Document 150b2Q3ra5LJYB7wzwg12GO6m

A REVIEW AND EVALUATION Of CARCINOGENICITY STUDIES IN MICK AND RATS AND MUTAGENICITY STUDIES WITH POLYCHLORINATED BIPHENYLS yt G*or? J. L*vln*k* MONSANTO COMPANY St. Loulti HiMouri October 14. 1911 HONS 015961 INTRODUCTION This Is review and evaluation of studiss which dl with th potential carcinogenicity and mutagenicity of polychlorinated biphanyis (PCBs). It ia subdivided into 4 factions! Chronic Rodant Studies, Metabolism Studios, Co-Carcinogenesis Studios and Mutsfanicity Studios. A brief suaaary of Epidtaiolofy Studios is added to complete covorafo of the issue of carcinogenicity. This review does not diseuss the effects of impurities or contaminants, particularly polychlorinated dibenzofurant (PCDF), which are reported to be present in sane PCS mixtures (Brinkman and deKok, 1980). The pretence and amounts of such impurities have not been specified in the materials used ia many studies. Thus, attempts to apportion the observed biological effects between impurities and PCBs would only add further conjecture to a subject which currently ia rife with speculation. By contract, specific chemical and trade names have been used to identify materials that wart studied instead of the all encompassing term PCBs. Thin was deemed necessary because there are too many one-sided generali sations in the literature. Every adverse finding is, by implication at least, sxtrapoUted to the entire class of materials designated as PCBs, Conversely, every report which has failed to find an adverse effect is carofhl in stipulate that it relates only to the substance studied. Even moro difficult to contend with are misleading refsrences to adverse findings which sre not substantiated by the actual publications referred to. An example of this occurred ia s discussion of in vitro netsboUen studies with liver nicrosossal easynes which stated that the formation of PCBnacroaoUwular adducts had been demonstrated. Among the references 1 HONS 015962 eiMd wii * papr entitled "Ths in vitro binding of 2.2',5,5,-tetrachlorobiphanyi mstabolitea to rat livar microsomal proteins". Tha Uttar paper claarly stataa "There U no elaar avidanca in tha data obtainad from the work raportad hara to aubatantiata covalent binding; however, it U also not poaaible to exclude tha nondiaiyzabla radioactivity aa being covalently bound". (Hargravaa and Allan, 1979). To emphasise tha point that dote ia important in evaluating safety, teat axpoaura conditiona have bean deeeribed ao that tha reader can compare them to ambient axpoaura lavaU encountered in tha literature and eUewhere. Hoopingarnar, at al. (1972) in their atudiea with ArocUr 12S4 on cultured human lymphocytes are among tha few authors who acknowledged that "The toxic does of the chemical was eeveral times greater than ia usually found biologically". Thoy uatd concentrations of 100 ppm of Aroelor 1254 in their atudiea discussed under Mutagenicity. Chronic Rodent studj.ee Difficultiee in compering and assessing different atudiea are compounded not only by problems of histopathologic diagnosis but also by variations in experimental design and animal strain difftreacat. Animal feeding atudiea sited in the literature at evidence far the carcinogenicity of FCBa and related studiee ef elmflar duration have been tabulated for mica ia Table l and fir rata in Table J. Studies have been grouped by the approximate weight percent of chlorine in tho materials tasted to facilitate comparisons betwosn products from different manufacturers with the asms chlorine content. HONS 015963 Mouse studies la T*bl 1 wr conducted with high diaury l*vU of PCBi. Kimbrough tad Linder (1974) reported t 52% mortality for mice fed 300 ppm of Aroelor 1254 for 8 month* end then held an additional S months. During that interval, control mice had a 32% mortality. This appear* to be quit* high for control mice about 1 year of age. In a report discussed under Co-carcinogen*sis, Boiler (1977) studied mice injected with Aroelor products and Moloney leukemia virus. Some data from hi* study, although not shown in Table 1, are quit* similar to those of Kimbrough and Linder (1974). Koller (1977) fed diets containing Aroelor 1221, 1242, or 1254 to groups of 25 Balb/c male mice for 6 months. After that time, some mice wore sacrificed for examination and others were returned to a control diet for another 3 months and then sacrificed. Dietary levels of each Aroelor were 375, 37.5, or 3.75 ppm. The only feeding regimen that was toxic was 375 ppm of Aroelor 1254. Only 9 mice of that group survived for 4 months, giving a mortality of 49%. Other authors cited in Table 1 (Xto, *t el,. 1973a,b and Nagasaki, et al.. 1972, 1974, 1975) did not indicate how many of the mice in their studies died when fed 500 ppm of Kaaechlor 500, which has a chlorine content similar to Aroelor 1254. XoDer (1977) reported that "PCS* did net produce hepatic neoplasia". There was marked liver injury is mice fed 375 ppm of Aroelor 1254, arild liver injury which persisted through the 3-month recovery period at 37.1 wm, mad no hepatic lesions at 3.75 ppm. Moderate liver injury was prodaw hy 375 ppm .of Aroelor 1242, but this regressed and no hepatic J& * ' Moms* were seen 3 months after mice were returned to e control diet. Other dietary levels of Aroelor 1343 end all levels of 1221 did not produce liver changes. Highly significant mean liver weight increase* after 8 months of feeding occurred in all Aroelor 1254 groups and in the group fed 375 ppm of Aroelor 1243. Threo months after mice were pieced on a 3 HONS 015964 control diet, Man Uvtr weights of each (roup had dscrested. The decreases war* highly significant for th# 37.5 ppm Aroclor 1254 and th* 375 ppm Aroclor 1242 groups. Ths* observations ar* (snerally consistent with those of other investigators which show regression of lesions when PCB dosing is ended, th* degree of regression depending upon the duration of th# recovery period. Data for mice reported earlier by Nagasaki, et si. (1972) appear to have been included in th* later publications of Its, et al. (1973a,b). It is interesting to note that the tumora war* called hepatomas in tbt fonaar publication and were labeled wU~di/f#r*atiatad hepatocellular carcinomas in the latter. Kimbrough and Under (1974) apparently also felt that these earn data on mice were reported in these 2 papers as they reference the production of hepetemae in mala 4d mica to th# later publication by Ito, ot al. (1973b). Stellar terminology troubles beset th* 1974 and 1975 Nagasaki publications. Data from the latar publication list 9/17 males and 4/17 females fed Kanecblor 500 aa having liver tumora. Table 1 shows data from th* earlier publication; among malee, 9/17 had nodular hyperplasia and 7/17 had hepatocellular carcinoma while the female incidence of 4/17 was described ss nodular hyperplasia. The hepatomas described by Kimbrough and Under (1974) were later referred to as "neoplastie nedulea (hematomas (sic], hyperplastic nodules)" by Kimbrough, et al. (191S). The use of the tern hepatoma has created confusion. With respect to terminology of tumor* in the mouse liver. "Hepatoma is a collective term used to describe the progressive stages of tumour development from the lesion called 'hyperplastic nodule" or "simple hyperplastic growth" to the morphobgically and biologically malignant neoplasms" (Turusov and Takayama, 1979). Novortholtss, whatever -4- MONK 015965 terminology is ussd to describe than, tumors wero attributtd only to PCBs containing 52-54 porcont chlorine. Lowor dots* of tbo S2-S4 ptrecnt chlorine materials, as well as lower chlorinatsd materials, wars not dascribtd as tumorigsnie. Tab!* 2 summarizes rssults of rat studios with PCBs. As in tho aouso studios, only soma studios with materials having a chlorine content of 52-54 porcont, or highor, woro roportod to produce carcinomas in tbo livers of rats, and oven for those materials this was not a consistent, reproducible observation. Odashima (1975) reported the results from a series of different testa used to evaluate potential carcinogenicity of several compounds, including PCBs. Two tests are of sufficient interest to mention here. One is the transplacental method in which rata were trotted for 3 days during pregnancy. These were days IS, 17, sad 19 or 14, 16, sad 19, depending on the strain used. The total dose was approximately the auudmum one that did not cause abortion or early death of the weanlings. In the other, the newborn method, pupa were dosed subcutaneously on days l, 9, IS. and 22 after birth. The maximum dose was one that did not cause early death of over 20% of the animals. The subsequent observation period in each teat wms "*>**> to one year after birth. For both Xanechlor 300 and ImiiMir 100, the transplacental test was scored negative tad the 4 '. ........ ". newborn ana as equivocal. Both of these tests gave positive results with erne known carcinogens such as 4-aminobiphenyl, benzo(s)pyrene, butyl-nitrosourea and N-methyl-H-aitrosouree. HONS 015966 Objective appraisal of chronic rodent feedinf studies is difficult. Scat of th* rodont studies in Tabit 1 and Table 2 art of tha type used to detect potent carcinogens. They uet relatively few animals, hifh doata of the teat material, and have a relatively abort duration. Studiea of that nature have beta included in Tablea 1 and 2 to illustrate the veriouj diagnoses of the rodent hepatic leaiona obaerved after dosing with PCBa and to aid in the atepwiee analytic of the reaulta of all the reported carciao(enicity studiea. la any short'tern teat with a few animals, the chance occurrence of acme tumors is e possibility which must be considered. The probability that an event is a chance occurrence is decreased if it is repeatable. Many of tha studies in Table 1 did act produce tumors. Those which were reported to produce hepatocellular carcinoma apparently were also reported in other publications as producing nodular hyperplasia, or tumors, or hepatomas. Since terminology of tumors and the use of those terms have subjective, l.e.. Judgemental, elements it may not be valid to makt direct comparisons between different studies on the basis of terminology alone. The rodent studies which wars of lass than lifetime duration raise e question as to whether or not cancers would have occurred had the feeding periods been extended. That question is speculative, and it eammet he answered Area the present daU. Nevertheless, negative studies even If they are of relatively short duration art part of the overall evidence which has to he considered. As e first step in the analysis of the data, the evidence shows that at e minimum, PCBe are not potent carcinogens to rodents because they do not product cancer* when tested In studies designed to detect potent carcinogens. . -- HONS 015967 With respect to further tnalyiia 0f the mouse studies, rroster signifiesncs can bo stuchod to the date of Kimbrough and Linder (1974) beesust their study bad a larfor number of animals, a longer duration, and a relatively high dietary level of PCB. In those respects, it more closely resembles conventional cancer studies. They did not observe any hepatocellular carcinomas. Thus, it can reasonably be concluded that PCBs are not carcinogenic to mice. Review of the rst data in Table 2 shows that there are 2 studies on PCBs with an average chlorine content of 40% (Levinskas, 1981 and Weitaan and Norback, 1979) and 3 on PCBs with an average chlorine content of 52*54% (Levinskas, 1981; MCI, 1978; Wasseraan, at al.. 1978). Results of those studios are consistent with respect to the absence of hepatocellular carcinomas, lids consistency reasonably suggests a conclusion that PCBs with those chlorine contents are not carcinogenic. Of the 3 studies with PCBs having an average chlorine content of 60%, one reported hepatocellular carcinomas (Kimbrough, et al. 1975) and 2 did not (Levinskas, 1981 and Wsltmaa and Norback, 1971). Since Kimbrough, et al. (1875) and Levinskas (1881) both uasd Lot No. AK-3 of Arocior 1980, the different conclusions they reached are not related to differences in the tent materiUL la addition to tha usa of a different strain of rat, IWbreugh, et al. (1875) used a different histologic diagnostic ertterit. Kimbrough, . ft 1. (1875) used the criteria for classification of specific hepatocellular lesions in rats developed et a National Cancer Institute Workshop (Squire and Levitt, 1978). That workshop recommended that the term "neoplastic nodules* replace to-called "hyperplastic nodules* because "Such nodules are proliferative lesions and . -T- HONS 015968 art known to be induced by carcinogens and, at tht leaat, they indie*tt *n increased probabiity for tha development of hepstoceilular careinems" (Squire and Levitt, 1975). However, Pitot, at al. (1978) in a diacuaaion of stages in tha prograaaion of hopatoeareinogancaia in rat livtr have ootad that "Tha demonstration of carcinoma call* that appaartd to aria* within tha noduJaa waa also reported (13)*, auggtating tha nodule waa a precursor to tha malignant neoplasm. On tha other hand, aa waa shown by Parbar and other*, the vaat majority of tha raganarating or hyper plastic nodule* diaappaarad on removing tha animal* from the carcinogenic diat. Thua, daapite tha more recant suggestion that the** nodule* ba termed "neoplastic nodule*" <14)*, it i* difficult to understand a precursor relationship of the nodula to carcinomas if tha existence of tha putative precursor is so transient." further, the use of those criteria for classifying experimental hepatic lesions waa considered and rejected by Ihiura, at al, <1976) in thair studies on tha co-carcinogenesis of Kanachlor 400 and 3*-nethyi-4-di**thylaminoaiob#na*n#. Thor* is concern that a presently benign leaien might at some future date er under other circumstances bo tranaformod into a malignant lotion. Kimbrough (1979) apparently had that concern when she stated "Although it hao been shown many times that the neoplastic nodules are part of the *' caniMfMie rispcmia they art not always included in the statistical imfcnf nf Mocasays, which may load to erroneously interpreted results, particularly when they arc classified as hyperplastic nodule* or "nodular hyperplasia" and whoa the number of animal* studied was small (Carcinogenesis Testing Program, 19T7)".* A similar concern appear* to hew* boon behind the statement in * recent review (Anon., 19*1) that "hexachtoroblphenyl administered to groups of SO mate and 50 female - - HONS 015909 Sprague Dawley rti at dietary levels of 0 and 100 ppm for 105 weeks was carcinogenic in female rats, producing an Increased incidence of liver hepatocellular carcinomas among the dosed animals (Norback and Weltaan, 1980. Personal communication)''. Contact with Dr. Norback (Ribelin, 1981) revealed that her data were available only as an abstract (Weltsan and Norbsck, 1979), and that the abstract and her oral presentation of the dau referred to the lesions as neoplastic nodules, i.e., net distinctly tumorous. These 2 examples illustrate the difficulty in establishing that cancer is not present, and they strongly suggest that the different findings reported by various researchers are a reflection of their orientation, training, and philosophical perspective. The latter is defined as the difficulty in separating whet is actually being observed under the microscope, from a concern over what it might have become if the animals had Uved longer. Since only an abstract has boon published, details on the studies by Weltman and Norback (1979) art limited . Their studies are of particular interest because they observed the sequential development of liver changes over a 2-year ported. They noted that hexacbloroblpbenyl was more toxic than tetrschlorobiphenyl, and that the more toxic, more highly chlorinated hexachlorobiphonyl produced neoplastic nodules only. Again, the weight of thm evidence leads to a reasonable conclusion that the carcinogenicity of bipbanyls with am average chlorine content of 60%... has not bean established. t HONS 015970 Overall, on* can surmise that th* inability to resolve th crucial issue of whether or not a Union la neoplastic in character was ono of th* factors which 1*4 to th* following statement from a recent Surgeon General's report: "Science and society have not yet arrived at a final consensus on th* definition of a carcinogen either in the huaaa population or in experiaental animals* (DHHS, 1980). Metabolism Studio* Several reviews (Goldstein, 1980; IARC, 1978; Matthews and Sato, 1979; Roberts, et al,, 1978; and Safe, 1980) discuss the metabolism of specific isomers as well as mixtures of PCBs. While there are some exceptions, th* following general conclusions can be drawn. Both th* degree of chlorination and th* positions of th* chlorine substituents determine the ease with which PCBs are metabolised. In general, th* lower chlorinated ones are metabolised and excreted mere readily while the mere highly chlorinated materials are stored hi fat. Th# process of metabolism converts th* fat soluble PCB into a water aohible hydroxylated derivative which can be excreted in the urine. This metabolism and excretion appears to occur vis area* oxide intermadiates, and th* carcinogenicity of some compounds bee been attributed to formation of arena oxide interne- dints* and their Muffing to aubceOular macromolcculae. Chlortnetkw at the 4,4* pmittloa bkeks th* metabolism and excretion of PCBs as illustrated Misti. . ............................ MOMS 015971 Studies to rata (HinssU, ft al., 1977) and to Diet (Moral** and Matthews, 1971, 1979) ara of interest baeauaa they report the metabolism of iaeaar* which were fad to rata for 2 year* by Weltaan and Norback (1979) HanaaU, it 1. (1977) gave rroupa of male rata single intraperitonea] injection* of 0.2 tuaoles/kg of 2,2',5,5'-t*tra- or 2,2\4,4',5,5'haxachlorobiphanyl (TCB and KCB, respectively). They aaaaurad changes to hepatic mixed function oxidases and the persistence of the PCB to the liver* of animals killed at selected intervals for 35 days after dosing TCB produced a transient, but significant increase in O-dcaethylase activity only at day 3 while HCB produced significant increases in O-denethylas* and aniline hydroxylase activities within 24*41 hours. Induced enzyme activities by HCB peaked at 4-6 time* control activity during days 7-14 and were about 3 time* control activity at the end of 35 days. Sven though equimolar amounts of each isomer were given, Uver residues of HCB one day after dosing were about 7 times higher than these of TCB. HCB residues decreased relatively slowly with time while TCB residues were mere rapidly aliminated and had almost returned to control value* by 35 day*. Livers from HCB treated rate had centrolobular necrosis at day 35. Thty also ware significantly Increased in sise, showed increased anouate of smooth endoplasmic reticulum (SER), and appeared la eontain increased numbere of microbodies and reduced tr amounts of 'rough andaplasmic reticulum throughout the 35 day interval. By oomtraat, TCB treated livers were similar to control ones except for occasionally increased aggregates of SIR on days 3 and 4. Hansel). at *1. (1977) stated "It would be interesting to speculate that...2,4,5,3*,d'.S'hexaehlorobiphenyl undergoes direct hydroxylation whereas 2,5,2\5*tetraehkrohipheayl undergoes bfotraasforoatiea via the arene oxide intermediate, thereby accounting for the different slope for the - U- HONS 015672 elimination curve of the latter compound." Thus, th* metabolism (Hansell. ot si. ItTT) and feeding (Weltman and Norback, 1979) study results load to somewhat dlfforont conclusions. Th# moro potent enzyme Inducing, loss readily excreted HCB which appears to resist hydroxylstion produces neoplastic nodules in rat livers. By contrast, the more readily exereud TCB, which apparently is excreted via an arena oxide intermediate, does not produce tumors when fed to rats for 2 years. Covalent binding to cellular oacromolecules also appears to be greater for the store readily metabolized PCS isomers. Morales and Matthews (1971, 1979) compared the covalent binding of 2 hexachlorobiphenyls; the more readily metabolized 2,2,,3,3,.5,5'-hexachferebiphenyl <2.3.5-isomer) and the mere elowly metabolised 2lr,4,4\S,S,>hesachferobiphsnyi (2,4,5-Isomer). Bach PCB. with a radiocarbon label, wea given orally to group# of nice at a dosage of 7.25 mg/kg on each of five successive days. Animals were killed 1, S, and days later. The concentration of each PCB was datarminad fat liver, muscle, and lddnay. All tissues had consistently higher concentrations of the loss readQy metabolized 2,4,5-isomer, The moro roadily metabolized 2,3,5-isomer showed a consistently greater binding to purified macromolecules. The binding was at least one order of augaitude graotor than that aeon with the 2,4.5-isomer. Results of sairnal foodinf startles with the 2,3,5-isomer would be of particular interest to rtztatmlBa the degree of corrslatioa, if any, between the carcinogenicity of this loaner and its covalent binding to ctlhtlar mscromolecule#. Taken as a whole, metabolism studios suggest that if PCBs are carcinogenic, it should bo those PCBs which are more readily metabolised and excreted, l.e., the lower chlorinated material*. This is in sharp - IB - HONS 015973 contrait to result! on animal studios in which questions of carcinogenicity arias regarding higher chlodnatsd materials only. On baianco, metabolism studios on tho formation of arant oxide intermediates would load one to axpset lower chlorinatad materials to show a more pronounced carcinogenic response in animals. Conclusions drawn from metabolism studies tre not supported by animal feeding studies. Co-Carcinogenesis studios The position end degree of chlorination of ?CBa also are important in inducing microsomal mixed function monooxygenases (Goldstein, i960 and Yoahimurt, et al.. 1979). Such induction of microsomal monooxygenases could altar tho metabolism of exogenous and endogenous substances in the body, as reported in e variety of studios which have been conducted to determine whether PCBa might be cocarcinogens. These an summarized in Table 3 and discussed in greater detail below. Dchlyama and Chiba (1974) inserted 20-methyleholanthrone impregnated threads into tho uteri of virgin mice and fed them diets containing up to 100 ppm of Kanechlor 400 or DOT. Animals wen killed at various times and the cervical epithelium waa examined for cancerous changes. Kanechlor 400 dosed mice showed no significant changes as compand to the eoatrple, but - these receiving 100 ppm of DOT "showed e remarkable fsmijpcy towards the induction of cancer." 40*- ' - Diets containing benaene hexichloride (BHC) isomers and Kanechlor 500 tepantely and in various combinations wen fed to male mice by Ite, et al, (1973a ,b). Concentrations of the BHC isomers ranged from 250 ppm to 13 HONS 015974 SO ppm. The amount of Kanschlor 500 was 2S0 ppm or 100 ppm. Tst groups eonsistsd of 20 to 30 animals. Whan fad lions, -only -8HC at 2S0 ppm produced a high incidanc# of nodular hyperplasia and a moderate incidence of hepatocellular carcinoma. A diet containinf 250 ppm each of e-BHC and Kanechlor $00 increased the number of hepatocellular carcinomas. In comparison, combinations of 100 or $0 ppm of e-BHC and 2S0 ppm of Kaneehlor $00 yielded a moderate incidence of nodular hyperplasia and produced only a few hepatocellular carcinomas. There were a few nodular hyperplasias in mice fed 100 ppm each of e-BHC and Kanechlor $00. A mixture of $0 ppm o-BHC and 100 ppm of Kanechlor $00 waa without effect. Diets container 2S0 ppm or 100 ppm of #-BHC and 380 ppm of Kanechlor $00 produced both nodular hyperplasia and hepatocellular carcinoma* at a lower incidence than that seen with comparable diet* of e-BHC. Ho liver nodules war* seen with $0 ppm of l-BHC and 2S0 ppm of Kanechlor $00 or with 100 ppm of each. y-BHC end Kanechlor $00 did not produce liver nodule* either sinfly or in combination. Hagaiaid, at *1. (1974, 197$) reported e similar series of experiments except that Kanechlor 400 was included. The same concentrations of the PCBs, ISO and 100 ppm, were used. Thtir tast groups consisted of 20 to 31 male mice. For e-BHC and Kanechlor $00, they presented tuentieny the same data as Ito, at *1. (1979a,b). While fsnarlInr 400 did mot produce liver nodules when fed alone at 290 ppm, * there was an increase, fat the incidence of hepatocellular carcinoma* whan it was fed hi combination with 2S0 ppm of e-BHC. The increase was similar to that reported by It, at al. (1973a,b) for e-BHC end Kanechlor $00. A combination of 100 ppm of e-BHC and 2S0 ppm of Kanechlor 400 produced only a fsw nodular hyperplasias and 100 ppm of each in the diat did not induce any liver nodules. - 14 HONS 015975 Tba affects of PCBs oo rumor induction by diethylnitrosamine (DEN) hive boon studied extensively. Milo nta were given 2$ ppm of DEN in their drinking water and 500 ppm of Kanechlor 500 in their diet concurrently for 20 weeks, returned to a control diet for 4 weeks, and then killed (Maldura, et tl. 1974). Neither Uver tumors nor nodular hyperplasia were seen, even though rata receiving the ease DEN treatment alone had a 92% incidence of liver cancer. The livera of rats treated with Kanechlor 500 only also had no tuaors. When administered after DEN, Kanechlor 500 markedly enhanced Uver tumor production as reported in a series of papers by Nishixuml (1976, 1979a, 1979b). He described studies in which sale rata were given 50 ppm DIN in drinking water for 2 to 10 weeks, after which they were intubated twice weekly with a com oil solution of Kanechlor 500 for 4 to 12 week#. Kanechlor 500 doses were either 0.2 ml of a 5% or 0.1 ml of a 10% solution. Since experiments were terminated at 20 or 52 weeks after doting, some animals received untreated diets prior to sacrifice. Only one paper (Nishixuml, 1976) contained results for animals doted only with Kanechlor 500 alone. That treatment produced enlargement of the ttrera of rota hot no neoplastic lesions. After pre treatment with DIN, however, hepatocareiaogeneeis wae enhanced as evidenced by the earlier appearance and e significant increase in the number of tumore. a theflar dosing pattern waa used by Preston, et l. (1901). Mala ran were given drinking water containing 66 pg DEN/ml (It ppm) for f woaks after which they were fed far 19 weeks on a control diet or one containing 100 ppm of cither of 2 Aroclor 1254 diets. One was Arador 1296 as received. The other was an ArocSor 1254 which the authors claimed to have purified by removing polychlorinated dibenaofursn (PCDF) impurities by adsorption onto end subsequent elution from activated riorisfl. . Ho analysis wae made of Aroelor 1254 as rsetivad for 15 HONS 015976 PCOf. The authors raportod recovery of PCDF at a level comparable to 30 PC of tatrachlorodibanaofuraa (TCDF) par 10 g of Aroclor I2S4 TCDF was used as a positive control and a standard for quantification for the purification process. Other animals received one of the 2 Aroclor 1254 diets only during the latter 18-week interval. No evidence of hepatic tumor formation was seen in control animals or those receiving 100 ppm of either Aroclor 1284 diet. However, using the diagnostic criteria of Squire and Levitt (1923). they reported that both Aroclor 1254 diets resulted in significantly greater incidence* of hepatocellular carcinomas in rate pretreated with DXN aa compared to those doted with DEN alone. Thus, they concluded that the hepatic tumor-promoting ability appears to reside hi Aroclor 1254 itself. Aa the authors of this paper pointed out, it cannot be concluded that PCDfs art not promoters of hepatoesrdaogenosis sine# appropriate studies have not been done on PCDfs alone. Similarly, their findings do not invalidate the hypothesis that some of the other effects reported for acme PCBi may have been due to PCDP impurities. The dependence of the inhibition or promotion effect of PCBs on DKN-induced tumors on the dosing sequence was illustrated in a different manner by Niahiauml (1990). Pregnant famals rats were given oral doses of 200 ag/kg or 10 mg/kg Xanachlor 500 on days 5, 10, and IS of geetattam and were allowed to deliver their pups. At 29 days of sge, pups were given SO ppm of DEM in their drinking water for 5 weeks. Groups of these pupa were sacrificed at 19, 20, and 24 weeks after the start of DEN dosing and their avers were examined. The number of liver tumors in the offspring from Kanechfer 500 treated dams was significantly decreased as compared to the oootroLs. The decreases were more pronounced in males. This decrease in DIN-induced tumors sppsrtntly resulted from induction of . - 19 HONS 015977 microsomal enaymea in the liver* of the pup*. Those livers contained Xanechlor S00 residues and electron microscopy showed an increase in the smooth endoplasmic reticuiua of hepatic ceils. Rainbow trout were fed 8 ppb of aflatoxin Bt (AFBi), or 100 ppm of Aroclor 1294, or a coabiaation of both for a year (Headrick*, et *1.. 1977). At intervals durtaf the year, soae fish were killed. The reaainder were killed at the end of that da*. AH livers were examined Crossly and microscopically. There were no tuaor* in those fed a control diet or Aroclor 1254 alone, and growth wa* not affected by 100 ppa of Aroclor 1254. The number of tumor-bearing fish in the group fed ATBX, phi* Aroclor 1254 was significantly reduced (to less than one-half) when compared to those fed AFB|, alone. There also were fewer tumors per liver and the tuaore were smaller in those fed the mixture. la the studies cited earlier, Makiura, et al. (1274) also fed combinations of 500 ppm Xanechlor 500 with 300 ppm of S'-aethyl-d-diaethyLsaiaoasobenseno (3'-M*-DAB) or ISO ppm of N-2-fluorenylacetsmide (2-FAA). Results similar to thoee whoa PCBs wore fed concurrently with DEN were obtained. Xanechlor 500 reduced the incidence of liver tumor* to aero from 81% for thoee treated with 3'-M*-DAB and from 54% for those treated with 1-PAA. However, the effect of PCBs on the tumorifonicity of i'-He-DAB eppeare M -depend on the doting sequence as shown for DIN. Xhaura, et d. (1878) used different sequence* to food groups of rats diets containing 400 ppm of Kanochlor 400 for six months and 800 ppm of S'-Me-DAB for 2 months. Some received Xanechlor 400 slon# and some only 3'-Me-DA. Others received on* diet, then the other, after a 2-month interval on control diet. ' A final group received Xanechlor 400 for , - 17 - HONS 015978 4 month* usd both materials for another 2 months. No hepatocercinomss were produced by Kanechlor 400 slone or whn it was given before or during the overlap with 3'-Me-DAB. No hepatocarcinoaas occurred in control animals. The incidence of liver cancer roae froa 13% in those receiving 3'-MeDAB alone to Ml in the group which received Kanechlor 400 after 3'-Me-DAB. The inhibitory effect of Kanechlor 500 on rat liver tumors was evident when rets were dosed with two carcinogens (Makiura, et al.. 1974). Combinations of 3'-Me-DAB and DSN or 2-FAA and DEM were adainistersd with and without Kanechlor 500 at the concentrations stated earlier. The liver cancer incidence fell from 92% to 1% when Kanechlor 500 wea given to rate doeed with 3'-Me-DAB end DIN. It went to aero from 52% for those receiving 2-FAA and DIM. Nishiiuai (1979a,b) studied the effects of varietur combinations of DDT and sodium phcnobarbltal (SPB). in the absence and in the presence of Kanechlor 500, on the induction of rat liver hepatocellular carcinoma by DKN. Pretreatment rtf rata with DIN followed by DDT, by SPB, or by e combination of both produced lew incidences of liver cancer whBa DEN pretreatment alome did net induce cancer. When Kanechlor 500 was tasMad with each of tha preceding dosing regimens, there was a marked tN|Pitpi ih ttvar cancers. Increases resulting from joint adminiatratioa of cempouada aftar pretreatment with DIN were lower than those observed for DIN followed by Kanechlor 500 alone discussed earlier. Its, et al. (1979) fad diets containing 200 ppm of 2-FAA to male rats for two weeks and than a diet containing 1000 ppm of an unspecified Kanechlor . - II HONS 015979 ixturt for weeks. Partial hepatactomias (PH) wart performed on some ***** during the third wstk of tho atudy. Feeding of 2-FAA w* without offoet, but Out diot plus PH produced a few hyperplastic nodule* in tho livor. Treatment with both diot* caused an even grtator incidonc* of byporp1**^ noduloa and both diot* combined with PH produced a narked riao in the number of liver nodule*. DiGJovanai, et al. (1977) and Berry, at *1. (1979, 1979) etudied ArocJor 1254 in a two-stag* mouse akin carcinogenesis aaaay to aee if it was a tumor initiator or promoter. The shaved akin of female mice was dosed with ArocJor 1254 at a level of 100 pg or 62$ pg per amuse. This was applied atone aa an initiator or from S minutes to 72 hours before initiation with 7,12-dJmethyIbeaz(a]anthracene (DMBA). On* week later, mice received twice weekly applications of 8 pg of the phorbol diester promoter 12-0-tetradecanoyIphorbol-13-acetate (TPA) far 32 weeks. Animals were observed for both papillomas and carcinomas. Aroelor 12S4 slant produced * few pmpOteaus. The authors of these reports apparently faced a dilasuia common to sdmntists, i.e., how much significance should bo attached to the obeervation of a low incidence finding which may or may not have a causal ralationship so treatment. On the basis of those few papillomas, Aroelor UM waa called a "weak tumor initiator" (DiOiovanni, 1171). Later, It mao described ea possessing "little or no tumor-initieting properties" (Berry* et al.. 1979). White Aroelor 1354 had a negligible effect am tumor induction when given 5 minutes before an initiating dots of DMBA, it markedly inhibited tumor induction when givan II to 72 hours before DMBA. In other studies, an initiating dose of 200 nmole of DMBA woo applied to tho shaved backs of mice. A/tor one week, 100 pg of Aroelor 1254 waa applied twice weekly for 30 weeks. Aroelor 1254 filled to 19 HONS 0159 promote tumors white 0.2 Hf doses of TPA applied in a similar manner induced aa average of 0 papiUomaa par mouss. Tha author* concluded that Arocior 1254 poaaasaad littls or no tumor-initiating or tumor-promoting proportio*. . Tha transplantsbilitp aad growth of Walker 256 carcinosarcoma la rat* was inhibited top Arocior 1254 (Ktrkvliet aad KJaaldorf, I977a,b). Diata containing up to 800 ppm of Arocior 1254 war* fad to rmts of both sexss for 30 dap*, aftar which thap war* given intramuscular injection* of Walkar tumor calls. Nina dap* later, during which time thap continued to receive Arocior 1254 in thair diata, animals war* killad and tha tumor* war* dlaaactad out aad weighed. Mean tumor weights of all Arcelor 1254 fad group* war* aigniflcaatip reduced aa compared to thair sex-matched controls, and the raductioaa war* do**-related to tha dietary level of Arocior 1254. Body weight gain was depressed in mala* readying 400 ppm or more of Arocior 1254. Parnates showed reducedbody weight at 100 ppm, tha lowest level fad to that mx. tatraparitonaal injections of SO to 200 mg/kg ovary other dap for 14 daps aftar injection of a small of Walkar 256 calla <10*) inhibitsd both the davslopmant aad growth of tha Walker tumor. The number of turner takes aad tha six* of tha tumors wars raducad and the tumor latency parted was increased. 14f weight gain was raducad only at tha 200 mg/kg dosags. Alttruata tep tetnparifionaal fnjaettena of 100 mg/kg and 200 mg/kg of Arocior 1254 aftar a largo inoculum of Walkar 256 calls (IQT) wart continued for 80 daps. Control recalling tumor colls only had 100% mortality bp dap 20 with a mean survival tha* of 13.5 days. Moan survival time* war* significantly increased to IT.? days aad 18.8 days for animals dosed with 100 mg/kg aad 200 mg/kg of Arocior 1254. A faw animals survived to 80 - 20 - HONS 015981 day*. end four receiving the higher doa(* of Aroclor 1254 showed total regression of their bilateral tumor*. Tumor growth ratoa in Aroclor-treated animals war* ligni/icantly reduced, Again, body woight gain waa dapreaaed only at tha 200 mg/kg doaaga. An experiment was undertaken to datanaiaa if thar* waa an optimum time period for tha antitiueor affact of Aroclor 1254. In trsperitones! injections of 100 g/kg of Aroclor 1254 wara given on 5 consecutive day* before tumor inoculation, on 5 day* aftar inoculation, and daily from 5 day* before until 10 days after inoculation. This atudy waa ended 14 day* after the initiating tiuaor inoculum waa given. While all doting schedule* reduced tumor growth, there were variation* in the reapoeaes. The greateat inhibition of tumor growth resulted from doaing anheals for 18 days. Ahaoet equally effective were pr*treatment and treatment atarting with inoculation of tha Walker call*. The delayed treatment starting aftar 5 day* waa least effective a* the tumor had become established. While early treatment reduced tunor growth, it was lesa effective in preventing metaatasea and death of the anhaala. Serkvllet and XoOer (1M0) offered group# of male mice diets containing 10, 100, or 500 ppm of Aroclor 1284 for 18 weeks prior to injecting them with MSS, an established tisane culture cell line derived from Moloney sareemn virus. They measured tumor growth and cell-mediated toxicity ever Mm next if days. A dietary level of 500 ppm of Aroclor 1254 'esuttad in markad weight loss end deaths. This waa reduced to 250 ppm t ll weeks, and a few weeks later surviving animals were placed on a central diet. They reported that Aroclor 1254 pratrsatment enhanced the frewth rate of the MSB tumor and inhibited or delayed the development of eettblar immunity against the tuawr. MOHS 015952 22 KoUtr (1977) fsd groups of talc* diets conuimnf 3.75 . 37.5. or 375 ppm of Aroclor 1221, Aroclor 1242, or Aroclor 1254 for Booth*. About 2 weeks after being placod on t*t dioti, some mice in each group war* inoculated intrsperitoneally with Moloney leukemia viru*. None of the dietary levels of any Aroclor affected, i.e., did not promote or induce, the oncogenesis of Moloney leukemia viru*. Since PCBs are enzyme inducer*, their biological effects resemble those of other enzyme inducers. Feralno, et al. <1578) noted that phenobarbital had a specific tumorigeuie enhancing effect that was restricted to the liver. With the exception of the mouse skin painting studies and those in which tumor cells or a virus were injected, all of the studies in Table 3 were concerned with liver tumors. Pertiao, et al. Cl*71) also pointed out resemblances between phenobarbital and PCBs. Both substances "enhanced hepatic tumoriganeeia" when given after DIN, and both "exerted a protec tive effect against hepatic tumorigenesis" whan administered concurrently with AAP or DIN. The comparison between PCBs and phsnobsrbiul is sxtsndsd by the observation of Berry, et al. (1979, 1979) that PCBs did not promote skin tumors In mice pr*treated with DMBA. Persino, et si, (1971) refer to s study4 in which skin tumorigsnesis was not enhanced in mice fed phenobarbital after stdn painting with DMBA. IMt; eld. (1971) described the induction of ornithine deosrboxylsse (ODC) In mouse epidermal cell cultures by TPA. Aroclor 1284, apparently et much higher concentrations then TPA, induced s low but reproducible stimulation of ODC In the same system. They also noted s report4 that s high intrsperiteneel dose of Aroclor 1284 induced ODC in the liver of rate. After commenting that PCBs "warrant further Investigation into their HONS 015983 possible tcHon it tumor promoters," thoy added that "These compounds are beiaf tested on carcinogen-initiatsd mouse skin (T.J. Slags, personal communication)". The series of publications by Berry, st al (1973. 1979} and DiCiovanni, st al. (1977) show that PCBs are not promoters on mouse skin. Authors of some of the mutagenicity studies to be discussed (Dana and Urban, 1980; Norback, et al.. 1981; and Stadnicki, et al.. 1979) have suggested that PCBs may act as promoters of carcinogenicity. In general, those comments appear to have been offered as hypotheses to aid in understanding the observations which had been made. This also appears to be the case for the NCI study on Aroctor 1284 which was reviewed by the Data Evaluation/lUak Assessment Subgroup of the Clearinghouse on Environmental Carcinogens. That group, chargsd with the responsibility of providing a peer review of NCI bioaasay reports on chemicals studied for carcinogenicity, mede and accepted a motion to add the following to the report summary: "Bated on the liver proliferative lesions in the treated rata and published reports, it is suggested that Arodor 12S4 may bt a tumor promotar" (NCI, 1971). With reapeet to tumor prossotion, Weisburgsr and Williams (1980) stats that "carttft Macaw of ttvor mataboiic enzyme eyetema, such as phsnobarMQ, IMP? and BUT, when administered after minimal doses of primary hepetocardnogens exerted a powerful promoting offset". Aa mentioned earlier, PCBs also induce Uver metabolic onxyme systasis. However, since the carcinogen alone produced tumors in animals in the co-ctrcinogenesis studio* summarised in Table 3, it appears that the doses were greeter than minimal. In addition, even though the co-carcinogenesis studies were of 015984 23 HONS shorter duration, several used Much higher dietsry ivU of PCS* thus thoss fd to rat* for 2 poor*. The latter studios sro the ones which gave rise to questions of carcinogenicity. Thus, while tho evidence indicates that PCBs are not carcinogenic, thsir reported effects in rodent livers following prolonged exposure in conjunction with an initiator aijr bs promotion or inhibition. When administered to animals together with s biological agent, PCBs show a promoting or inhibitory offset similar to that seen with chemical agents. Pretreatment with PCBs inhibitsd the growth of Walker 285 carcinosarcomas in rats and ineraaaed thsir survival Urns (Xerkvllet and Xkameldorf, i9T7a,b), enhancsd the growth of MSB tumor in mice (Xerkvllet and XoQer, 1979) and neither presMHted nor induced the oacoganesis of Moloney leukemia virus in mice (XoQer, 1977). Mutagenicity Studies Mutagenicity testing of PCBs has ranged from bacterial systems to intact animal studies. A aeries of studies with eleven bacterial teat strains (Saddle and Bruce, 1977; gain, et si. 1971; McMahon, at *1- 1979; OdashJma, 197S; Prebet, et al.. 19*1; Suginura, et si.. 1978; and Wyndham, et al.. 199*} are suaaaarised in Table 4. Haia, et H, (1978) used bolb pbenobarbital and Aroclor 1254 to induce liver enaymes in rata for prsgsritfcin of M activation system*. They also tested 4-hydroxy2,f,S,S'*tetrselilerobipbesyl and *,r,5.'*tetrachlorobiphenyl-3,4-oxide, a known and a presumed metabolite of 2,2\5,5'tetrachlorobiphenyl, respectively. All tiara* materials gave negative response* with each activation system. Odashlma 11979) presented data in tabular form from a aeries of screening teste. In addition to the result* shown in Table 9, . **' HONS 015985 bacterial strains WP2, TA100, TAM, H-17, M-4S, W3110, tad TA1978 ire shown ia groups in their Ttbit S with t text notation that not all chemicals were tested with etch ttraia. For Ktnechlor 300 tnd 500, such of the precedinr strain $ at were tested showed t nefttive response. The (roup consisting of H-17 A M-4S, WP, try" (her* A hcr") shows t plus sign for Xtaechlor 300. Presumtbly, one or more of those bacterial strains gave a positive response. Ktnechlor 500 wtt listed at giving t negative response with the Utter group. With the exception of Wyndham, at tl.. (1976), til of the studies were negative with tad without the addition of a mlcrotoeaal enzyme activation system. It ia somewhat difficult to reconcile the text tad the graphs la the publication by the hitter authors. They stated that their 'results dearly showed that as the degree of chlorination decreesed the mutagenicity increased, a concentration of 100 pg of 4-chbrobipheayl in the test medium gave over 2000 revertaat colonies per plate. The higher chlorinated biphenyls show very little activity as mutagens". While Figure 3 in their article shews their marked increase in revertanta for 4-chlorobiphenyl, the sane figure else shows results for Arodor 1221, which averages 1.15 chlorine units per molecule. The mutagenicity of Arodor 1231 W ant discussed in the text, hut Figure 3 shows that st a oonceatratisn of 100 pg per piste, it produced about one-tenth of the revertamt colonies that 4-chlorobiphenyl did. Thus, a 15% faicrsase ia chlorine content produced a 10-fold decrease in the reported mutagenic activity. In light of that sharp reduction of mutagenic activity with a alight chlorine increase, it ia difficult to understand the statement ia their text that Arodor 1254 "was only weakly mutagenic." Results from Arodor 1254 (avertga of 4.90 chlorine per molecule) are not shown in their - 25 HONS 015986 Table 3 but 2,2',3,5'-tetrachlorobiphenyl (average of 4 chlorine per molecule) was pretented. At 100 pg per plats. 2,2\5,5'-tetrachJoroblphtnyl was virtually devoid of mutagenic activity. Overall, primarily because McMahon, et *1. (1979) reported that 4-chiorobiphenyl was not uUfank, it can only be concluded that the finding of Wyndham, at al. (1978) are aberrant. A aimliar conclusion that the findings reported by Wyndham, et al. (1976) art unfounded because attempts to repeat them have been unsuccessful was reached by ths Sute of California (Anon. 1M1). la a variant of this tost, Stott and Sinnhuber (1978) usad Arcelor 1221, 1343, 13S4, and 1360 to induce the mixed function oxidase system of the liver in trout. The submitocfaondrisl fraction of those livers was used to activete the metabolism of A7Bt which was assayed for mutagenicity using strain TA 1S38 of 3, typhimurium. Ths mutagenic response was decreased compared to the concurrent control using untreated trout liver. A general pattern of decreasing response with increasing degree of chlorination was observed, except for Arocfer 1260. Induction of mutagen detoxifying ensymt systems was suggested as a possible explanation for the apparent conflict between the reported induction of trout mixed function oxidases and the decrease in mutagenic activity. These results are consistent with those at Hendricks, et al. (1977) discussed earlier who reported that Arocisr 1394 reduced hepatic tumors in trout fed AFBj. (hi the basis of slower sedimentation rates in alkaline sucrose gradients, StadnicU, et al. (1979) reported that 2,2' ,5,5'-tetrachU>robiphenyl (TCB), a mixture of the 3-hydroxy and 4-hydroxy derivatives of TCB and ths 3,4-epoxide of TCB induced single strand breaks in DMA of L-929 cells. 39 MOWS 015967 At 100 pf/*l, each of tho three materials caused all of the DHA to com out in fraction* at tha top of the gradient. The epoxide cauaad tone breakage down to 1 yg/ml, Tha sixcure of hydroxy derivative* caueed significant breakage at 30 pg/al and only alight breakage at 1 and 10 yg/al. TCI, tha leaet potent, cauaad lasaar breakage at 30 yg/ml and wa* without affect at 1 and 10 pg/ml. UQmon and Ramel (1974) conducted genetic testa on adulta and larvae of Droaophfla aelanorastsr fad Clophen 90 and Gophan SO. Thaaa FCB mixtures ware without affect on tha loss of sax chromosomes used to measure chromosome breaking action and nondiajunctfoa of the sex chroeeotomes. Taaine (1900) has described a (pacific locus teat using tha silkworm (Bombvx mort). Neither Kanachlor 900 nor Kanechlor 500 showed mutagenic activity in that ayatam. Koopingarner, at al. (1973) induced mitosis in cultured human lymphocytes with phytohenagglutiain and treated them with 100 ppm of Arodor 1334. There waa no effect on the mitotic index, satellite association, chromatid gap* or chromatid breaks in comparison to control human lymphocytes. Odashhaa (ItTt) also studied tho incidence of chromosomal aberrations. Thair la stm tmt mad Teshida ascites sarcoma cells cultured for $ to 72 hours hi tho presence of FCBa at concentrations producing a minimal or 80% growth inhibition. Kanechlor 900 gave a positive response and Kanechlor 300 a nagativt one. for an fn vivo ayatam, they examined bone marrow cells 8 to 49 hours after adult or newborn animals were given an approximately lethal dose of PCBs. This test gave the opposite result; Kanechlor 800 waa positive and Kanechlor 900 was negative. They noted - *7 mqnS 015988 that chromosomal aberrations frequently occurred in control* and that cheaticaii were caUed positive when they induced mere than twice the aberration* in control*- In a Syrian hamster cell transformation system (Pienta, 1980), Aroclor 1254 was ona of sevsral chtmicals teatod double-blind. It gava a nsfative result. Mouse embryo fibroblasts also have been exposed to different PCBa. Neanow, et al. (1981) studied the cocarcinofenic action of agants which increase microsomal mixad-function oxidase activity in the C3H10T*CIA transformation assay. After a 48-hour pretreatment with Arodor 1254. cells were then treated with benzo(a)pyrsne [BCa)P] and the gent for an additional 24 hours. Aroclor 1254 did not increase B(a)P-aediat*d transformation and no Type II or Type II! fed were observed. Norbaek, et al. (1974. 1M0, 1941) exposed C3K10TH cells continuously for 4 weeks to 10 pg Aroclor 1254 por ml of medium. Treated ceJQa developed Type 111 fed. Cells exposed to the same concentration of Arodor 1254 for. 24 hours or to 1 Mg of Arodor 1254 per ml of medium did not develop Type 111 fed. Continuous exposure of cells to Aroclor 1280 end 2.4.S,r.4%5'hexsehlorobipheayl also caused formation of Type 111 fed. A dome frees a focus transformed by Aroclor 1254 induced sar coma fomatiom wham inoculated in irradiated mice. Pod from Aroclor 1280 a*d |,4,l,T,4\l,-hexachk>robiph#nyl had not boon characterised further. MM8 tsumafomatioa occurred only after continuous exposure, Norback. e` ot >1. (HID suggested that the affects of PCBa in culture include promotion. Whom fod to rats, however, 2,4,5,2'..5'-hexachlorobipheny1 produced neoplastic liver nodules, not hepstoearcinomea (Woltman and Norback, 1875). HONS 015989 Woof, ft al. (1979) claimed that 4-chlorobiphenyl induced to increase in unscheduled DNA synthesis in Chinese hsaster ovary call cultural in tha praaanca of hydroxyurea, a chemical afant which suppraasas normal raplicativa DNA synthesis. Faw datail! wara praaantad regarding tha validation and raliabillty of thair taat system. By contraxt, Arodor 1254 fava a negative raaponaa in an unschadulad DNA aynthaaia in primary cultural of adult rat hapatocytaa (Probat, at al.. 1181). Tha latter autbora praaantad raaulta of an extended aariaa of compound! taatad in thair ayatam. Dans and Urban (1990) have propoaad using tha mitoganie raaponaa of tha rat adrenal cortex after dosing tha animals with a tost substance as a short*term test for evaluating tha promoting action of chemical compounds. Clophen AM, Dolor 103*. Odor 109*, and Phanodor DP9 all gave positive responses in their taat ayatam. At 3-9 days of incubation, embryos from ring doves (Streptooella riaorta) fad a control diet or one containing 10 ppm of Arodor 1254 wara examined for cytogenetic changes (Peaks!!, at al.. 1979). Tha raletive frequencies of chromosome aberrations In the largest chromosome pairs in meUphete calls of sQamboic amn and Bmb bud origin wore scored. Tha 9 control embryoe had s 'moan aberration rata of o.H (0-2.0). Tha aberration rata in IT mrtnryma from Arodor 1284 treated birds was i.<% (0-9.4). In tha latter group, therm was one chromosome rearrangemant, 13 embryos with sborration rates sxeaading tha moan control rate, and 4 embryos which axoadad tha higbaat control rata. Arodor 1243 woo injected into fertile White Leghorn eggs to give estimated final concentrations of 10 or 20 ppm (llasak and Marcun. 1978). After 4 or 8 days of incubation, eggs were I* HONS 015990 injected with coleaside, Incubated an additional 4S-60 minutes, and embryos ware harvested for examination. There was a hifh degree of early embryonic death, and a few live embryos showed drastically retarded development without malformation. The first five pairs of chromosomes, constituting over 50% of the chromatin material per cell, were examined for detection of clastofenesii. There were no chroaosoaal aberrations. Heddla and Bruce (197?) injected aice with Aroclor 1254 for five consecu tive days and then examined bone marrow preparations for chromosomal breakage and sperm cell preparations for sperm with abnormally shaped heads. Whila nonspecific factors cam indues sperm abnormalities, the m authors believe the latter also can result froai point mutations or small delations. Aroclor 1284 was judged to be neither carcinogenic er mutagenic- Dikshith, at al. (1978) gave mala rata oral dosages of SO mg/kg of Aroclor 1284 on each of 7 consecutive days. Animals ware kilted at intervals over the next 2 dsys. Thoee scheduled for cytogenetic analysis wars injected with colchicine 2 hours before killing, after which time the seminiferous tubules war* prepared for such study. At autopsy of the ether animals, testis, epididymis, and aver weight were recorded and urttMii ware prepared for microscopic study and histochemical deteralmetiome. Livers were markedly enlarged and shewed a significant increase in weight compared to controls. There were no differences in body weight or weight end'appearance of testis, epididymis, or vas deferens between control and treated rats. Aroclor 1284 treated rati showed a few meta phase figures with abnormal chrotsosomes whch appeared to be sporadic and not specific to any one typo. Histological examination showed teatie - 30 - MQNS 015991 and epididymis froa Arodor 1254 treated rau were comparable to controls although interstitial cells were increased in Aroclor 1254 dosed rats. These ceils showed an increase in scid phosphatase activity. The authors concluded that they "found no evidence to suggest that Aroclor 1254 causes significant chromosome daaage or histopathological changes in the rat testis.* Similar studies were conducted by Green, et al. (1913, 1975a) who gave wale rats single oral dosages of 5000 mg/kg, 2500 ag/kg or 1250 ag/kg or four successive daily dosages of 500 ag/kg of Arodor 1242. Other rau received five consecutive daily dosages of 300 ag/kg. ISO ag/kg or IS ag/kg of Aroclor 1254. Aniaals were injected with cotcaelde 3 or 4 hours before sacrifice which occurred about 14 hours after the single dose or the series of doses. There were sosm body weight losses and soae deaths occurred. Boat aarrow frees rata dosed with both PCB alxtures and spermatogonial preparations from Arodor 1242 treated rata were prepared for cytogenetic study. Repeated dosages of ISO ag/kg end 300 ag/kg ef Arodor 1254 produced decreases- in the number of mitoses in bene aarrow coils. Repeated dooagoo ef 75 ag/kg of Arodor 1254 and all dosages ef Arodor 1242 were without effect. Neither PCB aixturt at any desaga produced a significant number ef chromosomal abnormalities in bone aarrow eaOe. At As tower dosages, Arodor 1242 did not affoct mitoses of aporaatogoaisl eeOs,. but 8000 ag/kg or 4 dotsgas of 500 ag/kg significantly reduced the rate of cell division. No dosage of Arodor 1242 produced cytogenetic abnormalities ha spermatogonia! colls. Tbs authors concluded from their studies that Arodor 1242 and Aroclor 1254 did not possess mutagenic potential. Garthoff. et al. (1977) fed mala rata diets containing 5, 50, or 500 ppm of Aroclor 1254 for 5 weeks, after which they 11 * HONS 015992 examined boa* marrow ud testis samples, Th*r* was ao significant difference between teat and control arumala with respect to tha Incidence of chromosomsl abnormalities and th* number of calls in mitosis from bon* marrow and spermatogonial calls- la a dominant lathal study (Xaplingar, at ai.. 1972 and Cslandrt, 1978), albino mica war* given single iatraparitoaeal dosagas of 500 or 1000 mg/kg of Aroclor 1282, Arocior 1284, or Aroclor 1260 and natad oa successive waaks to virgin famalaa. Thar* was ao evldanca of mutagenic effects, Although datails oa thair stadias ar* limited, thair results are in general agreement with those from another dominant lathal study in rats conducted by Qreas, at al. (1978b) with Aroclor 1282 and Aroclor 12S4. Tha former was administered orally at a single dosage of 825, 1250, or 2500 mg/kf or ia five dally dosages of 125 or 250 mg/kg. Aroclor 1258 was given orally in five dally dosagas of 78, 180, or 300 mg/kg. After dosing, they wars mated with untreated females for 10*11 waaks. Another group of rats was gives 150 mg/kg of Aroclor 1254 for five succassiv* day* and starved overnight before admittance to famalaa. Other mala rats war* offered diets containing 28 or 100 ppm of Aroclor 1254 for 70 days, than autad with untreated famalaa for ono weak. TIM (trtathybmamalamiaa) was used a* a positive control. Tharo was a body weight loss and soma death* occurred is fear group*. Neither tha oral dosing nor th* dietary reading of Arodar 1242 ar Arodor 1254 had any affect on tha number of implantations or the number of dead implantations par pregnant female whila TEM produced significant poatlmpiastatlon buss in weak* 2, 3, and 4. Those author* noted that tha reduction In th* number of dividing spermatogonial ealla reported aarUer far Aroclor 1242 (Green, at al.. 1973, 1978a) did not Impair tha reproductive performance of mala astasia. - at M0NS 015993 Boviow cf the various mutagenicity tests ud their results prompts three comments. Chlorine ted hydrocarbons do net generally five positive results in Selaonelli strains. Therefore, the mutafenic responses with TA1538 (Wyndham, et al. 1978) are either aberrant or else the response is, indeed, limited to essentially monoehJerobiphenyl. The degree of correlation between in vitro mutagenicity testa end carcinogenicity depends upon tho initial classification of tho tost materials. Pienta (1980) classifies Arodor 1254 as a non-carcinogen. Rinkus and Legator (1980) regard Arocior 1254 and Kanechlor $00 as having known or suspected carcinogenic activity. Odaahime (1978) and Sugimura, at al. (1976. 1977) consider Kanechlor 500 to be carcinogenic and Kanechlor 300 to be ooo-carciaogenic. Apart from the difficulties they present for correlation, these different opinions about the carcinogenicity of PCBs are e reflection oI what data these Individuals considered and how they evaluated those data. Finally, in Ught of the large number of testa conducted to evaluate various mutagenic parameters, it is not surprising that an occasional suspicious or positive finding resulted, simply on s statistical basis. Those occurred in in vitro systems. In vivo- tests gave negative results sad provide a baste for concluding that ambient levels of PCBs do not present s mutagenic risk. With eaffact to Spidsmioloyic studies, it should be noted that there are frequent references tat the literature to an epidemiologic study of workers exposed to Arocior 1254 (Beha, et al.. 1978. 1977). Those articles are died tat two recent reviews. One state* "The epidemiological data provide suggestive evidence of a relationship between exposure to polychlorinated biphenyls snd tit* development of malignant melanoma. ...for practical 33 - MOWS 01599* purpose*. polycJbiorinatad biphenyl* should be regarded if they were carcinogenic to humans" (IARC, 197a). The other statea "No conclusive ovidanca haa thua far baan reported which demonstrates that occupational exposur* to PCBs ha* cautad an increaaed incidence of cancer" (Kimbrough, i960). The latter author than proceed* to diacuas the atudy by Bahn, at al. (1976). Among the reference* for the** 2 review* are N108H (1978) which haa th* following comment on the Bahn atudy, "PCB axpoaura hiatorie* were baaed on recollection* of two company employee*. gxposurss to other chemical* could not bo aacertalned.----- To correct theao deficienciea in th* preliminary study, a more intaaaive invaadgation is balng eoaductad (B.N. Kightlingar, written communication, November 1976). A substantial Chang* ha* occurred in the cohort since release of th* preliminary report by Bahn and bar eoworker*, and it seems likely that the finding* on this new cohort wBl differ significantly from thoas of th* preliminary study. The final report is not yet available." Recently, Gaffey (1961) reviewed and evaluated existing reports concern ing health effects and axpoaura to PCB*. Th* roports h# discu***d dealt with diverse potential health effects. With respect to liver effects, he concluded that "Alterations of liver function and fat metabolism associated with PCB exposure have bean observed in several studies, but are characterised by investigator* as mild and of no clinical significance." Ha also concluded that "Mortality studios concerned priaaarily with cancer present problems of interpretation due to th* email sample six* of eons of the studies, mid to the confounding effect of other exposures. However, they do exhibit a pattern, which it that none of the studies agree on the cancer sites at which an excess mortality was found, and th* excesses that 54 * HONS OI5995 wr found are in gsnsrtl not statistically significant. On* must conclude that tho findings of tho mortality studies reflect a sporadic pattern of axcaaa mortality at different sitae which is not consistent with a carcinogenic effect of PCBs. In addition, where an examination of duratioa and latency of exposure was possible, no association with these variables wea found {32]'. "Taken as a whole, the epidemiologic studies find that high occupational exposures to PCBs may cause dermatitis of various kinds, but that there are no other clinically observable effects, including the occurrence of cancor." SMB82SZ Seme remarks by Cole and Merletti (1S90), although written in a store general vain, appear to be particularly suited for ending a discussion on the potential carcinogenicity of PCBs. "In view of the difficulty of deciding whether a "suspect" carcinogen is in fact a weak carcinogen or in fact harmless, we point out one aspect of this scientific Judgement which, though well known, is often lost sight of; namely, that in prin ciple, it is mors likely that non-carcinogen will appear to be a weak carcinogen than the reverse. This asymmetry should be fully appreciated by legislators end regulators. It is summed-up succinctly, if not very accurately, is the oftrheard phrase "you can prove a positive but not a negative*. This is eiearly Qlustreted in the case of PCBs. Review of the results of s large number and wide range of chronic feeding and metabolism studies in anhasls and of mutagenicity studies in various systsaM has failed to establish that PCBs are carcinogenic. However, as - HONS 015996 presently available rodent data raise some suspicion* and load to disputts about tbo carcinogenicity of PCBs. Attoapts to compart and evaluate results of rodtnt studios reported in the Utorsturo art complicated by different usos of atallar torainolofy and variations in tho criteria oaployod for diagnosing hepatic tuaors in rodents. Co->carcinoftnosis studios hsvo roportod both promotion and inhibition of tuaors in rodent liver* following prolonged administration of PCBs in conjunction with an initiator. Thus, it is not likely that animal studies and other laboratory procedures will lead to a universal conasnsus on tho carci nogenicity or non-carcinogenicity of PCBs. While epidemiology studies of humans exposed to PCBs present problems of interpretation due to their small sample size and tho confounding efToot of other exposures, they present tho boat available evidence for assessing the carciaogenie potential of PCBs to humans. Epidemiology studies, including recent studies involving large cohorts with lengthy exposures to PCBs, demonstrate a pattern that is not consistent with a carcinogenic effect of PCBs. M MONS 015997 Footnotes 1. Farbcr, E- (1973). HyperplsaUc liver nodules. In: Methods in Cancer Research, Vei. 7. K. Busch, ad. Academic Press, N.Y. pp. 343-375. 2. See reference: Squire. R.A. and Levitt, M H. (197$). 3. See reference: NCI (1971). 4. Grube, O-O.. Peraino, C., and Fry, R.J.M. (197$): The effects of dietary phenobarbital on the induction of sldn tumors in hairless mice with 7,12* dimethylbon*(a)anthracene. 3. Invest. Dermatol., 64, 2$6*262. 5. Costa. M., Costa, t.R., Maaen, C.A., Sipes, I.G.. and Russell, D.H. (1976): Adenosine cyclic 3',5'-monophosphite-dependent protein kinase and ornithine decarboxylase involvement in the induction of cytochrome P-450 and hepatic hypertrophy. Mol. Pharmacol., 12, 71-179. S. Dolor is a tradename for PCBs made by Chemko in Czechoslovakia. 7. Broom, D P. and Jones, M. (1961). Mortality and industrial hygiene study of workers exposed to polychlorinated biphenyls. Arch. Environ- Health 36, 120-129. MONS 015998 References 1. Anon (1961). Th* Toxicology of PCB's An overview with emphasis on human health effects and occupational exposures- Hazard Ivalustion System. Epidemiological Studies Section- State of California Berkeley. 2. Bahn, A.K-, Grover, P., Rosenwalks, I., O'Leary, K., SteUaan, J. (1977) PCB? and melanoma. New Engl. J. Med. 296. 106. 3. Bahn, A.K., Rosenwaike. I., Herrmann, N., Grover. P., Stellaan, J. and O'Leary, K. (1976). Melanoma after exposure to PCB's. New Engl. J. Med. 295, 480. 4. Berry, D.L., DiGiovanni, J., Juchau, M.R., Bracken, W.M., Gleason, G.L., and Slaga, T.J. (1978). Lack of tumor-promoting ability of certain environmental chemicals in a two-step mouse skin tumorigeneais assay, Rea. Commua. Che*. Pathol. Pharmacol. 20, 101-108. *" 6. Berry, D.L. Siege, T.J., DiGiovanni, J., and Juchau, M.R. 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