Document KRgdQ6GGZk5VagQOe3wO7n8br

jjj. r,i Pathologic Changes in Animals Exposed to a Commercial Chlorinated Diphenyl I By J. SV. MIIXKK lL IlKI'RliNT No. 2573 FMOM Till PIML1C HEALTH ItKI'OKTS Voi.. 59, No. 33 Accust 18, 1944 Paws* JOBS-J093 r i RLE COPY MQNS 0961^4 KEDKHAL SECUMTY ACENCY UNITED STATUS PUBLIC HEALTH SKUVICE Thomas PahRa.v, Surgeon General DIVISION OK I't'UI IC HEALTH METHODS C. Sr. J. I*KHurr, Oit>J UJ Piii'inn UNITED STATES GOAEKNMKNT KHINTINC OFKICK. WASHINGTON i J94I r*f ulf L> ||m S*|iriiitrmltfl of f)<H-iiiurnl, 1*. S. Cotnnimni I'timing Office Warliinf dm JS, I). C. Price S c*m i i MONS 09812 5 PATHOLOGIC CHANGES IN ANIMALS EXPOSED TO A COMMERCIAL CHLORINATED DIPHENYL 1 By J. \V. Mitten, Surgeon (/?), United Stafee PuMie Health Service The demands of industry as a result of the war have greatly increased the use of chlorinated naphthalenes and chlorinated diphenyls. In the past few years the hazards associated with their application havo attracted much interest and a number of reports regarding tho systemic and dermatologic effects of exposure, including fatal cases, have been made. Only the pathologic changes in animals exposed to a commercial chlorinated diphenyl are given here. The chlorinated diphenyl used was viscous, almost water white, and clear at room temperature. It consisted of a mixture of isomers of diphenyl chlorinated in different positions and extent, with an ap proximate cldorino content of 42 percent and an approximate empirical formula of CHrCl,. It was insoluble in water but soluble in mineral and vegetable oils, other chlorinated hydrocarbons, and fat solvents. Its specific gravity was 1.374 to 1.393. Guinea pigs, rats, and rabbits were exposed to the above compound by subcutaneous injections, feeding, and applications to the skin and cornea. Tho survival times given here refer only to animals subjected to pathologic examination in each exposed group. Certain additional results reported are from groups of animals exposed solely to obtain pathologic data. I. SUBCUTANEOUS INJECTIONS (A) GUINEA riOB A series of 31 guinea pigs was injected subcutaneously with a single dose of 0.05 cc. (OD mg.) of the chlorinated diphenyl. Some of these animals were killed and examined at 2-day intervals up to 38 days after injection. Sections were made of the liver, lungs, spleen, kidneys, adrenals, floncroas, heart, and skin, and were stained with hematoxylin and eosin, cosin-polychromc methylene blue (/), and frozen sections with hematoxylin and Sudan IV (). , Skin.--In 2 days the skin lesion at the site of subcutaneous injection consisted of a central, faintly basophilic necrotic mass containing much nuclear debris, many polymorphonuclcnrs, and lymphocytes. Much of the nuclear material was present at the periphery. On the 1 From tbf IotfmfHi! n>)r/>f Jlrjrvrb J.aboialory, Nation*) Institute ol Draltb. Tbli maluUI la on f t perlntenU conducted by Suttcon Dcnjaioln f. Jours and rhyslologist D. D. Donahu*. l)OI9fr>44 ' 1 MUNS 098126 2 CHANCES IN ANIMALS EXPOS' X) CHLOHINATED DIPHENYL periphery wore, first a zone of fibrin, then one of monocytes, leucocytes, lymphocytes, and, externally, a few fibroblasts which extended into the adjacent muscle and subcutaneous tissue. In 4 days a poorlydefined zone of fibroblasts accompanied by numerous capillaries was noted in the periphery. In 6 days the necrotic material had bccomo eosinophilic. There was a central portion of finely granular and fibrinoid material surrounded by a zone of nuclear fragments, and & fairly well defined capsule of connective tissue and fibroblasts which extended into the adjacent fat and muscle. If, however, the injected material had been placed in the superficial portion of the derma, the inner portion of the capsule was partially lined by stratified squamous epithelium extending down from the epidermis. The layers of cells adjacent to the necrotic debris were strongly oxyphil, flattened, and lacked nuclei, thus presenting the appearance of early keratinization. These changes arc essentially those of chlorncno (S, 4, ), though the latter are thought to be due to mechanical occlusion of tho ducts resulting in accumulation of secretion within the glands. Encapsulation progressed from the eighth to the thirty-eighth day. When the lesion was superficial, the epithelium continued to develop until It lined the cyst wall, and the portion adjacent to the necrotic material was cornificd. The outer layers of fibrous tissue became increasingly collagenous. The necrotic, material remained eosinophilic in niotl cases and nuclear remains became less abundant. When tho lesion was deep, the capsule consisted of adult connective tissue; other wise the appearance was the same. Liver.--Eight to 10 days after injection, fat droplets were noted in tho liver cells. At early examinations, these wore very few in num bers, but after 10 days they were present in moderate or very largo numbers. Small fat droplets were generally scattered throughout tho lohulo, although in somo of tho sections they were present only about the portal canals. Central atrophy, usually slight or moderate in degree, occurred in practically nil of the animals with hepatic fat, and made its appenrnneo at about the same time os the fat. Con gestion of the sinusoids was absent in all but 2 of the animals. At irregular intervals and with no particular regard to interval after injection, a slight (o moderate amount of perinuclear, basophilic gran ulation was noted in the liver cells in sections stained with cosinmclhylcno blue. Focal necrosis occurred in only 4 of 31 ani mals. It was found in various portions of the lobules and may not bo significant. Spleen.--The spleen showed no particular lesions. Kidney*.--The kiducys were essentially normal in most of the series. A slight to modcrato congestion of the interstitial capillaries was noted. Fat was present in the cells of tho convoluted tubules in only two animals. r MQNS 098127 i CHANGES IX ANIMALS EXPOS .0 CHLORINATED DIPJIENVL 3 The adrenals, heart, lungs, and pancreas showed no noteworthy changes. In another series of 23 animuls receiving larger single doses of 0.1 to 0.5 cc. (138 to 600 nig.), which were killed or died from 2 to 24 days after injection, pathologic changes consistent with those found in the previous series were noted. Fatty degeneration in the liver was more marked, appearing on the second day in animals injected with 0.5 cc. (690 mg.). Approximately the same degree of central atrophy re sulted from the injection of 0.5 cc. (690 mg.) as from 0.05 cc, (69 mg.); central atrophy was more frequent in animals dying or killed before the tenth day. Changes in the spleen were essentially the samo except that lymphoid hyperplasia was more frequent and few to moderate numbers of hcmosidcrhvbeuring cells were seen in 12 of 23 animals. In the kidneys congestion of capillaries was moro frequent. Fat was absent. The adrenals showed cortical congestion in most animals, sometimes also medullary. Similar adrenal congestion has been observed with carbon tetrachloride (6). However in the latter it often goes on to hemorrhage and necrosis. The lungs in the animals which died during the test showed congestion of the alveolar capillaries and focal extravasation of red blood cells and scrum into alveoli in only 4 animals. The changes in the skin were essentially the snmo ns those noted before. However, in contrast with the findings fol lowing administration of smaller doses, with larger doses the necrotic material in the derma remained basophilic in reaction. The heart and pancreas were normal. A third series of 10 guinea pigs received n single 0.5 cc. (345 mg. ns chlorinated diph ayI) dose of the chlorinated diphenyl dissolved in on ciiunl amount of mineral oil. These all died within 13 days. The pathologic picture wos essentially the same as noted in the previous series. The lungs showed somewhat greater congestion and edema in 9 of 10 guinea pigs. (B) RATS A series of 20 rats received a single dose of 0.05 cc. (09 mg.) of chlorinated diphenyl subcutuneously. Animals were killed nt 2 and 7 days and at 10-day intervals thereafter up to 90 days. Liter,--Fat appeared in liver cells of rats much earlier than in guinet pigs. It was already present 2 days after injection and was fairiconstant throughout the remainder of the series. Central strop* was less marked. There was no greater periportal lymphocytic irfiltrution than in untreated rats. Irregular basophilic stippling >'f liver cell cytoplasm was noted, ns much in untreated controls a: n experimental animals. Spleen.-- Congestion of the cavernous veins was moro frequer.:. while rcticuiocndothcliosis was only slight and often absent. Pul? MONS 0981d 4 CHANGES IN ANIMALS EXPOSKI CHLORINATED DIPHENYL myelosis, most about trabeculae, occurred in oil but two of the ani mals am! varied from slight to marked, as commonly seen in rats. The lungs, kidneys, adrenals, and pancreas showed no changes. The lesions in the skin were identical with those noted in the. guinea pigs in which the materials had been injected deeply. In all but one of the eases the capsule about the necrotic mass consisted of fibrous tissue and fibroblasts. In one animal killed 60 days after injection, a partial epithelial lining of the cavity was noted. A second series of 10 rats, receiving a 0.5 cc. (090 mg.) dose, was killed at the same time intervals, the last at 40 days. In these, fatty changes in the liver were more marked. The spleen showed con siderable hypertrophy in most eases. The other organs were essentially the same. Two other groups of 4 rats each were given 10 doses (total of 690 mg.) on alternate days and 27 doses (total of 1.SG3 mg.) daily of 0.05 cc. (09 mg.), respectively. Half of each group was killed 00 and 90 days after the first injection. In nil of the rats receiving 10 such doses peculiar round or oval intracellular bodies were observed in the livers of the animals in both 00- and 90-day groups. However a negligible number were found in only 1 rat injected 27 times. These bodies varied considerably in size. Tho smaller were homo geneous and usually oxyphilic. Somewhat larger ones showed a denser periphery and paler center. A9 size increased the central portion became either grunulnr, reticular, or foamy in appearance and oc casionally contained a small faintly eosinophilic body about the size of a nucleolus. With tho increase in size the shell became sharply demarcated. It was hyalino and deeply eosinophilic, with a scant basophilic outer margin, and sometimes presented a concentric lamination. The thickness of this hyaline shell varied. Many of the bodies were often within a single cell. They were generally present in the cells in the central one-third to four-fifths of the lobule. The changes in tho other organs were consistent with the findings in the previous groups. (O BABBITS Two rabbits were given 0.5 cc. (090 mg.) and another 1.0 cc. (1,380 mg.) for 10 consecutive days. They died 10, 72, and 14 days after injection. Three more received 10 similar daily doses of 345 and 090 mg. as a 50 percent solution of the material in mineral oil and died in 40, 300, and 42 days. Doth groups showed essentially the same pathology. The liver contained numerous fine fat droplets accompanied by focal necrosis in 2 of tho rabbits receiving the undiluted material. No fat or other significant changes were observed in those injected with the 00 percent solution. Occasionally slight centrolobulnr atrophy MQNS 098129 CHANCES IX ANIMALS EXPOS CO CHLORINATED DIPHENYL 5 of liver cell cords was noted. Tho pathology of the other organs end of the skin lesions was identical with that encountered in the guinea pig and the rat. H. SKIN APPLICATION TESTS (A) QOIXfcA riOB One series of 11 guinea pigs received 11 daily skiu applications of about 1/40 of a cc. (34.6 nig.) of the'undiluted chlorinated diphenyl These died at various intervals up to 21 doys following first applicnlion. Tho pathologic changes in the liver, kidneys, lungs, adrenals, and heart were essentially those noted in the subcutaneous injection scries. The spleen showed less pathology. The skin lesions, however, differed from .those in the subcutaneous group. In another series of 2 animals receiving 11 (17 mg.) doses with an equal amount of mineral oil, similar changes were noted. Changes in the skin were not constant in the animals which died in 12 to 21 days after the first of 11 applications of the undiluted material. The principal changes were injection of the capillaries of the derma and occasional thickening of the epidermis. The con gestion was sometimes accompanied by edema of the derma adjacent to the small blood vessels. In one instance, a moderate infiltration of lymphocytes occurred. In a group of 14 animals painted daily with 10, 20, 30, and 50 per cent (3.5 to 17 mg.) chlorinated diphenyl in mineral oil, and whidi died or were killed 7 to 15 doys after first application, the prinury ehango was thinning and often destruction of the superficial, cormfrd layers of the epithelium. When destruction of tho epidermis oc curred! tho superficial cells contained fine, granular material, anti in some eosinophilic inclusions. The amount of involvement of the kin appeared to have no relationship with tho doso or length eff ex posure. The reaction was not that of an acute inflammation. () HATS treatment. Very little fat was found in the liver of 6 of the animals. TAy*. rats showed tho hyaline bodies in the liver cells at the GO- and intervals. * Slight to moderate degree of congestion of the cavernous veil* i tho spleen was noted. Pulp myelosis was present in a modre.' degree. Id 7 of the 10 animals, a slight to moderate amount a,' intracellular hemosiderin was noted. MOWS 096130 Public MciUK Rtponi. Ne. 2)7) Plat* 'h . ':* 3' I'I(il Mr l.--Imfuf' 1 ' ilif hyolloo limllff Id llvfrt of imi ti|niDl to i chloifosird dlfilirofl. MONS 098131 CHANCES IX ANIMALS EXPOSED ;HLOHIXATED DIPHENYL 7 No significant changes were found in the kidneys, heart, pancreas, and lungs. ' In sonic of the rets the treated skin was much thickened, and the hair follicles were swollen and poorly defined. (C) RABBITS Eleven rabbits received cutaneous applications, at 2-day intervals, of about SC mg. for the first 7 and 172 mg. for the last S applications of the undiluted chlorinated diphenyl' The animals were examined 09 death occurred between 17 and 98 days. The number of applica tions varied from 9 to 15. Sovon of the rabbits received the latter number. Fatty degeneration and central atrophy of the liver cells were moro prominent than in the previous scries of experiments. The spleen showed less pathology and in two of the animals (90 and 98 days) was normal. The changes noted were slight to marked con gestion of the cavernous veins and slight to moderate follicular lym phoid hyperplasia. Focal caseous necrosis occurred in four instances, but was probably due to intercurrent infections. Changes in the heart, lungs, adrenals, and kidneys were slight and consisted primarily in congestion. The skin showed thinning of the prickle cell layer and rclativo thickening of the outer cornifiod layers. . III. FEEDING EXPERIMENTS (A) 3CINEA TIGS A scries of 8 guinea pigs received 2 doses of 0.05 ce. (09 mg.) of iho chlorinated diphenyl 1 week apart. Death occurred in 11 to 29 days after initial feeding. The changes in the liver were more marked in this series than in the others. Fatty metamorphosis was extensive in all of the animals and wos of mixed fine, medium, and large droplet variety. Central atrophy was noted in only 2 animals dying IS and 29 days after the initial feeding. The changes in the other organs were of the same negligible char acter and degreo as observed in the other experiments. The gastroin testinal tract showed no abnormal histology. No changes were noted in tho central nervous system. () RAT* Twelve rats receiving 25 daily doses of 0.1 cc. (138 mg.) were killed and examined at 10-day intervals, beginning at 30 and ending at 90 days after initial feeding. The pathologic findings were practically idenlical with (hoso of the series of rats receiving skin applications of 25 daily doses. The intraccl- MONS 098132 8 CHANGES IN ANIV S EXPOSED TO CHLORINATED DIPHENYL lular hyaline bodies mentioned previously were found in the livers of 2 of the animals. No changes were noted in the gastrointestinal tract. IV. APPLICATIONS TO CORNEA HATS A small amount (1 drop from a 25-gnge hypodermic needle, approxi mately 1/80 cc. or 17 mg.) of chlorinated diphenyl was instilled into the eyes of 8 rat* for 25 consecutive days. Beginning 30 days after the initial dose, 2 of the animals were killed and examined each 10 days. The pathologic changes were very scant in degree. Slight central atrophy of the liver cords occurred in half of the rats at various irregu lar intervals. Slight to niodernto congestion of cavernous veins of the spleen was constant. The presence of blood pigment, both free and intracellular, occurred rather frequently in the spleen. The conjunctival tissue presented no gross changes when examined under magnification in the living animal. SUMMARY AND DISCUSSION Guinea pigs, rnts, and rabbits were exposed to a commercial chlo rinated diphenyl by subcutaneous injections and applications to the skin. The material was also administered to guinea pigs and rots by ingestion and to rats alone by corneal instillations. The doses varied from 17 to 1,380 mg. and were either single or were repeated at regular intervals. Two conspicuous pathologic findings were observed--liver damage in all series of experiments and skin changes in the animals receiving subcutaneous injections or applications of the material to the skin. Fatty degeneration nnd atrophy of the centiolobular cells were present in varying amounts and in varying numbers of animals in the different test groups. In the rnt an additional finding, bynlino bodies within tho liver cells, was noted in certain animals. It was possible to detect a difference in response of the three species to the material on the basis of liver damage. Most liver damage was found in the guinea pig, less in the rabbit, and least in tho rat. This same species order was followed, regardless of dose, duration of test, or mode of administration. Hepatic fat appeared in the guinea pigs receiving 0.05 cc. subcu taneous!}' in 10 days nnd remained in significant amounts throughout 38 days. Rats receiving the same amount showed fat in 2 days but only small amounts wero noted in some of the animals over a period of 00 days. Fat appeared earlier in both rats nnd guinea pigs re ceiving larger doses. The involvement was generally ccntrolobulnr. Fatty degeneration, when compared with tho other routes of ad ministration, was most marked in the guinea pigs fed the chlorinated MQNS 098133 ' CHANCES IN ANIMALS EXPOSED TV MLOKIXATED DIPHENYL 9 hydrocarbon but was absent in rats similarly treated. Central atrophy was more or less the same throughout the series of tests. Only slight central atrophy was present in about half of the rats treated by application of the material to the cornea. Intracellular hyaline bodies were found in the liver of the rat alone. They were present, usually in large numbers, irv all of the rats receiving 10 0.05-cc. doses ami in some of the animals receiving 25 doses by skin and corneal applications and ingestion, but were not observed in any of the animals subjected to single doses. These bodies were noted in the animals sacrificed 50, 00, and 90 days after first exposure. None were observed in rats examined prior to 50 days on test. They occurred in from 20 to 38 percent of the animals treated m the various ways. They were somewhat less marked in degree and in number of animals when the chlorinated diphenyl was ingested. These find ings agree with Bennett (7) who reported similar hyaline bodies in liver cells of white rots exposed to mixtures of chlomaphthnlcnea and chlorinated diphenyl, chlorinated diphenyl, and less frequently to mixtures of chlornaphthnlcncs. To date such bodies have only been observed in rats exposed to such chlorinated compounds. It is interesting to note (lint while the bodies appeared in all of a series of 4 rats receiving 10 0.05-cc. subcutaneous doses giveu on alternate) days, they were absent in 4 rats treated with the smne amount by 27 consecutivo daily injections after CO and 90 days following first exposure. The liver cells in the latter scries showed very slight deviation from normal, namely, areas of oxyphil granula tion of cytoplasm and occasional loss of nucleus. It is possible that the gradual accumulation of 1,$G3 mg. over a period of 27 days so damaged the liver cells functionally that they were unable to form iho hyaline material. The hyaline bodies are morphologically differ ent from those produced by butter yellow in hepatic tumor cells (8). They probably represent further development of the same general typo of hyaline degeneration as lias been observed with certain azobcnzencs (9). The skin lesions produced by subcutaneous injection were histo logically similar to those of chloracnc in man. The changes produced by direct application to the skin were not constant and were essentially those of low-grade irritation. The failure of local applications to produce acne lesions may be due to relatively early deaths from systemic toxic action before pathology could be produced in the skin or iho amount tolerated without causing death was too small to causo such lesions. Attention is called to the fact that the chlorinated diphenyl used in the abovo experiments produces liver changes in the rat having marked differences from those resulting from other toxic substances and that such changes were not found in the guinea pig and rabbit MGNS 098i3<r 10 CHANOHS IN ANIMALS 'OSKU TO CHLORINATED DIPHENYL REFERENCES (!) Lillie, It. D.: Iloiitnttowpky (ninliig with buffered eolufinn* m i* . aflVi) mCtI,0d t0 It')n,anov'skJ' 6tai,,s in general. Siaii) Tech^'Vg*^1-6 (*> "7^3)''"' Z"r rclirarb"''l!- Ccnlralbl. f. All*, pall,. Anat., M. S91 w ^srtoksffiS: ?i,>!b:.,?ru|,a,lo',al -- s- ** *' M-u, U) Sr,iri?i7V'i?-!7M7j;-ov. m; mS"'"0 rrom cu"`"oNs- '>> (0 I Iliu/n'nH (MaVlII'lMS1)?1' ' l.lllle, II. T).. Personal coin rnmmal ton. (?) Dennett G. A., Drinker, C. K,, ami Warren, M. K.: Morphological chain:,. |,, (lie llvcra of na n nanlting Iron, espoanre lo certain ch'orinalcl l,7,ir r(e8,, Er'adC\\anrrd>?n",*Vj. fKl.', Ianndidj.-it. I I vy,JattnlitrilpT: nP.vaitchool,l,o2g0ic: c0h7a-n1g2e3s (wPicthhnsimar-vrh1lTrIeKfeIr'ence to plKrocnladon and dacificalion of Impalie minor., in rals Tod p ,11 ncthvl(o&issr* (l",uwvci,o">- *Ka'- c*"" i!n s. iwis (5) Smith,M. I., I.iliie, It. n.. ami Sluhhnan, K. J?; The toklcilr and I,intoCmX,sTby diMnry |,fol'in- ,,ub- 1/ MOMS 098135