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H Cumrt Tmk1 V#l II pjv (VJ? hipiM* Pm* l(K Prtnm ( GMuia THE EFFECT OF POLYCHLORINATED BIPHENYLS ON RAT REPRODUCTION R. E. Linder*. T. B. Gaines and R. D. Kimbrough Bio-Effects Branch Prlntaie end Pesticides Effects Laboratory, Environmental Protection Agency, 4770 Bttford Highway, Chamblet, Georgia3034/, USA [Received 13 August 1973) _ . Abimc*--Reproduction, Flibology and acuio toxicity were ihMicrf in Rerniu nu exposed to the polychlorinated biphenyl*, Aroclor I2M md Aroclor 1260. Rah exf-ossd to Arocior 1254 at dietary level* of 20 ppm or more bad.fewer pops per litter (bau the control* in the F,k aad Fj generations. The 100 ppm exposure level of Arcelor 1254 increased mortality in the F,k offspring and markedly deci eased mating performance of the F, adults. The 500 ppm dietary level of Arce lor 1260 reduced litter size and decreased survival in the Ft litter*. Dietary levels of 5 ppm Arcelor 1254 and 100 ppm Aroclor 1260 had no effect oo reproduction m rate exposed through two geae> radon*. Liver weight* were increased in 21-day*old F, male weanling* at the 1 ppm level of Aroclor 1254 and in either sex of F, aad F, weanlings at 5 ppm or higher level* of both Aroclcr 1254 aad 1260. Histological changes in the liver and increased liver weight* were observed in adult rats exposed to the higher level*. Pregnant rats given Aroclor 2254 at the rate of 100 rag/kg/day oo day* 7-15 of gestctloo produced grossly normal litters, but only 301% of the pup* survived to weaning. Reproduction and pup survival were sot alTeeted at dosage rates of 50 mg. Aroclor l254A^dayor 100 mg Aroclor I260/kg/day. Oral LD} values in 5-4-wk-old male rats were 1295 aad 1215 mg/kg for Aroclor* 1254 and 1260, respectively, The iv LDj* for Aroclor 1254 in adult females was 25S mg/kg. - INTRODUCTION Reproductive defects due to commercial mixtures of polychlorinated biphenyls (PCBs) have been reported in experimental birds (Dahlgren, Linder & Carlson, 1972) and mam* mals (Ringer, Aulericb & Zabik, 1972). Porphyria (Vos & Kocman, 1970), microsomal- enxyme Induction (Street, Urry, Wagstalf & Blau, 1969) and liver damage (Kimbrough, Linder & Gaines, 1972; Miller, 1944) have also been reported in experimental animals exposed to PCB mixtures. - ' Hie present investigation was initiated in 1970 to study the effects on reproduction and pathology produced by two American-made PCB mixtures sold under the trade-names Aroclor 1254 and Aroclor 1260s. Aroclor 1234 contains 54% (w/w) chlorine and is com posed of 11% tetra-, 49% penta-, 34% hexa- and 6% heptaclilovobiphcny'.s; Aroclor 1260 has 60% (w/w) chlorine, with a composition of 12% penta-, 38% hexa-, 41% septa-, 8% octa- and 1% nonochlorobiphenyls (Thruston, 1971). An almost complete identification of isomers in Aroclor 1254 and Aroclor 1260 has been obtained (Sissons & Welti, 1971). Since 1970, the Monsanto Co., the only US manufacturer of PCBs, has voluntarily reduced sales or Aroclor mixtures and has taken steps to limit their use to closed systems (Mon santo Co., 1971) The present communication is an account of reproduction studies in rats Also included arc acute toxicity values from preliminary studies and comments on pathology and haema tology in animals from the reproduction elpen men Is A detailed account of the liver path ology of the F,, rats given 20 ppm and higher dietary levels has been published (Kim brough at. 1972) Ptesent address: United States Environmental Protection Agency, National Environmental Rcsreich .Center, Research Triangle Park, Norih Carolina 27711. Rcjinered trademark ot the Monsanto Company, tor. in - e 47 Exhibit 3 MONS 083842 64 R. E. Linder, T. B. Caines and R. D. Kimbrough EXPERIMENTAL Animals and tjiaierials. All animals used in the studies were Sherman-strain rats pro duced under spccific-pathogen-free conditions. Aroclor 1254 (Lot AK-38) and Aroclor 1260 (Lot AK.-3) were supplied by Monsanto Industrial Chemicals Company, SL Louis, Mo. Experimental design and conduct LDm studies. The single-dose oral LDJ0 was determined for Aroclor 1234 and 1260 in weanling male rats (3-4 wkold) The ivll}s0 was determined in adult females for Aroclor 1254. Between live and eight groups, each of ten rats, were used to obtain each LD50 value. The procedure described by Caines (1969) was followed for oral dosing; Arcdor 1254 or Aroclor 1260 was dissolved in peanut oil and the solution was administered by oral intuba tion at the tate of 5 ml/kg. For iv dosing, Aroclor 1254 was first dissolved in peanut oil. One part of this solution was added to nine parts of a 1% lecithin-saline suspension and homogenized. This formulation was administered in a single injection via the tail vein at the rate of 5 mJ/kg. A control group of live rats received 10% peanut oil in lecithin-saline in the same volume. Survivors were observed for 10--14 days. Twthgenerotion reproduction studies. For these studies the r&ts were fed either untreated ground laboratory chow (control) or ground chow fortified with Aroclor 1254 or Aroclor 1260. The Aroclors were dissolved in ether and mixed with cornstarch. The ether was allowed to evaporate and the Aroclor-comstarch mixture was mixed into increasing amounts of ground chow. Aroclor 1254 was fed at dietary levels of 0 (control), 1, 5, 20 and 100 ppm, while Aroclor 1260 was fed at levels of 0, 5, 20 and 100 ppm. Because expo sure schedules had to be staggered, a specific control group was added each time one or more treated groups were started on a dietary regimen. The F0 rats were started on the diets at 3-4 wk of age and the Flbrats at weaning. Exposure was continuous through mat ing, gestation and lactation until the rats were killed. Ten males and 20 females were fed at each dietary level in both the F0 and Flb generations. The F0 males at each dietary level and half of the females were caged individually. Other F0 females and all the Flb rats were caged in groups except during the reproduction cycle. Body weights were recorded weekly on F0 rats, except during the reproduction phase, and food consumption was measured during wk 2 and 5, the week before each mating and the week after weaning of the F,, litters. F,b rats were weighed when started on the diet, during the week before mating and at sacrifice. The F0 rats were pair-mated when 3 and 7 months old to produce the F,a and F,b gene rations, respectively. Breeding-stock F,b rats were selected at weaning from all available litters and pair-mated when 3 months old to produce the F2, generation. F.. rats on diet ary levels of 0, 20 and 100 ppm Aroclor 1254 were mated a second time when 8 months old to produce the F:b generation. Viability counts of offspring were made at birth, day 3, day 7 and day 21 (weaning). Litters were inspected daily for condition and the presence of dead pups. After the F,b offspring had been weaned, ten adult F0 rats of each sex were lulled and their livers were weighed and fixed for histological examination. Following the weaning of the Fib generation, for Aroclor 1254, and the F2m generation, for Aroclor 1260, haematological values (total leucocyte count, haematocrit, haemoglobin and differential leucocyte count) were determined on ten adult F,h rats of each sex at the dietary levels of 0,20 and 100 ppm. These rats were then killed and weights of the spleen, heart, lungs, brain, kidneys, testes and liver were recorded. Tissues were fixed for microscopic study. oa38<*3 KON* Effect! of FCBi on r*l reproduction S Al lower dietary levels, haematology was not done and only the livers were weighed. At dietary levels of 5 ppm or less of both Arodors, one male and one female weanling from each of ten litters from each generation were killed at 21 days of age and thc livers were weighed FJt weanlings at dietary levels of 0, 20 and 100 ppm Arocior 1254, and F,b and F2a weanlingsat levels of0,20and 100 ppro Arocior 1260 were killed in the same manner and the livers were fixed for further study. One-generation reproduction studies. Preliminary Studies were conducted on both Aroclorsat dietary levels of 0,100 and 500 ppm. The experimental design was the same as that described for the two-generation studies, except that only ten females were used. This study was terminated after the Fu offspring had been weaned Rats Ted 500 ppm Arocior 1254 were bred only once. Haematology values and organ weights were determined and tissues were examined microscopically on the F0 rats. Post-implantation exposure studies. Stock rats 100 days old were pair-mated Insemina tion was verified by micioscopic inspection of a daily vaginal smear. Counting the day of insemination as day 0, the females were dosed by oral intubation on days 7-15 of gestation (a total of nine doses) To maintain a constant dose volume on a body-weight basis, Arocior 1254 or 1260 was dissolved in peanut oil at appropriate concentrations and these formulations were administered at the rate of 5 ml/kg body weight, based on day 7 of pregnancy. Arocior 1254 was given at dosage levels of 0 (peanut oil only) 10, 50 and 100 mg/kg/day. Arocior 1260 was given at dosage levels of 0 and 100 mg/fcg/day. The females were allowed to deliver and the litters were observed through weaning. Tissue preparation. Tissues were fixed in buffered 4% formaldehyde solution and stained with haematoxylin and eosin for microscopic examination. An Oil Red O stain on fixed frozen sections was used on occasional livers to demonstrate lipids. Statistics and calculations LDjq values were calculated by the method of Litchfield & Witcoxon (1949) A r test was used for comparing the litter sizes, organ weights; body weights and haemalological parameters of the treated groups with those of the control groups. Survival percentages of offspring were compared by the Mahn-Whitney V test as proposed by Weil (1970) Food consumption values were arrived al by averaging the consumption of both males and females over the five periods in which food was measured. RESULTS LDh studies on Arocior 1254 and 1260 . The single-dose oral LD10 for Arocior 1254 in weanling male rats was 1295 mg/kg with 95% confidence limits of 1136-1476 mg/kg- The lowest lethal dose tested was 1200 mg/kg, and 1000 mg/kg produced mild diarrhoea. Signs of toxicity at lethal levels were diarrhoea, depression and salivation, with death occurring in 1-3 days. . For Arocior 1260,.the single-dose oral LD50 in weanling males was 1315 mg/kg with 95% confidence limits of 1174-1473 mg/kg. The lowest lethal dose tested was 1000 mg/kg, S00 mg/kg produced no signs of toxicity. Rats given lethal doses developed diarrhoea and depression, with death occurring in 1-7 days. Under the same test conditions the estimated oral LD0 for Arocior 1254 and 1260 in adult rats was 4-IOg/kg (Kimbrough ct al. 1972). The single-dose iv LDjq for Arocior 1254 in adult female rats was 358 mg/kg with 95% 66 R. E Lsnoca. T. B. Games ted R. D. Kimbrough confidence limits of 328-390 mg/kg. The lowest lethal dose tested was 300 mg/kg; 230 mg/kg produced dyspnoea, depression, diarrhoea and salivation soon after dosing. Most rats died within 25--110 min, but at the highest dose (400 tng/kg), death occurred as soon as S min after dosing in some animals. Rats that survived appeared normal after 48 hr. In the control group, which received the formulation vehicle only, ooc rat developed mild diarrhoea. jPreliminary one-generation reproduction studies on Aroclor 1254 and 1260 The reproduction summary lot the Aroclor 1254 feeding studies is presented in Table 1. Exposure for 67 days (at the time of pair-mating) to 500 ppm Aroclor 1254 (370 rag/kg/day) resulted in fewer litters, smaller litter sizes and 103% mortality by day 3 in the F,, pupa The survival of offspring of die rats fed 100 ppm (7-2 mg/kj/day) for 67 and 186 days before mating was reduced, only 85-9 and 681% of the pups surviving to weaning in the F), and Ftk generations, respectively. Litter sizes from both matings were- smaller, but the difference was not statistically significant The mean body weight of the 100 ppm pups at weaning was 7 8 and 10 g less than that of the controls for the F,, and Flb gene rations, respectively. This difference was probably dose-related, as the treated litters were smaller, but a statistical evaluation was not done. In general the pups appeared small, but othciwise were normal at weaning. Aroclor 1260 fed at a dietary level of 500 ppm (35-4 mg/kg/day) for 67 and 186 days prior to mating markedly reduced live-litter size and survival-to-wcaci.ng in the Ft,-and Fi generations (Table 2). No effect on reproduction was observed in rats fed 100 ppm (6-9 mg/kg/day) in either the F,, or F,b generations. The F0 rats in the one-generation studies were sacrificed after exposure for 8 months Organ-weight analysis indicated that the liver was the primary organ affected. As reported in the previous paper, the livers in male rats receiving either Aroclor 1254 or 1260 at diet ary levels of 100 and 500 ppm were heavier than in controls; this was also observed in females fed Aroclor 1254 at levels of 100 and 500 ppm or Aroclor 1260 at 500 ppm (Kim brough et al. 1972). The testes-to-body weight ratios of F0 rats fed Aroclor 1254 or 1260 at the 500 ppm level were greater than those of the controls (P < 0025). The difference in actual weight of the testes however, was not significant. This observation may reflect the reduced body-weight gain observed in all F0 rats fed the 500 ppm level of both Aroc- tors No difference in other organ weights was found. - After 8 moqths exposure, terminal haematofogical values in F0 rats indicated a reduc tion in haemoglobin and haematocrit (P < 0005) in both sexes fed 500 ppm Aroclor 1254 or 1260. The haematocrit was reduced in males (P < 0-05) on 100 ppm Aroclor 1260, but haemoglobin values were normal in this group. The total leucocyte count was normal in rats fed Aroclor 1260, but there was a shift in the differential count. The number of lym phocytes was increased in females (P < 0005) fed 100 and 500 ppin Aroclor 1260 and in males(P < 0-05) fed 500 ppm. A corresponding decrease (P < 0 05) in-polymorphonuclear leucocytes was observed. Differential values for rats fed Aroclor 1254 were normal, but an increase (P < 0-001) in total leucocytes was observed iu the $00 ppm females. ' Two-generation reproduction studies on Aroclor 1254 F0 rats fed 100 ppm Aroclor 1254 (7-6 mg/kg/day) for 62 or 188 days before mating pro duced smaller litters than the controls in both the F,,and F,b generations (Table 1). Survival-io-weaning was not affected in the Flt offspring. Only 73-6% of the 100 ppm F,b pups MONS 083845 Table I. Atprvdvetion andpap survival to groups ofratsJed Atodar 1254 ; *- :`y-~ : f; . a .' .. .*_ ' :' - .V ' -C ' '' V ^ . - SNOW ' . Total pups/group , ' *. Dietary. Parental No. of * . No. of fitters .. Liner *izrs$ . ... Mean body weight level exposure females At At . Survival Generation (ppm) ' MyK " mated 8ora$ Weaned birth weaning Dead' Alive ' ' Day 3 Weaning ..'CD W F,, F.. v\ F:v ' 050021110000Q00|| 001052l100;000J ' 0005223l111000000 . . 666666666222111211221111117072200277S77887288772222211168848999564453.`._,,*.1:`15v-.,.;'':*-..`.*..- 2.122222222112222222222211100000000000000000000000090 . > ' .. .. ' ' 9% <111799R> 7I11S67 . 17s -...- 20IS . 2I11S088 41m11111279577Ci). . . ' . 08767111111111979857897-. . I11S79 ' . 24111111IS276759,` - ' .. . 9491J9111U1111112110-11204021205*--------1741*34738268*-***^.v.>r.,. .91311I111122011-26l52-----1l675`'77*V*** ~. . 81104 1-- M 91U111-1H001--S386 SO , 389571U11n1111-U-120018405015-------115357220r - 63 703004J226632063573111110 * . - . . 85 97*22222222281223211I111S1140201200128227604881519556S93242723240 . v: ' . : . ; - ' .027282111<802894654950* ' 72221S7222111111i29900419286977!I13987444440!J ' - 02872103159196 Z11O84O35 . 6S148I9 . 7222211J1m111198007729197311S8332690*. . * . 09999999988189967889999971806635859MS34579809780178-0-------V0580644847906469325531-0*' * " - ' . . . . ' 3391-42 34334423333333333333333078178627757562HS852889-----------3115519230779899470293 .' t Crnccpticn to mating for Uni parents of the FJt and F- generations. ' - '. X Numbers in parentheses indicate numbers of Huns in which no live offspring were found . . ' ' . $ Number of live offprir.,li*e litter bom. . " r. f Onc-ccncration study. `: . "' * Values marked with aucrisks difkr Ejnificaaiiy from the control value'. *P < OOS:**P <0005. ; survived lo weening. hut this was not statistically significant IP -- 00.58) Because 01 me poor condition of a number of ihe MX) ppm 1',,. pups, all were held for 30-days posl-wcaning observation. Fifteen Of these pups died within 30 days of wcaniiig. The mean body weights or the MX) ppm )*', generation pups from cither mating was about 6 g less than the controls at weaning. This is probably significant, since Die treated litters contained, fewer pups A reduction in litter si/e was also observed in the F(h generation at 20 ppni (15 mg/kg'dayl . Fib rats hred after exjxssurc fiir 129 or'274 clays (conception lo mating) lo 20 or 100 ppm had smaller litters than the controls (F,, and f'jh generations) A marked decrease in mating performance was observed in Hie 100 ppm group, in which only seven and four litters were born in the K,, and F.h generations, respectively. In each ease two litters eontained no c iable oll'spring wjlcn first observed. Tile data also suggest a decrease in ma (ini performance at 20 ppm, at which level only 12 F.,, litters were produced. Survivnl-to-wcnrj? ing was reduced in the 100 ppm F2, offspring. AU 100 ppra FJb offspring survived to wean ing. but this is of little significance since only seven pups were found alive. No effect on reproduction was observed in ran fed 5 ppm (032 mg/kg/day) or 1 ppm (0 05 mg/tg/day) through two generations. "' ,, , Although reproduction was not aflected at lower dietary levels of PCBs, an increase in liver weight in 21-day-old weanlings was found at all levels tested (Table 2) At 5 ppm or highcr. lhe liver-to-body weight ratios of weanlings were increased in both sexes in belli Table i Memllrtr y.'ri'jksoj21 -ituy-oM riKsfrm imrmtsfiil dri'v/ur, l2Slor.lMB - : Liver weight . Dietary level : Aroclor. . Generation (ppm) ' Male* ' Female* % of body, 't " ' . / weight . . * - /oftady Wfiflit , 1254 . V . F,. -0 ' 1-40 ' 369 1-38 3 83 ` * ' 14 v * 181* 401* i . Ml . . 391 ' 5 . H2* . 4-34 ` 1-86* . 443* "'F,* 0 1-5.1 , ->W ' 1*58 >84 < .' 1 1*54 . 394* 1-49 392 . sr- ' .1-77 4*63* 1*72 . 475* F* . 0 I-J7 3-81 1*28 ' 3-74 ' 20 2*20* 5-78* 2-23* 6-W4*-? *: '- " 100 163* 6-97* 267* 710* v " Fj. 0 1- * M3: ' ! 1-4* ' .V78 w 3-74 . 142 . 3 63 W6 ' 1 >69 ,,v ; 5 156 4-23* . 1*18 - * 4-09* > -i I2W . F,, 0 154 . 3*66 * v 1-57 , 3 92 : $ 173 418* ' V 1*70 ' 4)9* , Flfc 0 l* 3 73 1*55 393 20 ` 2-06* 504* 214* 518* . 100 2-59* 6-33* 2* 59* 64K* Fu .0 >5 359 1-29 3-70 5 l*5N 401* 1-52* 4 07* ` F* .0 M5 3*82 : 1*4 402 20 ior.* 5-21* 1-94 5 On* . 100 2Mi* 612* 2-49* 6 85* I-V, 0 lUl >K5 r*3b 387 5 Hsj* 421* H-U* 425* Vulm-w marked will) un UNferisk iltlKr sipmticuml) fii>in ihccotimil vuluc: *P < IH)5, t ' , -v.; ., wohs 0 ->.ds : ''if?';K .i'i-.v v' v : ' " V' Effects or 1`CBj on rat reproduction 69 the F| and F2 generations, but at ! ppm the liver enlargement was observed only in the Fi and F,,, male weanlings. No liver-weight increase was found in either sex of the 1 ppm Fj, weanlings.. In 10-month-old Flb rats, an increase in liver-to-body weight ratios was observed in both sexes at 100 ppm, but at 20 ppm it was seen only in th'e males (Table 3). At 5 ppm or less no increase in liver weight was found in 6-month-old F,b-rats or in F,, rats exposed for 10 months. Both sexes of F0 rats fed 20 ppm or more Aroclor 1254 for 8 months had enlarged livers (Kimbrough cl at. 1972). The testes-to-body weight ratios were increased (F < 0-001) in 100 ppm F,b adult males. Body-weight gains of adults in all the test groups were comparable to those of the controls except in the F,b females at 100 ppm, which gained less weigh! than the controls (Table 3). Reduced haemoglobin (P < 0-005) and hae- matocrit (P < 0-025) were observed in female Fib adults at 100 ppm, while Flb males at this level had reduced (7' < 005) haemoglobin only. Haemalological values at lower levels were normal. No dose-related signs of toxicity were observed in adult F0 or FIb rats fed 100 ppm or less. . Two-gcneralion reproduction study on Aroclor 1260 No cflcct on reproduction was observed in rats fed Aroclor 1260 at levels of 100, 20 or 5 ppm (7-4,1-5 and 0-39 mg/kg/day) through twwgenerations (Table 4). The liver-io-body weight ratios of 21-day-old pups wero increased at all dietary levels (Table 2). This effect was observed at the lowest level tested (5 ppm) in all generations. An increase in liver-to-body weight ratios was observed at all dietary levels in Flb males between 5 and 7 months old, but only at 100 ppm in the Flb females (Table 3) After 8 months exposure, an increase in liver weight was observed at 20 and 100 ppm in F0 mates but not in females (Kimbrough ct at. 1972). No effect on liver weight was found in either aex of Fo (Ms fed 5 ppm for 8 months. T/ie tesies-to-body weight ratios of the 100 ppm F(b males were increased (P < 005); at 20 ppm the increase was not significant. Body- weight gnin in all test groups was comparable to the control groups. Haemalological values were normal in the 20 ppm F0 adults and in F.a adults at 100 ppm or less. No signs of toxicity were observed in any of the test animals. - Post-Implantation exposure studies on Aroclor 1264 and 1260 The data on reproduction and survival of offspring from females dosed during organo genesis is summarized in Table 5. Nine oral doses of Aroclor 1254 (100 mg/kg/day) given on days 7-15 of gestation resulted in a decrease in the survival of the pups. At this dose Only 30-1 %of the offspring of the treated group survived to weaning, compared with 98-2% in the control group, and the mean body weight of the test pups was 7-1 g less than that of the controls at weaning. No effect was observed in the groups receiving 50 mg/kg/day or less. v No effect on reproduction or survival was observed with Aroclor 1200 given at a rafev of 100 mg/kg/day. Summary o[pathology after treatment with Aroclor 1254 and 1260 A detailed account of the liver |>aihology in the F0 rats fed Aroclor 1254 or 1260 at 20 ppm or higher dietary levels has been published (Kimbrough el al. 1972). Of the rats consi dered in the current report, similar changes were observed in the liver of adult rats, particu larly in the F|b generations at the higher dietary levels. The incidence of the various Generation Exposure Sex (daysl Dietaiy level (ppm) Terminal body weight <K) liver weight %of No. of body livers Enlarged S weight examine!i hcpatocytes Liver pathology (no. efEven affected) Foamy Fibrous fnefusoas cytoplasm Pigment strands Adenofibrosis Nodules 3 Axorior 1254 F. M 310 0 601 15-22 2-53 10 0 0 0 00 0 0 1 602 15-88 2-60 to 0 0 0 00 0 0 5 614 15-37 2-51 10 0 0 l 20 0 0 F 313 0 370 10-34 280 SO 0 0 0 10 0 0 1 338 1014 284 10 1 0 0 40 0 0* S 362 1066 294 10 4 1 0 30 0 0 p F.. M 323 0 588 14-51 247 . 10 0 0 0 00 0 0r 20 602 16-11* 2-67* 8 8 3 7 10 0 0 100 354 19-43* >54* 10 (0 5 7 42 3 l F 328 0 349 9-92 285 10 0 0 0 0 0V 0 0 20 340 9-41 278 7 7 2 .< 70 0 3 I 100 300* 1053 360* iot 10 2 8 93 5 7 F,, M 190 0 475 1H3 2-34 10 0 0 0 00 0 0 l 453 1H4 244 10 0 0 0 0j 0 0 s. 1219 246 10 2 0 2 0t 0 0 F 190 0 287 8-27 2-87 10 0 0 0 00 0 0 1 294 844 286 10 1 l 0 0 4 *. 0 0 5 299 8-92 293 10 1 0 0 01 0 0 Arodor 1260 F. M 230 0 $63 1445 2-57 10 1 0 0 00 o 3 567 15-06 2-65 10 3 0 2 00 o F 250 0 322 1009 >13 10 0 0 0 00 0 * s 331 1080 >23 10 1 0 0 00 0 F,, M 179 0 472 12*68 2-63 10 0 0 0 00 0 20 494 14-72* 299* 10 9 3 4 00 0 0 I0Q 509 16-63* 3-27* !0 10 * 10 3 0o o F 170 *0 297 9-66 3-27 10 0 0 0 00 0 o X 20 304 1030 340 10 6 0 0 00 0 o o 100 304 IM4* >70* 10 9 0 1 52 z Fi> M 217 0 492 11*97 243 10 0 0 0 00 o o 5 505 13-16 2-60* 10 7 3 3 10 o 0- F 217 0 279 7-93 214 10 0 0 0 00 0 0' o a $ 286 849 2-96 10 2 0 0 00 0 0 u a t Necrosis in two liver* Values marked with an asterisk differ tgaiScantly from control (roup: *f < 0015. C o * s.3 s, ? a s .^5 ?a r $% ? 5S SrlC J S3 <TS S' -^--ns- t Macron So two Sun Valaca narked wibaatttriikd&r significaady from cootrol group. *F < 00851 u' t9 0 0 Table 4. Reproduction ml pup survival In groups ofratifed Aroclcr }260 Generation Dietary level (ppm) F,. os 1001 500{ 0 20 100 0 s Pn 05 1005 soos 0 20 100 '5 Pa . 0 20 100 0 5 Parental exposure (days)t 67 67 67 61 63 63 71 71 136 136 136 187 1S7 187 188 188 128 128 128 127 127 Mo. of females mated 10 10 1 20 29 20 20 20 10 9 8 20 20 20 20 19 20 20 20 20 20 Mo. ofHum Pont Weaned Utter size! At At birth weaning t 8 19 17 17 19 18 9 5 6 17 16 13 17 16 18 19 20 13 18 9 12-3 122 8 1HS 11*6 3 85** 3*3 19 ? 112 17 as 11*3 17 11-6 a*2 19 a-7 JH 18 121 as 8 121 103 5 11-0 IOI 2 6-7" 2*3 17 111 11-0 16 ll-y 11*4 13 KM IOI 16 103 9-2 15 11*2 106 18 10-9 103 19 tw Ul 20 101 9*9 13 lit 11*6 18 a-s IK Total pups/group Born (found) Ali vest Dead Alive Day 3 Wexmrtg 2 in 111 no 0 93 93 93 10 68 55 26 2 223 217 213 0 20i 193 191 1 197 192 191 Q 222 218 216 I 217 213 212 r 109 96 93 0 55 54 54 1 40 38 14 6 189 119 187 1 191 191 183 5 135 131 131 10 175 160 156 2 179 171 170 1 197 187 185 1 215 213 211 1 201 200 197 1 153 152 151 0 213 212 209 Survival <50 k Mean body weight at weaning (s) 99*1 1000 3S-2* 95*5 95-0 97-0 97-3 . 97-7 ' 85-3 98-2 350 989 95-8 97-0 89-1 ' 950 939 98-1 98-0 98-7 98-1 370 356 36-3 398 380 3>8 38-5 409 36-2 <07 408 410 39-2 36-1 39-3 403 33-7 3S-6 34 8 38-5 m f a 9 f Conception to mating for parents of the Fi, generation, t Numbers of live offspring/im Utter bom. $ One-generatioo stud/. . X Values marked with asterisks differ significantly from the control value: *P < 0025; < OOOL c z O a U* a vr o R. E. LfNDER,T. D. G aines and R. O. K im&mouch TableS. Reproduction end swrfco/ ofpupsfrom dams doxd orally with AroctorJ2S4 or 1260 on daps 7-13 ofpregnancy Dose (mg/fcg/day) No. of females treated No.oflitten Born Weaned Utter soef Total pups/ETO'Jp Born (found) Alive at At birth At weaning Dead Alive Day 3 Weaning Areclor 1254 0 99 9 12-3 121 0 100 97 5 11-9 3d 8 0 10 10 10 11-9 118 0 10 99 9 . 121 12-0 0 50 10 10 10 13-4 12-7 0 Arocier 1260 0 12 12 12 12-0 11-5 4 100 12 12 11 11-7 10-3 10 t No. of live ofTspring/littcr born. The value marked with an asterisk differs significantly front the control value: *P < 0-001. 111 S3 119 109 '134 144 140 1(0 64 119 109 131 140 135 109 25 US 108 127 138 124 Survival ` (*/J 98-2 30-1* 99-1 99-1 948 95-8 88-6 Mean body weight at weaning () 379 301 406 37-6 305 33-9 13-9 * O z Fie. 1. Section of ret liver, showing a hepatic nodule tr.e*s>iring0-2 cm Indiansater surrounded by narruil li .'sr cdb .(arrow at margin of hepatic nodule). The hepatccytes within the nodule are lerner than those of the surrounding tissue and stain lest well. Hacmatoxylin and eosin x 100. * muns ThcrestiHs oT our studies confirm file ci'.pitbiJily of some commercial PCB mixtures to or Koplini'cr, Fanchcr & Catandra (1971). who observed eiTeets on mating performance and/or pup survival in rats fed KK) ppm Aroclor 1242 or 12S4. hut found no effect at the same dietary level of Aroclor 1260. In our study, 20 ppm Aroclor 1254 affected litter size and probably decreased matins performance in the second mating of the F,k rats. The trend suggested the possibility that even lower levels might affect reproduction in sub sequent genera lions; but M. L. Kcplinger (personal communication 1973) found no effect through three generations in rats fed 10 ppm Aroclor 1254. Rcproducu'vc failure in mink exposed to PCBs has also been reported (Ringer cl til. 1972). Although actual PCB con sumption on a mg/kg basis was not reported, the mink is apparently more sensitive to PCB exposure than die rat since levels of I and S ppm Aroclor 1254 affected reproduction after only 4 months' exposure In the present study in rats, fewer pups per litter were observed at dietary levels of 500 ppm Aroclor 1260 and of 20 ppm or more of Aroclor 1254. Reported litter sizes are based on the pups found, but since it is common for the dam to cat dead or defective offspring, the reduced litter size probably reflects decreased fertility and/or increased perinatal and foetal mortality. ` . .' Aroclor 1254 (100 mg/kg/day) given to pregnant rats during gestation produced no effect on the viability or morphology of foetuses examined on day 22 of gestation (Villeneuve, Grant, Khera, Clegg, Baer & Phillips, 1971). In the present study under a similar dosing . regimen, dams dosed orally during gestation with Aroclor 1254 (100 mg/k'g/day) gave birth to litters that were grossly normal, but only 30-1% of the pups survived to weaning. Since PCBs are excreted in milk (Curley, Burse & Grim, 1973; Fries, 1972), it is possible that PCB ingestion via the milk contributed to the increased mortality of pups observed in both the post-implantation and dietary-exposure studies. The milk of rats 11 days after the hut of nine daily doses of Aroclor 1254 (50 ing/kg/day) contained 66 ppm of PCB-derived' material (Curley tt al. 1973). Kimbrough rt al. (1972) estimated the orai LDJ0 Tor Aroclor. 1254 and 1260 at 4-10 g/kg in adult rata From the oral LD30 values in weanlings in the present study (about 1300 mg/kg), it is evident that the Aroclor mixtures tested are con siderably more toxic to immature rats than to adults Although we have no acute toxicity data for PCBs in neonates, nor figures on their milk consumption, it is possible that lacteal exposure could approach a toxic level in suckling rats ut the higher exposure levels. In both the dietary and post-implantation studies, direct PCB exposure occurred in utrro, since PCBs have been shown to cross the placenta (Curley et al. 1973; Grant, Vil- leneuve, McCully & Phillips, 1971). PCBs have been reported in eggs (Peakall. 1971) and embryonic mortality has been observed in birds treated experimentally with commercial PCB mixtures (Dahlgren cr al. 1972; Peakall, Linccr & Bloom, 1972). Dahlgren rt al. (1972) also reported a decrease in survival and a depression of weight gain in 6-vvk-old pheasant chicks from hens treated with Aroclor 1254. In the present studies, similar effects were observed in young rats at the higher exposure levels of Aroclor 1254. Besides a reduclion in observed litter size, there was additional evidence of lute foetal mortality in the group given 100 ppm Aroclor 1254, no live offspring being found in four of the Fj liners. Thus it is apparent that some PCI) mixtures affect the embryo in both mammalian and avian systems, although the mechanism of this effect remains undefined. Although the suckling rats ate some of the Aroclor-trcatcd diet for 4-5 days before weaning, it is probable that the observed increase in liver weight in 21-day-old rats was k t | jt. [ fo l Q ' 73 Effects of PCBs on rat reproduction due to PCB exposure via the mothers* milk. Curley et al (1973) also observed a liver* weight increase in weanlings from dams dosed only during gestation. When Arodor 1254 was given during pregnancy to rabbits, maternal liver weight was increased but not foetal liver weight on day 29 of gestation (Vi)lencuve el al. 1971), thus eliminating the possibility that placental transfer of PCB was the cause of (he liver-weight increase seen in weanlings. When adult rats of the F(k generation were killed after dietary exposure for 5-6 months, liver weights at the lower dietary levels were comparable to those in the controls except in males given 5 ppm Arodor 1260. Presumably once exposure via the milk ceased, the livers returned to normal in spite of a continued low dietary intake of PCB. At a dietary level of 1 ppm Arodor 1254, only the malcjweanlings of the F,a and F,fc generations had enlarged liven The absence of this effect in the F2a weanlings is inconsistent One explana tion could be the credibility of the 1 ppm formulation. Problems inherent in consistently reproducing a 1 ppm formulation, as well as contamination of the basal diet with PCBs, are possible sources of error. Over the last 18 months this laboratory has analysed the basnl diet for PCB contamination at 6-wk intervals. Most values have been less than 0-1 ppm, but occasionally values of up to 0-5 ppm have been found. The latter level of contam ination would introduce a 50% error in a 1 ppm formulation. It is likely that I ppm in (he parental diet is very near a threshold effect level for the observed liver-weight increase in weanling rata. At the lower dietary levels, adverse effects were not observed in weanling rats, which exhibited only an increase in liver weight However, Arodor 1254 fed to 1-montb-old rats at the 100 ppm dietary level (approximately 7-9 mgAg/day) increased cytochrome P-450 and liver weight after 2 days and microsomal protein after 3 days, while a single oral dose of 10 mg/kg increased cytochrome P-450 after 24 hr (Goldstein, Hickman & Jue, 1973).. A 50-70% increase in nitroreductase activity was observed with Arodor 1254 and 1260 fed to rats At a dietary level of 0-5 ppm for 4 wk (Litterst, Farber, Baker & Van Loon, 2972) lit view of these reports if is very probable that offspring in the reproduction studies, even those involving the' lowest levels of PCBs, were subject to inductive effects. 4cJ.'newJ#'bm*r>--We wish to think Mr. R. L Moore for assistance with the animal tests. Mrs. Estelle C. Gray for statistical analysts and Mrs Linda \V. Anderson and Mrs. Annie R. Alford for preparation of tissues. - REFERENCES Bennett, O. A, Drinker, C K. & Warren, M. F. (I93S). Morphological chanf.es in the livers of rats resulting hom exposure to certain chlorinated hydrocarbons J. ind. Hyg. Toxicol. 20,97. Cuitey, August, Burse, V. W. & Grim. Mary E (1973). Polychlorinated biphenyls: Evidence of transplacental passage in the Sherirata ML W Comet Toxicol. II, 471. Dahlgren, K. B., Under. R. L. & Carlson, Q W. (1972) Polychlorinated biphenyls: their effect on penned phen- unit Envir. HUh Peripec. 1,89. _ Pries O. F. (1972) Polychlorinated biphenyl residues in the milk ofenvironmentally and experimentally contami nated cows nnr. HUk Ptrspec. 1,33. Gaines T. B. (1969) Acute toxicity of pesticides Toxic, oppl. Pharmac. 14, SIS. Goldstein, J. A, Hickman, P. ft Jue, D. L. (1973) Hepatic porphyria induced by polychlorinated biphenyls (PCBs): Relationship between porphyria, delta-aminolevulinic acid synthetase and cytochrome P-450. Pre sented at the FASEB 57tb Annual Meeting^ 13-20 April 1973, Atlantic City, New Jersey. OranL D. L., Villeneuve. D. C. McCully, K. A. ft Phillips W. E J. (1971) Placental transfer of polychlorinated biphenyls in the rabbit mir. Physiol 1,61. Keplinger, M. I... Fancher, O. E ft Calandra. J. C. (1971) Toxicologic studies with polychlorinated biphenyls. Presented at the Tenth Annual Meeting Society of Toxicology. 7-11 March 1971, Washington, D.G Kimbrough. R. D., Linder. R. E. ft Gaines T. 11. (1972) Morphological changes in livers of rats fed polychlor inated biphenyls. Arch em ir. Hlth 25, 334. R. E Ltsofn, T. B. Gaikes and K. D. KimaxouCii 76 Litchfield.). T., Jr. & Wilcoxon, F.(1949) A simplified method of evaluating dose-effect experimcnti J. Pharrmc. exp. Ther. 96,99. Litiertt. C L, Father, T. M., Baker, A. M. & Van Loon, E. J. (1972). Effect of polychlorinated biphenyls on hepatic microsomal enzymes in the rat Toxic, appl. Phamtac. 23,112. Miller, J. W. (1944) Pathologic changes in animals deposed to a commercial chlorinated diphenyl. Pubf. HUh Pep.. H'adi.,59,1085. Monsanto Co. (1971). Monsanto releases PCI! data. Chcm. Enyng AVws, 6 December, p. 15. Faakall, D. A. (1971). Effect of polychlorinated biphenyls on the egg-shells of ring doves. Bull. env. content. & Toxicol. (V.S.}6, 1U>. Peakall, D. B., Lincer. J. L. & Bloom, S. E. (1972) Embryonic mortality and chromosomal alterations caused by Aroclor 1254. rir. Hlth Perspec. 1,103. Ringer. R. K- Aulcrich, R. J. & Ztbik. M. (1972) Effect of dietary polychlorinated biphenyla on growth and reproduction in mink. American Chemical Society Reprinu of Paper* 12 (2) 149. Sissons, D. & Welti, D. (1971) Structural identification of polydiloriotted biphenyls in eommercitl mixtures by gas-liquid chromatography, nuclear magnetic resonance and mass spectrometry. J.XJiromot. 60,15. Street, J. C, Very, F. M, Wagstaff; D.). Si Blau, A. D. (1969) Comparative effects of polychlorinated biphenyla and organaeblorine pesticides in induction of hepatic mictosoreai enzymes. Presented at ISftlh Meeting of the American Chemical Society, 8-12 September 1969, New York. Thrustori, A. (1971) Quantitative analysis of PCBi PCB Newsletter. No. 3. 28 July. VUIeneuve, D. C, Grant, D. L.. Khera, K., Gegg, D. J., Baer. H. & Phillips, W. E. J. (1971) The fetotoxidty ofa polychlorinated biphenyl mixture (Arocloi^ 1254) in the rabbit and in the rat. Enetr. Physiol. 1,67. Vos, J. G. & Koeman, J. >1. (1970) Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residue! Toxic, qppl. Phoneme. 17,656. Weil C & (1970) Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or caretoogeneris. Pd Cosmet Toxicol. B, 177. L'eflet dcs biph&iyles po)ych)orur6s sur la reproduction du rat Rtaim+--On a itudk la reproduction, la pathologic et ks indtaet tTimoxiauioo aigui chei des rats auxquels on administrait Tun ou i'autre des biphtnyks polychlorurts Aroclor 1254 et Aroclor 1260. Let rats qui rccevaient TAroclor 1254 6 raison de 20 ppm ou plus du regime avaient des nkhiesmoinsnoinbreusesquelesammaux timoinsaux generations Flk el F,. La dose de ICO ppm d'Aroclor 1254 a fait augmenter la morulit* dans la generation F,k et fail nettemem diminuer les petformances d'aeeouplemem des adultes de cette feneration. Administri i raison de 500 ppm du regime, I'AtocJor 2260 a fail diminuer t'imponance des nichees e: k taux de survie des niches F,. Dec taux de 5ppcn d'Aroclor 1254 et de 100 ppm d'Aroclor 1260 sont reties sans effet sur la reproduction dcs rats toumis i ccs regimes pendant deux generations. Le poids du foie a augment* chez ks mules F, iges de 21 jours au regime i I ppm d'Aroclor 1254 et chex les males et femelfes F, et F, sevris dcs groupet k 5 ppm ou plus d'Aroclor 1254 ou 1260.' Dcs modifications biitologiquet du foie et des augmentations du poids de cel organe ont iti ohtrrvect chez (es rats adultes qui reccvaient ks plus fortes doses. Des femelles gravides qui avairnt rvcu 100 mg d'Aroclor 1254/kg dc poids vif el par jour du 7eme au 15*me jour de gestation ont mis bas des nichtcs k peu pres normales, mail seulement 30.1V, dc ees jeunes om autvecu au sevraga. La reproduction et le taux de survie des jeunes n`ont pas it* influences par In do* de 50 mg d'Aroclor 1254/kg/jour cl de lOOmg d'Arocloi 1260,'llg/jour. Les vakurs DL>0 oraies chez tee rate miles igis dc 5-4 Kmainet*taientde 1295 mg/kg d'Aroclor 1254 cl de IJISmg'T.'g d'Aroclor 1260. LaLDt inlraveincute d'Atocfor 1254 Cuiii dc 35S mpAg che* tee ArmeUrt adultes. MDNS 083855 A&enofibrosis in the Rat Liver Willi Persistence of Polychlorinated Biphenyls in Adipose Tissue Rcnatc D. Kimbrough, MD; Ralph S> Linder; Virlyn \V, Burse; Ralph W. Jennings, Chamblce, Ga Fifty mot* Shannon strain rats were fed 500 ppm of a polychlorinated biphenyl (PCfl) (Arocior 1254) for six months. Five each were Killed xero, one, two, three, four, six,eight,and ton months after exposure to Arocior had ceased. The livers of these rats woro examined by light and electron mi croscopy. liver lesions persisted although exposure to PCBs cessed. Ten months alter exposure ceased, 1,182 ppm PCBs wore still present In the rats' adipose tissue and 22.65 ppm in tbo rat livers. Arocior patterns found in the tissues by electron capture gas chromatography dif fered from patterns of dietary Aroctors. Mass spectra! analysis ol livor and adipose tissue revealed three major Arocior compo nents with masses ol 324, 356, and 392. These contained isotopic clusters indica tive ol tho presence of Cta, Clt, and Cl,, n a previously reported study' I Sherman strain rats were fed poly chlorinated biphenyls (PCBs) (Arocior 1254 and Arocior 1260). The rats developed characteristic mor phological changes in the liver. These changes consisted of hypertrophy of the liver cells, a brown pigment in Kupfler cells, lipid accumulation in the cytoplasm of hopatocyles and, at the higher dietary levels, adenofi- brosis. Because of the wide distribu tion of PCBs in the environment1 and Submitted for publication March 8. 1973; ac cepted April IS. Prom the Environmental Protection Agency, Bio-Effects Branch. Chamblce, Ca. Reprint request* to 11% Avery Dr NE. Atlanta their persistence, the hepatic lesion was studied further. The present study was undertaken to ace whether, once cxjxreuro to PCBs was stopped, the morphological changes produced in tho liver might disappear. Materials and Methods A total of 50 mate weanling, specificpnlhogen-free, randombird Shorman strain rats were tagged individually and groupenged, ten rati per cage. They were given Arocior 1254 in their diet for sis months. The dose of 600 ppm, which is equivalent tn an average daily intake of SS.4 mg/kg body weight,' was chosen be cause in the previous study all ten exposed rnqlc rats developed adcnofUn-osia within a period of eight months. Tltc Arocior n os mixed into ground laboratory chow as pre viously described.1 After six months of Fig I.-Arcs ol edenofibrosil in liver section consisting primarily of ducts surrounded by flattened epithelium. Duds are surrounded by little .............. ..............................................nx 150). rig 2.- Rat liver section showing cluster of small columnar cells resem bling pancreatic tissue adjacent to blood vessel. Hepatocytcs surround(ng lesion are vacuolated (hemaloxylin-eoatn, original magnification x 175). 390 Arch Cnvirou Hcatth/Val 27, Doc 1073 Rxhibit 5, 7 Adenofibrosis/Kimbrotigh et <l MONS 033856 Ukc* of the present method produces (in nvcrnj'c slope around J. Kor nick el, (he slope chanj'cs from 0.222 in Acid, l< 0,CC>(> in (his organic bone. Since copier nnd zinc conccntrn(ions ere determined after dilution, end therefore subject to a certain Amount of dilution error, a National Bureau of Standards sample (No. 1577) of bovine fiver whs subjected to the same procedure nnd analysed. Tho copper recovery was 11 3% 5.0%, and aloe, 112% 6% in three sepa rate nnolysea. Tho precision of the method in re peated analyses of control and ex posed lung tissue is presented in Ta ble 2. All lungs were divided into approximately five equal portions and oach portion analyzed for cad mium, nickel, and zinc. The standard deviations Tor each lung varied from a low of 0.02 ppm to 1.56 ppm, depend ing somewhat on the exposure to the inhaled metal. Within measured ranges or cadmium nnd nickel con centrations, 200 to 300 mg of wet tis sue from an exposed animal arc nec essary for precision. If the animal is unoxposed, 300 to 400 mg are re quired for the cadmium analysis. Although one would not expect any dillcronco in the nickel concentration before or afler exposure to cadmium, the nickel concentration xvus below the level of detection in the control animals. Sample weights can be re duced by os much as 90% to 95% for the analysis of zinc and copper/ Using the data given in Table 3, the total amount of cadmium re covered from the lung was calculated and compared with the total amount of cadmium present in the inhalation chamber during that, period, as wall as with the estimated amount or cadmium inhaled by the rats. It can be seen thul the percent retained (6% to 20%) of that "inholed," is larger by a factor of 50 than the amount re tained from the total exposure. These figures are somewhat comparable to those reported in the literature,1* which for short acute exposures var ied from 6% to 13% for rats, und un der chronic conditions, lasting eight months, are estimated at 2% for rab bits. Beth of theso studies were per formed nt higher exposure levels than those in the present experiments. Under tho conditions of the present study, all animals are exposed to a t. FrlltcrR )., IVcnlor M, Nmilbcr* C: Prob lems sf anniyaU, in Cadmium in the Environ- mtnf. Cleveland, Chemical Rubber Co, 1971, p IS. 2. Christian 0, Frldtnnn F: Atomic Abvorp- turn Si#ctro*mi>y. New York, Wiley-lnterscnmce Publishers, 1970. 9, Analythof Method* for Atomic Absorption Speetroftholamiitry. N'orm iOk, Conn, Pet kin- Elmer Co, 1971. 4. Pulido P.Fuwa K. Vnllie 0: Determination cf r:irffiuin in bioIty.'ir;il material* by atomic absorption spwlroplwtnmvlry. Analfliochcm 14: 3S3.10I, iww. &, Pmhtnd Tivhnii'iil Bulletin. publirutkvn 6031S. Downer* Orove. III. Pnekmd Instrument Co, 1970. References 6. MacCeo J: Characterization of mammalian tissues *nd microorganisms by Rus-tlquld chro- melii*raphv. J Gat Chromatagr 6:1672-1073, 1970. ' 7. Jackson AJ, Michael LM, Schumacher IU: Improved tissuo solubilisation for atomic absorp tion. Anal Client 44:1004-1065,1975? S. Mustafa MO. Cross DTI: Pulmonary alveo lar macrophage Biochemistry 10:4176-4185, 1971. 9. Bingham E, st al: Alveolar macrophages: Reduced number in rota after prolonged inhala tion of lend sesquioxide. Science 162:1207-1299, 1909. 10. llorstmnnS.eta]: Aerosols of lend, nickel, and cadmium: A method of generating soluble and Insoluble compounds. Arch Environ Health relatively small (turliclc size, (H9%, are less than 0.53/im in diameter)1* which for cadmium oxide would be relatively soluble. The uttimnls were killed 16 hours after rcntovul from the chamber, and the total exposures were not large. Experiments per formed In these laboratories after five- to six-week exposure periods, to be reported in the future, do not show much incrensc in lung retention. Under the conditions of these experi ments then, it would appear that of the cadmium oxide aerosol absorbed, a substantial portion remains in the lung. In summary, dole have been pre sented to illustrate a fast and rela tively precise method for the analysis of cadmium and nickel in animal tis sues.13 Tho technique has been used to measure cadmium concentrations in the rat lung after exposure to cad mium oxide aerosols. Thia investigation was supported by the Na tional (intitule of Occupational Safely nnd Health (rani 6-R01-OH 00347 02. Emil Pfitter, PhD. assisted in tho construction of inhutslibti experiments. William Barkley ran tho inhalation ohombora. Tony Horatman, PhD, performed the particle aitin*. 26:76-77.1973. 11. Sundermon FW: Measurements of nickel in biofafpoil mnteriol* by atomic absorption spectrometry. Am J Clin Pathol 44:181-199, 1066. 12. Friborg L, Tiftcutor M, Nordbrr* G: Me tabolism, in Cadmium in the Emironiteiit. Cleveland, Chemical Rubber Co, 1971, pp 27-66. 13. Murthy L, cl al: Atomic nbaorption deter mination of cine, cupper, yadniium and lend in tlssuci aolubilired by aqueous tetrsmelhylonimonlum hydroxide. Annl Diachem, to he publixhrd. Atch rnviron Health,'Vol 27, Dee 1973 Direct Determinotion/Kaplan et si 389 MONS 083857 fig 3.-Section of hepatic tissue in latter part of recovery phase illustrating numerous small lipid vacuoles (IV) (lead citrateuranyl acetate ormmai magnification* 51,300). * Tig 4. - Portion of hepatoeyte with tubulos of endoplasmic reticulum (ER) arranged In disorderly fashion. N represents nucleusi M, mitochondria (lead cltralt-uranyl acetate, original magnification x 20,235). Arch Environ Hualth/Vol 27. Doc 1973 Adcnofibrosis/KimhroiiQii et al 301 MCNS 083058 r-*f*T i jt td * .\ A a ) \ vr i 1 pr> ' `.i. i >P~ ; r, i 1 ,/ = 'T; U ... ; V- / , /'v'-' v -:o t ;( j)u v-rj Vvi.'t :'i 1 ' '< Fig 5.-Border of ire* of idenofibrosis in liver. Note round clusters of dark granules intermitted with web-Uke clusters el vacuoles. These lonnitions illustrate appearance of brown pigment (BP). C represents collagen; LC, liver cell (lead cilrate-uranyl aceiaie. original magnification * 20.235). receiving (ha exporimonta) diet, the rnta recoivcd no motti Aroclor end were Ted plain laboratory chow. At thin time five rate were killed. Additional killings of five randomly selected rats were mode onn. two, three, four, six,eight, and ten months after exposure to PCB had liceu discontin ued. At au(0|y the livers were removed, weighed, and the tissue fixed in photphate-buflenil 5$ glutnrnldehydc for three hours for electron micruxcopie exam ination, post-fixed in Id osmium tetrox* Ida, embedded in mnrag)nB. aectioncd with o glass knife, and stained with lead citrate and urnn.vl occtatc. Tietuo for the light microscope was fixed in 4*4 buffered formaldehyde solution and stnined with hematoxyliu ami cosin. Proven tissue of solected formalin-fixed hepatic ti*iue was cut o>\ the cryostat and stnined with oil red 0 to demon"!rale lipida, At the time of the lost sneriftev. ten month* after exposure to PCB-cimtaining diets had been diwvntinued, adi|x><M> itauc and liver from seven .previously exposed ruts and tin* adipose tissue and liver from two rats of the same age that hud hern fed only plain chow were analyzed for the presence of PCBs by electron capture iKCtgas clutmuuography according to methods descrilnxi by Curley ctnl* In order to determine which Aroclor COlistitllcntK ueivof suv)cheoieal uliibillt,v thill they ivniuim<ri at a measurable residuul level ten months after dietary in take hud ceased, adipose and hepatic com posite* were analyzed by gas chromatog raphy-mass spectrometry. The conditions for moss spectrol analysis were ax follow*: the mass spectrometer was interfaced with a gns chromatograph and equipped with a mass marker accurate within 0.3 mass units. The gas chromatographic column temperature was 220 C; flash heater, 235 C; glass-coiled column, 3.05 m x 0.64 cm; 1.5* OV-17/I.95* QF-1 on 60/80 moth chromosorb "W" (high performance, acid washed, dimethyldichlorosilane treated),' helium pressure; 0.84 kg/sq cm; flow rate (rotameter setting 3), 45 ml/min; separa tor temperature, 225 C; source energy, 70 ev; accelerating voltage 3.5 kv; trap cur rent GOpnmp; box current 50/znmp, nod leak current Spamp. . Results During the course of the experi ment three ruts died. The livers of the rats that were killed nt the time expo sure to Aroclor 1254 was discontin ued were enlarged, with average weights of 6.32ft of the body weight. When the experimental diets had been discontinued for four months, the liver made up only 3.6ft of the body weight, and after eight months recovery the average weight amounted to 3 34* of the average body weight. The fraction of the body weight for the liver in control adult male rats ranges from 2.44* to 2.59%. ` Forty livcr6 were studied micros copically. The hcpatocytes were en larged in 39 livers and nil 40 livers showed either vacuolated or foamy cytoplasm because of increased lipid accumulation. Cytoplasmic inclu sions similar to those described previously* were observed in the liv ers of 24 rats. Adcnofibrosis, which in many instances was very extensive, was present in 36 of the 40 livers, and a brown pigment was noted in the KupfTcr ceils and other macrophages in 25 livers. Of the four rats that did not develop adenofibrosis, one each wns killed two anil six months after the poisoned.diets were discontinued. Thu other two mis without ndrnoff* brosis were killed ten months after onset of exposure. The ndortofibrosw ranged in sire from grnjish while le sions measuring 0.5 to I cm to areas of 6 cm with pitted svnfnve. Quite often the lesion was imillicenlrie. The adenotibiosis* is illustrated in big 1 and 2. In this study the appearance of 392 Arch tiwiron Hcnlth/Vol 27, Dec 1973 Adcno(iL>fosis/Kinit)fOU|'l* et at HONS 083859 Fig 6. - Gat chromnlograpliic traces of Arector I2S4 standstd (A), hopalk ti**u 'tract (B), and extruct of adipose tissue (C). Response expressed as percentages, with 100%* 1.0 mv. the lesion, particiilnrly when il tvas extensive, varied a prent den). Sninll lotions consisted predominantly of glandular pnlo-slaining epithcliul Cells that formed ducts nml were sur rounded liy very little fibrosis. The larger lesion* had extensive fibrosis (tud contained collagen, am) often the UucIk which were formed by the epi thelial pale-staining cells wove markedly dilated. The dilated ducts contained mucus or necrotic debris, and nt times formed small cysts mea suring up to 0.S cm in diameter. Small dilated ducts were lined by co lumnar epithelium, and larger ones by flattened epithelium < Fig 1). In areas o( extensive fibrosis, and also udjacent to blood vessels in mvn* of normal hepatic parenchyma tKig'JV, clusters of small glandular evils were seen In 15 of the livers which resembled the epithelium observed normally in the pancreas. These small ( lusters ofpnncrvntic-like tissue have not previously been dcscrilx-d in the livers of rats or of other species. Special stains for esterase, esternse with sodium taurocholale, and tryp tophan showed a posit ivo reaction indicative of salivary gland tissue. The significance of these cells is pres ently not understood. Normally tis sue of a pancreatic typo is not ob served in the livers of our rats. Clus ters of small columnar epithelial cells resembling pancreatic tissue also occurred in portal triads without be ing surrounded by typical adenoftbrosis. In the present study, one to two rats at each killing showed these small columnar cells with highly aci dophilic cytoplasm and a small, round nucleus. In the older animals, tho fibrosis was quite extensive in some instances and the glandular proliferation was present mainly at the periphery of the lesion. Signs of regression of the lesion were not noted, except for the disappearance of epithelial cells from the center of the lesion. Furthermore the lipid accumulation and the hyper trophy of the hepatic cells remained constant throughout the study. Ex tensive areas of necrosis were ob served in throe of the 40 livers. In addition to the change described previously,' electron microscopic examinations of sections of liver tis sue showed numerous small lipid vacuoles in the cytoplasm of many heprtocytes, particularly in the latter part of the recovery phase (Fig 3 >. Tho smooth and rough endoplasmic retic ulum was arranged in a disorderly fashion (Fig 4). Many of the mito chondria were atypical and surround ed by double membranes. The electron microscopic nppearancc of the brown pigment present in the macrophages and Kupfler cells is illustrated in Fig 5. Large clusters of dark granular material, intermixed with lipid vacuoles, were observed, as well as round clusters of granular material about the sire of mitochon dria. In our pivvious studios il wa* found that at least sonic of Ibis pig- Arch Environ Mealth/Vol 27, [>cc 1973 Adenofibro&is/Kimbrouch et dl 393 0838fa^ HONS menl contained iron' but in addition high concentrations of urojjorphyrin (75% 8-carboxyprophyrihR and 25% 7-curboxyprophyrins) were found in the livers,* which could account for some of the pigment. In addition to those findings, oil red 0 stain demon strated granules of rod-staining lipid material in the area of the brown pigmont. Since the morphological changes in the liver did not disappear, the re maining seven rats were killed after* a ten-month recovery phase and their adipose tissue and livers analyzed for Aroclor. In adipose tissue, the concen trations of PCUs ranged from 924 ppm to 1,638 ppm, with an arithmetic I mean of 1,192 ppm. The concentra tions in the liver ranged from 17.30 ppm to 26.24 ppm, with an arithmetic moan of22.65 ppm. The PCB Icvol* in conlrol adipose and liver tissues wens <1.0 ppm. . The gas chromatogram obtained from the EC analysis of adipose tissue did not differ from that of liver, but was significantly different from standard Aroclor 1254. An average of seven peaks was observed as a resi due in the tissues. Under, these gas, chromatographic conditions* at least .18 poaks have been observed in the standard materia! (Fig 6). The differ ence is gas chromatographic patterns between stored and ingested Aroclor j has been observed before in this labo ratory and by other researchers in the ftold.*1** The tolnl ion current (TIC) chro i matogram for the liver tissue compoaitu in shown in Fig 7. Mass spec Fig 7.--Total ion current chromatogram of sample of composite liver. Response expressed as percentages, with 100%- 10.0 mv. tral analysis revealed the presence of only three major Aroclor components chlorinated moieties, while other* epithelial proliferation occurs in the with musses of 324, 358, and 392. peaks observed in the liver TIC con periphery, white the centra) portion These components contained isotopic tained the following molecular ions: of the lesions contains primarily clusters Indicative of the presence of peak 4 (324); peaks 5 to 7 (392 and fibrous tissue. The present study dews &, Cl*, and CJ,, respectively. Adi 358); and peaks 8 to 9 (392). not completely resolve the question of pose tissue analysis by mass spec reversibility of this lesion since tl* trometry revealed the same patterns. Although only (hive different mo lecular components were present, Comment The ndenofibrosis of the liver pro storage levels of PCBs in adipose tis sue and liver may have been suffi ciently high to stimulate continued .1 examination of the EC and TIC duced in this study nnd the one pre growth of tile tillered hepatic tissue. chromatograms coupled with mea viously reported1 is the result of Aro The levels in the liver suggest that a i1 sured intensities from multiple scan ning of eluting peaks indicates that clor consumption. Whether the Aro clor itself, or n contaminant such ns a constant low-level turnover of PCits tukes place in the rats. In a previous stereoisomer* of these three mole chlnriuulcd dibetv/ofuran, i* responsi study, Curley et al' found levels of cules are obviously present. Peaks 1 to 3 (Fig 7) did not contain any ble st ill needs to be resolved. A study of the lesion shows that *10,000 ppm Aroclor 125-1 in adipose tissue of rats that had heen fed 5(Mt 394 Arcti Environ Health/Vol 27, Dec 1973 Adcnolibiosis/KiMibreugli ct at HONS 063861 }>)>in of the mixture* in their diet for eight tm/nlhs. This finding indicates thnt n great deal of tin* Aroelnrn had been mobilized from the adipose tis sue and excreted in the present study. The storage levels found ten months after onset of recovery are in the same range of those of mis fed 100 ppm I'CDs in thoir diet for eight months.1 The dietary level of 100 ppm produces'a much lower incidence of adonofibrosis,1 and it is possible that adenofihrosis represents a |>ermanont lesion, particularly since the percent of body weight of the livers had de creased. The Aroclors found in the tissues differed from those fed in the diet. Metabolism of lower chlorinated bi phenyls is considered as a possible explanation for the differences ob served analytically between con sumed and stored PCB. Earlier work of Block and Cornish1 has shown that biphenyls and chlorobiphenyls can be hydroxylated and excreted in the urine of rabbits. A moro rocent study by Hutzingcr et al" indicates that the rat is capable of hydroxylnling mono-, di-, and totrachlorobi phenyls, but not hexachlorobiphenyls. Since Aroclor 1204 averages five chlorines per mol ecule, it is conceivable that the heav ier chlorinated moieties could remain as a residue. l'-dimelhylammunzobenzenc also produces adenofibrosis. Edwards and Whitow and Bennett ot si" described the lesion in rats when a mixture of chlorinated naphthalenes and chlori nated diphenyls were fed. Unfortu nately, the finding was not well de scribed in Bennett et al's report, but Edwards and White mentioned Ben nett et nr* finding as another exam ple of the induction of adenofibrosis by chemicals in an excellent descrip tion of the lesion. A critical review of tho histopntKogvncsitf of this lesion was made by Stewart and Snell1*. Tho presemv of the brown pigment in Kupfier cells and macrophages is of interest and was to a lesser extent also observed in the earlier study.1 Electron microscopic examination of the pigment shows that particularly tho large clusters of it resemble very closely illustrations of ceroid pig ment.1* Experimentally ceroid pig ment can bo produced by hemorrhage into fatty tissues rich in unsaturated fatly acids, particularly in the ab sence of an antioxidant (vitamin E deficiency). Ceroid pigment is com monly referred to as iipofuscin. These Kpid pigments arq at times associated with hemosiderin and may contain iron-positive granules as in the pres ent case.* Ceroid granules were also described by Edwards and White in the livers of rats fed butler yellow. Bennett et al also observed a brown pigment in rats fed a mixture of chlorinated naphthalenes and chlori nated biphenyls. These authors claimed that it failed to slain in a manner characteristic of either hemosidcrin or hemofuscin. The porphy rins demonstrated in the livers of our rats fad Aroclor may also contribute to the overall pigment deposits. Con oid pigment increases in people with age and has therefore been called *AOnuUungspigment" by the Ger mans. Attempts have been made to relate it to senility. It has been associ ated with neuronal ceroid-lipofusci nosis (Batten disease) in man. In minks, a disease called yellow fat disease11 is associated with the accu mulation ofexcessive amounts of cer oid in adipose tissue. The accumula tion of ceroid in mink can be prevent ed by addition of vitamin E to the diet. It occurs when the food rations contain high percentages of fish scrap. However, Gorham et al suggest that other factors may also he in volved, since high dietary percent ages of fish scrap did not promote growth as well ns the stock ration, evon when vitamin E was added to the diet. These observations raise the question whether high doses of vita min E, and possibly the addition of choline to the diet, could prevent or min inti/e tho observed effects of PCHst on the liver of our rats. Richard I. Moore a*i*i"d with llr animal IrMr. Lind-t W. Andorrun HrUlid with pri-p.iriHinn and inlcrjuvtution of the electminicrugraplw. Annin H. Alford perform'd tint iiviK- prepara tions. II. L Stewart, MD, and tlio theme laiwmtnry f lira Kallennl Cnnaar lm.iliui, BnhtMlv. Md, performed evaluations of liver alidcs. References 1. Kindmmj'h RD, Linder UK. Cninea TIP Morpin;logical chance* in liver of rni* fed pnlvchlorinated hiphenyla. Arch Uni-iron Ifmlth 24; 354.304,1972. 2. Rbchrotigh RW, et el; Pnlyi-lilorinotvd hiphenyl* in the global eci*y*lem Noluiy 220; 1078 1102. 1068. 5. Curley A, rt al: PolyrhlorlnnU-d UipbenvU. Dialrihuliun and itoragc in body fluid* end liouee of sltermnn rata. Cncimn Are 4:4SJ-49.V 1971. 4. Kimbrough RD, Gainey TR, Linder RE. The ullrastruclore of liver* of rut* fed DOT and dieldrin. Arch t'nviron Health 22:400-167,1071. 6. Coldatein JA. Hickman P. Juo D; Hepatic porphyiia induced liy polychlorinated biphenyl* fPCB'k): Relationship between porphyrin, 6eminolcvulinic ncld aynihttaae and cvtochrome (*-450, abstracted. Fed Rrve 32:702,1973. & Grout OL, Phillip* WKJ. VilUnouve DC; Metabolism of, polychlorinated biphmiyt t Aroclor 1254) mixture In the rat. Bull Ent-uon Confirm Toxhvt 6:102-112,1971. 7. Voo JG, Koemnn Jll. Comparative laximlegie study with polvchlocinated biphenyl* in chickens with epeeial reference to porphyria, edema formation, liver necroai* ami tissue midura Twsicol A/ipl Pharmacol 17:<tfa-659. 1070. 6. Block WD, Cornixh HI): Metabolism of bi phenyl nnd 4-chlorubiphcnylin the rabbit. J /ho/ Cketn 234:330) 3.102. 1030. 9. HtrttingtrO, c*t al: Polychlorinated bi|ihrnjtIk metabolic behavior of purr luomcn in pi geon*, rats, and hruok trout- Science 175112 913.1972. 10. Edward* JE, While J:Pulholugic ehungv* with special reference to pigmentation And daaifadion of hepatic turnip in rat* fed p-dimethylnnimaunbcim'iie (butler yelltml. J Xml Comeer Inti 2:157 )51,.Hl | 1. 11. Bennett GA. Drinker CK. Wnrrtn MF: Morphological chnngea in the liver* of mu re mitting Trom rtpo-uic In cvrtnin vhlnrimitnl Iiy'drvcorbnii*. J tnd lt?K Tvxicoi21MJ7-1:.|. |tM8. 12. Stewart HI.,Snell KC: Thv hitlopath..lt>a.v ofexperimental tumors if tin* liver uf tin- rut; A tdliml review uf the hininpnthnpeni^i*. Arm iJnioIntCnntio Cnm-riiui 11l:770-lto:i. 1>>57. 13. llsutrofi WS: Olnv-rviilMm and intcrprvt.i<lion of lipid pimrnt i l.ipr*tuwin> in tin- ptIh*1>l'v of Inliointnry iminudx. ('nt Ret- T>*wt>l 3:378 411, 1972. 14. Cnrlmm JK. Baker (5A. line N: Utwrv.i- Cion* on the elinliv.jy of yellow f.u iHmiim* in mink; Preliminary report. IVt Mill 4C.100-1U_> last. HONS 083862 Arch Environ Ho.iith.'Voi 27. Dee 1973 Addnofibro&is/Kinibrouch et al 305