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* ' Environmental Health Perspectives gly of for wo md the her our nal use the W71 jiyht iron. si of Lion, sled Den )PO0 Ills). freat lUnl long ives Toxicology of PCBs for Mammals and for Birds byJ. G. Vos* Introduction In the early days of its use, little work was done on the toxicology of the PCBs, and this was only in relation to the risks of occupational exposure. As will be shown many more studies were made as soon as it appeared that the extremely 6tablc PCB'8 became a threat, to the environment and its wildlife, and accidents occurred of acute poisoning in man and animals. These, studies have been made with material w'ith different contents of chlorine, from different manufacture, and--as we can now say in retrospect--with different and unknown contents of toxic im purities. For tliis reason the toxicological infor mation of PCB'6 is difficult to summarize, but since the character of some important impurities has recently been elucidated, it is well to discuss these first in order to be able to consider their contribution to the overall toxicity of the dif ferent. preparations studied. ' These studies were started because of the analogy between the effects of the PCBs (1-6) and 6omc toxic effects associated with toxic factors in crude chlorophcnols and in "toxic fat". Effects of the latter arc liver damage (7), chloracnc (7,8) and edema formation (9). toxicity of three commercial PCB samples (con taining 60% chlorine on the average): Phcnoclor DP6 (sample I), Clophen A60 (sample II) and Aroclor 1200 (sample III). These three mixtures showed a marked resemblance in their gas chroma tograms and mass spectra (10). In this compara tive feeding test in one-day-old chicks (11), it was found that 100% mortality, subcutaneous and abdominal edema, and ccntrolobular liver necrosis occured in only two groups (fed the samples 1 and II). Hydropericardium (Fig. 1), a common effect of these two mixtures, was only occa sionally seen in the chicks fed the sample III. The Chemical, Toxicological, and Pathological Identification and Evaluation of Toxic Impurities in Technical PCB Preparations The first indication of the presence of toxic impurities was obtained in a comparison of the Institute of Vctcrinnry Pathology and Institute of Veterinary Phnrmnrology and Toxicology, State Uni versity of Ulrcchl, The Netherlands. Figure 1. Hydropericardium In a chick fed 400 ppm of sample I. April 1972 DSW 031145 10S STLCOPCB4015107 1 ?A h . ,/i b rf. j- ^ muMiriiaj TaMc 1. Mortality, and Pathologic Observation* of OilcVi Fed 400 ppm VCIJ for 60 I>ny. PCB sample Number of Number of birds with edema Number of N birds with Hydro Abdominal Subcutaneous liver necrosis pericardium 1 II III Control 24 24 18 8 6 e 22 22 20 9 7 9 20 3 3 0 0 0 20 0 0 0 0 0 mortality in this group was only 15% (Table 1). The excretion of coproporphyria and protopor phyrin in the feces was increased. Examination of tissues under Wood's light showed the presence of red fluorescence indicating porphyrins in the liver and other tissues of especially the birds that died. This hepatic porphyria was found in all three experimental groups. In the subsequent study (12) by means of column and gns-chromatography the presence of relatively more polar compounds was demon strated in the 25% dicthylcthcr fraction of samples 1 and II. In a chick embryo assay (Tabic 2), the difference in toxicity between the three samples was confirmed; the high toxicity of the 25% dicthylcthcr fraction of sample II is demon strated by the similarity between the mortality levels in the group injected with 3.5 sample II/ egg and the group injected with the 25% dicthyl cthcr fraction from 3.5 mg sample ll/egg. Mass spcctromctric analysis revealed that Table 2. Chick -Km bryo Assay of Three PCH samples and the 25% Dicthylcthcr Fraction from Sample II PCH sample Dose (mg/egg) Number of eggs treated Percentage batch of fertile eggs I 11 111 Fraction from ample 11 Ethanol control Untreated control 8.5 3.5 8.5 35 8.5 0.85 0.035 0 -- 15 20 15 15 16 15 15 20 20 0 6 80 0 7 92 100 94 90 identical chlorinated compounds were present in the 25% dicthylcthcr fraction from samples I and II but not in that from sample III. They included compounds with mass number 304 and 338.. The proposed identity of these compounds, tctrachlorodibcnzofuran and pcntachlorodibenzofuran, is indicated by the following chemi cal-analytical, pathological, and toxicological data. From exact mass measuremen's it was found that the formulae of these peaks were Cpl^O^Ch and CjiHjO^Ch. The formulae of the fragment ions 241 and 275 were CnHi,5C! and CnIljS5Cl. From this data it can be concluded that the parent ion has a preferential fragmentation for the loss of a single C1CO unit. Mass spectra were compared with the spectrum of a chick edema factor 1,2,3,7,8,9-hcxachlorodibcnzo-p-dioxin. The mass spectrum of this compound has a similar fragmentation pattern, i.c. the loss of two sue- cessivc mass units of 63 suggests a loss of two C1CO units. With microcoulomctric analysis a certain maximum level could be indicated. This maximum level was found to be five ppm of the compound with mass number in sample II and 20 ppm in sample I. ~ ^Polychlorinated dibenzofurans are strong hepa- totoxic and acnegcnic compounds. Tri- and letra- chlorodibcnzofurans in a single oral dose of 0.5 -- 1.0 mg/kg caused severe and often lethal liver necrosis in rabbits (7,13), and application to the ear resulted in chloracnc. Tctrachlorodibcnzo- dioxin was about 10 times more toxic (7). In jection of the 25% dicthylcthcr fraction obtained from 35 mg sample II into the aircell resulted in 100% mortality (Table 2). It can be calculated that the maximum dosc/cgg is 0.2 pg penta- chlorodibcnzofuran (taking five ppm as the maxi- 106- Environmental Health Perspectives ' , | 1 I ! ( i I | ' ! j . DSW 0311*6 STLCOPCB4015108 mum value). This confirms (he order of toxicity found by Higginbotham ct nl. (9) in the case of chloro-dibenzodioxins. Tetra and pentacldoridibentofurans were considered responsible for the higher toxicity of samples I and II. Confirmation is also obtained from the sub sequent comparative toxicity study in rabbits (14). Again samples I and II were more toxic; the liver and skin lesions were more severe. Por phyria, especially of the liver, was present in all three groups (Tig. 2). A remarkable finding was the intense red fluorescence of 6mall foci inside hepatic cells. They probably represent nuclei. This was confirmed in an additional cell culture experi ment with the Aroclor sample and with 2,4,5,2',4',5,-hcxachlorobiphenyl. (This experiment was carried out by my colleague Dr. J. G. Wit of the Biochemical Section). Application of the 25% diethylcther fraction on the 6kin of rabbits resulted also in differences in toxicity (Fig. 3). So the presence of the hepatotoxic and acncgenic polychlorinated dibenzofurans as impurities in samples I and II was found to be established. In another experiment (15), the toxicity of the Aroclor (60% Cl) sample was compared with the toxicity of 2,4,5,2')4',5'-hcxachlorobiphcnyl. From the increased fecal excretion of copropor phyrin in both experimental groups (Table 3), it is very likely that PCBs themselves arc responsible for the porphyrogenic action of crude prepara tions. From the presence of slight skin lesions induedd by 2,4,5,2',4',5'-hcxachlorobiplicnyl when compared with the Aroclor sample, it can be concluded that the major acncgenic action of crude mixtures comes from a possible contamina tion with chlorinated dibenzofurans. It can also be concluded that l'CBs themselves have a slight acncgenic action. Liver damage was es sentially the same after treatment with both 2,4,5,2',4',5'-hexachlorobiphcnyl and the Aroclor mixture. The conclusion that the liver injury, caused by crude preparations, is pre dominantly due to the contaminants, is based on the differences in liver toxicity between the three FCB preparations (11,14). The probable contribution of polychlorinated dibenzofuran (PCF) and pure polychlorinated biphenyl (PCBs) in the toxicity of crude prepara tions is summarized in Table 4. A proper evalua tion of toxicity data and residue data can be hindered by the possibility that PCB samples may Figure 2. Fluorescence of porphyrins under ultraviolet light in livers from rabbits treated with 60% chlorinated PCIi'a, - 1, Aroclor; 2, Clophcn; 3, Phenoclor; and 4, Control liver (14). April 1972 107 OSH 031147 STLCOPCB4015109 '1 t* 't$4 I Fioure 3. Itcs]K>nsc of the inside of the rabbit's ear after topical application of the 25% diethylcther fractions from tech nical PCIVs. Haemotoxylin and eosin. X60, (a) Skin of control animal treated with ethanol. Note the hair follicle at 1, scabacoous gland tissue at 2, and cartilage at 3. (b). Ear skin of the animal treated with the fraction from sample 111. Some hyperplasia and hyperkeratosis of the follicular epithelium can be seen. (c). Ear skin of the rabbit treated with the. fraction from sample II. Considerable hyperplasia and hyperkeratosis of the follicular epithelium, (d). Ear kin of the rabbit treated with the fraction froip sample I. Part of a section that shows the most severe lesion. Tho gravity of the response was in general the same as Been in (c). Noto the cystic dilated hair follicle with prominent hyperplasia and hyperkeratosis of the follicular and epidermal epithelium (14). i 108 Environmental Health Perspective* DSW 031148 STLCOPCB4015110 i ltti- ollnr Miyir t*xd . Far Tit im ) difTrr in an important respect: the presence of It is very probable that, the results of these i Ionic impurities. The possibility of distinguishing tatween the effects of PCBs and their impurities studies may have been influenced by the presence of polychlorodibenzofurans or other toxic im can be further improved by using pure isomers purities. Tor example, in the study of Kelt fold with known positions of the chlorine atoms. ct al. (23) general edema was already found in chicks fed 30 ppm Aroclor 48% Cl. while Mortality, Liver Effects, Edema Formation and Other Kohanawa and co-workers (22) noted edema Effects formation at the 100 ppm level of another 48% These data, as presented by different authors, arc summarized in Tables 5, 6, and 7. As can be chlorinated mixture. Mortality in the latter study was also lower (Table 6). seen in Table 5, the acute and 6ubacutc toxicity data of PCB's are poor. The established values Liver Effects arc high. Scmichronic oral toxicity studies are summarized in Table 6. Dermal and inhalation studies arc given in Table 7. The most important liver effects, summarized in Tables 5, 6, and 7, are weight increase, fatty degeneration, hyalin degeneration and necrosis. Table 5. Coproporpliyrin Contents (^g/g Dry Weight) Increased liver weights, as noted in several studies, of Feces of Rabbits Treated with fCB for 4 weeks, nd of Controls.* Table 4. Probable Contribution of Polychlorinated Dlbcnzofuran (PCF) and Pure Polychlorinated 2,4,6,2',4', 6'-Hcxachlorobiphcny1 Aroclor Control (60% Cl) Diphenyl (PCB) In the Toxicity of Crude PCD Mixtures. 45.0 6.2 20.8 80.6 Mean 27.9` 24.1 6.0 29.4 16.0 18.6` 4.6 3.7 3.6 3.6 3.8 Figures are the contents of feces, collected from the cecum of the individual animals. Significantly different from controls, P <0.025. Polychlorinated Dibeniofuran Polychlorinated Biphenyl Chtor- Edema Liver Hepatic acnc forma damage porphyria tion ++ + + + + + ++ Table S. Acute and Subacute Oral Toxicity Studies of PCB Preparations. Preparation Animal Treatment Mortality Liver effects References Unknown Aroclor 64% Cl M0U6C Rat Aroclor 42, 64, 60, and 68% Cl 42% Ct Mallard Rat 42% Cl Guinea pig 66% Cl Rat single dose of LD50 approx. 2000 mg/kg single dose of 0% Increase of weight and 600 mg/kg lipid; potentiation of CCU toxicity single dose of 0% 2000 mg/kg 20 daily doses of 0% in 3 Hyalin bodies in liver cells 138 mg months 2 doses of 69 mg 100% between Fatty metamorphosis; 1 week apart 11 and 29 central atrophy days 6 daily doses of 70% in 14 Increase of weight; cell 800 mg days swelling: hyalin granules (16) (17) (18) (19) (19) (20) April 1972 109 DSW 031L49 i STLCOPCB4015111 1* " " f * " "1 1,1 ( Table 6. ScmlcliToidc Oral Toxicily Sludies of PCR Preparations. Prepnration Animal Treatment Mortality Liver effects Other effects Refer ences 65% Cl Hat 48% Cl 48% Cl Cynomolgus monkey Squirrel monkey 48% Cl Mouse Aroclor Chicken 42% Cl Aroclor Chicken 42% Cl 48% Cl Chicken Aroclor Chicken 48% Cl Aroclor Chicken 64% Cl Aroclor Bengalese 64% Cl finch Fhenoclor Japanese 60% Cl quail Doses of 50 mg every second day From 641 mg in 40 days to 348 mg in 230 days From 320 mg in 40 days to 67 mg in 48 dayg 60% in 5 weeks not given not given Daily doses of 0.001 ml for 13 to 26 weeks 0% 100, 200, 400, and and 1000 ppm in diet for 4 weeks 0, 0, 60, 90, and 90% respectively 200 and 400 ppm in diet for 3 weeks 0 and 12% respectively 1, 6, 10, 25, 50, 0% from 1 to 100 100, 300, 600, ppm; 100% from 1200, 2400, and the 100 ppm level 4800 ppm in diet for 20 days 10, 20, 30, 50, 100, After 3 weeks: 0, 0, and 160 ppm in 30, 30 and 20%>; diet for 4.5-5 at the end 0, 0, weeks 80, 60, and 80% respectively 250 and 150 ppm in diet for 6 to 13 weeks Estimated dose rale at 56 daya of 251 mg/kg/ day 2000 ppm in diet 250 ppm 100% botween 3 and 10 weeks; 500 ppm *ome mortality at the end 60% 100% between 6 and 65 days 33% weight increase; cell swelling; byalin globules Enlargement; SKR Mnin cause of death: proliferation pneumonia or Enlargement; SEIt diarrhen Mnin cause of death: proliferation pneumonia or 6ER proliferation diarrhea; palj>c- bral edema in 1 animal Enlargement; SER Skin: loss of hair, proliferation erosion and RER reduction; myelin figures; ulceration after 3 months increase of micro bodies, )y80somes and lipid Enlargement; Edema formation damage at the from 200 ppm; at higher levels high levels internal haemorrhage and tubular dilatation in kidneys Pronounced edema at 400 ppm; en larged kidneys; small spleen; defcathcring and dermatitis Edema formation 100 ppm level Enlargement General edema and depression of the secondary sexual characteristics from the 30 ppm level 600 ppm at end: comb weights 20 fold and testes weights 2-fold lower than controls Hydropericardium in some birds Hydropericardium (20) (21) (21) (21) (6) (6) (22) (23) (24) (25) (10) 110 Environmental Health Perspectives w " r am w r m' STLCOPCB4015112 Tabic 7. Dermal Toxicity and Inhalation Studies of PCD Preparations. Prepa ration Animal Treatment Mortality Liver effects Skin effects Refer ences 42% Cl Guinea pig 42% Cl Rabbit Aroclor Rabbit Aroclor Rat 65% Cl 11 daily ekin applications of 34.5 mg 100% between 11 and 21 days Skin application at alternate days, total dose from 946 to 1980 ing Daily ekin appli cations of 0.3, 0.6, and 0.9 g 100% between 17 and 98 days High dose died be fore liver necrosis developed Inhalation of . 0.57 mg/cubic meter for 16 hours for 37 to 134 days 0% Fat; central atro phy; pcrinuclcnr basophilic granu lation; focal ' necrosis in a few animals Fatty degeneration; central atrophy Moderate doses: mottled liver, sub acute yellow atrophy, fatty degeneration, and marked necrosis Pale and yellow; cell swelling; hyalin degenera tion ; potentiation of CCli and CjHiOII toxicity Occasional thicken ing of the epidermis Thinning of prickle cell layer and thickening of outer cornificd layers Reddening; forma tion of small papules and blisters; finally desquamation of external epidermal layers (19) (10) (26) (20) are well explained by the proliferation of smooth surfaced membranes of the endoplasmic reticu lum (SER) as was found by Nishizumi (21) in mice and monkeys and by Norback and Allen (27) in rats. The latter workers found a prolifera tion of the SER in rats fed PCB for 1 to 5 weeks. Concomitant with the structural changes, the activities of measured drug metabolizing enzymes (nitroreductase and aromatic hydroxylase) were increased. The induced level of drug metabolizing activity persisted as the proliferation of the SER decreased and concentric arrays pervaded the cytoplasmic reticulum (27). These concentric membrane arrays, probably representing the hyalin bodies described by Bennet et al. (20) and Miller (19), could have an enzymatic func tion similar to that associated with the SER (27). Similar formations, the so-called myelin fig ures, were demonstrated in mouse liver by electron microscopy; in monkey liver they were not found (21). In both mouse and monkey liver a proliferation of the SER was found. In our comparative dermal toxicity study (15) in rabbits with 2,4,5,2',4',5'-hcxachlorobiphcnyl and Aroclor (60% Cl), the light microscopic findings included necrosis, hydropic degeneration (Figs. 4 and 5) as well as a peripheral and perinuclear shift of cell organelles (Fig. 5) and focal cyto plasmic hyalinization. In electron microscopy, the shift was found to be due to a proliferation of the SER resulting in a displacement of rough surfaced membranes (RER) and mitochondria. The focal cytoplasmic hyalin degeneration, often seen in hydropic cells, was recognized as tightly packed tubules of proliferated SER (Fig. 6). This very probably represents hypertrophic, hypoactive SER. Sublethal effects caused by induction of hepatic enzymes have been noted by several authors. Increased steroid metabolism in pigeon liver homogenates has been demonstrated by Risebrough et al. (28). Lincer and Peakall (29) confirmed the effect of PCB on the hormone metabolism in birds at very low dose levels. They fed kestrels for 5 months with Aroclor 54% and 62% Cl at levels of 0.5 and 5.0 ppm. The higher April 1972 OSW 0311.51 111 STLCOPCB4015113 % - Fiouhk 4. Liver damage in a killed rabbit treated with 120 mg Aroclor (60% Cl), 5 times per week, for 28 days. Note the ecntrolobular necrosis (1) and the hydropic cells (2) at the margin of the necrotic and vital tissue. Haemotoxylin and eosin. X160. Fioure 6(a). Liver cells of a control rabbit. Clear areas may represent negative images of glycogen (arrow). Totuidinc blue. X04O. (b). Liver cells of an Aroclor treated killed rabbit. Note the hydropic cells (I) and the perinuclear and peripheral displacement of cell organelles with sometimes hyalin foci (2) inside hepatic cells. Toluidine blue. X04O. 112 Environmental Health Perspectives DSW 031152 STLCOPCB4015114 --- -- ----T-. -... -7 --narrhiitoin m t ,, 1 i FlOimr. 0. Hydropic liver cells from the same animal as seen in Figure 5b showing a large number of vacuoles (V) and a porivacuolar localization of mitochondria (arrow). Note the strong proliferation of the SliH, consisting of lightly packed tubules. Uranyl acetate and lead citrate. X6500. dose being roughly equivalent to 2 mg/kg PCBs to each kestrel. A dose dej>cndenl in vitro break down of estradiol to a more polar metabolite occurred in the livers from kestrels fed cither Aroclor 1251 or Aroclor 1262. No such conversion took place in the livers of the control birds. The increase in hepatic enzyme activity correlated with an increase in cytoplasmic RNA, as was measured cylophotomctrically. A shortened sleep ing time after treatment with hcxobnrbital, and enhanced in vitro rates of aniline hydroxylation and p-nitroanisolc dcmcthylnlions were demon strated by Street and coworkers (30). These authors also found an increase of these effects with increasing chlorine content of the different PCB preparations (Aroclor 21 to 68% Cl). Using enzyme induction as parameter, no effect levels of some PCB preparations were established in the rabbit, rat, and Japanese quail. Oral administration of Aroclor 21% Cl and 54% Cl (1.0 and 10 mg/kg) for 28 days to pregnant rabbits resulted in liver enlargement and in creased activities of the drug metabolizing en zymes aniline hydroxylase and aminopyrine n-demelhylase at the 10 mg/kg level of the 54% chlorinated Aroclor. The no-effect level for enzyme induction in the pregnant rabbit appeared to lie between 1.0 and 10 mg/kg in the case of Aroclor 54% Cl, and higher than 10 mg/kg for Aroclor 21% Cl (31). Another parameter for enzyme induction was used by Komatsu and Tanaka (32). They found that the hcxobarbital induced sleeping times in rats were reduced by pretreatment with PCBs. April 1972 DSW 031153 113 I J STLCOPCB4015115 The minimal effective dose was 5 mg/kg for 3 days with Knnpchlor 400 (48% Cl) nnd 2 ing/kg for 3 days with tlic higher chlorinated Knncchlor 600. The porphyrogonic action of PCBs was further evaluated in a study with Japanese quail (33). The results (Tnblo 8) indicate that the hepatic porphyria is closely associated with an increase of mitochondrial ALA synthase activity. A significantly increased activity of this enzyme was already noted after administration of daily doses of 1 mg/kg Aroclor 60% Cl 'for 1 week, r Mean PCB content of the liver at that dose was 1.41 ppm. A less sensitive parameter is tissue fluorescence due to excess quantities of porphyrins. Liver fluorescence was only seen at the 100 mg/kg level. It develops, probably, only in animals showing clinical symptoms, such as loss of weight. A similar finding was done in the prior experi ment with chickens (11). Edema Formation The most striking finding in birds is the ac cumulation of fluid. The pathogenesis of the edema formation is discussed by Flick and co workers (6). The primary site of the edema caus ing factor could be the heart by increasing the permeability of the vascular bed, leading to cardiac congestion. Pulmonary edema could be the result of the cardiac congestion. The pul monary edema might be followed by a flow of fluid into abdominal and subcutaneous air sacs. Decreased scrum protein values (34) could also contribute to the edema formation. Liver damage can be responsible for reduced scrum albumin levels. A6 mentioned in Table 4, the edema formation is probably due to the presence of polychlorodibenzofurans. In our study the edema formation by the G0% chlorinated Aroclor sample was minimal at the 400 ppm level. As can be seen in Table C, chick edema-like lesions were noted at low feeding levels and were caused by lower chlorinntcd Aroclors. Therefore the presence of toxic impurities in these Aroclor samples has to be considered. Other Effects An interaction of PCBs with duck hepatitis virus was found by Friend nnd Trainer (35). Ten-day-old ducklings were fed a 54% chlorinated Aroclor mixture at levels of 25, 50 and 100 ppm. The birds suffered no apparent clinical intoxica tions. Five days later they were challenged with duck hepatitis virus, and they suffered signifi cantly higher mortality than birds which were not exposed to PCBs. Effects of PCBs on the lymphoid system were noted in some studies. Feeding of PCBs to chick ens resulted in small spleens (6,11). Lymphopenia, atrophy of the cortex of the thymus, and a re duction in the number of germinal centers in spleen and lymph nodes was found in rabbits (14). Therefore, an immunosuppressive action could be present. In an experiment with guinea pigs, this was established (36). Feeding of 10 ppm Aroclor 60% Cl, for 8 weeks resulted in a Table 8. Formation of i-AmlnocvuUnlc Acid by Liver Mitochondria, Liver Residues, and Tissue Fluorescence in Female Japanese Quail Orally Dosed with PCB for Seven Days. Aroclor (00% Cl) ALA formed (m^ moles (mg/kg body weight) ALA/g liver/hr) PCB content liver (ppm) Tissue fluorescence incidence Macroscopic Microscopic (liver) 0 0.1 1 10 100 6.401.64 8.76A4.06 10.60il.211> 17.3A6.4k 118.9' * Mean values A-Sl), 5 birds per group. Significantly different from controls, 1'SO.Oi. Pooled samples 0.15' 0.450.27 1.41 0.67 27.0A9.4 478A294 0/5 0/5 0/6 0/5 3/5 0/6 0/6 0/6 0/6 2/6 114 Environmental Health Perspectives DSW 031154 i i i ii ` * { r i STLCOPCB4015116 *. -v . *w tv fn rr > v * r- * I U* `v f'-'.i if' . fl --1!.*.X V lev: '.: I 1C-- ,):-J ' e-n *- u -.. i. ,i: tii--' a :i tv * f k". 3 h r .:<-v lir: - rrr tr i l i i l Fiounr. 7. Representative areas of tetanus toxoid stimulated popliteal lymph nodes of guinea pigs, (a) Large number of antibody forming tells in a control animal, (b). Reduced number of antibody forming cells in an animal fed JO ppm Aroclor (00% Cl) for 8 weeks. Direct fluorescent antibody technique. Cryostat sectiota. X 370. (36), April 1972 DSW 031155 115 STLCOPCB4015117 decreased number of antibody-forming cells in the popliteal lymph node, after stimulation of tho humoral lymphoid system with tetanus toxoid (Fig. 7). This suppression may explain the higher sensitivity of PCB-fcd ducklings for duck hepatitis virus (35). In a comparative toxicity Btudy in guinea pigs, an indication for an effect of PCB (Clophen and Aroclor 60% Cl) on the cell-mediated immunity was obtained. Feeding of these, mixtures at 50 ppm levels for 6 weeks resulted in a decreased number of circulating lymphocytes (unpublished data). An estrogenic activity of PCBs (Aroclor 21-48% Cl) was demonstrated by Bitrnan and Cecil (37). The estrogenic activity was evaluated using the 18-hr glycogen response of the immature rat uterus after r single subcutaneous injection. The minimum effective dose was 8 mg. The higher chlorinated PCB mixtures were inactive at the 8 mg level. In the above mentioned subacute feeding 6tudy of 60% chlorinated mixtures in guinea pigs, we found significantly increased uterus weights in the PCB treated animals. Both increased steroid metabolism, as mentioned by Itehfcld and coworkcrs (23), and the estrogenic activity could be responsible for the depression of secondary sexual characteristics (decreased development of comb and wattles) noted in cockerels (24). Administration of Aroclor (54% Cl) at levels of 12.5, 25, and 50 mg/kg body weight during the first 28 days of gestation had ernbryotoxic effects in the rabbit (31). Edema and beak deformities in chicken embryos have been described after yolk-sac injection of 10 and 25 mg 42% chlori nated Aroclor, resulting in respectively 95 and 100% embryonic mortality (38). An effect of PCB on the nervous system was noted by Ogawa (39). Oral administration of PCB (0.3-O.5 ml/kg/day) to rats for 14 or 21 day6, resulted in marked or moderately impaired motor function, decreased motor conduction velocity and loss of large nerve fibres. He con cluded that PCB caused neuropathy in rats. Conclusion Because of the possible presence of poly chlorinated dibeneofurans (PCF) or other toxic impurities in crude PCB preparations, it is difficult to interpret many of the toxicity studies. Pure samples arc required for comparative in vestigations. Also the fate of the toxic impurities in the environment hns to be determined. As presented here, l'CBs have several subletlial effects, such as microsomal enzyme induction, porphyrogenic action, estrogenic activity, and immunosuppression. Since porphyria seems to be an effect of PCBs themselves and not from PCF, the induction of ALA synthase could be used as criterion in the approximation of a nocffcct level (at least for the 60% chlorine type of PCBs). The no-cffect level could be about 0.1 mg/kg (mean PCB content of the liver in Japa nese quail about 0.2 ppm). This is in the same order of magnitude as found in the other studies. Additional research is needed to determine fully the significance of these sublethal effects. More over, chronic and reproduction studies arc neces sary. The present results also make clear that manufacture of commercial PCB mixtures that are free from impurities is urgently requested. Acknowledgment The author gratefully acknowledges the helpful suggestions and critical reading of Prof. H. van Genderen, Head of the Institute of Veterinary Pharmacology and Toxicology, Many thanks are also due to colleagues of the working parly of the Institute of Veterinary Pathology and the Institute of Veterinary Pharmacology' and Toxi cology: Dr. J. H. Koeman, Mr. H. L. van dcr Maas, and Dr. J. G. Wit. The author also thanks the students who studied for their degree in biological toxicology and Mr. M. 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