Document Rp7k64rVrX0N8K34jrKEokQKB

qly of for wo md the her our nal UK . thr IW71 aiyls trun. m of Lion, i*(ed l)cn moo itU). rrwit lUnt long lirci v, iTCftmaa.'.:.! Environmental Health Pcrtpectivct Toxicology of PCBs for Mammals and for Birds by J. 6. 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 stable PCB'a 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 with 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 this reason the toxicological infor mation of PCB's is difficult to summarize, but since the character of 6omc 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 some toxic effects associated with toxic factors in crude chloropbcnols and in "toxic fat". Effects of the latter are 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 1260 (sample 111). 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 I and II). Hydropericardium (Fig. 1), a common effect of these two mixtures, was only occa sionally scon in the chicks fed the sample 111. 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 * lnititute of Veterinary Pathology and Institute of Veterinary Pharmncology and Toxicology, Bute Uni versity of Ulrcclit, The Netherlands. ' Fiodre 1. Hydropericardium In chick fed <00 ppm of sample 1. April 1972 105 PLAINTIFF'S EXHIBIT ___________ 7 -ZS'06 MONS 207260 006726 WATER PCB-SD0000047054 Table 1. Mortality, nd Pathologic Observations of Chicks F*H 400 ppm PCII for 60 Days. PCB sample Number of Number of birds with edema Number of N deaths - birds with Hydro Abdominal Subcutaneous liver necrosis pericardium I 11 111 Control 24 34 22 22 20 3 20 0 18 20 3 0 8 '6 e? 00 00 e e 0 0 mortality in this group was only 15% (Table 1). The excretion of coproporphyrin 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 csj>ccift!ly the birds that died. This hepatic porphyria was found in all three experimental groups. In the subsequent study (12) by means of column and gas-chromatography the presence of relatively more polar compounds was demon strated in the 25% dicthylcthcr fraction of samples I and 11. In a chick embryo assay (Table 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 11/ egg and the group injected with the 25% diethylether fraction from 3.5 mg sample II/egg. Mass spcctronictric analysis revealed that Table 2. Chick-Embryo Assay of Three PCD samples and the 25% Dicthylcthcr Fraction from Sample 11 PCD sample Dose Number (mx/egg) of eggs treated Percentage hatch of fertile eggs 1 11 111 Fraction from sample 11 Ethanol control Untreated control 1.6 1.6 8.6 36 1.6 0.36 0.035 0 16 20 . 16 IS 16 16 15 20 20 0 5 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 301 and 338.. The proposed identity of these compounds, tctrachlorodibcnzofuran and pontachlorodi- benzofuran, 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 CitlhO^Ch and CnHiOMCl*. The formulae of the fragment ions 241 and 275 were CnH**Ch and Cnlli^Cli. From this data it can be concluded that the parent ion has a preferential fragmentation for the loss of a single C1C0 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 suc cessive mass units of 63 suggests a loss of two ClCO units. With microcoulomctric analysis a certain maximum level could be indicated. This maximum level was found to be five Ptnn of the compound with mass number ft38 in sample II and 20 ppm in sampIcT * ' _ Polychlorinated dibenzofurans arc strong hepa- totoxic and acnegcnic compounds. Tri- and tetra- 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. Tctrnchlorodibenzo- dioxin was about 10 times more toxic (7). In jection of the 25% dicthylcthcr fraction obtained from 35 mg sample II into the airccll resulted in 100% mortality (Table 2). It can be calculated that tlic maximum dosc/cgg is 0.2 /<g penta- chlorodibcnzofuran (taking five ppm as the maxi- 106- Environmental Health Perspectives HONS 207261 I I i i II \ OR 727 WATER PCB-SD0000047055 mutn value). This confinns the order of toxicity found by Higginbotham ct nl. (9) in the case of toxicity of 2,4,5,2',4',5'-hcxnchlorobiphcnyl. From the incrcnscd fecal excretion of copropor cliloro-dibcnzodioxins. Tetrn and pcntachlori- phyrin in both experimental groups (Tnble 3), it dibenzofurans were considered responsible for the higher toxicity of samples I and II. is very likely that PCBs themselves arc responsible for the porphyrogenic action of crude prepara Confirmation is also obtained from the sub tions, From the presence of slight skin lesions sequent comparative toxicity study in rabbits (14). Again samples I and II were more toxicj the induced by 2,4l5l2',4',5'-licxachlorobiplicnyl when compared with the Aroclor sample, it can liver and skin lesions were more severe. Por phyria, especially of the liver, was present in all be concluded that the major aencgcnic action of crude mixtures comes from a possible contamina three groups (Fig, 2). A remarkable finding was tion with chlorinated dibenzofurans. It can also the intense red fluorescence of small foci inside bo concluded that PCBs themselves have a nt hepatic cells. They probably represent nuclei. This slight aencgcnic action. Liver damage was es < 1 was confirmed in an additional cell culture experi sentially the same after treatment with both >(*y ment with the Aroclor sample and with 2,4,5,- 2,4,5,2',4',5-hcxachlorobiphcnyl and the nd 2',4/,5,-hcxRchlorobiphcny). (This experiment was Aroclor mixture. The conclusion that the liver ds, carried out by my colleague Dr. J. G. Wit of the injury, caused by crude preparations, is pre di- Biochemical Section). dominantly due to the contaminants, is based ni- Application of the 25% dicthylcther fraction on the differences in liver toxicity between the caI on the skin of rabbits resulted also in differences three PCB preparations (11,14). in toxicity (Fig. 3). So the presence of the hepato- The probable contribution of polychlorinated nd ra, loxic and aencgcnic polychlorinated dibenzofurans dibenzofuran (PCF) and pure polychlorinated as impurities in samples I and II was found to biphenyl (PCBs) in the toxicity of crude prepara .nt be established. tions is summarized in Table 4. A proper evalua X In another experiment (15), the toxicity of the tion of toxicity data and residue data can be Jie Aroclor (60% Cl) sample was compared with the hindered by the possibility that PCB samples may for ere inn in. !nr no- wo a hLf Llif II un ra vel ti* to- In- ltd tod ted La- ixi- Figure 2. Fluorescence of porphyrins under ultraviolet light in livers from rabbits treated with 60% chlorinated PCI!'*, 1, Aroclor; 2, Ciopheu; 3, Phenoclor; and 4, Control liver (14). April 1972 107 MQNS 207262 WATER PCB-SD0000047056 Florae 8. Response of the inside of the rabbit's esr after inpleat application of the 25% diethylelher fractions from techDical PGlt'a. Ilsemotoxylin and eosin. X60, (a) Skin of control animal treated with ethanol. Note the hair follicle at 1, seabacoous gland tissue at 2, and cartilage at 3. (b). Ear akin of the animal treated with the fraction from temple 111. Some hyperplasia and hyperkeratosis of the follicular epithelium can be seen. (c). Ear akin of Utc rabbit treated with the frartion from sample II. Considerably hyperplasia and hypcrkeratotla of the follicular epithelium, (d). Her akin of the rabbit treated with the fraction from sample I. Part of a section that shows the most severe lesion. Tho gravity of. the response was in general the same as seen in (e). Note the eyslio dilated hair follicle with prominent hyperplasia and hyperkeratosis of the follicular in4 epidermal epithelium (It). 108 Environmental Health Perspectives HONS 207263 006729 WATER PCB-SD0000047057 {ji/Trr in an important respect: the presence of loxie impurities. The possibility of distinguishing between the effects of PCBs and their impurities rn be further improved by using pure isomers Hj(h known positions of the chlorine atoms. Mortality, Liver Effects, Edema Formation and Other Effects These data, as presented by different authors, fjc summarised in Tables 5, 6, and 7. As can be seen in Table 5, the acute and subacute toxicity data of PCB's are poor. The established values are high. Scmichronic oral toxicity studies are summarised in Table 6. Dermal and inhalation studies are given in Table 7. Table S. Coproporphyrln Contents ittg/g Dry Weight) of Feed of Itabblts Treated with PCI! for 4 weeks, and of Controls.* 2,4, 5, 2', 4\6'-Hcxachlorobiphonyl Aroclor Control (60% Cl) 45.0 6.2 20.8 19.6 Mean 37.9* 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 eoeum of the individual animals. * Significantly different from controls, P0.025. It. is very probable that the results of thcec studies may have been influenced by the presence of polychlorodibcnrofmans or other toxic im purities. For example, in the study of ltdifeid ct al. (23) general edema was already found In .chicks fed 3Q. DPm Aroclor 48%, Ol, i,;i,, Kohanawa and co-workers (22) noted edema formation at the 100 ppm level of another 48% chlorinated mixture. Mortality in the latter study was also lower (Table 6). Liver Effects The most important liver effects, summarised in Tables 5, 6, and 7, arc weight increase, fatty degeneration, hyalin degeneration and nccrosi*. Increased liver weights, as noted in several studies, Table 4. Probable Contribution of Polychlorinated Dlbensofuran (I'CF) and Pure Polychlorinated Diphenyl (PCH) In the Toxicity of Crude PCD Mixtures. Chlor- Edema Liver Hepatic ftcnc forma damage porphyria tion Polychlorinated Dibeotofuran Polychlorinated Biphenyl ++ 44 44 44 44 Table 5. Acute and Subacute Oral Toxicity Studies of PCB Preparations. Preparation Animal Treatment Mortality Liver effects Refereeecs Unknown Aroclor 54% Cl Mouse Rat Aroclor 42, 54, 60, and Mallard 68% Cl 42% a Rat a43% Guinea pig 65% a Rat tingle dose of LD50 approx. 2000 mg/k(t single dwe of 0% Increase of weight and 500 mg/kg lipid; potentiation of OCh toxicity ingle dose of 0% 2000 mgAg 20 daily dotes of 0% in 3 Hyalin bodies in liver cells 138 mg 2 doses of 69 mg months 100% between Fatty metamorphosis; 1 week apart 11 and 29 central atrophy days 6 daily doses of 70% in 14 Increase of weight; cell 100 mg days swelling: hyalin granules (10) (17) (18) (19) (19) (20) April 1972 109 rw^wr^ijaggnyyi^ t! MONS 207264 006730 WATER PCB-SD0000047058 Table 6. Sentlclironlr Oral Toslrlty Studies o( PC.H I'rrparetiona. Prepa ration Animal Treatment Mortality Liver effects Other effects Refer ences 65% Ct Hat 48% Cl 48% Cl Cynomolgus monkey Squirrel monkey 48% Cl Mouse Aroelor Cliickon 42% Cl Aroelor Chicken 42% Cl 48% Cl Chicken Aroelor Chicken 48% Cl Aroelor Chicken 64% Cl Aroelor Bengalese 64% a finch Phenoelor Japanese 60% Cl quail Doses of 50 mg every eocond day From 041 mg in 40 days to 348 mg in 230 days From 320 mg in 40 days to 67 mg in 48 days 60% in 5 weeks not given not given Daily doses of 0.001 ml for 13 to 20 weeks 0% 100, 200, 400, and and 1000 ppm in diet for 4 weeks 0, 0, 50, 00, and 80% respectively 200 and 400 ppm in diet for S wooks 0 and 12% respectively 1, 6, 10, 25, 50, 0% from 1 to 100 100, 300, 000, 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 150 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 kt diet for 6 to 13 weeks Estimated dose rate at 50 days of 254 mg/kg/ day 2000 ppm in diet 250 ppm 100% botween 3 and 10 weeks; 500 ppm some mortality at the end 60% 100% between 6 and 65 days 33% weight increase; eeil swelling; hyalin globules Enlargement; SER Main cause of dentil: proliferation pneumonia or diarrhea Enlargement; SER Main cause of death: proliferation SER proliferation pneumonia or diarrhea; pal;>c- brai edema in I animal Enlargement; SER Skin: loss of hair, proliferation erosion and RER reduction; ulceration after myelin figures; 3 months increase of micro bodies, lysosomes and lipid Enlargement;, Edema formation damage at the from 200 ppm; at higher levels high levels interns] haemorrlisge and tubular dilatation in kidneys Pronounced edema at 400 ppm; en larged kidneys; small spleen; defeathcring and dermatitis Edema formation 100 ppm level Enlargement General edema and depression of the secondary sexual characteristics from the 30 ppm level 500 ppm at end: oomb weights 20 fold and testes weights 2-fold lower than controls Hydropericardium in eomo birds Hydropericardium (20) (2!) (21) (21) (6) (6) (22) (23) (24) (25) (10) no Environmental llcallh Perspectives MONS 207265 oor.731 WATER PCB-SD0000047059 to/er- etirra (20) (2!) (21) (21) (6) (6) (221 (23) (24) (26) (10) fives Table 7. Dermal Toxicity and Inhnlot Ion Studies of PCD Preparations. Prepa ration Animal Treatment Mortality Liver effects Skin effects Refer ences 42% Cl Guinea pig 11 daily (kin application* of 84.6 mg 100% between 11 and 21 days 42% a Rabbit Aroclor Rabbit Skin application al alternate days, total dose from B4G to 1980 mg Daily skin appli cations oi 0.3, 0.6, and 0.9 g 100% between 17 and 98 days High dose died be fore liver necrosis developed Aroclor Rat 65% Cl Inhalation of 0.67 mg/cubic meter for 16 bourn for 37 to 134 days 0% Fat; central atro phy; perinuclear basophilic granu lation; focal necrosis in a few animals Fatly degeneration; central atrophy Moderate doses: mottled liver, sub acute yellow atrophy, fatty degeneration, and marked necrosis Pale and yellow; eel) swelling; byalin degenera tion; potentiation of CCi and CtlI,OII toxicity Occasional thicken ing oi the epidermis Thinning of prickle cell layer and thickening of outer cornified layers Reddening; forma tion of small papules and blisters; finally desquamation of external epidermal layers (19) (19) (20) (20) arc 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 SKIl in rats fed PCD 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 Bcnnct et al. (20) and Miller (10), 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 Been in hydropic cells, was reoognized as tightly packed tubules of proliferated SER (Fig. 6). This very probably represents hypertrophic, hypoactivc SEK. 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). Linccr and Pcaknll (29) confirmed the effect of PCB on the hormone metabolism in birds at very low dose levels. They fed kestrels for 6 mouths with Aroclor 54% and 62% Cl at levels of 0.5 and 5.0 ppm. The higher April 1972 111 HONS 207266 006' j2 WATER PCB-SD0000047060 Pioimi: 4. Liver damage in a killed rabbit treated with 120 mg Aroclor (60% Cl), 5 times per week, for 28 days, Note the ewilrolobular necrosis (1) and the hydropic cells (2) at the margin of the necrotic and vital tissue, ilaemotoxylin and wain. XI60. |'VnW . blue, XWO. (l>). Liver ceils o( an Aroclor treated killed rabbit. Note the hydropic cells (1) and the perinuclear and peripheral displacement of cell organelles with sometimes hyalin foci (2) inside hepatic cella. Toluidine blue. X940. 112 Environment!*! Health Perspective* MONS 207267 006733 WATER PCB-SD0000047061 Flown: 0. Hydropic liver cells from the came animal as seen in Figure 5b showing a largo number of vacuoles (V) and a pcrivacuoUr localization of mitochondria (arrow). Note the strong proliferation of the SEH, consisting of lightly packod tubules. Uranyl acetate and lead citrate. X6500. dose being roughly equivalent to 2 mg/kg PCBs to each kestrel. A dose dependent in vitro break down of estradiol to a more polar metabolite occurred in tire livers from kestrels fed either A rodor 1254 or A rodor 1262. No such conversion took place in the livers of the control birds. The increase in hepatic enryme activity correlated with an increase in cytoplasmic RNA, as was measured cytophotomctrically. A shortened sleep ing time after treatment with hcxobarbital, and enhanced in vitro rates of aniline hydroxylation and p-nitroanisolc dcnicthylations 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-demethylasc at the 10 mg/kg IcycI 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 ca.sc 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 steeping times in rata were reduced by pretreatment with PCBs. April 1972 113 HONS 207268 00R734 WATER PCB-SD0000047062 The minimal effective dose was 5 mg/kg for 3 day8 with Knnechior 400 (48% Cl) and 2 mg/kg for 3 dnys with the higlier chlorinated Knncchlor 600. TJic porpliyitigcnic action of PCBs was further evaluated in a study with Japanese quail (33). Tile results (Table 8) indicate that the hepatic porphyria is closely associated with an increase of mitochondrial ALA synthase activity. A significantly increased activity of tin's ensyme was already noted after administration of daily doses of I mg/kg Aroclor 60% Cl for 1 week. . Mean PCB content of the liver at that dose w'as 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 develop, probably, only in animals showing clinical symptoms, such as loss of weight. A similar finding w&s done in the prior experi ment with cliickcas (II). 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 (0). 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 Uic result of the cardiac congestion. The pul monary edema might be followed by a flow of fluid into abdominal and subcutaneous air sacs. Decreased serum protein values (34) could also contribute to the edema formation. Liver damage can be responsible for reduced scrum albumin levels. As mentioned in Table 4, the edema formation is probably due to the presence of polychlorodibciuofurans. In our study the edema formntion by the 60% 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 chlorinated 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 and 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 (30), Feeding of 10 ppm Aroclor 00% Cl, for 8 weeks resulted in a Table 6. Formation of t-Aininoevulinic Acid by Liver Mitochondria, Liver Reaiduea, and Tiaaue Fluorescence in Female Japanese Quail Orally Doacd with PCB for Seven Days. Aroclor (00% Cl) ALA formed (m*i 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 6.464:1.64 8.764:4.00 10.604:1.21* 17.34:6.4* 119.9* Mean valucsi-RD, 5 bird* per group. *Significantly different from controls, Pf 0.01. * Pooled samples 0.16* 0.464:0.27 1.414:0.67 27,04:9.4 4784:294 0/5 0/5 0/6 0/5 3/5 0/6 0/5 0/6 0/6 2/5 114 Environmental Health Perspectives HONS 00673a WATER PCB-SD0000047063 ft ;S V' Kk .it rilTtoifc Fioonr. 7. Representative areas of tetanus toxoid stimulated popliteal lymph nodes of piinee pip. (a) Larne number of antibody, forming cells in a control animal, (b). Reduced number of antibody formate cells in an animal fed 10 ppm Arodor (00% Cl) for 8 weeks. Direct fluorescent antibody technique. Cryostat sections. X 376. (30). April 1972 HONS 207270 115 006736 WATER PCB-SD0000047064 decreased number of antibody-forming cells in the popliteal lymph node, after stimulation of the humoral lymphoid system wit h tetanus toxoid (Fig. 7). This suppression may explain the difficult to interpret many of the toxicity studies. Pure samples are required for comparative in vestigations. Also the fate of the toxic impurities in the environment has to be determined. As higher sensitivity of PCB-fcd ducklings for duck presented here, PCBs have several sublet lial hepatitis virus (35). In a comparative toxicity effects, such as microsomal enrymo induction, study in guinea pigs, an indication for an effect porphyrogcnic action, estrogenic activity, and of PCB (Clophcn and Aroclor 60% Cl) on the immunosuppression. Since porphyria seems to cell-mediated immunity was obtained. Feeding be an effect of PCBs themselves and not from of these mixtures at 50 ppm levels, for 6 weeks PCF, the Induction of ALA synthase could be resulted in a decreased number of circulating used as criterion in the approximation of a no lymphocytes (unpublished data). effect level (at least for the 00% chlorine type An estrogenic activity of PCBs (Aroclor 21-48% of PCBs). The no-effect level could be about 0.1 Cl) was demonstrated by Bitman and Cecil (37). mg/kg (moan PCB content of the liver in Japa The estrogenic activity was evaluated using the nese quail about 0,2 ppm). This is in the same 18-hr glycogen response of the immature rat order of magnitude as found in the other studies. uterus after a single subcutaneous injection. The Additional research is needed to determine fully minimum effective dose was 8 mg. The higher the significance of tlicac sublcthal effects. More chlorinated PCB mixtures wore inactive at the over, chronic and reproduction studies arc neces 8 mg level. In the above mentioned subacute sary. The present results also make clear that fooding study of 60% chlorinated mixtures in manufacture of commercial PCB mixtures that guinea pigs, we found significantly increased are free from impurities is urgently requested. uterus weights in the PCB treated animals. Both increased steroid metabolism, as mentioned by Acknowledgment Itehfcld and coworkers (23), and the estrogenic The author gratefully acknowledges the helpful activity could be responsible for the depression suggestions and critical reading of Prof. H. van of secondary sexual characteristics (decreased Genderen, Head of the Institute of Veterinary development of comb and wattles) noted in Pharmacology and Toxicology. Many thanks cockerels (24). are also due to colleagues of the working party Administration of Aroclor (54% Cl) at levels of the Institute of Veterinary Pathology and the of 12.5, 25, and 50 mg/kg body weight during the Institute of Veterinary Pharmacology and Toxi first 28 days of gestation had embryotoxic effects cology: Dr. J. H. Kocman, Mr. H. L. van dcr in the rabbit (31). Edema and beak deformities Maas, and Dr. J. G. Wit. The author also thanks in chicken embryos have been described after the students who studied for their degree in yolk-sac injection of 10 and 25 mg 42% chlori biological toxicology and Mr. M. C. ten Noever nated Aroclor, resulting in respectively 95 and dc Brauw, Mr. It. H. de Vos, and Dr. R. J. C. 100% embryonic mortality (38). Klcipool of the Central Institute for Food and An effect of PCB on the nervous system was Nutrition Research, T. N. O., Zeist. noted by Ogawa (39). 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