 22, 1965 2- - mlw.G'j .h Cftruoabmliinf ogW((>hr, n1i)lkfi aiCIivnneesj(enNctfIornetCitfoseeuotrtlrgu1uemiI1mnnnaes,)lne.e1trn/tg8,ant-hidPnycpRrheaexcskOeega.dlDrac.swh, s,it0hfI.no0rcr8e.laA,- ig-n6Wec0nah0tl-nD5Iu..(tDc C)..A,revesaktia,lainb le le s s steel 5. Reagents 0 TrmI(enNheaecsogh.teD,eCo(ti(n(((dgewacbWflhti))))))(rbaoCglmS)C2CrCSPnha.o,oueohoaad3dlmtsullerrjoiioum7rdtiCrrcimijboents8ariofesrol-ohWpdeuTnrCgiyak.pumeiaod,cnmptm,srkrgoACoa,ipnrhAx.avtaCgcnay,inadSlhd,giyitieCf,lrleroaoogf,ohniMrbrrxvrgtoano1i,leideeddmdiaenNeSilobap.f,.Esrneiseoo-ndcn'orrm)boc,0zluemo,oMWnWnt-mieSpt,oiiceli-nklbhl6rdiic.ciyi0tingiooe/aas8wxln0DnrieenIg.inism,ogrsasghfetduoartsmweulvhv.om,aaerftiueJleabS4onrb0.bEhtle-nega3rsrn0-a-mfdMmrooesRnamthne6oysv0felil/al8er0.ch, 6. Chromatographic Conditions - icbsnyoc anotlthenaee(i[ndicsmmi))nogACIoOivnlbvtialn1teltie0earglnri0nittineusteeemotraedrmaitmcpgiporoafoprenbsfoger,umarrcfbaatlhsoumbtuaulrlwoescerrhe1,orot,efa02tfohc22trehma265mn,0W,t iC.3icqCa7.,ru75.oer,elm8.ist-plet.eortnprsaseearcmhmoploflienraouottdefilbeaaeasnsszdttaoen5t-ed0pra#-mdrdoiinofexdin 7. - Preparation of Standard .Steeet.rVaFc((i(igahcdbu)l)))orerWDIAongiedIjlaeuiigbintcfheoet,nrreuatzoaasod1i-ntt.phy0gS-epdemaiiccomtmiaxiaooilrinnrkcocrohwli3inr-t.iobe'ttomhar laaacstnoahgcmtleorepanr,lmoefmo.onirnle.mt-om.voitllhuliegm. reacthmrricomoffalat2osg,k3ra.*p7h*8. - 8 Procedure to CO mM o ,cphro.i*.cp:-:e*.*((u(erdba.(fee)b)))si.aTUWAPliadslealdniaincgcygeh2ine0ga1rt.h0n,04ea.'.0s.emc** syepgeoinrlo-laltduirsmlirtsifoiatuoiepbgnpolreeefsirni,ftnhooetdarforaccfswiaahevmlneootpwtrrflomoeifff-uooiaingrnusmuenttocmeeastuunac.bhbd feooos.tu.hfatraln-ektdohe,.ue. n7f occ;elre bonoet aivr;'f thl:e_o^.u7r. m 7 8 Io June 22, I 9G5 3- CJD Ml.W.G'i.1 I sbteot(hhhogfxeoit)tstwcro.asrhmev(c(((i(lsogfheTaojit)p))r)))clhuoorahimesfnUEICTnlonospdeavsreerjfk.ramieonineespnfgcth.osoorauatefAirrfpkaalmudne1ttdghtesfw.a0eeilfo2tvaioitt5lvhoyeum2freoet,m-i3i1dcrromc5c,rhln.7ryahfloinoims,rvlo8oerrllieioemfi-sintlstefpsiueao1tmrrtttereeopirNmniagssirnonrecussatnatsh(tosmooNeytdlisohbs.oriuio.5trltnehemoot-ege0ddne#1i.hinbc,1ytghoecodrrdfna)orr.omamzatxwhosaiesd-topmooegv-ffadrofatllsiolupoaamhsvmxteihipnmaaelen.nuld.occphtheFmeldNoiroegofraumoitsnertfuhelor.eersmIteI p 9. Calculations Let: A Tthhee saaremaploe.f the .2,3. ,7>8-tetrachlorodibenzo-p-dio-xin in B = The attenuation of the'chromatograph fo r the sample. C = Tchhelomroidcriobgernazmos-pp-edriomxinilliinlitteher sotfanthdeard2.,3*7 ,8-te tra D = dThibeeanrzeoa-po-df iothxein reinspothnesesftraonmdatrhde. 2,5,7 j8 -tetrachloroE = The attenuation of the chromatograph fo r the standard. Then. . . -..'; .-..r.'? ' : , ppm of 2,3i7i'8-teAtrachlorodlbe- n*z"o-p-diox'in = p^ cyTTBlTxToC \ ! 10. ' Av;"ccuracy ` _'v. " ' V". '-.'.v ';-v ; . . ' The accuracy of th is method is - 5jg, or less,` ^relative. o o 1 1 . ICotes 2TA6TT-/Vtoo June 22, 1965 -b MLW.6 5 . l l aands ditnhieo ttxhaRcienht((eicbro.so)ce)nmhaaAlraorcntyrhyoo,gfp2o.rIhaS,rn4apmic,lshi5ec.ic-,e.ot rxnWaitceIrnhaatallFcnolaSyturls-otsm1ipo2sCuh6rseei5otsenf bk(oeaft,xhlvuyeCraeramoai2lcalo),ei3bfvtoe,lih7ecdran>safa8irascob-.teimediettnrYwawf/hcoiilhckui llhinlondrshionadttIsoneibsrdweftireonsursrzmkeooel-wvpwnee-tidtllh as ************ C _ . irtsCTldqatnieohhotacuuqeeemctaiaurcurtpeploaeaaraipawrfnsnntboircyuetnhoacyieiddrlecmwlieycuhtdfatyrtocuaikiioectaserrntenoosareaesotuflnlhnwyrrcpbootseihepprtertiohesreurlceirtrebheemmpitonvlhedsirreestieuyseeuthdhdsrsnlieoateeirdossdtrtvneohneusetdegtsaloobfbeeitvrppmeiyx.ooeebupmnnaderarepUetcpeuohTooltspeirpsheebraridoeearortbnasweslpDitl.inaeornatoiinheraw.rredeHhyateaCwioneIbvacwhthneynlataeodeeduslmvewbsttiehsieraitohcreeseso,aansnso.rlteuhTsufmdahCoailnneteAedeosdsatxnmvosDpyepppnoootareaewooncfrnrdecoiftymCols.niafwnsuahefrihocenirfsboemmfmthiaurciinillclniditiaetnhglynge t 0001385 ' r-. i . . ij'. *V ri_ ;v.r - = : r i- - .... . S --- r. w- v. - ' rw'V.v.' '-.i-0 * ** .- .. . wrf? . . *' -';Vf . : - 11 dioxin r DOW H 9713 99CIOOO * 3 \k Jui.e 22, 196'3 Ltenuation J2 x 10 FIGURE I I MLW.G>.I1 w 1197X4 R ecorder Response (m. r>co co o o o ''" 5 4 .8 2 58 5483 D O W 765061 Ey Houard C. Alesondar Toiry L. Eatohaldar Uasta Control Tha Dow Chanloal Company Midland, Michigan! Por presentation on April 14, 1966 at the annual meeting of the Midwest Benthologloal Sooloty at Central Mlohlgan Uhlverslty. 0G05G91 ^ 5484 DOW765062 \ "1 INTRODUCTION The past few years ouoh attention has been focused on the effects of paaticides, end in particular Insecticides, upon natural habitats. Sens lnaeotloldoa have been observed to be toxic In very snail amounts not only to lnoooto, Its primary victims, but also to other living animals. The ecology of the aquatic ocmmunlty may likewise be upsot by postloldos. Suggested toxicity test procedures for birds, fish, and marine mollusks have been outlined by the U. S. Pish and Wildlife Service In their "Procedures for Evaluation of Acute Toxicity of Pestloldes to Fish and Wildlife", revised Deoember 14, 1964. Tho purpose Is to aid Industry In the determination of pesticide toxicity on fish and wildlife for data to support requests for label registration. The techniques for fish are similar to those used by the research stations of the Bureau of Coomerclal Fisheries and Sport Fisheries and Wildlife, and are considered to give a reasonable basis for Judging acute fish toxicity. One of the main objectives Is to proteot fish and wildlife resources from hazardous compounds, Including those which cause aoute and chronic toxldtles. Consideration of the total hazard must lnolude the stability of the compound In nature and the proposed use pattern. Those utilized repeatedly, or that persist, may causo chronic toxic ity and thus have a greater significance In total potential toxicity 0005032 <> 5485 2- DOW 765'63 even though the compound exhibits no aouto toxicity at low concen trations. All compounds that may contaminate our uater resources should at least be tested for acute fish toxicity. Three Dow pesticides, known under the trademarks Daxtron^ Tordon^? and Dursban^ were tested for aoute toxicity to three spoolas of fresh water fish. Daxtron, a general herbicide, is still in the experimental stagej Tordon horbleide is used for killing a wide variety of deep-rooted perennial herbaceous weeds and woody plants; Dursban insecticide is in the experimental stage. Rainbow trout ( Slmo galrdneri Richardson) were used as cold water representatives; bluegllls (Lepomls macrochlrus Raflnesoue) and channel catfish flctalurus nunotatus Rafinesque) as warm water fish, lhe blueglll data is acceptable for evaluation of possible effects on marine fish. The common goldfish (Carassius auratus Linnaeus ) may also be used as a warm water fish. *, METHODS AND MATERIAL Water used for the test was aerated distilled water with the follow ing additives: 30 mg/1 CaS04, 30 mg/1 MgS04, 48 mg/1 NaaCOa, and 3 mg/1 KC1. This distilled water solution was made up in a 35 gallon plastic ooated drum and aerated. Water was added to the test vessels and aeration was continued until the addition of the test oompound. There was no aeration during the tea>; period. 000S092 ; 5486 k 3- DOW 765064 Test fish were acclimated for at least 13 days in constant flow storage drums. Dry trout pellets were fed for 10 days; three days prior to testing, no food was given so as to empty the digestive tract. Dead or diseased fish wore removed Immediately. After . acclimation, five fish were placed in each five gallon wide mouth glass Jug or 10 fish in each 10 gallon aquarium. The fish were introduced into the test vessels 24 hours prior to the addition of the test compound. The compound was added in the following manner: 15.5 liters of wator were placod in eaoh five Ii. gallon Jug, five fish added, then at the end of 24 hours the i chemical was added with two more liters of water making a total I of 17.5 liters. Eaoh ten gallon aquarium was filled with 31.5 liters of prepared water and ten fish were added. After 24 hours, i the compound was introduced with four more liters of water. The vessels were then placed in constant temperature water troughs: i 6oF for the cold water fish, and 80P for the warm water fish. i Plastic mesh was laid across the tops of the containers to prevent fish loos. Ten fish were exposed to eaoh concentration of the compound. The fish were observed and the data recorded dally at tho cone time " I for four days (96 hours). A reference DDT toxicity was made for each test lot on each species of fish. GG05094 i, 548 7 DOW 765065 ? - 4- After eaoh test the floh vooaolo were thoroughly scrubbed and rinsed twice using a commercial "chlorine" oloansor. Caro oust be taken to Insure that only vessels of non-porous materials be used. Several slate-bottomed aquaria had to be discarded because of contamination that could not be removed by this method. All test solutions vere prepared on a weight-volume basis Immedi ately prior to administering the test pesticide. Suitable test fish are not as readily available as statod In the Floh and Wildlife procedure. Rainbow trout In the proper size range may be obtained most months of the year from several sources within Michigan. However bluegllls and channel catfish In this size range are available for only a oouple of months during the year and a source for these fish could not be found within the state. Trout were obtained from the Michigan Department of Conservation Harriotta Hatchery and Baldwin Rearing Pondsj the bluegllls and channel catfish from the TJ. S. Fish and Wildlife Service National Fish Hatcheries at Hebron Ohio and Senecaville Ohio. 0G03095 D W 76506b' - 5- RESULTS The test solution concentrations and determination of TZ^ values were In accordance with the bloassay methods stated In "Standard Methods for the Examination of Water and Wastewator", 11th Edition* I960, pages 457-471. The 24, 48, 72, and 96-hour TL^ for each species of fish was determined for eaoh compound. The TI^ Is the median tolerance limit or the concentration of the material being tested at which 50 percent of the tost animals are ablo to survive for a speoifio time Interval. The TI^ corresponds to the 50 percent lethal or LC^0 used in the fish seotlon of the Fish and Wildlife test procedure. GGG503S : 5489 2 DOW 765067 DAXTROH Candidate pooticide: Manufacturer - 'Rio Dow Chemical Company A. Trade Ilarlx - Daxtron Actice Ingredient - 2,3*5-triohloro-4-pyridinol potaaslua salt B. Reference - 3*1350-11 91# potassium salt Solvents used: A. Identity and quantity used for reference pesticide: 0.1 mg/ral in acetone B. Identity and quantity used for candidate pesticide: 100 mg/ral In water Fish: A. Rainbow trout Souroe - Baldwin, Michigan Average weight (g.): 1.8 Date: 7-23-65 Range (g.): 1.5 - 2.1 Blueglll Source: Hebron, Ohio Average weight (g.): 1.2 Date: 9-14-65 Range (g.): 1.0 - 1.3 Channel catfish Souroe: Senocavllle, Ohio Average weight (g.): 1.9 Date: 9-14-65 Range (g.): 1.8 - 2.0 Bioassay v essels: A. V e s s e l typ e and s i z e : g la s s - 5 g a llo n j a r g la s s - 10 g a llo n aquarium B . Loading (grams o f f i s h / l i t e r o f t o s t medium) less than 0.5 gmfish/liter of water 000503? - 5490 - 3- DOW 765068 C. Number of fish/vessel: 5 gallon Jar - 5 *ioh 10 gallon aquarium - 10 f l B h Water: Distilled Hater with the following compounds added: 30 mg/ 1 calcium sulfate 30 mg/ 1 magnesium sulfate 48 mg/1 sodium carbonate 3 mg/ 1 potassium chloride Results with pesticides expressed as TI^ on each species Species Rainbow trout at 60*F Bluoglll at 80P Channel catflBh at 80F Daxtron TL^ mg/1 24 48 72 96 hours hours hours hours 142 100 100 82 615 425 320 320 138 134 130 130 Reference pesticide (p,p* - DDT) Species Rainbow trout at 60F Blueglll at 60F Channel catfish at 8oF 24 hours .0160 .0078 .0145 * ng/ 1 48 72 hours hours .0143 .0125 .0066 .0066 .0134 .0128 96 hours .0115 .0046 .0128 Results with controls expressed as survival over 96 hours Species Rainbow trout at 60F Blueglll at 80F Channel catfish at 80F Total Pish 10 20 20 Number of fish surviving at 24 48 72 96 hours hours hours hours 10 10 10 10 19 19 18 18 20 19 19 19 GG05033 -4- ' 'r o n r o n Candidate pesticide: Manufacturer - The Dow Chemical Company A. Trado loris: Tordon Active Ingredient: 4-Amino-3,56-trichloropicolinie acid as the Lot 4*962 potassium sait DOW 765069 Solvents used: A. Identity and quantity used for reference pesticide: 0*1 mg/ml In acetone B. Identity and quantity used for candidate pesticide: 12.5 mg/ml as formulated In water Pish: A. Rainbow trout Source: Harrietts, Michigan Average weight (g.): 1.6 Date: 6-22-65 Range (g.): 1.7 - 2,1 B. Bluegill Source: Hebron, Ohio Average weight (g.): 1.2 Date: 9-14-65 Range (g.): 1.0 - 1.5 C. Channel catfish Source: Senecavllle, Ohio Average weight (g.): 1.9 Dato: 9-14-65 Range (g.): 1.8 - 2.C 0005033 5492 DOW 765070 5- Bioaosay vessels: A. Vessel type and size: 5 gallon glass Jar 10 gallon glass aquarium B. Loading (grams of flsh/llter of test medium) less than 0.5 gra flsh/llter C. Number of flsh/vessel: 5 gallon Jar - 5 fish 10 gallon aquarium - 10 fish t Mater: Distilled water with the following compounds added: 30 mg/1 calcium sulfate 30 mg/1 magnesium sulfate 48 mg/1 sodium carbonate 3 mg/1 potassium chloride Results with pesticides expressed as TI^ on each species Species Rainbow trout at 60*P Blueglll at 80? Channel catfish at 8o*P Tordon TI^ mg/1 24 48 72 hours hours hours 27 13 13 69 69 45 4l 24 16 96 hours 13 24 14 'Reference pesticide (p,p' - DDT) Species Rainbow trout at 60F Blueglll at 8o#F Channel catfish at 80P 24 hours .0091 .0078 .0145 TI^ mg/1 48 72 96 hours hours hours .0074 .0068 .0063 .0066 .0066 .0046 .0134 .0128 .0128 GG05100 5493 i -6- Results with controls expressed as survival over $6 hours Species -Rainbow trout at 60P Bluogill at 8oP Channel catfish at 8oP Total Pish 10 20 20 Hicnbsr of fish surviving at 24 48 72 96 hours hours honra 10 10 10 10 19 19 13 16 20 19 19 19 DOW 765071 OGOSlOi -7- DURSBAN Candidato pesticide: Manufacturer - The Dow Chemical Company A. Tracio Ilark - Duraban Active Ingredient - 0,0-dlethyl-0,3,5#6-trichloro-2-pyridyl phoaphorothloate Lots 2 and 3 99$ pure ^ C 7 Pnon Solvents used: A. Identity and quantity used for reference pesticide: 0.1 ng/ml in acetone B. Identity and quantity used for candidate pesticide: 0.1 og/ml Ip acetone Fish: A. Rainbow trout Source: Baldwin, Michigan Average weight (g.): 1.8 Date: 7-23-65 Range (g. ): 1.5 - 2.1 B. Blueglll Source: Hebron, Ohio Average weight (g.): 1.8 Date: 8-12-65 Range (g. ) 1-6 - 2.0 C. Channel catfish Source: Senecaville, Ohio Average weight (g.): 1.9 Date: 9-14-65 Range (g.): 1.8 - 2.0 Bloassay vessels: A. V e ss e l type and B lz e : 5 g a llo n g la s s Jar. 10 g a llo n g la s s aquarium o 5' 5 DOW765073 - a- . -Loading {grams of fiah/lifcer of toot medium) less than 0.5 n floh/liter of water C, Number of fioh/veosel: 5 gallon Jar - 5 fish 10 gallon aquarium - 10 fish Water: Distilled water with the following compounds added: 30 mg/1 calcium sulfate 30 mg/1 magnesium sulfate 48 mg/1 sodium carbonate 3 mg/1 potassium chloride Results with posticIdes expressed as TZ^ on each species jo to Suecles Dursban TLm mg/1 48 72 noure hours hours Rainbow trout at 60F Bluegill at 80*F Channel catfish at 80SF 0075 .0040 .0198 .0047 .0036 .0142 .0033 .0033 .0134 .0030 .0033 .0134 Reference pesticide (p,p* - DDT) Species Rainbow trout at 60F Bluegill at 80P Channel catfish at 8oF 24 hours .0160 .0128 .0145 h ">s/l 48 72 hours hours .0145 .0125 .0056 .0042 .0134 .0128 96 hours .0115 .0042 .0128 ) ' 5 436 0G051Q3 Results with controls expressed as survival ovor 95 hours Spoolrs -Total Floh Rainbow trout at 60F 10 Bluegill at 80*P . . 10 Channel catfish at 8oF 10 Nuabor of fish surviving at 24 48 72 95 houro hours heurn hours 10 10 10 10 10 . 10 10 10 9999 D O W 765074 CGQ5I04 5497 * 4. * % DISCUSSION AND CONCLUSIONS The reoults show that Daxtron and Tordon herbicides are not particu larly toxic to the fish. Duraban insecticide is toxic to the fish studied in low concentrations parts per billion. Howovor, it should be remembered that laboratory tests using clear water and standard conditions serve only as a preliminary indication of the possible problems that need to be evaluated under practical use conditions. DOW 765075 Environmental factors such as the effect of temperature, sunlight sorption and biological stability of the compound can only be evaluated by an actual field test. Formulation controlling release of the toxicant; pH organic sediment and foliage density may all modify the selectivity of the chemloal to various aquatic faunal species. The hazard of a chemical under specified uses in specified locations may be greatly different than the toxicity shown in laboratory conditions. Such factors are being determined for Dursban insecticide at the present time. Pesticides used improperly can cause serious damage. Always read the label and follow the instructions given on the label. U3e recommended procedures and equipment for application. Avoid con tamination of any other areas. Never allow spills or water for cleanup to contaminate sewers ditches or the ground. Properly dispose of unused application mixtures contaminated clothing and containers. To be sure Road the LabolI 0G0S105 5498 Y.bUYb R eport Ko. OS-781 >r <- Iir Jt'OVV Cl II: ICAL CO MPA UV 4 Vwl/Wi ( V/AUNwT C#*CC/(t A lif O n M A \j Ci '* pnOLcM no. 3 -^ q. C. R. Youngson / . v /z y /f'1 A HIOASSAY STtllJV Oil THE DETOXIFICATION OF AQLTOUK SOLUTICL'S: Or TORDOII I'Y suksjiike' iii nn:r containers under still and cifculatimg conditions * ^ v a lu a tio n o f th e e f f e c t o f s u n lig h t on p h o to d e to x ific a tio n of Torclon in w ater in 1 2-foot deep c o n ta in e rs has shown: 1. C ircu latin g Water ** T **** *" a , Rate o f lo s s c a lc u la te d f o r 9*4 d ay s' exposure during Kay to October was a t .least 0.35 lb . p er a c re p e r day. b . Rate o f lo s s c a lc u la te d f o r 56 d ay s' exposure during September and October was 0.35 l b . p e r a c re p e r day. P ercen t d e to x ific a tio n was roughly proportional to exposure period during the course of the experiment. * (Continued) Work by: C. A. I . Goring arid C. R. .Youngson ** i Record books: GS-1U50, 14157, 1172, Ii75, W 80, l `i65, 1501, 1598 , 1 O *s M4 ro oo o TY?r OF REPOST! Research PAGES! 9 STAGE: '5 D istribution: Midland CRI Texas CRI P itts b u r g CRI Executive Research Bioproducts Dept. Xgr. < v:rd ) . P lan t Science Manager . PSR5D D ire c to r 2.C. B ritto n Res. Lab. (( CKCEOR )) .( TR,EHR.) ( 2) 3iop. In fo . Center R egistration Section ' D ir. Product Planning ( JDE ) ( GFL ) ( DEP ) Product Management Teem Xgr. ( ESS ) P roduct T ech n ical S p e c ia lis t ( EG" ) Animal Science Section Synthesis Form ulations . P aten t Department Bioproducts Midland ( TAII ) (KR . ) (JVfV ) ( JLS ) ( ajv: ) 0G0510G (Complete Report) Davis ( PLG) Lake Jackson ( RVJ ) Pitman-Moore ( AJS ) L. A. Doan S tage F ile (K -38323 ( DG ) Piochcw, In v e s tig a tio n s . ( VRB ) H^iochcm. Ros. Lab. ( EHA ) Bicmechanisms Group H erbicide Res. Group ( G'iS ) ( TV'H ) Midland F ield Group* ( ERL ) Midland H erbicide bev Gp. ( J!:C ) W inter P ark, F lo rid a ( ELC,,CTL Wayside, M ississippi Davis Oklahona C ity ( KAM,,v:om : ( LEU ) ( JUG ) M inneapolis, Minnesota * S c a ttl c j Washington ( csw ) ( JPT ) Ways'*do F ield Group ( RCK ) V'a s h i r r ton ( RGH ) Dov: L atin America Dow In te rn a tic n a l ( Ah'S ) ( ETC ) Dow Canada Dev: Fiiror.e ,,q q U4 o o ( LJ!,, m : ) ( LLC ) - 765077 2. Calm Pater Lops was uniform throughout th e depth o f th e c o n ta in e r, even though su n lig h t ra re ly penetrated to the bottom. > A survey o f d a ta from 4 re p o rts has shown t h a t r a te o f p h o to d e to x iflo a tio n o f Tordon in w ater i s markedly a ffe c te d Ly such `fa c to rs in th e environment as brightness o f sun, geographical lo catio n , time of year, cloudiness, du stin ess, haziness, e tc . P hotodetoxification in water can occur ir. a v a rie ty of water environm ents, probably in clu d in g th e su rfa c e o f le a v e s, however, when w ater ev ap o rates lea v in g dry film s o f Tordon on s u rfa c e s , the lo s s r a t e i s about 100 tim es le s s th an th a t in w ater. Furtherm ore, when Tordon i s p resen t in muddy w ater o r s o i l , so rp tio n o f UV r a d ia tio n by the s o i l p a r t i c l e s e ith e r d ra stic ally c u rta ils or elim inates photodetoxification. 0GQ5107 5500 \ i 5 5501 ' \ar ~ "*'/ ' iU ' z ,4-D (2,4-Dichlorophenoxyacetic acid) and related compounds rv^< A ii-juLcU<M A/o. 96 /966 In feeding studies of 2,4-D with dairy cows and steers (Lisk et al., 1963; Gutenmann et al., 1963a,b; Bache et al., 1964a,b), 2,4-D was found unchanged in the urine only. No evidence of beta-oxidation was found. * Similar findings were obtained with sheep. Ninety-six percent of an orally administered dose of 2 , 4 - D - C ^ t o a sheep was excreted unchanged in the urine in 72 hours and slightly less than 1.4% in the feces. Very little residual radioactivity was found in edible tissue (Clark et al., 1964). Cows fed 2,4,5-T and silvex excreted both as soluble salts in their urine. Kuron was hydrolyzed to silvex prior to elimination (St. John et al., 1964). Treatment of lemons with C ^ labeled 2,4-D isopropylester indicated that the ester was hydrolyzed and that part of the 2,4-D then reacted with some plant constituent to form an ester-like complex. Ester-like residues were also found after treatment with the sodium, diethanolamine, or triethanolamine salts (Erickson and Nield, 1962; Erickson et al., 1963). Hydrolysis (Crafts, 1960; Morre and Rogers, 1960) and decarboxylation (Edgerton and Hoffman, 1961; Basler, 1964) of 2,4-D by other plants has also been shown. Plants are capable of hydroxylating phenoxyacetlc acids (Wilcox et al., 1963; Thomas and Loughman, 1963). When bean plants were treated with 2,4-D, three compounds were found (Crosby, 1964).' One corresponded roughly to that of 2,4-dichloroanisole; one was a water-soluble, ether-insoluble ester derivative; and the third, an ether-soluble compound with a basic 61 .> 0002214 5502 structural change. The methyl derivative was less volatile than 2,4-D methyl ester, but more volatile than the 5-hydroxy-2,4-D methyl ester. It might be one of the other two hydroxy derivatives; however, 6-hydroxy- 2,4-D was not detected (Holley, 1952; Jaworski and Butts, 1952; Jaworski et al., 1955; Evans and Smith, 1954; Bach, 1961). ./- * The free acid has been recovered from bean and corn plants after 1 treatment with 2,4-D butoxyethanol and propylene glycol butyl esters * (Hag4n et al., 1949; Fang e't al., 1951; Fang and Butts, 1954; Hay and Thimann, 1956; Szabo, 1963). On cotton, cucumbers, beans, and grain sorghum, ! & labeled 2,4-D gave rise to (Holley et al., 1950; Weintraub et al., 1952a,b). Pea and tomato plants have also been studied (Fang, 1958). In CO CD young leaves and bolls of cotton, material chromatographically different cn CO from 2,4-D was formed. Sorghum converted 2,4-D to a complex different CP CO i f than that found in cotton (Morgan, 1963; Nencki and Giacosa, 1880; Weintraub ( et al, 1950, 1952a,b, 1953, 1954, 1956; Slife et al., 1962). Amino acids have been implicated, in the formation of some compounds, as in the case of 2,4-dichlorophenoxyacetylaspartic acid (Andreae and Good, 1957; Bach and Fellig, 1961). Evidence indicates that 2,4-D moves thrbugh plants as a protein complex, which can be recovered after aqueous extraction and NaOH hydrolysis, into the roots where most of the degradation occurs (Canny and Markus, 1960). Glucose esters have also been suggested (Klambt, 1961; Crosby, 1964). Recent studies have shown that glucoside complexes are formed. From stem tissues of oats (Avena sativa). 1-0- (2,4-dichlorophenoxyacetyl)-f3-D-glucose was isolated (Thomas et al., 1964b), and from stems of the kidney bean (Phaseolus vulgaris), the 2,5- and 2,3- 62 0002215 X5 5 0 3 DOW 365940 ) b I II j li dichlorophenoxyacecic acid glucosides have been obtained (Thomas and Loughman, 1964a). , Degradation of compounds related to 2,4-D has also been studied (Vain, 1954). MCPB gave rise to MCPA when fed to dairy cows (Bache et al., 1964a, b, c). In laboratory tests, J)luegill (Lepomis gibbosus) converted 4- (2,4-DB) to 2,4-D (Gutenmann and Lisk, 1965). When 4-(2,4-DB) was fed to dairy cows, however, 2,4-D was not found, although it does not decompose in the rumen. The major portion of 4-(2,4-DB), therefore, is presumably degraded by a mechanism other than 6-oxidation (Lisk et al., 1963; Gutenmann and Lisk, 1963c). In silage (Linscott and Hagin, 1963; Linscott, 1964), timothy, birdsfoot trefoil, or pea plants (Fertig et al., 1964), decomposition of 4-(2,4-DB) has been shown to proceed by 0 -oxidation. It also has been shown that related compounds could be degraded by a- and 0-oxidation in plants (Levey and Lewis, 1947; Fawcett et al., 1954, 1958; Moore and Rogers, 1960). y-(2,4,5-Trichlorophenoxy)butyric acid was metabolized to its acetic acid derivative in wheat but not in pea stems (Salayannis et al.-, 1965b). _ The biotransformation of 2,4-dichlorophenoxyalkanoic acids and related compounds by soil microflora has been extensively studied (Audus, 1949, 1950, 1951, 1952b; Audus and Symonds, 1955; Newman and Thomas, 1950; Steenson and Walker, 1956; Byrde et al., 1956, 1957, 1958; Jensen and Peterson, 1952; Evans and Smith, 1954, 1957; Fawcett et al., 1954; Webley et al., 1955, 1957, 1958; Bell, 1957; Henderson, 1957; Thomas and Loughman, 1957, 1963; Faulkner and Woodcock, 1961; Thiegs, 1962; Clifford and Woodcock, / 1964). Phenoxyalkanoic acids with an even number of carbons in the fatty 63 0002Z li, 5504 acid were converted by 9 -oxidation to products with an even number of I j carbons (Levey and Lewis, 1947; Webley et al., 1955, 1957, 1958; Gutenmann | et al., 1964a, c). A second mechanism involved cleavage of the ether linkage f /i (Canny and Markus, 1960; MacRae et al., 1963a b, 1964; Audus, 1964; Bocks et al., 1964). ,, V r Evidence has been obtained that 2,4-D is dissimilated by a variety t of microorganisms (Reid, 1960; Alexander and Aleem, 1961) through 2,4- dichlorophenol and 4-chlorocatechol and that MCFA is dissimilated through OOW 365941 4- chloro-2-cresol (Audus, 1952b). MCPB was degraded by the bacteria Nocardia opaca via crotonic and 9-hydroxy acid to 2,4-D (Webley et al., 1957, 1958). A product from the degradation of 2,4-D by bacteria of the genus Pseudomonas has been identified as 9-ehloromuconic acid. A second species of Pseudomonas gave rise to a-chloromuconic acid (Fernley and Evans, 1959). Pure cultures of a Nocardia species and an Achromobacter strain of bacteria rapidly degraded 2,4-D and the presence of 2,4-dichlorophenol, chlorohydroquinone, a monochlorophenol, an unchlorinated phenol, and three other unidentified compounds have been demonstrated (Newman and Thomas, 1950; Audus, 1951; Bell, 1957, 1960; Steenson and Walker, 1957, 1958; I t [ < ; Faulkner and Woodcock, 1961; Taylor and Wain, 1962). The main product of 2,4-D metabolism by the mold Aspergillus niger van Tiegh was 2,4-dichloro- : l> j 5-hydroxyphenoxyacetic acid. By means of infrared and mixed melting points, a second metabolite was identified as the 2,5-dichloro-4-hydroxyphenoxyacetic acid - the first time such a rearrangement was reported (Faulkner j i j and Woodcock, 1964, 1965). Another unidentified acid, not the 3- or 6-. ; hydroxyacid, was also found. Under similar conditions, MCPA gave rise to 64 0002217 5505 ige I -8. 2-methyl-4-chloro-5-hydroxyphenoxyacetic acid (Faulkner and Woodcock, 1964, 1965). Studies with 4-(2,4-DB) showed that it is detoxified by. Flavobacterium sp. (MacRae and Alexander, 1965), releasing chlorine and cleaving the ring (Burger et al., 1962). In natural surface waters, 2,4-D, its salt and i-propyl or butyl esters, were hydrolyzed by microorganisms to 2,4-D acid and the corre- sponding alcohol (Aly and Faust, 1964). Under the influence of ultra violet, aqueous solutions of 2,4-D sodium salt gave rise to 2,4-dichlorophenol, and subsequently to 4-chlorocatechol. Complete dechlorination and polinerization followed (Hansen and Buchholtz, 1952; Tutass and Crosby, 1965). In the presence of riboflavin, compounds containing more than one aromatic nucleus were probably also formed in addition to 2,4-dichlorophenol. Products differed according to the original pH and concentration of the treated solution (Bell, 1956; Hansen and Buchholtz, 1952). Zf639C oo $ 0 -CH2-CH2 -CH2 -jjH Plants L 1'Cl 11 Plants " V ' Achromobacter Cl Silage Decomposition 2,4-D 4-(2,4-DB) 1 -0-(2,4-DichlorophenoxyAcetyl)-0-D-glucoside 2,4-Dichlorophenoxyace ty1 Aspartic Acid 65 00022L8 5506 i 2,4-D 2,5-Dichloro4-hydroxyphenoxacetic Acid 2,3-Dichloro4-hydroxyphenoxy- acetic Acid (As Glucosides) .365.043 J Sw * 2,4-D 2,4-Dichloro5-hydroxyphenoxyacetic Acid 2,5-Dichloro4-hydroxyphenoxy- acetic Acid 2,4-D Chlorohydroquinone or 4-Chlorocatechol An Unchlorinated Phenol + 3 Unidentified Compounds 66 19 DOW 365,044 *2,4-Dichlorophenol a-ChloroMuconic acid 5-Dichlorocatechol Me Cabo1zed With Release of Chlorine 67 4-Chlorocacechol 6 -Chloromuconic Acid 5508 B 0002220  C H CR,-CHi-<2 OH rf^ V c H . Cows Va 1 MCPB 1t i \I I i II iI Cl mcpa' o< I i \lI fIl *tII ir Cl 2-Methyl-4-chloro-5hydroxyphenoxyacetic Acid 68 i 5509 0002221 < i t ltI 4-(2,4-DB) \ P i i i ! i i I i it ii HjCOOH rifi e i I i ( I ii i IiI I Mechanism Other Than $ -oxidation 5510 0002222 365940 5511 BOW 121507 FROM TU K NATIONAL VETEIUNAIIY INSTITUTE, STOCKHOLM. SWEDEN STUDIES ON THE ANALYTICAL CHEMISTRY AND TOXICOLOGY OF PHENOXY HERBICIDES BY KURT ERNE C STOCKHOLM 1966 V 0001374 5512 DOW 121508 Contents F o r e w o r d .......................................................................................................................................5 I n t r o d u c t i o n ............ ................................................................................................................. 7 D ev elo p m en t o f th e a n a ly tic a l m e th o d .................................................................... 11 E xtraction of the s a m p l e ................................................................................... . . . 11 P urification of the e x t r a c t .......................................................................................... 13 C hrom atographic separatio n ..................................................................................... 14 D e te r m in a tio n .................................................................................................................. 15 A ccuracy and precisio n ............................................................................................. 16 Sensitivity ......................................................................................................................... 16 Selectivity ......................................................................................................................... 17 D istrib u tio n a n d e l i m i n a t i o n ........................................................................................... 19 Plasm a levels .................................... '........................................................................... 19 Tissue levels .................................................................................................................... 22 M etabolism ............................................................................................................................... 27 T o x i c i t y ...................................................................................................................................... 29 Acute t o x i c i t y .................................................................................................................. 29 Subacute to x icity ........................................................................................................... 29 C hronic toxicity ............................................................................................................. 32 Pi* .............................................................................................................................. 32 Hals .............................................................................................................................. 33 Chickens ..................................................................................................................... 33 S u m m a r y ..................................................................................................................................... 37 H e f e r e n e e s ................................................................................................................................. 39 3 0001375 5513 Foreword Thu present survey covers studies m ainly accounted for in the following publications. I. l i m e , A'.: D etection a n d d e te rm in a tio n o f ch lo ro p h c n o x y a c c lic a c id d e ri vatives in w alcr. Acta cheni. scand. 1963, 17, 1663-- 1676. II. l i m e , A'.: D e te rm in a tio n o f p h c n o x y a c lic h e rb ic id e resid u e s in b io lo g ical m aterials. A cta vet. scand. 1966, 7, 77-- 96. III. E rn e, A'.: D istrib u tio n a n d e lim in a tio n o f c h lo rin a te d p h e n o x y a cc tic acids in anim als. Acta vet. scand. 1966, 7, 240-- 256. IV. lim e , K.: Studies on the anim al m etabolism of phenoxyacctic herbicides. A cta vet. scand, 1966, 7, 264-- 271. V. B j r k ltin , X .-E . & K . E rn e : T o x ic o lo g ica l stu d ie s o f p h e n o x y a c c tic herbicides in anim als. A cta vet. scand. 1966, 7r " v - jrt-'. In this survey these papers will be referred to by the ro m an num erals. T he investigations have been carried out at the N ational Veterinr}" Institute, Stockholm , during the years I960-- 1966. To professor H ans-Jrgen H ansen, D irector of the Institute, I wish to express m y sincere gratitude for his keen interest in m y w ork and for his steady support and encouragem ent. To the D ean o f the Royal V eterinary College, P rofessor Carl Schm ilcrlow , an d to the B oard of Professors of the college, I am indebted for perm ission to subm it this thesis a t the College. My im m ediate chief, Professor Mans W annlorp, H ead of the C hem istry D epartm ent, has furthered m y w ork by m any helpful discussions and by Inking an extra share of ponderous w ork during m y preoccupation w ith this study, fo r w hich 1 am deeply grateful to him . Prosektor N ils-lirik H jrkluud earns m y sincere appreciation for a m ost pleasant and fruitful collaboration throughout this w ork. F u rth e r 1 am deeply indebted to Ni!s-C)lof L in d g rc n . V.M .D., Tor c o n s tru c tiv e critic ism o f th e m a n u sc rip ts o f th is a n d Die p re c e d in g p u b lic a tio n s, as to b o th su b je c t m a tte r a n d lin g u is tic form , 5i c c to B crnl T h afv clin , v eterin ary surgeon, fo r expert advise an d for v alu ab le help in co n n e d ion w ith the an im al experim ents, to A drian F ran k , civil engineer, fur enlightening discussions, to Airs. G un S allc rstro iu a n d Miss lle lg a llo lz, w hose skilful an d devoted technical assistance lias been of invaluable help to me, to Mrs. C arin A nsgard for excellent secretarial help and to Mrs. K crstin isaksson for conscientious illustrative and librarial help. Miss G erda S ch effer offered v aluable help in typing p a rt of th e m an u scrip ts, for w hich I am m ost grateful. The staff of the C hem istry D epartm ent has invariably and willingly helped m e; I should like to thank particularly Mrs. A rija Strom berg, licenced pharm acist, M rs. B rilt-L ouisc S chm idt, and Mrs. H elga R euterw all for valuable aid in various phases of the work. Mr. E rn st N ilsson an d Mr. K nul Iiu llk v ist aided in carefully looking afte r the experim ental anim als, and Mr. Inge Ericsson skifully prepared the photographs. To all of them I extend m y hearty thanks. C raw ford G rant, V.M.D., a n d G eoffrey F airlu irsl, M.S., ow e m y g ra titu d e fo r linguistic revision of (lie m an u scrip ts. T h is w o rk w a s ec o n o m ic ally s u p p o r te d b y g ra n ts fro m ''A lb ert H ja r r e fonden", "lim it och Alice W allenbergs S tiftelse", and the Sw edish A gricultural Research Council. Svcnska AB Philips, Stockholm , and AB Ew os, Sodertulje, generously supplied herbicide form ulations and reference grade chem icals. Stockholm , O ctober, 1966. Kurt Erne ! .O O 121510 6 OOOl^r? Introduction In th e b a ttle a g a in s t th o se liv in g o rg a n ism s (``p e s ts '') w h ic h m a y in te rfe re w ith m an 's production o r storage of food, feed, or oilier com m odities, or \vit)i h is h ea lth o r co m fo rt, chem ical m eth o d s o f co n tro l a rc being used to an increasing extent. Som e 200 basic chem icals are in cu rren t w orld-w ide use as pesticides (or "biocides"), com m ercially presented in thousands of d ifferent fo rm u latio n s an d u n d er n um erous trad e nam es. A ccording to the intended target organism , these m aterials m ay be classified as insecticides, acaricides, fungicides, herbicides, rodenlieides, etc. O n a quantitative basis, the herbicides constitutes the m ajor group. Broadly speaking, herbicides m ay be defined as agents intended for the chem ical control of weeds or other unw anted vegetation. Such agents are being used for a m ultitude of purposes in agriculture, forestry, horticulture, an d m any other areas of hum an activity. Typical applications include the control of weeds com peting w ith food or forage crops, o r w ith other useful crops, of low -value arboreal vegetation in forests, of p lan t vectors fo r insect, fungal, o r viral pests, an d of terrestrial or aquatic vegetation interfering w ith traffic, drainage, or irrigation. At lower dosage, herbicidal agents m ay be used for regulating physiological processes such us root induction, leaf abscission, fruit setting and m aturation, and seed developm ent. H erbicides were used in Sw eden d u ring 1905 to a value of m ore th an 25 m illion Sw edish crow ns, the total q u an tity am ounting to som e 4000 Ion of form ulated products. Am ong m odern herbicides, chlorinated phenoxyaliphnlic acids (phenoxy herbicides o r "horm one weed killers") hold a dom inating position, although a variety of other organic com pounds also are being w idely used, such as hnlogonatcd aliphatic an d arom atic acids, halo- and nilrophenols, substituted am ides, carbam ates, ureas, nitrogen hclerocycles, and q u atern ary nitrogen c o m p o u n d s (see <*.;/. C ra fts 19 0 I a n d A u d its 1964). T he application of phenoxy acids as herbicides followed the im portant d isc o v e ry o f S la d e , T e m p le m u n S e x to n in 1940 (re p o rte d in 1945) th a t certain sy nthetic plant horm one (auxin) analogues w ere toxic at very low co n cen tratio n s to certain broad-leaved p lan ts w ith o u t causing d a m age to cereals. T he subsequent intensive search for o th er com pounds ex ertin g a selective p h y lo lo x ie action resulted in the in tro d u ctio n of 2,4-dichloro- and 2-m elhyl-4-ehlorophciio.\yacclic acids (com m only abbreviated as 7 CQVy 121511 OCCIC 2TSTST ^ 0 9 c 2,4-D a n d M C l'A, respectively) as h ig h ly p o te n t selective h erb icid es, v astly s u p e rio r to a ll o th e r w eed k illers p rev io u sly used. T h e ir effect is p r e f e r e n tially directed against dicotyledonous plants, m onocotyledons usually being m ore resistant. Som ew hat later, the m erits of 2,4,5-lrichlorophenoxyacetic acid (2,4,5-T), p articularly in com batting Iigniform vegetation were disco vered. In recent years, derivatives of other phcnoxyaliphalic acids, such as 2-phcnoxypropionic and 4-phenoxyhulyric acids have been introduced as highly selective herbicides. T he butyric acid analogues derive their activity fro m a m etab o lic conversion w ith in the p lan t to the corresponding phenoxyacctic acids. T he structural form ulae and the com m on nam es of the phenqxy acids r e fe rre d to ab o v e a re listed iu T a b le 1. T a b le 1. P h c n o x y a lip h a lic acid s o f im p o rtan c e a s herb icid es. C hem ical nam e Common nam e S tru ctu ral form ula P henoxyacclic acids 2,4-D ichlorophcaoxyacetic 2,4-D CH.-COOH / 0 o d d (R, = Cl, R , = H) c 2-M cthyl-4-cliorophenoxyacctic c id (R, - C H ,, R , - H) 2,4,5-T richloxophenoxyace-tic a d d (R , = CI, R , = Cl) MCPA, 4K-- 2M 2,4,5-T P h cn o x y p ro p io n ic acids 2-(2,4-D id ilo ro p h cn o x y )-p ro p io n ic o d d (R , - Cl, R . H ) 2.4-D P. D iclilorprop. CH'.3 CH-COOH / 0 P hcnoxybtilvric a d d s 4-(2,4-D iclih>rophcnoxy) Im lyric a d d (R , " Cl, R . " il) 4-(2-M clhyl-4-chloroplicnoxy)I m ly r ic a c id (R , = C I I 3. R . = II) 4-(2,4,5-T richlorophcnoxy)-liulyric a d d (R , = Ct. R , = Cl) 2,4-D R 2 ,4 ,5 -T B M C I'Il 8 Cl CH.--CH.--CH.--COOII / 0 Cl . \ 5517 * ( Tin- m arketed herbicide p rep aratio n s contain eith er w ater-soluble am ine or alkali sails, or oil-soluble esters, or Ihc various plicnoxy acids. A flcr b ein g a p p lie d lo llie p la n l, Hie p lic n o x y h e rb ic id e s m a y b e ta k e n up from the foliage and Iransloealcd w ithin the planl, thus exerting a syste m ic actio n . U p tak e from the roots is also possible. W ith in the p la n l Ihc plicnoxy acids are m ainly transported along w ith Ihc assim ilation products to the m erislem and young grow ing tissues and also to the seeds an d other storage organs. T h e persistence of th e plicnoxy acids in vegetative tissues is u su ally n o t very high, being of the order of a few weeks. In storage organs, how ever, they m a y p e rsist fo r c o n sid e ra b le p e rio d s o f tim e (A a m ise p p 1961). It should be rem arked that also in p lant tissues killed by phenoxv herbicides the chem icals m ay show a re m a rk a b le sta b ility : e. g. in th e b a rk a n d leading shoots of aspen plants exposed to toxic concentrations o f 2,4-D, the h erb icid al activity rem ained apparently unaltered for m ore than a year (report from Dr. L. Eliasson, the Royal College o f F orestry, S tockholm ). At low dosage levels Ihc phenoxv herbicides elicit pli}'siological effects sim ilar to those of the n atu ral grow th horm ones; at higher dosage they induce an excessive, uncontrolled grow th, ultim ately leading to death o f the p lan t. T h e effect upon p la n l m etabolism is com plex. M etabolic processes shown to be affected include respiration, nitrogen an d phosphorus m eta bolism . an d salt uptake, as well as form ation of carbohydrates, RNA, and indoleacetic acid (see c.g . A udus 1964). Som e of the physiological effects o f phenoxv herbicides seem to he associated w ith binding of SH -groups. D espite intensive research efforts, how ever, the specific biochem ical m echanism underlying their action has not been definitely established. T he toxicity of herbicidal phenoxv com pounds to terrestrial an im als appears to be m oderate. P ublished acute oral LDso values fo r 2,4-D an d 2,4.5-T range betw een approxim ately 300 and 1000 m g/kg body w eight in several lab o rato ry an im als; the acu te toxicity in cattle an d sheep ap p aren tly is o f th e sa m e o rd e r. D ogs seem to h e m o re su sc ep tib le, th e a c u te o r a l L D jo being about 100 m g/kg. T he results of feeding experim ents w ith various, dom estic an im als suggest th at acu te or su b acu te poisoning is u n lik ely to occur after ingesting crops sprayed w ith phenoxv hcrhicdics at a recom m ended ra le |scc review s by Home & H y m n s 1934 a n d b y D aigaard-M ikkclscn & Paulsen 1962). In contrast, the piscicidal activity of plicnoxy herbicides m a y be a p p re c ia b le . W ith 2.4-1), a w a te r c o n c e n tra tio n o f 10 p .p .m . h a s p r o v ed fatal fo r various species of fish, oil 24 h o u rs' exposure, a n d w ith 2,4,a -T ester, le th a l effects h a v e been o b se rv e d at levels as low a s 1-- 2 p .p .m . (B a u er 1961; A n d rex so n it* B e rzin s 1961: D anis Jc H u g h e s 1963). 9 *8 121513 0001330 55i 8 In a ssessin g th e p o ssib le h a z a rd s involved in Ilit* u se of p h eu o x y h e rb ic id es, allcn lio n should also he paid lo indirect h azard s. F o r instance, interference of the h erb icid es w ith liant nielaholisin m ay lead lo an aecninulation in the p lan t of endogenous toxic m etabolites, such as n itra te and cyanide, lo levels as high as lo en d an g er the health of anim als feeding on the treated herbage (S ta b le r A W h ite h e a d 1930; S w a n s o n & S h o w 1954; F r a n k & G rig sb y 15).">7}. In the last few years, several reports of fatal phcnoxy herbicide poisoning in h u m a n subjects have been published, som e of the cases being due lo acci d e n ta l e x p o s u re (G oldstein, J o n e s & B ro w n 1959; M o n n rcn & l) i V ila 19(51: T o d d 1962; B e r k le y & M agee 196:1), a n d o th e rs a p p a r e n tly to in te n tio n a l ingestion, in suicidal purpose, of undiluted com m ercial preparations (Carry 1962; H erbich & M achata 1963: Popliam & Davies 1964; Nielsen, K aem pe & Jenscn-H olm 1965: Johnson & K oum idcs 1965; Geldm acher-v. M allinckrodt & lA utcnbach 1966). Cases of accidental, at tim es fatal, phcnoxy herbicide poisoning in cattle have been investigated by. o r com e to the know ledge of, the N ational V eteri n ary In stitu te a t several occasions during recent years. A ccording to available inform ation, the affected anim als m ostly have h ad access to toxic am ounts of the herbicide, c.g . in the form of spraying solutions or form ulated pro ducts. U nintentional contam ination w ith herbicides of w ater or feed supplies has also been observed repeatedly, som etim es associated w ith toxic effects in anim als. In addition, there have been rather frequent reports to the Institute of cases of dam age to dom estic anim als and w ildlife suspected o f being caused b y acu te o r ch ro n ic phcnoxy herbicide poisoning. A lthough in certain of these cases phcnoxy acid residues, indicative of exposure, have been detected in the tissues, the aetiological role of the herbicides has not often been convincingly established. U ndoubtedly, the w ide-spread use of phcnoxy herbicides provides am ple possibilities fo r (lie direct o r indirect exposure of h u m an beings an d anim als to these m a teria ls, a n d this, to g eth er w ith th e g ro w in g co n cern in wide, circles fo r environm ental hazards, lias accentuated th e need for m onitoring our environm ent for herbicide residues, and for a better understanding of the physiological significance of prolonged exposure to these residues. Tlic purpose o f the investigations under review w as: to develop m ethods for isolating, detecting and quantitatively m easuring sm all am o u n ts o f p h cn o x y herbicides in various biological m aterials, suitable for diagnosing cases of poisoning and for residue analysis, to stud}* th e tissue d istrib u tio n , elim ination a n d -- to sonic ex ten t-- the m e ta bolism of these com pounds in experim ental anim als, an d to stu d y the toxicological effects of phcnoxy herbicides in an im al feeding experim ents. 10 0001331 5519 DPW 121515 Development of the analytical method A p rereq u isite fo r the successful analysis of a given com pound in a com plex m ix tu re is its q u an tita tiv e isolation from the b u lk o f the sam ple m aterial, an d its seperation from soluble sam ple constituents w hich m ight interfere in the subsequent detection and determ ination of the com pound. In toxi cological a n d p e stic id e re sid u e a n a lj'sis, th e iso la tio n a n d se p a ra tio n m a y involve considerable difficulties because of the com plexity of the sam ple m aterial, and the usually sm all am ount present of the com pound for w hich o n e is search in g . A possible b io tran sfo rm atio n o r p o stm o rtal degradation o f the co m p o u n d m ay also co n trib u te to the an aly tical difficulties. Conse quently, procedures for toxicological and pesticide residue analysis m ust meet high dem ands for separating efficiency, as w ell as fo r selectivity and sensitivity. Studies on the isolation of phenoxy acids from various biological m aterials, and their separation from extractives were reported in papers I and II. EXTRACTION OF THE SAMPLE Anim al tissues w ere m inced and acidified w ith su lphuric acid and then subjected to altern ativ e extraction techniques, including extraction by sh ak in g o f a sam p le--sodium sulphate m ixture, Soxhlet extraction of a sample-- sodium su lp h a te , o r sam ple--c c litc m ix tu re , direct hom ogenizing of sam ple w ith organic solvent in a m ixer, and hom ogenizing w ith aqueous buffer. O f these techniques, hom ogenizing w ith organic solvent show ed m ost p ro m ise, being effective, rapid, an d convenient. A pplying this technique to tissues w ith an d w ithout added plienoxv acid, solvents of varying polarity were screened for selectivity and extracting efficiency as revealed by thinlayer chrom atography, em ulsificaliou-tendcncy and photom etric deter m ination of the extracts. T he results obtained indicated 2-propanol to be the solvent of rhoi.se for anim al tissues. T h e course o f the extraction w as follow ed by hom ogenizing liver tissue, fortified w ith 2,4-D, w ith successive portions of fresh propanol and delcrm iuing the am ount of phenoxy acid ex tracted in each fraction. As seen from p ap er II, Fig. 1 (low er curve), the cum ulative extraction curve at first rose hut then levelled o ff com pletely 11 5520 0001302 after the second extraction. the c o rre sp o n d in g 1) :m o v era ll reco v ery o f a p p ro x im a te ly 9.1 p e r se n t o f ad d e d 2. 4-1) (if co rrec ted fo r losses in Ilie p o sl-c x ira c lio n slops). F ro m llie u p p e r c u m ; o f (lie fig u re is seen llial 2. 4-1), w hen incorporated in tissues un d er physiological conditions, m ay lie equally effectively extracted. W illi o th er typos o f sam ple m aterial, conditions for (pianlitalivc and selective extraction w ere elaborated in an analogous way. T he hom ogeni zing technique using 2-propanol as a solvent, proved em inently satisfactory for extracting most anim al tissues, ingesta, faeces, and a variety of vegetable m aterials. From vegetable m aterials of a low m oisture content an d from o th er dried-in sam ple m aterials the phenoxy acids m ay lie less easily ex tractab le, unless the sam ple is reh y d raled before extraction. A sim ilar experience has been reported by B evcnue, Z w ciy & X ush (1962). Most likely, the ad d ed w a te r serves, at least in p a rt, as a d cso rp tiv c agent, since th e effect is m ost strik in g w ith slight]}' p o lar solvents. A lso fo r soil th e rc h y d ra tio n pro v ed h elp fu l. In th is case, ex tra ctio n is preferably perform ed by shaking w ith 2-propanol. W ith sam ple m aterials rich in lipids, analytical difficulties m ay arise w hen using propanol, because of co-extraction of em ulsion-prom oting sam ple constituents. In such instances, aqueous acetone proved m ore suitable, and w as applied successfully to m aterials as different as b rain tissue, eggs, and bees. L ipid-containing body fluids such as blood and m ilk were conveniently extracted w ith acetone. In o rd er to split possible p h en o x y esters, w hich, if p resent in the sam ple, would be extracted along w ith the acids, an alkaline hydrolysis step was inserted in th e analytical procedure. E xperim ents proved the butyl and propyleneglycolbutylethcr esters o f 2.4-D to be quantitatively hydrolyzed under the experim ental conditions. This hydrolvis step w as also expected to cleave possible carbohydrate esters and am ino acid conjugates of phenoxy acid s w h ic h m ig h t be fo rm e d in g ro w in g v eg etab le tissu es (see e. g. A n d rrtic <S Good 1957 an d K lnm bt 1961) an d are likely to be extracted w ith aqueous 2-propanol. E x tracts o f an im al tissues w ould not be expected to contain stable conjugates of chlorinated phenoxy acids, since these acids have been found to b e excreted largely unchanged in several anim al species (com pare M eta bolism, below ). Possible protein com plexes of the phenoxy acids, w hich m ight be present both in an im al and vegetable m aterials, should hot in ter fere w ith the extraction of the acids because of the generally reversible nature o f such com plexes, and of the denaturing action of the solvent an d the added m ineral acid. 12 9TST2T Moo 0001333 c PU RIFICA TIO N OF TH E EXTRACT O w ing lo the relatively h ig h acid stren g th of the phcnoxyacclic acids (the pK . values being of the o rd er of 3), an extensive separation of these acids from co-exlracled neutral or w eakly acidic m aterials should be attain able by partitioning the extract betw een an organic solvent and an aqueous p h ase of relatively low pH . T h is p rineiplc lias been applied also by Mtirtjiiu n it & L u c e (11)51, 1955). A ccordingly, the distribution o f phcnoxyacclic, as well as analogous phenoxypropionic an d phen o x y b u ly ric acids, in various system s an d at various pH values w as studied using ultraviolet spectrophotom etry. T he partition ratio or apparent portion coefficient, D, was calculated as the ratio o f total concentration o f the acid in the organic phase lo total concentra tion of the acid in the aqueous phase, an d then the partition coefficients of the undissociated acids (Ks ) w ere calculated by m eans of the follow ing equation log K d = log D + p H + log [ (h) + K J (1) In deriving this equation, the association of the acids in the organic phase was neglected. Since the dim erization constants of carboxylic acids in organic solvents such as benzene an d ch loroform usually are of the o rd er of 103 m ole-1 (see e. g. B ro w n & .V athieson (1954)), it can be sh o w n th a t d im e riza tion will not appreciably influence the partitio n coefficient a t concentrations C below 10'4 M. In the presen t experim ents, the overall concentration w as in th e r a n g e 10** to 10*4 M. T h e re fo re , D w ill b e d e fin e d b y [HA.,,J [HA..J D = [HAJ + (AJ " (HA] 1 + [A] (HA] (2) w here [HA,] denotes th e concentration of undissocialed acid in the o rg a nic phase, [HA] th e concentration o f uudissociaied acid in the aqueous phase, a n d [A] the co n c en tra tio n o f ionized acid in the aqueous phase. A fter substituting KD for an<l t T w h e re K , is th e a p p a r e n t d iss o c ia tio n c o n s ta n t o f th e a c id a n d (li) is th e h y d ro g e n ion a c tiv ity o f th e a q u e o u s p h a s e , e q u a tio n (2) c a n b e re d u c e d to D = KD.(h) (h)+K. (3) B y tra n s p o s in g in to a lo g a rith m ic fo rm a n d re a rra n g in g , e q u a tio n (1) will b e o b tain ed w hich is b e tte r su ited fo r calcu latio n s th a n is eq u atio n (3). 13 DOW 121517 4 =* a T a b le 2. P a rtitio n co effic ie n t] (lo g arilln n ic) o f p h c m isy acid s lielw cn t o rg an ic M jtvcids and aqueous buffers. C om pound 2,4-D f* n n MCPA 2 ,4 ,5 -T M CPP MCPB O rganic solvent Benzene llichlorum clhm ic C hloroform C h lo ro fo rm -- clliy i ellic r (3:1) & liy l ellicr C hloroform C hloroform C hloroform C hloroform Log Kd (in ) 0.0 0.0C 1.4 0.1 1 .3 1 0 .1 2.1 0.1 2.5 0.1 1.5 0.1 2.0 0.1 c. 2.2 c. 5 T h e p a rtitio n coefficients Unis calcu lated arc su m m arized in T ab le 2. Conditions for quantitative extraction of the plienoxy acids into the different organic phases and back into aqueous phase were calculated, and a "clean -u p " procedure w as developed, w hich involves extractive tran sfer o f th e a c id s in to b en z e n e a t p H 2, th e n in to a q u e o u s b u f fe r a t p H G.2, a n d finally into chloroform at p ll 2. In model experim ents, this procedure yielded q u a n tita tiv e recoveries w ith Utc p h en o x y acetic acids a n d also w ith p henoxypropionic acids. T he 4-phenoxyhutyric acids, being w eaker acids (pK, values around 5), w ould require a higher pH for a quantitative extraction into aqueous phase. E xtraction a t pH 9 proved useful for isolating these, us well as o th e r, w ea k acids, e. g. the d in itro p h e n o l herbicides. A dm ittedly, how ever, the selectivity in the b u ffer extraction step w ill be reduced at the higher pH values. D O ty 121518 CHROM ATOGRAPHIC SEPARATION W ith the object of separating the plienoxy acids from extractives passing the p artilo n in g step, and at the sam e tim e of obtaining evidence as to their id e n tity , a p a p e r c h ro m a to g ra p h ic sy stem w as d ev elo p ed (see p a p e r 1). T h e procedure em ploys developm ent by the circular technique, sec-butanol, satu rated w ith M aqueous am m onia, serving as the solvent. This technique is ra p id , a n d it pro v ed to fu n ctio n excellently w ith ex tracts low in ex tractives such as those from n atu ral w aters and m any body fluids. T he IlF values 14 ODOI3C5 5523 o f 2,4-l>, .MC1M a n d 2,4, j - T fall in llic ra n g e O.G-- 0.7, th e l t ,, values (m ove m ent relative to 2,4-1)) being 1.00, 0,07 an d 1.10, respectively. M ore heavily eonlaininaleil extracts require a system of higher tolerance against overloading, and therefore a thin-layer chrom atographic m ethod w as subsequently developed. The system , silica gel G: kieselguhr G (3:2)/ ethyl acetate: n-hexane: form ic acid (20:30:0.3 by volum e), show ed a good to lerance for extractives, and proved to effect a clean sep aratio n w ith extracts of m ost biological m aterials. U sually, ex tract aliquots equivalent to 5 g of sam ple, or m ore, could be applied w ithout overloading the chrom atogram . In rare instances, as w ith extracts of heavily decom posed anim al m aterials, resolution was unsatisfactory. The separation often could be im proved, how ever, by repealing the developm ent w ith the sam e solvent. T he R r values of the phenoxyacetic acids in this system (w ith a double developm ent) arc in the region 0.3-- 0.4, the R D values for 2,4-D, MCPA a n d 2,4,5-T being 1.00, 1.25 a n d 1.31, resp e ctiv ely (co m p a re p a p e r II, T a b le 1.) F o r delecting the phenoxy acids on the chrom atogram s, the silver nitrate-- phenoxyethanol reagent of M itchell (1053) proved em inently suitable, the chlorinated acids appearing as dark spots against a pale background. The lim it o f d etec tio n w ith th is re a g e n t is a b o u t 0.1 /g o f p h e n o x y a c id o n th in layer plates an d ab o u t 0.3 //g on p ap e r ch ro m ato g ram s. A rough estim ate o f tin: am ount present m ay be based on m easurem ent of the spot area, b u t obviously, elution of the spots and quantitative deter m ination by an independent m ethod will be m ore reliable and, in addition, provide co m firm ato ry evidence as to the identity. T he silver reagent cannot be used for locating the spots to be eluted, b ut experim ents proved the fluo rescent reagent 4-m elhylum belliferone to be suitable fo r this purpose. Ill u Iion experim ents proved the phenoxy acids to be effectively an d rap id ly .extracted from paper, as well as siliceous adsorbents, by am m oniacal m ethanol. DETERM INATION T he quantitative determ ination of the isolated phenoxyacetic acids was stu d ied in ex p erim en ts rep o rted in p ap e r I. A p h o to m etric tech n iq u e based on the colour reaction w ith chrom olropie acid (4.5-didhydroxy-2,7-naphthnlenedisulphoiiic acid) in su lp h u ric acid, first described by F reed (1948), w as developed. T he erilieal factors of the reaction were assessed by system atically varying the experim ental conditions (tem perature and lim e of reaction, 15 0001386 121519 )OW 121520 an d com position or reagent). From ttie results, as presented in paper I, Figs. 2-- 5, m a y b e c o n c lu d e d Unit th e c o lo u r d ev elo p m en t w ill In- co m p le te d w ith in 13-- 20 m in u te s, if th e sa m p le is h e a le d in an a ir oven a t I4 0 C w ith a reagent of u w ater content below 5 per cent w/w. At a chrom olropic acid co n cen tratio n of 0.4 p e r cent w /w , Hour's law will be valid fo r concen tratio n s u p to at least 0.3 .mol of phcnoxyacclic acid (approxim ately 110 n g o f 2,4-D) p er 3 m l of reagent (paper I, T ab le 1). T he absorption spectrum of the coloured product (identical for all the plienoxyacctic acids) has m axim a at 480 an d 380 m/( and a m inim um at 304 m /i. T h e 580-m/< m ax im u m w as selected fo r photom etric m easu rem en t. I t w as found th at som e irregularities in th e results could be attrib u ted to an u n co n tro llab le d eg rad atio n of the reag en t d u rin g heating, giving rise to a v ariable background absorption. In o rd er to m inim ize this error, the m easurem ent was supplem ented w ith a geom etric three-point correction, w hich proved to enhance the precision o f the procedure considerably. The corrected m o lar absorptivilics at 580 m u of 2,4-D, MCPA, and 2,4,5-T were a ll n e a r to 1.3. 104 litre m o le '1 cm*1. ACCURACY AND PRECISION T he reliability' of the m ethod was checked in recovery' experim ents. Thus, after adding 2,4-D in concentrations dow n to 0.15 p.p.m . to n atu ral w aters, recoveries of 84-- 102 p er cent of the added am o u n t w ere o b tain ed w ith the p ap er chro m ato g rap h ic m ethod (paper I, T able 2). Sim ilarly, 2,4-D, MCPA, a n d 2,4,5-T, w h en ad d ed to a variety o f biological m aterials a t levels o f 4 p.p.m ., o r above, w ere recovered to approxim ately 70-- 90 p er cent, using the thin-layer chrom atographic version of the m ethod (paper II, Tables 3 an d 4). In both sets of experim ents the standard deviation w as of the order o f 4-- 5 p e r cent a n d the coefficient of v ariatio n approxim ately 5-- 7 p er cent. SEN SITIV ITY T he sensitivity of the silver reaction, w hen applied in paper chrom ato g r a p h y , is a b o u t 0.3 /g o f c h lo ro p h c n o x y a c id , w h e re a s o n th in -la y e r p la tes a m o u n ts a s sm a ll as 0.0 5 -- 0.1 /<g c a n he d e te c te d . lie n e e , u sin g Ih in -la v e r chrom atography and applying the equivalent of 3 g of sam ple, concentrations 1C 0001337 5525 DOW 121521 o f 0.1 p .p .m . in tlio sa m p le s h o u ld he d electab le. W illi e x tra c ts low in e x t r a neous m atter the lim it of detection rail he suppressed by increasing llic aliipiots taken to ch ro m ato g rap h y . T hus, in n atu ral w aters, concentrations as low as 0.000*2 p .p .111. m a y h e d etected . T he low er lim it o f d eterm in atio n of the m ethod should he defined in term s of probability of differentiating a residue from a random variation of the sam ple blank value. T he blanks obtained, w hen control sam ples supposedly free of phenoxy acids w ere carried through the full procedure, correspond to 0.2 //g of ap p aren t 2,4-D (range 0-- 0.5), or a concentration of 0.07 p.p.m . (range 0-- 0.17) (com pare p ap e r II, T ab le 3). T herefore, th e practical low er lim it of determ ination w as set at 1 ug of phcnoxyacetic acid, o r 0.3 p.p.m . (calculated o n a 3-g sam ple aliquot). SELECTIVITY P artic u lar care w as devoted to checking the selectivity of the individual steps of the analytical procedure (sec paper I). In a com prehensive study of the scope of the chrom otropic acid reaction, it w as found, in corroboration of earlier results (Lc T ourncau Krog 1952), th a t th e reactio n is restricted to com pounds cap ab le o f sp littin g o ff a onecarbon fragm ent equivalent to form aldehyde, un d er the experim ental condi tions. F u rth e r, the violet-red chrom ogen proved to be identical in spectral ch a racteristics in each .case a n d to be form ed in stoichiom etric proportions. As appears from paper I, Table 4, positively reacting com pounds include, in addition to phcnoxyacetic acid derivatives, glycolic acid, 3-indoleacetic acid and com plcxoncs. O nly acidic com pounds have been considered here, since others w ill not be present in the purified sam ple extract. F ain t red colours w ere obtained also w ith alloxan, tryptophan, an d dialuric, hippuric an d uric acids, p ro b ab ly due to im purities in the test com pounds. In terestin g ly, several p otentially reactive com pounds, such as the haloacetic acids, oxalic acid, and glycine, did not react under the conditions of lest. In co n trast, p recu rso rs o f a few o th er aldehydes w ere found to give coloured products, although the absorption m axim a of the products were definitely displaced, w hen com pared w ith the form aldehyde chrom ogcn, and the colour in ten sity w as m ostly low. E xperim ents fu rth er proved that all of the chrom otropic acid-reactive com pounds w ere effectively separated from the chlorophcnoxyacctic group in the p ap er ch ro m ato g rap h ic step-- if not rem oved at an earlier stage in the analytical procedure. Com plem entary chrom atographic studies w ith a 17 0001338 5526 ( w ide v ariety o r organic, acidic co m p o u n d s w hich m ight lie presen I in p r e sum ptive sam ple m aterials (or processed extracts thereof), also show ed that only a few h ad ItF values close to those o f the phenoxvacetic, acids (see P a p e r 1, T a b le 3.). O f these, o n ly h a lo g e n -c o n ta in in g co m p o u n d s re a c te d w ith the silver reagent to form the characteristic, dark-coloured spots. None of th e c o m p o u n d s rese m b lin g p h e n o x y a c e tic a c id s in IhiIIi c h ro m a to g ra p h ic m obility and silver reactivity proved to re a d w ith chrom olropic acid. T he selectivity of the thin-layer chrom atographic system , w hen tested in a sim ilar w ay, proved to be excellent. From the experim ents review ed m ay be concluded that an analytical procedure involving a partition "clean u p " of the extract, as described above, in conjunction w ith a paper or thin-layer chrom atographic separation, and a photom etric determ ination, w ill afford an adequate degree of selectivity. C 12152 18 V 00013:9 5527 0 w 121523 Distribution and elimination Studies of (he distribution of phcnoxyacctic acids in, and their elimination from, the animal organism are reported in paper III. PLASMA LEVELS After giving 2,4-D amine as single oral doses to calves, pigs, rats, and chickens, blood samples were withdrawn at intervals, and plasma was ana lyzed for 2,4-D using the chromatographic--photometric method described in the foregoing section. Some of the animals were sacrificed at intervals -and tissues analyzed (see below). Plasma concentration was plotted against time to give curves of the type shown in Fig. 1 A (compare paper III. Figs 1--4). The initial sleep rise, apparent on all curves, indicates the phenoxy acid to be fairly rapidly absorbed from the alimentary canals of the species studied. Maximum plasma concentrations, ranging between 100 and 250 /eg/ ml for a dose of 100 ing/kg, were usually attained within 2--7 hours after dosing. The declining portion of the curves proved to be exponential; when plotted on a semi-logarithmic scale the graphs approximate to linearity, as is shown in Fig. 1 B, thus indicating a first-order rate of dissappcrance of 2,4-D from plasma. From the slope of the semi-logarithmic plots the biologi cal half-lives of 2,4-D in plasma were calculated (Table 3). As seen, the half-lives differ among species, the lowest values being found in rats (about 3 hours for 2,4-D amine) and the highest in pigs (about 12 hours), calves and chickens taking intermediate positions. Similar experiments were performed with an alkali salt of 2,4-D and with 2,4,5-T amine and 2,4-D butyl ester. The similarity of the plasma concentration--lime curves obtained with the different 2,4-D salts indi cates, as would be expected, that the cation of the salts does not appreciably influence the absorption, distribution, or elimination of the phenoxy acid. Further, a comparison of the 2,4-D and 2.4,5-T data docs not suggest the physiological properties of 2.4-D to be fundamentally altered by introduction of another chlorine atom in the molecule. The 2,4-D ester differed strikingly from the other compounds tested in giving only low and variable plasma 10* 'fVt1 5528 HOW 121524 F ig . 1. A . P l a s m a le v e ls o f 2 ,4 -D in a p ig g iv e n 2 .4 -D a m i n e (5 0 m g /k g I. n \ ) a s a s in g le o ra l d o se . B . S c m i-Io g aritlm tic p lo t o f (lie sa m e cu rv e. levels of 2,4-D, the cslcr no! being delectable in plasma al any lime after dosing. The results may be attributed lo a low absorption or hydrolysis rale of tlio ester (sec Metabolism, below). As shown by the data given for pigs and calves in Table 3, the plasma 20 000139! 5529 LOW 121525 lo zero lin n -. F ro m Hie ex tra p o la te d "zero lim e p la sm a levels" o b ta in ed , th e a p p a r e n t d istrib u tio n v olum e, VD, ex p re ssed in p e r cen t of th e b o d y w eight, w as calculated by m eans of Ihc form ula (4) w h ere A is Ihc dose adm inislered. in m g/kg bo d y w eight, an d ca is the "zero lim e plasm a coneenlralion", in /g/nil. T hese calculations arc based on Ihc assu m p tio n s (hat the o ral dose given is com pletely absorbed, w hich does not seem unreasonable in view of the relatively sm all residues found in faeces of orally dosed pigs, and lh:il diffusion equilibrium will be atlain cd relatively rapidly, w hich seem s lo be b o rn e o u t by the com paratively sm all differences between plasm a and tissue half-lives. T he VD values thus calcu lated , w ere fo u n d lo ran g e betw een 35 a n d 60 p er cent of th e body w eigh!, depending on Ihc species. H ow ever approxim ate, these values clea rly exceed Ihc ex tra ce llu la r flu id volum e (ra n g in g betw een 15 a n d 25 per cent of the body weight for m ost species), and therefore suggest some in tracellu lar distribution of the phenoxy acids. W ore direct evidence of the phenoxy acids penetrating cellular m em branes w as obtained from analysis of blood cells. In m odel experim ents 2,4-D w as a d d e d lo w hole blood, an d the 2,1-1) levels in blood cells an d plasm a d eter m ined separately on aliquots w ithdraw n after various limes. T he blood cells w ere w ashed w ith saline before analysis, hut only once and for a few m inutes, in o rd er to avoid excessive leaching of possible intracellu lar phenoxy acid. Hlood cells and plasm a obtained form experim ental anim als receiving 2,4-D w ere analyzed in the sam e w ay. Inv ariab ly a substantial fraction of the to ta l 2,4-D , u s u a lly 10-- 30 p e r cen t, a p p e a re d in th e b lo o d cells (c f . p a p e r III, T ables 3 an d 4). Analysis of w hole blood gave concordant residts. TISSUE LEVELS P igs, rats, and chickens w ere given single oral doses of 2,4-D am ine, an d th e an im als w ere sacrificed at intervals an d specim ens of tissues laken lo a n a lysis fo r 2.4-1). (Dead and killed anim als w ere exam ined hislopalhologically; see T oxicity, below.) R epresentative results obtained w ith a pig are show n graphically in Fig. 2. 22 5530 Ootv 12152G T a b le .7. P l a s m a h a lf - lif e v a lu e s n f iih i'iu ix y a c v lic a c id s in i l i f f r m H a n im a l s p e c ie s a f l v r a single u ra l dose. S |icrics C alf M P ig H at, fem ale "n m ale nn * C hicken M aterial 2 ,1 -D a m i in* 1 ' M " K-N'a snH " ester 2 ,4n-D a miiin e " ester 2,4-D a m in e II t " K-N a u H " ester 2,1,5-T am ine 2,4-D a m in e D ose ing/kK l.w. 50 100 200 100 100 50 100 100 100 100 100 100 100 100 P la sm a Ita+f-Iife. h o u rs (m ) 7 1 .5 8.5 1 7 1 8 1 10 1 10 I 12 1.5 11 2 3 J 0.5 2.9 0.5 3.5 0.5 6 2 3.0 0.5 7 1 d isappearance rate o f 2,4-D docs not seem to be significantly influenced by dosage, a result w hich is reconcilable tfith the feeble b inding of the phenoxy acid to p lasm a proteins d em onstrated in p a p e r IV (sc M etabolism , below ). In ex perim ents w ith repeated ad m in istra tio n o f 2,-f-D, the plasm a d isa p p ea rance rale w as seen to increase grad u ally in som e of the anim als. In a typical case a pig w as givn 2.4-D am ine orally as daily doses of 50 m g/kg. No clinical effects w ere seen, but the plasm a level of 2,4-D (at 24 hours a fte r dosing) w as found to decrease d uring the first 8 days from 50 to 8 /rg/m l, an d then to rem ain a t the low er .level. Since a co n co m itan t increase in u rin ary o u tp u t of 2,4-D w as noted, an d there w as no indication of a m eta bolic transform ation being involved, the finding m ost likely reflects an en hanced renal excretion. Conversely, a reduced disappearance rale w as usually encountered after doses sufficiently high to produce toxic sym ptom s, an effect w hich probably w as due to an im paired excretion. Conceivably, also a delayed absorption m ight have influenced the result. An attem p t w as m ade lo estim ate the ap p a ren t d istrib u tio n volum e of p h enoxy acids from plasm a levels. T h e term inal lin e a r |>orliou of the log p la s m a c o n c e n tra tio n -- lim e p lo ts (Fig. 1 B) w a s e x tra p o la te d b a c k w a rd s 21 000139j 5531 DOW 121527 Fiij. 2. T issu e lev els o f 2,4-D m a pig (al C h o u rs a flc r d o sin g ) g iven 2,4-D a m in e (100 m g /k g ) as a single o ral dose. As seen, a rap id an d high u p tak e of 2,4-D occurred in the excretory organs, Uic liv e r a n d k id n e y levels re la tiv e lo p la sm a (al G h o u rs a f te r dosing) a m o u n tin g to 70 a n d 85 p e r c e n t, resp e ctiv ely . R a th e r h ig h levels (GO-- 70 p e r re n t) w ere also rap id ly attain ed in the lungs, adrenals, m yocardium , an d ovaries. S o m ew h at low er, yet c o m p arativ ely h igh, levels (30-- 50 p e r cent) w ere e q u a l ly rapidly attained in a variety of tissues, including the lym ph nodes, spleen, thym us, thyroid, pancreas, and salivary glands. A pparently, the phenoxy acid distributed itself fairly evenly over the body w ater. T he relative concentrations found in skin, skeletal m uscle an d cartilage w ere m oderate (20-- 30 per cen t), an d those in adipose tissue an d b ra in low (5-- 10 p er cent). T h e low brain levels observed suggest a lim ited rale o f tran sfer across the bloodb rain b arrier. T h is w ould not be unexpected in view of the cu rren tly accepted lip id -p artitio n m echanism of d ru g tran sfe r across body m em branes (sec e. g. Sclum krr 1902). Because of the extensive ionization of phenoxyacetic acids at plasm a pH . only a m in u te fraction (less than 1/10.000) of the acids will be present in the undissocialed form , the concentration of w hich determ ines the rale of diffusion across the lipoid m em brane. It should he noted, how ever, that in cases o f acu te o r su b acu te poisoning, ra th e r high concentrations, in an absolute sense, m ay be attain ed in the b rain (up to 05 /rg/g), as evi den ced b y th e re su lts s u m m a riz e d in p a p e r 111, T a b le 9. A lso in cases o f 23 0001394 . 5532 DOW 121528 fatal phenoxy h erb icid e poisoning in liiim nn stihjce.ls, m oderate to h ig h p h en o x y ac id levels in c e n tra l n erv o u s tissu e h av e been re p o rte d (I lr r b ic h & M a ch id a t Oti.'l; X lc lsc n cl al. ItMi.V. G elilniachcr-- n. M n llin ckro ill & L a itlc n bacli 19f>(>). As judged from the rapid appearance and the high concentration of 2,4-D in the u rin e an d kidneys of pigs, renal excretion seem s to be a m ajo r route o f elim in a tio n of 2.4-1) in this species. T h e acid ap p a re n tly is excreted in an unchanged form (see M etabolism , below). Both phenom ena m ay be attributed to the low lipoid m em brane perm eability of the phenoxy acid, at norm al p h y siological p iI. T his w ould he expected to im pede not only the p en etratio n into the m icrosom al system of liver cells responsible for detoxication pro cesses, but also the renal tubular reabsorption of the glom erularly filtered p h e n o x y ac id s (c f . B rotlic, C oxm ides & R ail 1965). M oreover, a tu b u la r se c re tion of phenoxy acids does not seem unlikely, in view of the dem onstration in various anim al species of a tran sp o rt m echanism of low specificity (the hippurate transport m echanism ) capable of secreting a variety of acidic com pounds, including arom atic an d arvlsubstitutcd carboxylic acids (sec review by Spcrbcr 1959). T h e observed biliary excretion of 2,4-D lends som e support to the po stu lated tu b u lar secretion of phenoxyacclic acids, since tubular and biliary secretion o f m any organic anions seem to be governed by principally sim ilar m e c h a n ism s (S p e r b e r 1959). T h e distribution of 2,4-D in representative tissues of pigs, rats, an d chickens a f te r a sin g le o r a l dose is illu s tra te d in F ig s. 3 A, B, a n d C. T h e d ia g ra m s a re based largely on results presented in p ap er II, T ables 5-- 7, b u t data obtained subsequently have also been included. As seen, the relative kidney levels w ere hig h er in rats a n d chickens. T h is resu lt, w h ich is consistent w ith the h ig h p lasm a d isap p earan ce ra te observed in these species, m ay be an indication of an effective renal excretion. O therw ise, no striking species differences in distribution pattern were apparent. T he rate of disappearance of 2.4-D from tissues after being given in subtoxic doses w as fairly high in all species exam ined, the peak levels alw ays being attain ed in less than 24 hours, and the levels th ereafter usually decrea sing to a few ,g/g w ithin 2-- t days. T he tissue half-lives ranged betw een 5 a n d 10 h o u rs in ra ts a n d betw een 10 an d 30 h o u rs in pigs an d chickens. In o rd er to study the effects of prolonged exposure to phenoxy acids, 2.4-D am ine w as ad m iu slered in the diet to pigs and in the d rinking w ater to rats and chickens, and the anim als sacrificed after varying tim es of exposure (sce T oxicity, below). Plasm a and tissues w ere analyzed for 2,4-D (Fig. 4). 24 0001305 5533 300 200- _L 6 hrs, A. Pigs 24 hrs. 48 hrs. JL 100- 1 1 X IL 1 JJ X h 11 DOW 121529 100- V ix ill 123456 1 1 2 3 4 5t L6 _ 1 2 3 4 5 6 P ig s. 3 A , I t a n il C. A v erag e tissu e levels o f 'X X D . a I d if fe r e n t lim e s a f te r losing, in d iffe r c n l s p e c ie s g iv e n a s in g le o r a l d o v e o f 2.-I-1) a m in e 1100 m g /k g i. I : p la s m a . X k id n e y , if: liv e r , 4 : lu n g , a : s p le e n . 6 : s k e le ta l iiu im -Ic . V e r tic a l li a r s d e n o t e s t a n d a r d d e v i a t i o n . 25 0001326 5534 Dow 121530 F uj. 4. A verage tissu e levels of 2 ,4 -0 in d iffe re n t species a fte r rep eated oral a d m in istra tio n o f 2 ,4 -0 am in e. 1: p lasm a . 2: k id n ey , 3: liver, 4: lung, 5. b ra in , 6. sk eletal m uscle. V ertical b ars denote stan d ard deviation. A com p ariso n of F ig. 4 w ith Figs. 3 A, B, a n d C docs n o t reveal an y m ark ed change in distrib u tio n pal lorn of 2,4-D w ith repealed dosage, except fo r som e elevation of the tissue levels relative to plasm a in the latter case, reflect ing the slightly low er elim ination rale of phenoxy acid from tissues. A bsolute tissue levels, how ever, w ere not seen to increase d u rin g these experim ents. T herefore, prolonged ingestion of phenoxy acids, at the dose levels tested (corresponding to daily intakes betw een 25 an d 100 ntg/kg body w eight), w ould not be expected to involve a n y h az a rd of accu m u latio n in the species studied. It can be noted that laying hens evidently arc able to excrete som e 2,4-D w ith the eggs. R esidues of u p to 2 ,g/g w ere found in the yolk, the album en co n tain in g o n ly traces below 0.4 ag/g. In an o th er long-term study, a sow w as given 2,4-D am ine during the gestation period (sec Toxicity, below ). On p artu ritio n fifteen piglets w ere b o rn , ten out o f w hich died w ithin 24 hours. T hese piglets did not ingest 2.4-D -conlaiiiing m ilk or feed before death. On analysis of their tissues, ap p reciab le co n cen tratio n s of 2.4-D w ere found, ran g in g from 80 to 300 p er cent, relative to plasm a of the dam . w hich seem s to indicate a fairly unhindered passage of the phenoxy acid across the placental b arrier of sw ine. Considering the apparently reslriced penetration into the brain and the observed u rin ary and biliary excretion of phcnoxyaeclic acids, suggestive o f a lim ited m e m b ra n e |H Tinenhility at plasm a pi I. a low ra le o f p la ce n tal tran sfer would be expected. 20 0001307 553- TCST2T M o o Metabolism As reported in p ap er IV, som e experim ents w ere perform ed in o rder to study the possible b io lran sfo rm alio n of phenoxyucetic acids in the an im al organism . In prelim inar}' in uifro-studics using the gel-filtration technique, the m obility of 2,4-D in Scphadcx gel w as found to increase in th e presence of plasm a proteins. The result, w hich m ust he interpreted as a definite, though w eak, in te ractio n of 2,4-1) w ith th e p ro tein s, is in line w ith the d ata o b tain ed b y T ercsi &. L u c k ( I VI X) , in d ia ly sis ex p e rim en ts w ith u n s u b stilu lc d phenoxyacelic acid and serum album in. Several findings reported in the p re vious section of this review , such as the m oderate plasm a levels of 2 .4 -0 attained in various species a fte r oral dosing (suggesting a large distribution volum e) an d the h ig h p lasm a d isa p p ea ra n ce ra te o f 2,4-1), also p oint to a slight plasm a protein-binding of 2,4-D. If the plasm a protein-binding w ere extensive, high an d persistent plasm a levels of 2,4-D w ould be anticipated. F u rth e r, the plasm a d isappearance rate would be expected to increase at high plasm a levels, ow ing to the pro tein binding capacity being exceeded. T his w ould be accom panied by a m ark ed increase in unbound phenoxy acid in plasm a, and thence by an enhanced rale of diffusion from plasm a. From the results of Tercsi & L uck, the concentration of unsubstituled phenoxya c c tic ac id c o rre sp o n d in g to s a tu ra tio n o f p la sm a a lb u m in c a n be e s tim a te d In ap p ro x im ately 3 niM (or about 450 /rg/m l). F o r the m ore bulky ch lo rin ated dcrivates, low er values w ould be expected. As judged from differential hydrolysis studies, u rin ary conjugation 2.4-D in pigs w as not extensive. Acid hydrolysis of thin-layer ehrom utograplucnlly se p arate d fra ctio n s o f u rin e ex tracts w as round lo release 2,4-I)-like m aterial in a m o u n ts not exceeding 10 p er cent o f lolal phenoxy acid. Likew ise, b iliary 2,4-1) w as sho w n lo be essentially unconjugaled. T h e failure to delect si/.able am ounts of conjugales o r other m etabolites o f 2,4-1) by th e a u th o r, as well as bv o th er w orkers (Ask cl ni. 1003; liachc et al. 1004; S i. J o h n cl til. 1004; ('.lurk cl nl. 1004), is co n sisten t w ith o th e r findings, m entioned above and suggesting a low m em brane perm eability of phenoxyaeelic acids al plasm a piI. O ral ad m in istra tio n of a 2,4-1) ester lo various an im al species resu lted in on ly trace am o u n ts o f ester a p p e arin g in p lasm a, tissues, o r urine. In co n tra st, 27 0001338 Or/ 12153 2,4-D acid w as p resen t in lody fluids a n d tissues. a lth o u g h al relativ ely low levels. It m ay lie concluded th a t the ester is eith er slow ly ab so rb ed an d rapidly h y d ro lv /e d , or slow ly hydrolyzed, and the liberated 2,4-1) acid rap id ly ab so rb ed . T h e a lte rn a tiv e possibility th a t the ester is rap id ly ab so rb e d an d then slow ly hydrolyzed seem s rem ote, since In th at ease in ta ct ester sh ould he delectab le In p lasm a. 28 0 0 0 133 5537 OO\y 121533 r Toxirilv T h e acute, subacute, and chronic loxicilics of phenoxyacclic acids were stu d ie d in a n im a l e x p e rim e n ts rep o rted in p a p e r V. ACUTE TOXICITY In acute toxicity experim ents carried o ut in connection w ith the d istrib u tion studies, phenoxyacctic acid derivatives w ere given orally by stom ach tube to calves, pigs, rats, and chickens (Table 4). Blood sam ples w ere w ith draw n at intervals for phenoxv acid determ ination. As seen, definite, although reversible, toxic effects from 2,4-D w ere observed in calves at a single dose of 200 m g/kg body w eight. M ain sym lom s of poiso ning w ere anorexia, tym panites, m uscular incoordination and dysphagia. In pigs, adverse effects ap p eared at a single dose o f 100 m g 2.4-D /kg; the clinical signs included anorexia, diarrhoea, a stilled gait and, in severe cases, vom iting, m arked m usclar weakness and general depression. On autopsy, signs of gastro-inteslinal irritation and pulm onary lesions w ere the m ain findings. R ats and chickens ap p aren tly tolerated 2,4-D as single oral doses of up to 100 a n d .'100 m g /k g , resp ectiv ely , w ith o u t c lin ic a l a n d m orp h o lo g ical! effects. In a c c o rd a n c e w ith re su lts o f o th e r w o rk e rs (H ill &. C arlisle 1947; H ow e H ym n s 1954). no difference in toxicity betw een d ifferent salt form ulations of 2.4-D w as observed. Likew ise, the effects of 2,4.5-T, as an am ine salt, ap p aren tly w ere sim ilar, both qualitatively and quantitatively, to those of 2,4-D. An ester form ulation of 2.4-D, how ever, proved to be less acutely toxic by the oral route (ban the w ater-soluble salts, probably ow ing to a low rate of absorption o r hydrolysis. SUBACUTE TOXICITY R epeated daily oral doses o f 2.4-D am ine o r ester w ere given to pigs an d ch ick en s, th e do sag e sch ed u le an d m ain resu lts being su m m arized in T able 5. D ead, as well as surviving, anim als w ere autopsied an d tissues, 29 0001400 5538 o S p e c ie s Ci. If * M aterial 2.4-1) a m in e "" " " K-Xn salt 1 l.itly l e s te r P ig 2.4-1) iim inc 1 M M Rnt 1 It " C hicken I M M M * " b u ild elcr 2.4.5-T nniinc 2,4-1) n nrinc " K-Nn w ih " lintyl ester 2.4.5-T n niinc 2.4-1) n n iin c H Table 4. A cute to x icity o f p lien o x y ncct lc acid d eriv ativ es. X iim licr Ire n le d 1 2 2 2 2 2 * 2 2 2 2 35 III 14 10 3 6 2 D osage ins/liK 50 too 200 loo 100 50 too 600 1000 100 to o 100 ' 100 100 100 100 200 200 C linle.il effects A iiin|isv fim lhigs T ra n sie n t d y s|ili.isi.i in o n e nnin i.it A no rex ia; liloiitiug in o n e niiiui.it A n orexia, th irs t, m u sc u la r xveiikness, su b sid in g in 3 -- 1 d ay s T ra n sie n t d y sp h a g ia in o n e a n im a l C iiaffeH ed A norexia in on e an im n i A norexia, stilled gait, tran sien t d ep res sion V om iting, severe m iiscukir w eakness. C O IIK I V om iting, cvcrc m uscu lar w eakness, i`o m n > U m iffeclcil A norexia, vom iting, d iarrh o ea liH -oiiinlury d lslu rh a iio e s U iH iffected W M 1 n . -- --* -- -- G nslro-eiilerllis M I* G aslro-cnleritis. ren al nm l h ep atic congestion G nslro-eulcrilis. ren al an d hepatic rongestinn Slight g aslro -en lerilis tiastro -rn lerilis No significant gross changes It It II W It # N 1 (| H 1 tt It If II If P ro re n lric i.tilis in o n e b ird n ) KHhrd In a m o rib u n d M ule n t 24 h o u r n ite r d o lin g . 0001401 {'3121 M O o o tn cn fk. o OM S|H'CCS l*B ** ** ft 1 * 1 II C hicken M Co Table i . Siihncnlc loxlcily of 2,4 D am ine orni osier. M iilcriol DosiltfC niB /kc/ilny `2.4-1) lim in e I * ** " M osier II 1 1 1 It II II am ine " I* 30 TlO SO 50 50 100 100 :ioo 50 50 50 50 .100 300 .100 .100 X m illier nf doses given 3 H 10 15 51 3 7 2s 7 1*2 `2.1 3 6 13 24 C.Kiik h I o f fe e l s A u to p sy hifliiif** t'n a ffc e lc d A norexia, (liorrlioen, (lepre W o n l.'i i a f f r e lc d general A norexin, rein riled |r(i lli V oiinling. general depression V nuiiling, ilh trrlio n t, m iisn iltir n isi kuisvt (iciHTiil ilcprc.M ion l.'n n ffeelcd A n o rrsiii, litirrlm cu, miisciiki-r w nikiirvs t'lM iffccled M nseiilnr w eak ne vi .A norexia, ilinrrliooji, m tixruhrr w rn k n rv i, te n o rili <lr|rvv*inn D ead on fifth day l-'iK iffeeled No significant fn i.v changes Ini-drie n icer, cn ln rrh n l e n le rills X'n s ig n if ic n n l g r o s s e lim in e s llrnncliiiil lym ph node hyp erp lasia, renili fully degriienrlinn ( `. iliirrlin l e n l e r i l is (iiivtrie n icer, cnln rrlm l colitis ( iis lr ic n l i i T , <*;ii;irrl;i 1 m i r r i l i * IM iriiiiio n iii, r n i ; i l f;l 1y i l r ^ r m r a l i o n llcnnl fully d cgrncrnlinn Xo significan t Kross ch an g es C.nl-.irrlnil K o v tro -n H crilis, IM iriinioiiiti N'o slg n ific iH rt g r o s s c h a n g e s llronelihrl lym ph nolle liy p rrp lath i, kidiirv rnlartfrinriil Cm slric n icer, en larrln if en le rilis. p n e n in o n ia llennl a n d vise rra i goni Siigli! k id n e y e n larg em en t Il M M sesisi M o q 4 ex am in ed m icroscopically. 2.1-D levels w ere determ in ed in plasm a an d r e |)resell I1 live tissu es (p a p e r III). As will Ik * seen, both 2,4-D am ine an d 2.4-D esler evoked toxic effects in pigs al Ihe low dosage level o f 50 m g/kg/day. T he lesions w ere observed m ainly in Ihe digestive, resp irato ry , and exerelory organs. Il m ay he noted that Ihe gas lie nlceralions w ere consistently localized in Ihe fundus rath er th a n Ihe c a rd ia c region o f Ihe s lo in a c h . th u s d iffe rin g from Hie lesions seen in sp o n tan eo u s cases of g astric u lcer in pigs. In instances o f m acroscopic p n e u m onia. Ihe histopathologic picture w as dom inated by alveolar cell p ro lifera tion. T he renal fatly degeneration involved varying tubular sections and H c n lc 's loop. T h e chickens seem ingly tolerated 300 m g/kg/day of 2,4-D am ine w ithout clinical effects or gross m orphological changes. T he results of these studies seem to suggest a correlation betw een the ap p earan ce o f clinical sym lom s and the plasm a level o f phenoxv acid. T hus, in pigs, plasm a 2,4-D ap p aren tly h as to exceed a threshold value o f about 200-- 300 ,g/ml fo r th e signs of acu te poisoning to appear. T h ere seem s to be a sim ilar threshold level in calves. CHRONIC TOXICITY T he effect o f prolonged exposure to orally adm inistered 2,4-D w as in vestigated in pigs, rats, an d chickens. Pigs. A group of five 2 m onths-old pigs (litter-m ales) w as m aintained on an experim ental diet containing 2.4-D am ine at a level of 500 p.p.m . fo r up to 12 m o n th s, tw o o th e r litte r m ates serv in g as controls. T h is dose level w as calculated to correspond to a daily in tak e of about 25-- 50 m g/kg body w eight. In all anim als depression of Ihe grow th rale, not associated w ith a reduced feed intake, was evident. Docomolory disturbances were com m on, and two anim als exhibited a unique elongation of the anterior lateral hoofs. O llier significant clinical effects w ere not noted. H aem atologieal and b io chem ical exam ination revealed a m oderate hyperchrom ic anaem ia, slightly elevated plasm a GOT, slight hypoalhum inncm in and m oderate album inuria. T h e a n im a ls w ere k illed at in te rv a ls b etw e en 2 a n d 12 m o n th s. N o c h a r a c teristic gross changes were observed, but m icroscopically, parenchym atous degeneration of the liver and slight degenerative changes in the kidneys w ere delected. T he reduced feed efficiency and Ihe clinical-chem ical and hislopatholngical evidence point to a m ainly hcpntoloxic action of the phenoxy acid in 32 0 0 0 1 4 G3 5541 DOW 121536