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tough >rt it: effect gnethose icon967). tium n obtsium vhich ttion aug- mini urine ak>fI and . ppott (turn i rets, t and Ubo- y md A. V. n#or, snort pt lh h C Uo TOXKXtCOOY AhO AfrUfP rHA*MACOtOOV If, til 1911) wBP Dermal Toxicity Studies of Technical Polychlorinated Biphenyls and Fractions Thereof In Rabbits J. G. Vos AND R. B. BfEMS ; hutllntr of Veterinary Pathology and Institute of Veterinary Pharmacology and Toxicology, University of Utrecht, Bihstraat 172, Utrecht, The Netherlands , Received October J, 1970 Dermal Toxicity Studies of Technical Polychlorinated Biphenyls and Fractions Thereof in Rabbits. Vos, i. G,, and Beims, R. B. (1971). Toxicol. Appt. Pharmacol. If, 617--633. A significant difference in toxicity between 3 polychlorinated biphenyl (PCB) preparations was found-in a prior study: : Clophcn A (50 and Phcnoclor DP6 showing the highest, Aroclor 1260 fhe lowest, toxicity (Vos and Koeman, 1970). A subsequent study revealed the presence of tetra- and pcntachlorodibeneofuran In,Phcnoclor and Clophcn (Vose/o/., 1970). In the present study, application of 118 mg of the 3 PCB's . (3 times per wk, for 38 days) on the back skjn or rabbits also resulted in i differences in toxicity. PCB-Induced skin lesions were hyperplasia and hyperkeratosis of the epidermal and follicular epithelium. Histoflithology of the liver included centrolobular degeneration, oentrolobular liver cell atrophy, focal necrosis, and cytoplasmic hyalin degeneration. Definite hyperplasia and hyperkera tosis of the follicular epithelium of the ear skin were seen after the topical i application of fractions of Phcnoclor and Clophcn eluted from chromato graphic columns with 25 % diethylether in hexane. The fraction from Aroclor caused a minimal hyperplasia and hyperkeratosis of the follicular epithelium. PCB-Induced kidney lesions were hydropic degeneration of the convoluted tubules and tubular dilatation with the presence of casts. This dilatation was demonstrated also by a significantly increased relative percentage of the diameter which corresponds to the space of Bowman. Moreover, thymus atrophy and lymphopenia were found. Fecal copro porphyrin and protoporphyrin excretion was increased. Polychlorinated biphenyls (PCB) have been identified in tissues of fish and wildlife in tr)any countries (Jensen, 1966: Holmes el at., 1967; Holden and Mareden, 1967; Koenjaneffl/., 1967; Risebrough el at., 1968; Koeman el at., 1969; Jensen el at., 1969; Duke et^t., 1970; Prcstt el at., 1970). PCB preparations are extremely stable, oily fluids with very low aqueous solubility. They arc uaod as lubricants, as heat transfer media, in protective coatings for wood, rr)etal, and concrete, and for many other applications. Which of these applications has contributed to the (Jfeaenj environmental contamination has not yet been established. Inhalation and feeding experiments in rats with 65% chlorinaterlPCB resulted in liver injury (Drinker el at., 1937; Bennett et at., 1938). Liver damage and skin lesions have bpen described after inhalation, ingestion, and application to the skin using rabbits, guinea pigs, and mice (von Wedel el at., 1943). Hydropericardium occasionally accom panied by abdominal edema was found in chicks fed PCB (McCune el at., 1962; Flick .617 r ... i' DSW 029307 STLCOPCB4013269 618 VOB AND BECMS rr'fl/,, 1965; Vos and Koeman, 1970). Epithelial and follicular hyperplasia and hyper* pteratosis were found after application of PCB on the akin of rabbits (Adams tt /., 11941). Subcutaneous injection of 42% chlorinated PCB in rats, guinea pigs, and rabbits Iresulted in liver injury and skin lesions, which were essentially those of chloracne (hjlilkr, 1944). Follicular hyperkeratosis is an important feature of the occupational disease known as chloracne, which is characterized by the appearance of papules, come dones, and cysts. It may develop after exposure to some highly chlorinated aromatic 'pompoundt. Examples are chlorinated naphthalenes (Grcenburg era/., 1939; Hembrick, |957; Shelley and Kligman, 1957) and chlorinated phenols (Hofmann, 1957; Bauer tt pfy 1961; Bleiberg etal., 1964). The active compounds in chlorinated phenols have been identified as chlorinated dibenzodioxins (chlorinated diphenyiene dioxides) which knjay appear as contaminants in the synthesis of chlorophenols (Bauer tt ol., 1961).' )Liver injury (acute yellow atrophy), whether accompanied by chloracne lesions or not, |s jriso described in people engaged in the manufacture of the chlorinated hydrocarbons (Sfrauss, 1944; Braun, 1955; Behrbohm, 1959). Also for the PCB's there are early re ports of occupatiorfal chloracne (Jones and Alden, 1936; Schwartz, 1936). The presentday use of PCB docs not seem to have led to this kind of difficulty. A significant difference in toxicity between 3 samples ofcommercial PCB preparations Vv^s found in a previous study (Vos and Koeman, 1970), despite the marked resemblance Of the gas chromatograms and the mass spectra (Koeman ttaf,, 1969). One hundred per pejit mortality, centrolobular liver necrosis, and sc and abdominal edema were found pnly in chicks fed the samples of Clophen A60 and Phenoclor DP6. Hydropericardium was recorded in nearly all chicks fed these PCB's, and was only occasionally seen in $hjcks fed with Aroclor 1260. Porphyria was found as a general PCB effect (Vos and Koeman, 1970). ` jrhc hypothesis that in some technical PCB mixtures toxic factor(s) responsible for (h6 chick edomalike lesions and the liver necrosis were present has been the subject of further work, By meaps of column fractionation, gas chromatography, and mass spec tremetric techniques, |tetra- ahd pentachlorodibenzofuran were idantified as the toxic (actors in the samples of Cld^hen A60 and Phenoclor DP6 (Vos #( <|f.. 1970). These results suggest that PCB's contaminated with polychlorodibenzofurans may 4I10 produce chloracne and associated lesions when applied to the skin of experimental Animals. To test this possibility the present study was undertaken. METHODS The PCB samples, which contain an average of 60% chlorine, were obtained from frodelec in Fraftce (Phenoclor DP6), Bayer in Germany (Ciophen A60 Lot No. 912434) gnd Monsanto in the United States (Aroclor 1260 Lot No. AK*3). ^dult female New Zealand rabbits, 5 mo old and weighing 2500-3050 g, were used in (his study. They received pelleted food (Cunicon 1, Trouw and Co., Amsterdam) and yzater ad libitum. The animals were distributed at random into 4 groups of 4 animals, ind were weighed weekly. An area of approximately 10 x 15 cm on the backs was clipped, the remaining hair Ws shaved with a shaving powder (90 g barium sulfide, 105 g zinc oxide, and 180 g Starch), and the resulting small skin abrasions were allowed to heal for 3 days. 'v 1 \ 1 1 ! .PWWPH DSW 029308 -JSfW STLCOPCB4013270 hyperi et at., rabbits loracnc ational ,comcomatic nbrick, aoer et et been which 1961). or not, arbons irly rercKnt- rations iWance red per found irdium icon in ot and ble for ject of i spect toxic ii may mental I from J$434) ised in i) and >ima It, g hair 160 g mmjww DCRMAL TOXICITY Of TECHNICAL fC MIXTURES 619 The 3 PCB'* were dissolved in isopropanol (118 mg PCB/ml). One ml of PCB solution or <jf isopropanol as a control was dropped daily, 5 limes a wk, on an area of 5 x 10 cm by p>eans of a calibrated syringe. In order to minimize eventual ingestion of the PCB, tl>c animals were held for 7 hr in restraining boxes, after which time they were returned t<> their wire cages (2 animals per cage), In order to atudy the porphyrogenic action of the PCB preparations In the same rabbits, pooled samples bf the feces of the 4 group* were analyzed weekly for copro porphyrin and protoporphyrin contents (Rimington, 1961), using a Beckman spectro* photometer. The animals which survived the 38-day test period (27 applications of 118 mg PCB) were killed. At necropsy, performed on all animals that died or were killed, a grow pathologic examination was carried out, including macroscopic examination in ultra violet light using red fluorescence as an indication of porphyria. In order to get infor mation on the fecal excretion of coproporphyrin and protoporphyrin of the individual animal, feces collected from the cecum were analyzed, Hematologic examination included hemoglobin and hematocrit determinations, leukocyte and differential leukocyte counts. Clinical chemical determinations were tjiade of serum glutamic-oxaloacetic transaminase and glutaipic-pyruvic transaminase (Reitman and Frankel, 1957) using a Beckman/Spinco 151 spectrocolorimeter. The weights of body, liver, kidneys, spleen, heart, and adrenals were recorded. The skin, liver, spleen, kidney, pancreas, ovary, heart, lung, thymus, mesenteric lymph i)ojie, stomach, small intestine, cecum, large intestine, and skeletal muscle were Axed in (0% buffered formalin and embedded in Paraplaat. Sections (7 p and 2 fi) were stained ^vith hematoxylin-eosin. For detailed histology, selected sections were stained with Pefls, Azan, PAS, Zichl-Neelsen, Congo red, and thioflavine. Moreover, cryostat petitions of formalin-fixed livers and kidneys were stained with Sudan black. Tbioft* vinegained sections, unstained Paraplast-embedded sections, and unmounted cryostat *e<|tions of liver, kidney and small intestine were examined in a fluorescence microscope. prompted by the observation of tubular dilatation in the kidneys in previous studies gfter PCB ingestion (McCune et al., 1962; Vos and Koeman, 1970), measurements mere made on random samples of capsules of Bowman and glomeruli in 2-/* kidney ^tions. Measurements of one axis of the greatest diameter of both capsule and glomerulus were made of 25 renal corpuscles in a straight traverse across the cortex of path kidney. The relative percentage of the diameter which corresponds to the space of (Bowman, as an index of dilatation of the nephron, was calculated from the measure ments of each renal corpuscle. ;An additional experiment was made with the chromatographic fraction (column plated with 25% diethylether in hexane) of the 3 PCB's, containing tetra- and penuIctybrodibenzofuran in the case of Phenoclor DP6 and Clophen A60(Vos et at., 1970), Ito determine the dermal toxicity of this fraction. Aliquots of 600 mg of the 3 PCB mix'Xurcs were fractionated (Vos et a!., 1970). The 25% diethylether.hexane fractions were evaporated and dissolved in 0.6 ml ofethanol. Four female albino rabbits (3 test animals land 1 control), 2 mo of age and weighing 1550-1750 g, were uaed at the experimental lanJmala. A 0.2-ml portion of the ethanol solution was applied weekly, for 3 wk, to an laiW of 4 cm1 on the inside of the rabbit ear. The opposite ear served as a oootrol. The DSW 029309 ; STLCOPCB4013271 \ \ _..................................... ............................. _ TABLE 1............................... - ' . ^onaKmjnrtum and protoporphyrin Contents 0*o/o Dry Wqokt) or ftxuo 5>Mrtrs or Fra* mom Raswtj Treated with PCB pc* 38 Days, and or Control Animals 'r#tew*of 7*) Phenockx4 Cofrropbrphyrin Clophen4 Arodor* r ' Control . . Protoporphyrin Phenoctor4 i i ml? Arodor' 0 7 1422 29 , 36 0.6 28 20 33 3.1 8.7 03 43 1.6 17.2 43 9.8 ' 03 29 1.1 113 33 10.0 0.4 03 * ,0 1.0 1.4 1.7 113 243 263 133 10.1 243 5.4 ... 283 . 103 183 193 193 53 173 133 29.4 213 27.7 4 Sgniflamtty tttfeieut from controls, F < 0.05, according to the sign test. Control 7.7 6.7 7.1 4.7 73 U TABLE 2 CorworoRPHYMN and Protoporphyrin Contents (jta!o Dry Woomt) or Facn or Kkud RAaam Treated with PCB for 38 Days, and or Control Animals' Coproporphyrin Protoporphyrin Phenodor Clophen Arodor Control Phenodar Mean: . 8.1 144.1 443* 58.0 63.7* 74.0* 33.2 303* 90.0* 56.9* 4.7 313 333 21.7 228* 29 23 29 4.9 33 28.8 126.9 383* 383 5tb* * Fignret ire the content* of faces, collected from the cecum, of the individual anamh. * Animals that died during the experiment. . * Significantly different from cootrob, F < 0.05. 4 SigaiAcantfy different from controls, F< 0.025. Clophen 39.0* 30.4 493* 94.0* 533* Arodor 25.0 233 123 324 233* Control - 21.4 73 9.7 11.7 126 DSW 0 2 9 3 1 0 : DERMAL TOXKTITY Of TECHNKTAL l*C MIXTURES Ml tnirrul* were kitted after 3 wk. Body and liver weight* were recorded. Section* of the ear hnd the liver were itaincd with hematoxylin-eosin. Cryoitat sections of the liven wen) ilo stained with Sudan black. Sfetittical significance, on a one-tail significance level, was determined using the i WiK|on test for 2 unrelated umpkt (van der Waerden, 1937). I < . RESULTS . Experiment with Commercial PC6 Preparations in Rabbits The kin, especially of the Clophen- and Phenoclor-treated animals, became rodd-ehed after 2 day* of PCB treatment. This rednesj was more pronounoed after 1 wk of treatment, together with clear desquamation of the external epidermal layers and rodeoed regrowth of hair. Thickening of the skin and prominent transverse wrinkles of the1 treated skin developed during the experiment. 1 * t I i4 . A. f* II The results of the speclrophotometric determinations of fecal coproporphyrin and l prot.oporphyrin are given in Table I. The, test animals shoived, In geheral, a gradual loss of weight. One Phenoclor- and 3 Oopben-trealed animals died, with severe weight loss, after 10, 31, 32, and 32 days, respectively. At necropsy, carried out on a|i animals, it was found that i animal in each of the 3 test group* showed definite edema formation, with fluid in the abdominal and thoracic cavity, subcutis, and pericardium. AM je*t animals, except for I rabbit of the Clophen group that died after 32 days of treatment, showed Uuore*oence in ultraviolet light. Liver (Fig. 1) and bone fluorescence DSW 029311 Ip.. ! i : i STLCOPCB4013273 ........ ................... ill' r lit i^n--i i ' V06 AND M H S STLCOPCB4013274 Results or the Hematouxhc Examinations Mat* in Killed Ramus Treathj with PCB ro* 38 Days, amo at Control Ammais* - Hemoglobin Hematocrit Leukocytes N ('100 ml) (%> (lO'/mm*) SGOT (U) 90PT (U) Treated animals GootroCi " 8 92 (6.1-12.7) 27.0(20-44) 73 (3.3-11.1)* 10.7 (7.0-143) 47.1 (18-96) 4 9.0 (8.2-10.0) 33.3 (27-38) 15.0(93-26.2) 82 (6.3-10.0) 23.8 (17-27) * Mean vetoes; the range is gjren in parentheses. * SBgwftaandy different from controls, P < 0.023. - TABLE 4 O*oan Weights and Organ: Boor Wekjht Ratios of Killed Rawt* Treated with PCB for 38 Days, and of Controls' Organ Weight (g) Compound N Body (g) Heart Liver Spfaen Kidneys Adremis Pheoodor _3 2300(1900-2900) 4.74 (4.16-5.78) 133(85-169) 0.72(021-135) 14.0(13.1-14.6) 031 (024-034) Gopher 1 2900 5.93 . 137 1.18 143 027 O 4/5 Axodor Control ' _4 4 2312 (1930-2600)* 4.31 (3J8-5.00) 138(106-715) 0.61 (0.33-0.92) 14.3 (I27-t6.5) 02T?)23-037) 2912 (2700-3100) 5.09 (4.60-5.95) 124(112-152) 0.64(0.52-0.76) 133(120-15.1) 0.37 (0.28-0.43) 3E Organ:body weight ratio (g/1000 g body weight) -' O IV Ptwoodor 3 *0 Gophen 1 U I-- IV Anodoc Control 4 4 207 (138-225) 205 IM (1.63-222) 1.74 (138-202) 583 (44.8-80.5) 47.1 59.0(45.0-826) 43.0 (37.8-562) 028(0.10-033) 0.41 026(0.17-0.42) 022 (0.18-025) 62 (5.0-6.9) 5.0 62 (52-6.7)* 4.7 (3.8-5.1) 0.13 (0.11-0.16; 0.09 0.12(0.09-0115) 0.13 (0.09-0.15) Mean vahies; the range is shown in parentheses. Sqjmffcantiy different from controls, P < 0.023. DERMAL TOXICITY Of TECHNICAL K# MIXTURES 623 was Observed most frequently; fluorescence of incisors, kidneys, small intestine, and *ubci)tis was found in about half of the lest animals. There was no fluorescence in the bone rrjarrow. Fluorescent microscopic examination of cryostat sections of the livers reveale^ that porphyric animats had porphyrin diffusely distributed throughout the whole |obule. Small foci inside parenchymal cells, probably nuclei, revealed an even more intense red fluorescence. Kidney sections showed, when positive, a weak red fluorescence, which was found in the cytoplasm of the tubules in marrow and cortex. Fluorescence of granules (perhaps nuclei) in glomeruli was also noted. PASH, wC'i'Shs Fo. 2. Back skin of* kilted rabbit from the Fhenockxr-trealed (roup. Hyperplasia and hyperkeratosis of the epidermal am) follicular epithelium. Follicular plugsing is prominent. Note the hyalin foci in the cytoptssm ofepidermal cells (t), and the restlna (telojen) phase of the hair fottklcs (2). Hematoxylin and ecjshv *46, Fro, 3. Normal akin of back of a control animal treated with the solvent Isopropanol showing epi- dermb e( / and dermis at J. Several groups of hair follicles, cut somewhat tangentially to their long axes, are se^n jet J; groups of hair bulbs are seen at 4. Hematoxylin and eosin. x46. Cojproporphyrin and protoporphyrin content* of feces collected from the cecum at necrojptjy gre given in Table 2. The degree of excretion was not related to the intensity of porphyrin fluorescence; for example, the Clophen- and Aroclor-treated rabbits with a coproporphyrin excretion of 30.5.and 4.7 pg/g (Tabic 2), showed, respectively, no fluo rescence at all, and fluorescence of liver, bone, incisors, kidneys, small intestine, and subctili*. .. Tbp results of tike hematologic determinations are given in Table 3. The PCB-treated anim^U of the 3 gropps have been taken together since there were no differences be tween the groups. The total number of leukocytes was significantly reduced in ths test DSW 029313 STLCOPCB4013275 $24 VOS AND BEHMS animals. Mean SOOT and SGPT values were increased, but the large variation in Individual values and the small number of animals made the apparent differences statistically indistinguishable. The differential count of leukocytes was not significantly titered. ' , jDrgan weights and organ .body weight ratios, are given in Table 4. A significant decrease in body weight and a significant increase in the relative weight of the kidneys 1*as found in the Aroclor-treated animals. Mean absolute and relative liver weights of the test animals were increased, but the variation was also very high. (At microscopic examination of the skin, thickening of the skin due to hyperplasia and ftyperkeratosis of the epidermal epithelium was apparent (Fig. 2). A pronounced hyperplasia and hyperkeratosis of the follicular epithelium was also present, with the ("ofmation of comedolike structures (hair follicles which become dilated and filled with k keratinous plug) (Fig. 2). These changes were found, in general, in ail treated animals, hijt the Phenoclor-treated animal which died after 10 days showed less advanced altera tion! Follicular changes in the Aroclor group were generally less deep; the hyperplasia knd hypefkeratosis were more restricted to the upper part of the hair follicles. ]ffyickening of the surface and follicular epithelium was accompanied by the presence pf large quantities of keratohyalin granules in the stratum granulosum. In general, FA.S-positive, diastase-resistant hyalin foci were seen in the cytoplasm of epidermal pc|ls, located above the cutis papilla (Fig. 2). Dedifferentiation of the sebaceous gland (issue was found in primarily the Phenoclor and the Clophen groups. Hair follicles in (he resting phase (telogen phase) were predominantly seen in the same 2 experimental groups (Fjg. 2). The differences in response between Aroclor-treated rabbits on the one (land and the Clophen- and Phenoclor-treated rabbits on the other are quantitative, not ftualilativt. No differences in response were found between ihe two latter groups. In Control rabbits treated with the solvent Jsopropanol, these changes did not occur (Fig. 3). [Histope thology of the livers of the treated animals showed a considerable diversity |n lesions (Figs. 4 and 5), among them centrolobular degeneration (degenerated cells With nuclear pycnosis. rhexis, and lysis), focal hydropic degeneration (foci of swollen pejls with a light cytoplasm that did not stain with Sudan black and PAS, suggesting a prpnounced cellular edema), focal necrosis (foci of necrotic cells that were found in Various portions of the lobules), centrolobular liver cell atrophy (atrophy of the cyto plasm of liver parenchymal cells in the centrolobular area), cytoplasmic hyalin degenera tion (foci of intracellular eosinophilic hyalin material), and pigment in Kupffer and to a lesser degree in parenchymal cells. The pigment stained positive with Ziehl-Neelsen, FAS and Peris and showed a yellow-brownish fluorescence; these are strong indications For ceroid (Porta and Hartrofl, 1959). The results of the microscopic examination are |given in Table 5. In general, PCB-induced liver lesions were most pronounced in the Clophen group and least in the Aroclor group. Periportal fibrosis was a common finding |n the treated animals, but it was not found in the controls. Since some coccidia were ''Found in a bile duct in (liver, it is quite possible that the fibrosis has to be considered ju1 a healed coccidiosis. Material positive to Sudan black was seen in some fibrotic portal triade. Kidney damage was Found in all PCB-treated animals, though with a wide individual Variation. Hydropic degeneration (negative to PAS and Sudan black) of the convoluted l DSN 029314 \ tri. fit \ cy fir i t I ! "9* if STLCOPCB4013276 STLCOPCB4013277 MM 6tm& VO* AND IM M S STLCOPCB4013278 TABLE 5 Patholoosc Findings in Lrvcxs orJUam Treated with PCB for 3* Days, and of Control Anmau* Fatty degeneration CentFotobufaur loss of glycogen Ceotroiobular degeneration Focal hydropic degeneration Focal necrosis Centxolobular liver cell atrophy EqfarpeuKfit of nuclei Cytoplasmic hyalin degeneration Ceroid pigment in Kupffer cel s Periportal fibrosis OnRw-B--o-o1oc ft 22 1 I --1 2 1 1 2-- 1 -- 1---- -- 1 ---- 21 12 2 1-- I 1 -- I 2-- 1-- 2 2 -- 2' 1 Oopben ft b 1 I 1-- 21 12 12I2 -- 2 2-- 2222 21 21 22 22 2 1 22 1111 I I 22 Arodor- Control . ---- I 2 ---- 1-- --1 1 1 -- I-- 2 ---- 1 2 ------ 1 J-- 1 ------ --21 2 1 2 1-- 1 1 -------- --_ -- -- -- -- -- -- -- _------ ------_ -------- ------ 1 -- -- ---- '" -- -- -- -- -------- * Severity of lesion: 1 - slight, 2 - marted. * Animals that died during the experiment. o 00 SET O tv 'O ui "* O' 1 I -.. -v < Nr>*,-r ' ,v' /"I ' sr ? STLCOPCB4013279 rr* rv > tr uvyrT T V V ri *2* VOS AND BEM$ TABLE 6 Duwrit* Miasuxtmlnt* (/i) or Rsnal Coarutcus or Kilud Rawti TutATio with PCB rm 38 Day*, a no or Contkoc Animals' Capauteof N Bowman Glomerulus Treated animal* iCpolrolt 4 94.3(76.4-105.8) 82.2 (69.4-96.2) 91.3 (86.0- 96.3) 85.2 (79.4-90.4) Mean valuta; the range la thown in perenthtM*. Significantly different from control*, P < 0.005, flJioC the diameter ling to the pace of Bowman 12.3 (8.1-22.1)* 6.3 (5.0- 7.9) liie tubular lesion* explain the results of the diameter measurements of the renal cor- ipuacles (Table 6). ` . I Hemotiderin was found in the splenic macrophages of the test animals in larger (quantities than in the controls. Moreover, a reduction in the number of germinal cen- iters in the spleens as well as in the lymph nodes was found in the PCB groups. Atrophy loathe cortex of the thymus was a common finding in the PCB-treated animals. No evi- idcnce of PCB-induced damage was found in the sedions of the other organs. Experiment with the Dlethylether-Hexane Fractions on the Ear Skin of Rabbits . : The four animals did not differ in weight gain during the test period. After two weekly Topical applications of the fraction from the 3 PCB samples, follicular keratosis was Wen in both rabbits treated with the fraction of Phenoclor and Clophen. After 3 wk, 'When the animals were killed, the changes were more apparent (Fig. 8). The polar frac- jtion of Aroclor did not give rise to gross changes. . ( f* - -a t t C Fio. 8. Chloracoe-tike lesion* on the Inside of a rabbit'* ear tkln treated weekly, for 3 week*, with the 25Y. diethylathec in hexane fraction from Clophen. Note the follicular keratosis in the treated car (/) aji compared with the control (7). i .wbnm* ' ' > i ' , , t mmm OSM 029318 l t hex ( am am fra <di sex let pi STLCOPCB4013280 DCKMALTOXICITY Of TECHNICAL PC M1XTVHE* 429 l cor- Urfer it certlrophy io tvi- Weekly if wg 3 wk, r frac- > '4 i i I *tUt the I f (/) Fio,. 9. Response of the Intklt of the rabblt'i e*r after topical application of the 2J% dlethyiether- heuihe fraction* from technical polychlorinated biphenyl* a* teen In hematoxylin and eo*in lection*. *<o. ! 1. (a) kln of control animal treated with ethanol. Note the hair follicle at I, eebeceou* gland tissue at i, and cartilage at J (b) Ear at In of the animal treated with the fraction from Arodoe. Some hyperplasia and hyperkeratosis of the follicular epithelium can be aeen. (c) Ear skin of the rabbit treated with the fracth^n from Ctopken. Considerable hyperplasia and hyperkeratosis, of the follicular epithehuea (d) Ear skin of the rabbit treated with the fraction from Phervoclor. Part of* section that shows themoet revert lesion. The gravity of the response ww b general the same a* wen ta Fig. 9c. NoSa the cystic di lated hah foAMe wHh prominent hyperplasia and hyperkeratosis of the foflicufar and epidermal \ DSW 029319 qqwsnii)i|gw WO VOS AND Mtttt \ Histologically, hyperplasia and hyperkeratosis of the follicular epithelium with the con i presence of many keratohyalin granules (Fig. 10) were evident in the ear skin of animals nur itreated with the Phenoclor and Ctophen fractions (Fig. 9c and d). This comedolike | formation was topically most pronounced in the rabbit treated with the Phenoclor frac tion (Fig. 9d). Sebaceous gland tissue was decreased by ^reagmcnt with both fractions ofl of c prr |(Fig. 10). Only mild hyperplasia and hyperkeratosis of the follicular epithelium was whi inoted in the animal treated with the fraction of Arodor (Fig. 9b). Hyperplasia and I hyperkeratosis of the epidermal epithelium were also seen in the rabbits treated with the on jPbcnodor (Fig. 9d) /spd Clophen fractions but not in the animal treated with the F Arodor fraction.] and cor san san pri( si hi cav fra< in < dcf cor per the stu |Fkj, 10. Higher magnification of the ear skin shown In Fig. M. Hyperplasia and hyperkeratosis of the ifpillcular epithelium with the pretence of many keratohyalin granules (/). Note also a diminution (deHHfferteUatlon) of the sebaceous gland tissue (3). Hematoxylin and cosln. *37}. No red fluorescence was recorded at examination of the total body and of liver sec tions in ultraviolet light. L(ver sections of the test animals showed centrolobular 'vacuolisation, which stained positive with Sudan biack. ,;I I . i . DISCUSSION jAn increase in fecal excretion of coproporphyrin and protoporphyrin induced by PCB is as readily established in the rabbit (Tabic I) as in the chicken (Vos and Koeman, ' J970). Absence of porphyria in animals treated with the toxic fractions indicates that PCB itself is porphyrogenic.1 Liver and kidney lesions, except the loss of glycogen, the enlargement of nuclei, the Ceroid deposits, the periportal fibrosis, and the dilatation of the space of Bowman, am tox Co fur pre to! el i in cut I cor of sen D (D evi ..... 0SW 029320 T STLCOPCB4013282 DERMAL TOXICITY Of TECHNICAL ECU MIXTURES 631 j wilh (he , confirm the observations of Miller (1944). The lymphopenia (TaWe 3). the reduced tftnimals nurhber of terminal center* in the ipieeni and lymph node*, the atrophy of the cortex tmcdollke j of thymut, and the atrophy of the white pulp and lymphoid foci found in the spleen* >clor frac- { of Chick* fed PCB (Vo* and Koeman, 1970) are strong indication* for an immunosup- fraction* i prewive effect. Thi* may need further itudy, particularly with regard to the extent to ilium wii whlclji the*e observation* are due to trct* (release of glucocorticoid*). 4a*ia and If can be concluded that dermal application of PCB mixture* cause*, betide* the effect d with (he on |ht tkin, systemic lesions of liver, kidneys, and lymphoid tissue. with (be f' rdm the response of the back skin and the liver of the rabbit to the 3 PCB mixtures, ancS from the response of the ear to the 25% diethylether-hexanc fractions, It can be concluded that there are dehnlte quantitative differences in toxicity, at least between the sandpits that were used in the present and the prior studies. The extent to which these samples are representative of the normal commercial output has not been studied. In a pripr'sludy (Vos ft at., 1970), it appeared that the fraction of Clophen A60 was respon sible (for the toxicity of the PCB mixture, which also can be assumed to be true in the case (>f our samples of Phenoclor and Aroclor. From the response of the ear skin to the fractions it can be concluded that the presence of tetra- and pentachlorodibenzofuran in (Upphen A60 and Phenoclor DP6, found by mass spectrometry (Vos et at., 1970), is definitely proved. The presence of a smaller quantity of toxic impurity in Aroclor, when cornered with the Clophen and.Phenoclor samples, is likely, considering the hydro- peiicardium found in chicks fed with Aroclor (Flick et at., 1965) and also considering I the present result*. Preparation of pure PCB is therefore necessary for a comparative j study. Uncontaminated PCB should be available. ' These findings suggest that chloracne lesions induced by chlorinated naphthalene* an(J ^hick-cdema-like lesions in chickens (Pudelkiewicz et at., 1959) may also be due to (Mb of the mitton (de to)(ic impurities, as in the case of PCB'* and chlorinated phenols (Bauer ei at., 1961). Coal tar, which is an important source for naphthalene, may contain traces of dibenzofuran (Fieser and Fieser, 1956). Chlorination of technical naphthalene could then also produce chlorinated diben^ofuran. Since PCB is a porphyrogenic chemical, it is possible that the skin lesions in man due to PCB are a combination of chloracne and acquired porphyria cutanea tarda. Bleiberg et pi. (1964) found an etiologic relationship between the chloracne in worker* engaged in the manufacture of dichlorophenol and trichloropbcnol and acquired porphyria cujajtea tarda. liver sec* rolobular )Ftom the present and prior studies (Vos and Koeman, 1970; Vo* et at., 1970) it is collided that it is important tp take into account, in the evaluation of toxicity data of PCB, the possibility that PCB samples may differ in an important respect; the preBence of toxic impurities. The interpretation of residue data in wildlife is very compiicafed for this reason. duced by Koeman, ate* that odd, the lowman, ACKNOWLEDGMENTS tThe author* are grateful to Mr. H. Baart de la Faille, dermatologist (Department of Dermatology, Hospital of the State University, Utrecht) and Dr. P. Kanaar, dermatologist (Department of Dermatology, Hospital of the State University, Leiden) for their hstp in the evjdyatlon of the Ain lesions. 0SW 029321 ***** mmmmmm mmm mm - STLCOPCB4013283 <32 . . 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