Document VJ74KZ2q76pxJJ3k3gZJL0ZMo

.j must be taken into consideration, there is little evidence to support it: administration of magnesium sulfate to rats under withdrawal had little apparent effect on behavior, weight, or excretion of magnesium in these rats; bone and muscle magne sium concentrations of morphine-treated rats were not statistically different from those of control animals. Nevertheless, it is not unusual to observe normal magnesium s centrations in tissues in the presence of either hypo or hypermagnesemia (Walser, ! Vt. i. Since hypermagnesemia is often associated with an augmentation of magnesium clearance (McCance and Widdowson, 1939), the increase in magnesium excretion ob served here could be explained on that basis. The augmentation in urinary magnesium excretion could also be due to the diuretic effect of chronic morphine treatment, which is associated with an increase in sodium, potassium, calcium, and urea excretion (Marchand and Denis, 1968); jt is .well known that an osmotic diuresis causes an aug mentation in urinary magnesium excretion (Walser, 1967). Since morphine was admini stered in the form of the sulfate, the anion could be responsible for the increase in urine output (Walser and Browder, 1959). However, it has previously been shown that nalor phine ' oks the diuretic effect of chronic morphine administration (Marchand a J Denis, .^68). ACKNOWLEDGMENTS The authors wish to thank the Medical Research Council of Canada for financial support and Miss Claudette Lamoureux for her able technical assistance. REFERENCES McCance, R. A., and Widdowson, E. M. (1939). LXIV: The fate of calcium and magnesium after intravenous administration to normal persons. Biochem. J. 33, 523-529. Marchand, C, and Denis, G. (1968). Diuretic effect of chronic morphine treatment in r,. J. Pharmacol. Exp. Ther. 162, 331-337. Martin, W. R. (1963). Strong analgesics. In: Physiological Pharmacology (W. S. Root and F. G. Hofmann, eds.), Vol. I, pp. 275-312. Academic Press, New York. Vachon, M., and Marchand, C. (1970). The influence of morphine on magnesium metabo lism in rats. J. Pharmacol. Exp. Ther. 172, 122-127. Wacker, W. E. C., and Parisi, A. C. (1968). Magnesium metabolism. New Engl. J. Med. 278. 772-776. WalS' M. (1967). Magnesium metabolism. In: Reviews of Physiology, Biochemistry and Exp^ rtental Pharmacology (R. Jung, K. Kramer, O. Krayer, E. Lehnartz, F. Lynen, A. V. Mural t, U. Trendelenburg, H. H. Weber, and O. Westphal, eds.), pp. 252-254. Springer, New York. Walser, M., and Browder, A. A. (1959). Ion association. III. The effect ofsulfate infusion on calcium excretion. J. Clin. Invest. 38, 1404-1411. PLAINTIFF'S exhibit Dermal Toxicity Studies of Technical Polychlorinated Biphenyls and Fractions Thereof in Rabbits J. G. Vos and R. B. Beems s Institute of Veterinary Pathology and Institute of Veterinary Pharmacology and Toxicology, University of Utrecht, Biltstraat 172, Utrecht, The Netherlands Received October 5, 1970 Dermal Toxicity Studies of Technical Polychlorinated Biphenyls and Fractions Thereof in Rabbits. Vos, J. G., and Beems, R. B. (1971). Toxicol. Appl. Pharmacol. 19,617-633. A significant difference in toxicity between 3 polychlorinated biphenyl (PCB) preparations was found in a prior study: Clophen A 60 and Phenoclor DP6 showing the highest, Aroclor 1260 the lowest, toxicity (Vos and Kocman, 1970). A subsequent study revealed the presence of tetra- and pentachlorodibenzofuran in Phenoclor and Gophen (Vos et at., 1970). In the present study, application of 118 mg of the 3 PCB's (5 times per wk, for 38 days) on the back skin of rabbits also resulted in differences in toxicity. PCB-induced skin lesions were hyperplasia and hyperkeratosis of the epidermal and follicular epithelium. Histopathology of the liver included centrolobular degeneration, centrolobular 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 application of fractions of Phenoclor and Gophen eluted from chromato graphic columns with 25 % diethylether in hexane. The fraction from Aro clor 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 v- porphyrin and protoporphyrin excretion was increased. ychlorinated biphenyls (PCB) have been identified in tissues of fish and wildlife in y countries (Jensen, 1966; Holmes et al., 1967; Holden and Marsden, 1967; Koe- etal., 1967; Risebrough et al., 1968; Koeman etal, 1969; Jensen era/., 1969; Duke al., 1970; Prestt et al, 1970). - PCB preparations are extremely stable, oily fluids with very low aqueous solubility, are used as lubricants, as heat transfer media, in protective coatings for wood, and concrete, and for many other applications. Which of these applications has tributed to the present environmental contamination has not yet been established. Inhalation and feeding experiments in rats with 65 % chlorinated PCB resulted in liver ijitry (Drinker et al., 1937; Bennett et al, 1938). Liver damage and skin lesions have described after inhalation, ingestion, and application to the skin using rabbits, aea pigs, and mice (von Wedel et al, 1943). Hydtpp^ricardium occasionally accom- ed by abdominal edema was found in chicks fed PCB (McCune et al, 1962; Flick 617 WATER PCB-SD0000038511 Vos and Koeman, 1970). Epithelial and follicular hyperplasia and hypcrrc found after application of PCB on the skin of rabbits (Adams et al.. laneous injection of 42% chlorinated PCB in rats, guinea pigs, and rabbits ivcr injury and skin lesions, which were essentially those of cblor- nc 1). Follicular hyperkeratosis is an important feature of the occupati il <n as chloracne, which is characterized by. the appearance of papules, cc-meysts. It may develop after exposure to some highly chlorinated aromatic Examples are chlorinated naphthalenes (Greenburg et al., 1939; Hambriek, y and Kligman, 1957) and chlorinated phenols (Hofmann, 1957; Bauer et ribergef al., 1964). The active compounds in chlorinated phenols have been ; chlorinated dibenzodioxins (chlorinated diphenylene dioxides) which as contaminants in the synthesis of chlorophenols (Bauer et al., 1961). (acute yellow atrophy), whether accompanied by chloracne lesions or not, bed in people engaged in the manufacture of the chlorinated hydrocarbons 14; Bra"n, 1955; Behrbohm, 1959). Also for the PCB's there are earh rLjpatio ,'hloracne (Jones and Alden, 1936; Schwartz, 1936). The pre-i m'CB does not seem to have led to this kind of difficulty, int difference in toxicity between 3 samples ofcommercial PCB preparations a previous study (Vos and Koeman, 1970), despite the marked resemblance romatograms and the mass spectra (Koeman et al., 1969). One hundred per ly, centrolobular liver necrosis, and sc and abdominal edema were found ;s fed the samples of Clophen A60 and Phenoclor DP6. Hydropericardium d in nearly all chicks fed these PCB's, and was only occasionally seen in ith Aroclor 1260. Porphyria was found as a general PCB effect (Vos and 70). thesis that in some technical PCB mixtures toxic factor(s) responsib! cmalike lesions and the liver necrosis were present has been the subjc. t >>l <. By means of column fractionation, gas chromatography, and mass speci hniques, tetra- and pentachlorodibenzofuran were identified as the toxic ie samples of Clophen A60 and Phenoclor DP6 (Vos et a!., 1970). ults suggest that PCB's contaminated with polychlorodibenzofurans may ? chlo 'e and associated lesions when applied to the skin of experimental test ti..,, possibility the present study was undertaken. METHODS samples, which contain an average of 60% chlorine, were obtained ' ''i f ranee (Phenoclor DP6), Bayer fn Germany (Clophen A60 Lot No. 912434) nto in the United States (Aroclor 1260 Lot No. AK-3). lale New Zealand rabbits, 5 mo old and weighing 2500-3050 g, were used in I hey received pelleted food (Cunicon 1, Trouw and Co., Amsterdam) and >itum. The animals were distributed at random into 4 groups of 4 animals, eighed weekly. nf approximately 10 x 15 cm on the backs was clipped, the remaining hair with a shaving powder (90 g barium sulfide, 105 g zinc oxide, and I HO g I the resulting small skin abrasions were allowed to heal for 3 days The 3 PCB's were dissolved in isopropanol (118 mg PCB/ml). One ml of PCB solution of isopropanol as a control was dropped daily, 5 times a wk, on an area of 5 x 10 cm means of a calibrated syringe. In order to minimize eventual ingestion of the PCB, animals were held for 7 hr in restraining boxes, after which time they were returned their wire cages (2 animals per cage). ; In order to study the porphyrogenic action of the PCB -preparations in the same bbits, pooled samples of the feces of the 4 groups were analyzed weekly for copro- rphyrin and protoporphyrin contents (Rimington, 1961), using a Beckman spectro- tometer. .' The animals which survived the 38-day test period (27 applications of 118 mg PCB) ; killed. At necropsy, performed on all animals that died or were killed, a gross thologic examination was carried out, including macroscopic examination in.ultralet light using red fluorescence as an indication of porphyria. In order to get infortion on the fecal excretion of coproporphyrin and protoporphyrin of the individual al, feces collected from the cecum were analyzed. Hematologic examination included hemoglobin and hematocrit determinations, kocyte and differential leukocyte counts. Clinical chemical determinations were ie of serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase 'tman and Frankel, 1957) using a Beckman/Spinco 151 spectrocolorimeter. ie weights of body, liver, kidneys, spleen, heart, and adrenals were recorded. The liver, spleen, kidney, pancreas, ovary, heart, lung, thymus, mesenteric lymph , stomach, small intestine, cecum, large intestine, and skeletal muscle were fixed in % buffered formalin and embedded in Paraplast. Sections (7 ft and 2 ji) were stained hematoxylin-eosin. For detailed histology, selected sections were stained with i, Azan, PAS, Ziehl-Neelsen, Congo red, and thioflavine. Moreover, cryostat tions of formalin-fixed livers and kidneys were stained with Sudan black. Thioflavine- 'ned sections, unstained Paraplast-embedded sections, and unmounted cryostat 'ons of liver, kidney and small intestine were examined in a fluorescence roscope. ' -prompted by the observation of tubular dilatation in the kidneys in previous studies " PCB ingestion (McCune et al., 1962; Vos and Koeman, 1970), measurements - made on random samples of capsules of Bowman and glomeruli in 2-/x kidney bns. Measurements of one axis of the greatest diameter of both capsule and merulus were made of 25 renal corpuscles in a straight traverse across the cortex of kidney". The relative percentage of the diameter which corresponds to the space of 'man, as an index of dilatation of the nephron, was calculated from the measureof each renal corpuscle. An additional experiment was made with the chromatographic fraction (column ed with 25% diethylether in hexane) of the 3 PCB's, containing tetra- and pentaJtorodibenzofuran in the case of Phenoclor DP6 and Clophen A60 (Vos et al., 1970), determine the dermal toxicity of this fraction. Aliquots of 600 mg ofthe 3 PCB mixi were fractionated (Vos et al., 1970). The 25 % diethylether-hexane fractions were orated and dissolved in 0.6 ml ofethanol. Four female albino rabbits (3 test animals l control), 2 mo of age and weighing 1550-1750 g, were used as the experimental _ mals. A 0.2-ml portion of the ethanol solution was applied weekly, for 3 wk, to an 1 of 4 cm2 on the inside of the rabbit ear. The opposite ear served as a control. The WATER PCB-SD0000038512 620 VOS AND BEEMS o s-. S-* -- S-* v* oo g ^ \o` ^ u o ^I vq co -V av - I c>i U I 1. o C^^ 8 8*-* < sz & o a o c a> ^vNnNo jz a <o co C oo 0\ On U < 02 ' _o onv)t u d r4 r4 fs| fv| -- ro & < 9 JZ au J& 5O &. O COwu W< U. 5 c V JZ i twi!) ! o CL o ovOOv^t fn n r> On I sn 0 to. Z O< ?g _o n n n - oo 8 c c^i vc^di n-- 6 r^i O JZ a. o uj 2 s O 00 ON N N b 8 e r4 nno an aw R u JZ a. oz 11 co ^ t-* ic o ~ -1 ~ 8 U w n C\ -- n O d c4 -- ~ <~i O O U) c -o sr n no M rs co d ^ -- t*** ^ on V-i d v 0* o 3*2 to < ED On on On co r4 r4 r4 ^ U o tc u S CO I _o r-* vr t-^ co if^ec XP u -d ~ r^i mm x>:v < r4 RH O 2P 2 & O QZ< H e_ a o U c V JZ v-n b Cl O ^t*--fndn oon NO VN U C O Cu I O e CL O U *L o vO CO O n -- O ri n n n co JZ a. *o O 82 Q. o U oI * ~Oc ; ioo "4 *0 VoNo xao:. ! _ i*^ cNoO > I o t-" ^-- N NOs n'D 8 IS DERMAL TOXICITY OF TECHNICAL PCB MIXTURES animals were killed after 3 wk. Body and liver weights were recorded car and the liver were stained with hematoxylin-eosin. Cryostat secti were also stained with Sudan black. Statistical significance, on a brie-tail significance level, was deteri Wilcoxon test for 2 unrelated samples (van der Waerden, 1957). RESULTS Experiment with Commercial PCB Preparations in Rabbits The skin, especially of the Clophen- and Phenoclor-treated a reddened after 2 days of PCB treatment. This redness was more prono of treatment, together with clear desquamation of the external epide reduced regrowth of hair. Thickening of the skin and prominent tra of the treated skin developed during the experiment. Fig. I. Fluorescence of porphyrins under ultraviolet light in livers from PCB-tr< /, Aroclor; 2, Clophen; 3, Phenoclor; and 4, control liver. The results of the spectrophotometric determinations of fecal copi protoporphyrin are given in Table 1. The test animals showed, in general, a gradual loss of weight. One I Clophen-treated animals died, with severe weight loss, after 10, 31, . respectively. Nt necropsy, carried out on all animals, it was found that 1 anim; 3 test groups showed definite edema formation, with fluid in the abdoni cavity, subcutis, and pericardium. All test animals, except for 1 rabbit of the Clophen group that died treatment, showed fluorescence in ultraviolet light. Liver (Fir n K WATER PCB-SD0000038513 oo rOcoIs crn-I *>r- < Q v> O oO' o u. CQ aU. r- cn d od 5 >oO> Ect 3O -- ?N ~ so --I CNI m v> rn Os CN o OE g>g E~ u bQ Xw P* CN o' cn o On On HU >- < Q oO' u. CQ id ae t * P sD2 H 50 ftfl 5 0$ QQ 3d 3 c K 51 8* Po * pO>Qa 2 < Oo' O Q Z < sHo X o E 2 < o0o' rn m gg rn CO CN CN oo oo SO -- NO so d) CN CN O a n *ci v> CN so 50 On r- ~i i di -- CO CN CN CO 0 CN oo -- 75 . rI1 ~I eo -- * co oo n o n cn v-> so SO 4 44 -- co d t^ChnO N* 80 N* 60 g gg On vD -- lU ">ov CQ g g CN CN n n On CN CN CN CN *o C Do Q. Eo U si o 2 C a" r ^so o. U < U 1/lmvial iumv 11 t Oh Shl'UNICAL PCB MIXTURES was observed most frequently; fluorescence of incisors, kidneys, : subcutis was found in about half of the test animals. There was no bone marrow. Fluorescent microscopic examination of cryostat s revealed that porphyric ^nipaqls had porphyrin diffusely distribu whole lobule. Small foci inside parenchymal cells, probably nude more intense red fluorescence. Kidney sections showed, when jx fluorescence, which was found in the cytoplasm of the tubules in r Fluorescence of granules (perhaps nuclei) in glomeruli was also n I ig. 2. Back skin of a killed rabbit from the Phenoclor-treated group. Hyperplas of he epidermal and follicular epithelium. Follicular plugging is prominent. N< the cytoplasm ofepidermal cells (/), and the resting (teiogen) phase of the hair follii and eosin. x46. Fig. 3. Normal skin of back of a control animal treated with the solvent isopr dermis at / and dermis at 2. Several groups ofhaif follicles, cut somewhat tangentir are seen at 3; groups of hair bulbs are seen at 4. Hematoxylin and eosin. *46. Coproporphyrin and protoporphyrin contents of feces collected ft necropsy are given in Table 2. The degree of excretion was not related porphyrin fluorescence; for example, the Clophen- and Aroclor-treat coproporphyrin excretion of 30.5 and 4.7 fig/g (Table 2), showed, resf re cence at all, and fluorescence of liver, bone, incisors, kidneys, sm su xmtis. The results of the hematologic determinations are given in Table 3. animals of the 3 groups have been taken together since there were n tween the groups. The total number of leukocytes was signify-."M,. ~ WATER PCB-SD0000038514 624 VOS AND BEEMS DERMAL TOXICITY OF TECHNICAL PCS MIXTURES animals. Mean SGOT and SGPT values were increased, but the large vari: _ individual values and the small number of animals made the apparent dH&a^I^v statistically indistinguishable. The differential count ofleukocytes was not altered.' ' Organ weights and organ:body weight ratios, are given in Table 4. A t decrease in body weight and a significant increase in the relative weight of the was found in the Aroclor-treated animals. Mean absolute and relative liver the test animals were increased, but the variation was also very high. At microscopic examination ofthe skin, thickening of the skin due to hypeipli^j hyperkeratosis of the epidermal epithelium was apparent (Fig. 2). A hyperplasia and hyperkeratosis of the follicular epithelium was also present, formation of comedolike structures (hair follicles which become dilated and a keratinous plug) (Fig. 2). These changes were found, in general, in all treated but the Phenoclor-treated animal which died after 10 days showed less advanced tions. Follicular changes in the Aroclor group were generally less deep; the h; and hyperkeratosis were more restricted to the upper part of the hair: Thickening of the surface and follicular epithelium was accompanied by the. of large quantities of keratohyalin granules in the stratum granulosum. Ip: PAS-positive, diastase-resistant hyalin foci were seen in the cytoplasm of t u . > t -i cells, located above the cutis papilla (Fig. 2). Dedifferentiation of the sebaceous tissue was found in primarily the Phenoclor and the Clophen groups. Hair f< the resting phase (telogen phase) were predominantly seen in the same 2 expenm^g^> groups (Fig. 2). The differences in response between Aroclor-treated rabbits on "" hand and the Clophen-and Phenoclor-treated rabbits on the other are quand qualitative. No differences in response were found between the two latter control rabbits treated with the solvent isopropanol, these changes did h (Fig. 3). C." Histopathology of the livers of the treated animals showed a considerable in lesions (Figs. 4 and 5), among them centrolobular degeneration (degen with nuclear pycnosis, rhexis, and lysis), focal hydropic degeneration (foci bf cells with a light cytoplasm that did not stain with Sudan black and PAS, su pronounced cellular edema), focal necrosis (fod of necrotic cells that were various portions of the lobules), centrolobular liver cell atrophy (atrophy of plasm ofliver parenchymal cells in the centrol obul ar area), cytopl asmic hyalin tion (foci of intracellular eosinophilic hyalin material), and pigment in Kupffer lesser degree in parenchymal cells. The pigment stained positive with Ziehl-N^lllfv PAS and Peris and showed a yellow-brownish fluorescence; these are strong iodicawtf for ceroid (Porta and Hartroft, 195,9). The results of the microscopic examinational given in Table 5. In general, PCB-induced liver lesions were most pronounced iri fi* . Clophen group and least in the Aroclor group. Periportal fibrosis was a common finding in the treated animals, but it was not found in the controls. Since some coccidia wim. ' found in a bile duct in 1 liver, it is quite possible that the fibrosis has to be consider^ as a healed coccidiosis. Material positive to Sudan black was seen in some fitewfc portal triades. ' .'Tvl , Kidney damage was found in all PCB-treated animals, though with a wide individual. variation. Hydropic degeneration (negative to PAS and Sudan black) ofthe convoluted ' Fi<;. 4. Liver of a Clophen-treated rabbit that died after 31 days of treatment she triac .' at /, and degeneration and atrophy of the cytoplasm of the hepatocytes in the cei at 2. Hematoxylin and eosin. xI50. Fiu. 5. Higher magnification of the liver shown in Fig. 4.1: central vein; 2 : hydror cytoplasmic hyalin degeneration. Note also the enlarged nuclei and the karyopycnos nd eosin. x375. WATER PCB-SD0000038515 626 VOS AND BEEMS II II II - I I I e a CN| 0 1 < 4 CM CM CM | CM - CM CM cCJ - - M ri N N N jac o --< cm cm cm cm -- cm -- | ri n n n in CQ < H -- I cm cm --< cm --< -- -- --< t -- _c a cm i -- O 2 Q u_ 01H < a. O>boO 'W) & >v jac o cO g 8 %) u OCobOj (3OE. c *> 8 2 73 ^ -a ObO TOt3iJl .2 cn s2* ^2 8 c ---- ^ ^P>3>` *C"% 5-= .S E O _c,t-) e C aa -5 ^ j a o o oa (0)> OcJ >-v 4> UU CJ CL * Severity o f lesion: I - slight, 2 - marked. * Animals that died during the experiment. DERMAL TOXICITY OF TECHNICAL PCB MIXTURES jubules (Fig. 6) was found in half of the animals. Nuclear pycnosis, rhexis the tubular epithelial,cells were seen in all animals. Tubular dilatation, aco the presence ofcasts ofnecroticpithelial cells (Fig. 7), was found in half ol Fig. 6. Kidney ofa killed rabbit from the Aroclor-trcated group showing hydropic dc nuclear pycnosis of the convoluted tubules (/). A slightly distended space of Bowman ?). Hematoxylin and eosin. x375. Fig. 7. Kidney section of the same animal as shown in Fig. 6, show-in- ` WATER_PCB-SD0000038516 TABLE 6 Diameter Measurements (ft) of Renal Corpuscles of Killed Rabbits Treated with PCB for 38 Days, and of Control Animals 1 ", Capsule of N Bowman Glomerulus Relative % of the corresponding to tM&pxj space of Bowtftw&ifg Treated animals 8 94.5 (76.4-105.8) 82.2 (69.4-96.2) 12.5(8.1-22.1!- Controls 4 91.5 (86.0- 96.5) 85.2 (79.4-90.4) 6.5 (5.0- 73) ----------------- :------ --i-aHato incan valuer, me iau^c 15 aiivwu m paiwmuvoM. * Significantly different from controls, P < 0.005. The tubular lesions explain the results of the diameter measurements of the j puscles (Table 6). ' - - :'?'- Hemosiderin was found in the splenic macrophages of the test animus*' quantities than in the controls. Moreover, a reduction in the number off ters in the spleens as well as in the lymph nodes was found in the PCB groups.:* of the cortex of the thymus was a common finding in the PCB-treated animals^ dence of PCB-induced damage was found in the sections of the other orginsf<f Experiment with the Diethylether-Hexane Fractions on the Ear Skin of Rabbit} The four animals did not differ in weight gain during the test period. After twos topical applications of the fraction from the 3 PCB samples, follicular ke seen in both rabbits treated with the fraction of Phenoclor and Clophen., when the animals were killed, the changes were more apparent (Fig. 8). The j tion of Aroclor did not give rise to gross changes. . MB : iiyj i- _JP' Fig. 8. Chloracne-like lesions on the inside of a rabbit's ear skin treated weekly, for 3 weeks. wittUfcF 25% diethylether in hexane fraction from Clophen. Note the follicular keratosis in the treated tuif}:. as compared with the control (2). - T Fig. 9. Response of the inside of the rabbit's ear after topical application cf the 25% i hexane fractions from technical polychlorinated biphenyls as seen in hematoxylin and eo *60. ia) Skin of control animal treated with ethanol. Note the hair follicle at 1, sebaceous glan am cartilage at 3. (b) Ear skin of the animal treated with the fraction from Aroclor. Some and hyperkeratosis of the follicular epithelium can be seen, (c) Ear skin of the rabbit trea fraction from Clophen. Considerable hyperplasia and hyperkeratosis of the follicular (dtEar skin of the rabbit treated with the fraction from Phenoclor. Part of a section that sht severe lesion. The gravity of the response was in general the same as seen in Fig. 9c. Note i 1 " '' hair follicle with prominent hyperplasia and hyperkeratosis of the ' WATER PCB-SD0000038517 630 VOS AND BEEMS Histologically, hyperplasia and hyperkeratosis of the follicular epithelium m presence of many keratohyalin granules (Fig. 10) were evident in the ear skin of treated with the Phfcnoclor and Clophen fractions (Fig. 9c and d). This coi formation was topically most pronounced in the rabbit treated with thePbenocti tion (Fig. 9d). Sebaceous gland tissue was decreased by treatment with both (Fig. 10). Only mild hyperplasia and hyperkeratosis of the follicular epithefi noted in the animal treated with the fraction of Aroclor (Fig. 9b). HypcrpI' hyperkeratosis of the epidermal epithelium were also seen in the rabbits treated* Phenoclor (Fig. 9d) and Clophen fractions but not in the animal treated.' Aroclor fraction. . . \\ -V<- Fig. 10. Higher magnification of the ear skin shown in Fig. 9d. Hyperplasia and hypakeratc follicular epithelium with the presence of many keratohyalin granules (/). Note also a dnninii differentiation) of the sebaceous gland tissue (2). Hematoxylin and eosin. x375. No red fluorescence was recorded at examination of the total body and of liv tions in ultraviolet light. Liver sections of the test animals showed centrolofi vacuolisation, which stained positive with Sudan black. \ \ DISCUSSION ' An increase in fecal excretion of coproporphyrin and protoporphyrin indue PCB is as readily established in the rabbit (Table 1) as in the chicken (Vos and Keen 1970). Absence of porphyria in animals treated with the toxic fractions indicates: PCB itself is porphyrogenic. Liver and kidney lesions, except the loss of glycogen, the enlargement of nuclei, DERMAL TOXICITY OF TECHNICAL PCB MIXTURES confirm the observations of Miller (1944). The lymphopenia (Table 3). the r number of germinal centers in the spleens and lymph nodes, the atrophy of the of the thymus, and the atrophy of the White bulp and lymphoid foci found in the of chicks fed PCB (Vos and Koematt,~197.Q) are strong indications for an immu pressive effect. This may need further study, particularly with regard to the ex which these observations are due to stress (release of glucocorticoids). It can be concluded that dermal application of PCB mixtures causes, besides th on the skin, systemic lesions of liver, kidneys, and lymphoid tissue. I rom the response of the back skin and the liver of the rabbit to the 3 PCB mi and from the response of the ear to the 25% diethylether-hexane fractions, it concluded that there are definite quantitative differences in toxicity, at least betw samples that were used in the present and the prior studies. The extent to whic samples are representative of the normal commercial output has not been studii prior study (Vos et al., 1970), it appeared that the fraction of Clophen A60 was i sible for the toxicity of the PCB mixture, which also can be assumed to be trui case of our samples of Phenoclor and Aroclor. From the response of the ear skii fractions it can be concluded that the presence of tetra- and pentachlorodiben; . in Clophen A60 and Phenoclor DP6, found by mass spectrometry (Vos et al., 1 definitely proved. The presence ofa smaller quantity of toxic impurity in Aroclo co n pared with the Clophen and Phenoclor samples, is likely, considering the pericardium found in chicks fed with Aroclor (Flick et al., 1965) and also com the present results. Preparation of pure PCB is therefore necessary for a comp study. Uncontaminated PCB should be available. These findings suggest that chloracne lesions induced by chlorinated napht and chick-edema-like lesions in chickens (Pudelkiewicz et al., 1959) may also bt loxic impurities, as in the case of PCB's and chlorinated phenols (Bauer et al. Coal tar, which is an important source for naphthalene, may contain traces of d furan (Fieser and Fieser, 1956). Chlorination of technical naphthalene could tl produce chlorinated dibenzofuran. fiince PCB is a porphyrogenic chemical, it is possible that the skin lesions in r to PCB are a combination of chloracne and acquired porphyria cutanea tarda. 1 et al. (1964) found an etiologic relationship between the chloracne in workers t in the manufacture of dichlorophenol and trichlorophenol and acquired pc cutanea tarda. f- - I . 9 ,. From the present and prior studies (Vos and Koeman, 1970; Vos et al., 19 concluded that it is important to take into account, in the evaluation of toxic of PCB, the possibility that PCB samples may differ in an important respect: sence of toxic impurities. The interpretation of residue data in wildlife is very rated for this reason. ACKNOWLEDGMENTS The authors are grateful to Mr. H. Baart de la Faille, dermatologist (Depan Dermatology, Hospital of ihc State University, Utrecht) and Dr. P. Kanaar, derm ffVo a orient of Dermatology, Hospital of the State University, Leideni r WATER PCB-SD0000038518 M632 VOS AND BEEMS REFERENCES Adams, E. M., Irish, D. D., Spencer, H. C., and Rowe, V. K. (1941). The response off skin to compounds reported to have caused acneform dermatitis. Ind. Med., Ind. j 2,1-4. '1 Bauer, H., Schulz, K. H., and Spiegelbero, U. (1961). Berufliche Vergiftungen/ Herstellung von Chloiphenol-Verbindungen. Arch. Gewerbepathol. Gewerbehyg. II 555. . . Behrbohm, P. (1959). 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