Document ykBbxkoMj5vX1YNYmwyw2wGn4

:* > WAL HEALTH dio^raphic trac^jonS'of'the ga|J ' tract, and i percentage of >ence of, symp'.ent may not be the findings are institution of basis for early develop. Sigindispensable, olyposis alone se as a routine ical examinaestablishment ons in whom oratory studsent when it 3% of those d. and electroluable when ingle abnord with cau>ut verifica- ^^h:hi:is examiitly most for occult acid and is a valuiy in disyps." !! bladder, colon reiptomatic n, 0. T, winations, 5SL ation of lucose, J. nination. (Chronic Orai ^Joxtcitty 2-^tliy.lliexifl f-^htlialate In $atd and ROIERT S. HARRIS, H.D., Cwnbridge, Mm HAROLD C HODGE, Fh.D. ELLIOTT A. MAYNARD, Pfc.D. sod HARVEY J. RLANCHET JR, M.D., RedtMtw, N. Y. INTRODUCTION Among the useful and versatile plasticiz ers in the manufacture of films and tubing from synthetic plastics, few are more widely used than 2-ethylhexyl phthalate, known in the plastics industry as DOP, dioctyl phthal ate. Large volumes of this material are being handled in manufacturing and industrial ap plications. Of special interest is the use of DOP as a plasticizer in films for wrapping nonfatty foods and in the manufacture of intravenous vinyl tubing. The low toxicity and low leachability by aqueous media are important properties justifying these uses. In 1943 Hodge1 characterized the acute toxicity of 2-ethylhexyl phthalate in rats and mice as "very low.'' Shaffer and associatesa confirmed and extended these observations in other species and by other routes of adminis tration. From metabolic studies these authors concluded that "such injurious action as it (DOP) does exert within the body appears to be due to the alkyl part of the molecule rather than to the phthalate portion." Later Received for publication Sept 19, 1955. This work was supported in part by a grant from the Dewey and Almy Chemical Company. From the Department of Food Technology, Massachusetts Institute of Technology (Dr. Harris) sod the Division of Pharmacology and Toxicology, University of Rochester" School of Medicine and Dentistry (Dr. Hodge, Dr. Maynard, and Dr. Blanchct). chronic feeding studies in rats, guinea pigs, and dogs were reported by Carpenter and associates * and "no effect" levels of 0.06 gm/kg/day (or somewhat higher for rats) were established. The current studies con firm these results and add data for higher daily doses. For completeness the available information on the acute toxicity of DOP is summarized in Table 1. The low acute toxifcity in several species by two routes is evident. RAT FEEDING STUDIES Chronic Orai Toxicity.--From a large group of weanling albino male and female Wistar-strain rats three groups, each comprising 43 males and 43 fe males, were assembled. The basal diet1 contained 82% whole wheat, 16% full-fat milk powder, and 1.6% sodium chloride; 600 1. U. of vitamin A was added per 100 gm. of diet Diets and distilled water were supplied ad libitum. Record was kept of the food intake of each group. DOP was added to the diets in the following percentages; 0% (control groups) ; 0.1%, and 0.5%. Each group of 43 male and 43 female rats was further subdivided in a serial killing program: (a) 4 rats were killed after 3 months; (6) 4 rats were killed after 6 months; (c) 10 rats were killed after 12 months, and (d) 24 rats were to be maintained on the diets for 24 months. At each killing period the body weights, food consumptions, and organ weights of liver, testes, kidneys, lungs, brains, stomach, heart, and spleen were recorded. A number of tissues were sampled for histological study; these included heart, lungs, spleen, esophagus, stomach, small and large intestine, liver, kidney, testes, brain, and bone marrow. In addition, any pathological tissue was sampled and prepared for histological study. RESULTS Mortality.--The over-all mortality during the two-year period was high. During the two-year test period 85%-96% of the rats died. There was no adverse effect on mor- 259 ucc 041210 ***., V * * * * . t. #*' .* * # 0` (r . I? ,, * *, I ,,* i t, ' t* . ' * 1* ' * *h \ l` r* 'Ur ' 1 \*. 7 4' '-' i . : % .. 7^ A. M. A. ARCHIVES OF INDUSTRIAL HEALTR tality with increasing percentages of DOP in the diets. This confirms the observation of Carpenter and associates.* Body Weights.--During the initial weeks growths of the several groups were roughly equivalent. The rats given the 0.1% diet grew a little more rapidly than the control groups. The addition of 0.5% DOP to the diet ultimately depressed growth somewhat. For example, at the end of a year the body weights of the control and the 0.1% group were practically identical, whereas the aver age for the 0.5% group was approximately 50 gm. less; In the second year, as the groups grew smaller, there was a general trend for the average body weights of all three groups to become more alike, and just before the of the group given the 0.5% diet had fallen to about 75% that of the control group. The DOP intake calculated for the group given the 0.1% diet ranged between 0.05 and 0.08 gm/kg/day and for the group give the 0.5 % diet between 0,3 and 0.4 gm/kg/day The higher figures were observed during the earlier part of the experiment, i. e., in the third to the sixth month. In the study of Carpenter and associates* the group given the diet containing 0.4% DOP ingested about 0.2 gm/kg/day and exhibited no retardation of growth. The rats in the current study ingested nearly*twice as much DOP daily and exhibited a growth reduction. The difference may well be due to the higher dietary intake. Hefenm X 1 1 t % Species fiat* Bats Bata Babbits Does Humana 1 Mk Mice Bata 1 Rsts Sat* Rabbit* Table 1.--Acute Toxicity of Di(2-Etkylkexyl) Pktkalate Sea Male Mak Mala Hall Mak Mak Bale Mak Bamale Strain WBto, dOyn. Oral AdmioktraUon Wlatar Wktar Wiatar SMB 1S6-U4 Ui Albino ....... M004400 Obacrration Period. Daya 14 ,, I 14 .f. ..rntraparltonaal Administration Albino IMS ...... Wlstac ..1 ....... 149-191 a. ....... MMS1 1 ....... *4004,000 tl -----' Comment* 9U |Dr/kr.-nl.l>4t Bona dkd with M cm./kf. Bonadladwltb t ee./kc. tU cre.i'k*.--L.D.i, S *m./k./day vftbnot afleet A am. stack doss, no effect 10 cm. slock doss, mild ssthsnis US cm./kf. raw o% mortelity None died with 100 ae./kf. M.T fm./kc---Z*D.m Boos dkd with M cm./kf. Bono dkd with 71 tx.ftf. Cl ace/kf. tolerated 1 !3 f ; * ` ]t 1 I : end of the second year the averages for the three groups were numerically practically identical. This phenomenon is attributed to the small number of rats surviving in each group. Food Consumption.--Records of food dis appearance maintained to the third month and again to the sixth month showed prac tically no differences between the control group and the two experimental groups re ceiving DOP. This confirms the observation of Carpenter and associates * that food utili zation was the same in all their groups. By the end of the first year, however, the evidence indicated that the food consumption 260 Organ Weights.--Only in the case of the liver and kidney was there any question of a systematic variation in average organ weights, either fresh weight or calculated on the basis of body weight. At three months and six months, for both the males and the females, fresh weights of liver and kidney* and weights calculated on the basis of body weights were elevated for the 0.5% group (Table 2). These data confirm the finding by (Carpenter and associates * that livers and kidneys were enlarged when the diet con tained 0.4% DOP. At 12 months and 24 months, however, the fresh weights of the * Except the male kidney weights at six months. a * Conti 04%.. 0.5%. Cant 04%. 04%. organs basis c candy there t end of Path killed ' above. ination variou: from t sional -- 1 Oman Uvr Kidney Teetle Long* Brain Heart Spleen dTra Bladde ucc 041211 .... ,, l HEALTH At,h>had^faJJen roup. The sroP given n 0.05 .and Tonp given gm/kg/day. ved during . i. e., in the '.e study of roup given ested about retardation rent study X)P daily i. The difler dietary tf. c. *fl*et tot lutSuvt* e of the stion of organ Icukted months md the idney * f body groupEnding rs and t connd 24 ->l the tenths.. ORAL TOXICITY OF Z-ETHYLHEXYL PHTHALATE Table 2.--Organ Weights of Rats Fed DOP (Milligram/Gram Body Weight) Diet Groups ----------- -------- --------s Mo. t--------- * \,f, e Mo. ** \r-"m *----------\,, ,,u Mo. 34 Mo. --------------------- *--------------------- -------------------- *-- Liver Kidney Liver Kidney Liver Kidney Liver Kidney Mules Control......................................................... 30.2 7.4 #6.4 7.0 82.4 7.1 41.9 8.1 0.1%................................................................. 38.9 7.7 30.4 6.9 30.0 7.0 34.4 7.7 0.5%........................................................ . 4S.3 7.9 32.3 , 6.7 28.7 7.0 41.9 8.1 Females Control........................................................... 27,0 7.3 *7.2 7.0 31.8 7.4 42.8 8.6 0.1%...................................................... 33.93.1 29.9 7.5 38.0 7.1 36.0 8.0 0.6%................................................................. 40.4 3.1 33.3 . 7.9 27.3 7.6 37.6 8.0 organs and the weights calculated on the basis of body weights did not differ signifi cantly from group to group, mainly because there were only a few rats surviving at the end of the second year. Pathology.--Three Months: Rats were killed by decapitation at the times indicated above. The results of-the histological exam inations will be reported separately for the various feeding periods. Scattered foci of round cells were seen in the liver sections from the-control group-and also from both groups fed DOP. In the control rats occa sional dilated renal collecting tubules were seen. One rat on the 0.1% DOP diet showed some tubular atrophy in one kidney section, and one rat on the 0.5% DOP diet exhibited occasional casts. One 0.5% DOP-fed rat showed marked tubular atrophy in one sec tion of testis, and another showed mild changes. Six Months: In the Infers of rats from the three groups there was an occasional focus of round cells. One rat of the 0.1% DOP group showed slight changes in some of the cord cells of the liver. In the kidneys of certain rats in each group there were occasional dilated tubules and casts. Calculi Table 3.--Pathological Findings: Twelve Months Organ Liver Kidneys Testis Lancs / 1 Control Mato (8) Casts, 1 Nephritis, 1 Slight tubular atrophy, 2 Females (8) Occasional locus of round cells, 1 Small areas ot tubular dila tion, 2; piement In tubule epithelium, 1 Broncho* pneumonia, 3 Diet 0.1% DOF Males (8) Females (8) Few hyaline casts, 2 round-cell focus, 1 Occasional round-cdl focus, 1; casts, 2 Bronchi ectasis, 8 Broncho pneumonia, 3 Brain He*n Bpleen PolrarUrlUi of aoronur rtaiT.l Gl Tract Bladder foe! In suh* mucosa ot stomach, l Debris, x Rad cell phagocytosis, l; excessive pigment, l Cyst, I Debris, l Parasite, 1 0.6% DOF Males (8) Sllfht foamy cytoplasm, 1 Female* (8) Casts, 3 Cyst, i: chronic Inflammation, 1 Bronchi ectasis, 2; broncho pneumonia w/abscess, 1 pigment, 1; red cell pbafo' cytosis.i foci in submiicosa of large intestine, 1 Thin, homo geneous eosino philic coat over epithelium, 2 261 ucc 041212 A, M. A. ARCHIVES OF INDUSTRIAL HEALTH \ were observed in the bladders of one of the male rats on the 0.1% diet and two of the male rats on the 0.5% diet; such calculi are found from time to time in control rats of our colony. One rat of the 0.5% group exhibited a small focus of round cells in the heart muscle. Twelve Months: The pathological findings at 12 months are presented in Table 3. Tis sues from eight males and eight females from each of the three diet groups were ex- and excessive pigment were seen occasionally in the spleens, suggesting that there had been at some time an infection by Salmonella. Twenty-Four Months: A thorough histo logical examination of selected tissues from the relatively few animals that survived the full 24-month period revealed no evidence of any pathological change that was attrib uted to the administration of DOP. Among the unrelated pathological findings listed in Table 4, the commonest were (a) lung Table 4.~Pathological Findings: Twtnty-Four Months Orfia Tumor Liver dd7 r------------------Control Mata* (l> Pamela (1) * Cutl Caita OOMdf Luofi Artartal rtianfa Bronebl* setssla Acut, and chronic codoHMtritf, Bronchi* eetaite Brain Heart Spleen nicht mroeaidlal flbreaii (II Tract Bladder Marrow isenaw POlj In aabameoaa Diet 0.1% DOP Mala ) Pcm,1a, (t) 0S% DOP ' Mala (t) females (4) Watt, on loot Chronic iMphrltifl, 2; cutl, f Minimal bron chia, and bnoaiMitelc, 1 o 1; tod of round uDf11 Cbiwlt nephritic, 1; cortical aearrias, 1; cacti, t Manhritia.t; caaw, t Acut, and chronic andomctrltl,, 1 Atrophj, l; local MeroaU,l ante bron chitl,, 1; minimal bronchlactaal*, t Brondriataali, 1; amt, bronchltlc, 1; broncho pneumonia,! Myocardial Sbioaia, 1 Kaphritta, S: cortical marring, 1; cut*,* in fallopian tube, 1 Brooebiectaafa, 1: acute duoua bronebltic, 1; bronchopneu monia, l; arterioaderodi, 1 Pod ot braBnc tblckcnlttf ot manlnca, 1 Pod ol eoalnopbUalneobmneoaaof ftomadi,l Fnrarit*. 1 Xxeaelve piement,l Pod of ooahiopMhalnmbmnooaaof email bowd, 1 r i i i k amined. Despite a number of findings of obscure pathogenesis, there were no changes in the tissues that were attributed to DOP. The findings are those observed customarily in rats of this age in our colony. Specifically, in the control rats there were typical ex amples of bronchiectasis and of chronic nephritis. A slight testicular atrophy was found in two males. A rare periarteritis of the coronary artery was observed. In the rats given DOP, a bladder concretion was seen in one male. Red blood cell phagocytosis 262 disease, frequently fairly well advanced, and (b) chronic nephritis. Three of the rats showed warty tumors of the hindfoot, de scribed as fibroliposarcomata. A rare mild cardiofibrosis was observed in two rats. Acute and chronic endometritis was seen in two rats. An example of a rare arteritis of the testis and an occasional focal atrophy or necrosis of the testis were seen. Small granulomata were found in the fiver of one animal presumably as a result of some infec- ORAL T DDWOtP, %ia Control 0,01* OJ OJS* M* * Dtta tnr In Tabk form a si observed ii The data ; associates * increased 1 the diet. J the diets c was a ret; 0.5%. Foo that f con to and ind penter and tion in die the diet con even thou| comparable to 100% gi mat when including 0. were obset 0.4% or 0. Don, No. 4 4 1 r l * Dtt troc ucc 041213 health _ ccasionally ^khad been onella. ugh histo ries from *vivtd the > evidence as attrib?. Among ' listed in () lung >(4) Mon toot iittli,*; ell stafcis oiir Uoplaa dilMtiili, ito chronic iihooitaliw.l;- l; arte* eroala, i )f hyaHn+ Cl&Mf,f1Of at* l fCOltno* 4I>n0O1sf*u1b,*\ ed, and Te rats *bt, dee mild a rats, seen in ritis of itrophy Small of one infec- j*.- - - ` " ORAL TOXICITY OF 2-ETHYLHEXYL PKTHALATE Tabu 5.--Summary of Chronic Feeding Studies: Rats DOP in Dfet.% Control 0.M* 0.1 0.13* 0.4* 03 Mortality 70(* 00 flame at control flame at control flame ae control flame ae control flame as control Body Wt. Normal flame as control flame as control flame as control Lower Lower Pood Consumption Normal flame as control flame as control flame as control flame as control Lower Dally DOP Intake, Gm./K*. 0 0.02 0.04-0,08 0.00 030 OJ-03 Organ Wt. Norma] Normal Norma] Normal Enlarged liver and kidney Enlarted liver and kidney at 8 and 8 mo. Patholorr Sima u control rate Same aa control rate Same as control rata Same aa control rata Same aa control rata Bamaaa control rata * Data from Carpenter, WU, and Anyth.* Suuuaiy of Rat Studies In Table b is presented in an abbreviated form a summary of the effects of DOP observed in chronic feeding studies of rats. The data from the work of Carpenter and associates * are included. Mortality was not increased by feeding 0.5% DOP or less in the diet. Body weights were normal when the diets contained up to 0.13%, but there was a retardation in growth at 0.4% or 0.5%. Food consumption was the same as that of control rats when diets contained up to and including 0.4% in the study by Car penter and associates,* but food consump tion in the current study was lower when the diet contained 0.5%. In the current study, even though the dietary percentages are comparable, the intake of DOP was 50% to 100% greater. Organ weights were nor mal when the diets contained up to and including 0.13%. Enlarged livers and kidneys were observed when the diets contained 0.4% or 0.5%, At the end of the current study definitive comparisons of organ weights could not be made because of the very small number of rats that survived the two-year period. There were no specific histological lesions that were attributed to the adminis tration of DOP.i* DOG FEEDING STUDIES Chronic Oral Toxicity.--Two dogs were maintained for a period of 14 weeks on daily doses of DOP as follows: One was given 5 gm/kg. daily by stomach tube; the second was given 0.1 gm/kg. mixed in the diet. The male dog given the larger dose lost little weight during the 14-week period (initial weight--27.1 kg.; final weight--25.5 kg.). The female dog given 0.1 gm/kg- gained a little weight (initial weight--10.4 kg.; final weight--11.3 kg.). Blood samples were taken for hematological examination prior to the study, at the midpoint, and at the end; all the values lay in the normal ranges. When the dogs were killed, samples of a number of Tablc 6.--Summary of Chronic Feeding Studies: Dogs Don, No. 4 i 1 1 1 Dally Dose Control* 0.08 mL/kr. fori* doe** fc0tmL/kv. for *10 doem AMmL/k*. for Y7 doses* 0.<*ml./k*. for 18* doaes 0.1 cm./kff, for 14 wk. StOfm./kr, for 14 wk. Body Wt. */4 veined 4/4 reined 4/4 sained 4/4 raised flUfbtkwa Hamatoloar Normal Normal Liver and Sidney Wt. Normal Normal Patbolofy Normal Normal Normal Normal Normal Normal Not recorded Not recorded Patty ebanvee In Urerj cloudy aweHtnv in kidney Normal Chronic ebolccjetltte: bcmoaldcraala ot aplean ' Date froTn Carpenter, WeD, ud flmytb.1 263 UCC 041214 '& r -f . t. 0 * A. M. / . ARCHIVES OF INDUSTRIAL HEALTH tissues were prepared for histological exam when the diets contained about 0.1% or ination. These included the following organs: less. No specific histological changes were lung, spleen, stomach, small and large in found in rats maintained on diets containing testine, liver, gall bladder, pancreas, adrenal, 0.5%. Mild toxic changes occurred in three kidney, urinary bladder, thyroid, gonads, months when a dog was given 5 gm/kg brain, and bone marrow. The female dog daily; 0,1 gm/kg. was without effect. The given 0.1 gm/kg. daily had entirely normal absence of serious toxic effects when rela tissues. The male dog given 5 gm/kg/day tively substantial amounts of DOP were ad showed some chronic cholecystitis. There was ministered for protracted periods to rats and also some hemosiderosis of the spleen, a con dogs is good evidence that no serious injury dition not unusual in control dogs of our will accrue to the workmen handling DOP colony. in manufacturing or in plastics fabrication. The data available on the effects of DOP The minute traces that may appear in non on dogs have been compiled in Table 6, in fatty foods wrapped in plastics in which DOP cluding the work of Carpenter and associates is the plasticizer, on the basis of the animal on dogs given up to 0.09 ml/kg. daily for v studies, pose no threat of toxic hazard. a period of one year. Body weight gains were recorded, except for one control dog and REFERENCES for the dog given 5 gm/kg/day. Normal 1. Hodge, H. C. j- Acute Toxicity for Rats and hematological findings were observed. Liver and kidney weights were normal where re corded. The only pathological changes re lated to treatment were in the liver and kid ney of the dog given 0.09 ml/kg. for 169 *Mice of 2-Ethyl Hexanol and 2-Ethyl Hexyl Phthalate, Proc. Soc. Exper. Biol. & Med. Sl:20 (May) 1943. 2. Shaffer, C. B.; Carpenter, C. P-, and Smyth, H. F, Jr.: Acute and Subacute Toxicity of Di(2Ethylhexyl) Phthalate with Note upon Its Metabo doses and in the liver of the dog given 5 lism, J. Indust. Hyg. & Toxicol. *7:130 (May) gm/kg. daily for three months. In the 14- 1945. week studies 0.1 gm/kg. was a "no effect" 3. Carpenter, d. P.; Weil, C S., and Smyth, H, level, whereas in the study of Carpenter and F., Jr.: Chronic Oral Toxicity of Di(2-Ethy!hexyl) associates 0.09 ml/kg/day for one year was Phthalate for Rats, Guinea Pigs, and Dogs, A. M. associated with toxic changes in the liver and A. Arch, Indust. Hyg. 8:219 (Sept.) 1953. kidney. SUMMARY Feeding 2-ethylhexyl phthalate for two years to rats was without detectable effect 4. Caldwell, A. B.; MacLeod, G., and Sherman, H. C.: Bodily Storage of Vitamin A in Relation to Diet and Age. Studied by Means of the Antimony Trichloride Reaction Using a Photoelectric Color imeter, J. Nutrition 80:349 (Nov.) 1945. I si ! I t 264 A I. Veati HUK REG and AAR Thi functii *ers, 3t with t ice in try, w to eva conne cause roentf Int which posur lated osis i repor Vigli; in tw expo: of ca mace from prod- A. Ft versit Ri custr, Annu atior.. Ti Rese; Instit Heali 1 *r ucc 041215