Document N2pXpx66yb1j0GaRJLpYwN5bD

RECTIVID LH S BHAT 12 AE 310 Genotoxicity, Carcinogenicity, Developmental Effects and Reproductive Effects of Perfluorooctanoate: A Perspective from Available Animal and Human Studies Prepared for the Association of Plastics Manufacturers of Europe and the Society of the Plastics Industry John L. Butenhoff, Ph.D., 3M Company Gerald L. Kennedy, Jr., Dupont Sandra R. Murphy, Ph.D., Atofina John C. O'Connor, MS., Dupont Geary W. Olsen, D.V.M,, Ph.D., 3M December 19, 2002 0 Introduction `This document will describe the experimental database for genotoxicity, carcinogenicity, developmental, and reproductive effects of perfluorooctanoate (PFOA) and will provide our current understanding of the potential relationship of these toxicological endpoints to man, as supported by studies of worker populations. In addition, it provides perspective on the relationship of these toxicological endpoints to human exposure and potential human health risk. PFOA and its salts are fully fluorinated organic compounds that are used as reactive intermediatesoras processing aids and surfactants. A large toxicological and epidemiological database exists for PFOA. Most of the toxicological data have been developed using the ammonium salt of perfluorooctanoic acid (APFO); however, since APFO readily dissociates and is soluble in aqueous solution, the designation PFOA will be used throughout this document. The reader is referred to the U.S.E.P.A. document, "Revised Draft Hazard Assessment of Perfluorooctanoic Acid and its Salts" (U.S.EP.A., 2002), for a detailed presentation of the toxicological and human-health databases for PFOA. Laboratory studies designed to identify `potential health hazards of PFOA demonstrate that PFOA can produce effects in animal models. By contrast, the health effects observed in laboratory studies have not been observed in worker populations either under current or past exposure conditions. Therefore, we believe that PFOA does not present an unreasonable risk to human health at the levels encountered in the workplace. Background on Worker Studies `Throughout this document, reference will be made to several worker studies. Studies in workers include cohorts from a PFOA production facility (Cottage Grove, MN) and two facilities that used PFOA in manufacturing processes (Decatur, AL and Antwerp, Belgium). The workers from the Cottage Grove facility are considered likely to have the highest potential for exposure since this facility manufactured PFOA since the 1940's and employees have been shown to have higher serum concentrations of PFOA than either Decatur or Antwerp plant populations. The. Antwerp plant also manufactured PFOA but began in the mid 1970's. The Decatur facility routinely used PFOA but did not manufacture it until the late 1990's.and Antwerp plants are facilites that manufactured other fluorochemicals and routinely used PFOA. The types of studies performed include evaluations of mortality, medical surveillance, and episodes of care. `The mortality studies examined observed versus expected causes of death in the study `populations. Medical surveillance included standard worker health assessments as well as evaluations of biochemical parameters that had been affected in laboratory animal studies. An episodes-of-care study examined health insurance claims data. An episode of care is defined as a series of events all related to a particular health problem that exists continuously for a period of time. Developmental Toxicity `The developmental toxicity of PFOA has been studied in rats and rabbits by the oral exposure: route and in rats by the inhalation exposure route (Gortner, 1981; Gortner, 1982; Staples et al. 1984). In those studies, pregnant animals were treated with graded doses/exposures of PFOA during organogenesis. Observations of the structural integrity of the fetuses was evaluated both 1 extemally, internally, and by skeletal examination of the fetuses obtained prior to natural delivery. For one set of oral and inhalation studies in rats, dams were allowed to litter and pups were observed through the lactation period. These studies, as discussed below, allow the conclusion that matemal exposure to PFOA during organogenesis is not uniquely hazardous to the fetusor to preweaning rat pups. `The developmental study conducted in rats by Gortner (1981) was the first to be conducted with PFOA. In thisstudy, matemal toxicity was observed at the highest dose (150 mg/kg) and consisted of group mean body weight reductions and mortality (3 of 22 dams). Reproductive organs were unaffected by treatment. Fetal examination did not reveal any increase in embryofetal toxicity or structural abnormalities that were attributable to PFOA treatment. Lens abnormalities, originally attributed to PFOA treatment, were found subsequently to be an artifact of the sectioning technique. In another oral study, rats were given 100 mg PFOA/kg of body weight by gavage from gestation da6y through 15 (Staples efal 1984). One group of 25 pregnant rats and their liters were examined on day 21 of gestation. Another group of 12 treated dams gave birth and the resulting pups were examined on day 35 post-partum. Matemal effects including death and decreased `maternal body weight gain were seen in both groups. No developmentaltoxicityor abnormalities were seen in the fetuses, and offspring showed normal lactational viability. By the inhalation route, groups of pregnant rats were exposed to concentrations of either 0.14, 1.2,9.9, or 21 mg PFOA/m', 6 hrs/day from day 6 through 15 of gestation (Staples er al., 1984). Exposure to the highest concentration resulted in the death o3f of 12 rats with the remaining rats showing reduced weight gains and clinical signs including lethargy and chromodacyorhea. Reduced weight gains were also seen in rats exposed t0 9.9 mg/m'. No effects were seen in those exposed toeither0.14 or 1.2 mg/m'. Mean fetal body weights of surviving dams exposed 1e0xp2o1sumrge/mgr'owupesrethraetdcucoeudl.d bTeheasrseocwiearteednowistthruPctFuOraAl aebxnpoorsmuarlei.ties in fetuses fromanyof the Tn a rabbit developmental study (Gortner, 1982), rabbits were given oral doses of either 1.5, 5, or 50 mg PFOA/Kg from gestation day 6 through 18. The number of rabbits producing litters in this study was low in all groups, a fact that affects interpretation of the study. A reduction in `maternal body weight gain was observed in rabbits given 50, but not 1.5 or 5 mg/kg. No other signs of response to PFOA were observed in the pregnant rabbits. Fetuses from all treatment `groups were present in the expected numbers, were structurally normal, and weighed essentially the same as their untreated counterparts. No evidence of either embryotoxicity or teratogenicity was seen. An increase in the number of fetuses with the natural and stress-related variant of thirteenth ribs was noted. This latter finding is knowntobe quite variable (Christian, 1987), is not a malformation per se, and is not likely to be relevant to humans. Regarding reproductive development, the multigeneration reproduction study with PFOA in rats showed delays in the age at preputial separation (mean = 3.7 days) in males and the age at vaginal opening (mean = 1.7 days) in females (York,2002). These delays are believed to be secondary to toxicity and do not representa primaryeffecton organ development, as will be further discussed in the "Reproductive Effects" section that follows. 2 Reproductive Effects A two-generation reproduction study in rats was conducted with PFOA (York, 2002). Rats were treated with oral gavage doses of either 1,3, 10, or 30 mg/kg of body weight/day. In the parental rats, signs of toxicity were observed at all dose levels in the males and at 30 mg/kg in females. In males, body weight gain suppression was observed at all doses (except | mg/kg in the Py generation) along with organ weight changes (liver, kidney, and spleen). Female parental rats were relatively unaffected by treatment, with decreased kidney weights seen in Py females and decreased weight gains in Fy females only at 30 mg/kg. There were no effects on any of the mating or fertility parameters in either generation. At 30 mg/kg, a numberofeffects in the offspring were observed including decreased pup weights, increased pup mortality (F; generation only), and delayed vaginal opening and preputial separation. These findings were not observed at any of the lower doses. Clearly, the effects observed in the two-generation reproduction study did not compromise the reproductive success (i.., mating and fertility) of the rats at dosages of up to 30 mg/kg under the conditions of this study. `The two-generation reproduction study found decreased pup weights during lactation and increased pup mortality in the Fy but not the F; generation. The increases in pup mortality occurring pre- and post-weaning at 30 mg/kg may be suggestive of the beginningof adoseresponse curve. It is important to note that, while post-weaning mortality was not evaluated in the F; generation offspring (all F; offspring were necropsied at weaning), there were no effects on pre-weaning mortality in the F; offspring (pre-weaning mortality was increased in F; pups, but was not statistically significant). In addition, there were no effects on pup weights in Fz generation offspring through weaning. `The increased incidence of pup mortality at 30 mg/kg is most likely aresultof a general failure to thrive of the offspring, suggesting a compromised nutritional status of the offspring at preandor post-weaning as reflected by reduced body weight. In support of this hypothesis, eleven of the thirteen F; offspring that died post-weaning died before post-weaning day eight, and these included the nine lightest pups. Although not statistically significant at al time points, pup weights were consistently decreased throughout the lactation period (90, 90, 89, 92, and 95% of control on postnatal days 1,5, 8, 15, and 22, respectively). These effects have also been observed in reproduction studies performed with other peroxisome proliferators such as `gemfibrozil, RMI 14,514, and hydrochlorofluorocarbon 123 (HCFC-123) (Fitzgerald et al., 1987; Gibson ef al, 1981; Malinverno et al, 1996). It seems likely that the compromised nutritional status of some offspring is responsible for the increased pup mortality observed in the two-generation reproduction study with PFOA. `The data from this study, discussed in more detail below, shows delayed age at preputial separation in males (mean = 3.7 days) and delayed age at vaginal opening (mean = 1.7 days) in females in the F; offspring. The delays in sexual maturation may have been the result of delayed `growth of the F offspring. As noted earlier, pup weights were consistently decreased throughout the lactation period. While the body weights of the F generation offspring were similar to the. controls at the time of sexual maturation, it is plausible that the delayed growth that was 3 observed early in lactation may have contributed to the delays that were observed in sexual `maturation of the F; offspring. Decreased body weights can result in non-specific delays in puberty (Camey et al, 1998; Glass etal. 1976; Glas&s Swerdloff, 1980; Kennedy and Mitra, 1963; Marty ef l., 1999, 2001a, 2001b, 2001c; Ronnekleiv et al, 1978; Stoker ef al, 20003; 2000b; Widdowson & McCance, 1960). Ina recent report by Lewis and co-workers (2002), variability of sexual maturation data was evaluated in control populations of Sprague-Dawlraetys. They found that the typical variability among control groups was approximately two days, a finding that was also consistent with the typical variability in age at sexual maturation reported by others (Ashby & Lefevre, 2000; Clark, 1999; Marty et al., 1999; Stoker ef aL, 2000b). Since non-specific effects on body weight can cause general delays in sexual maturation, interpreting delays in sexual maturation can be problematic in studies where generalized delays in growth occur, such as those that were, observed in the current study of PFOA. Clearly, PFOA. do not compromise reproductive success (i.., mating and fertility) in rats at dosages of up to 30 me/ke. In summary, in the two-generation reproduction study with PFOA, paternal toxicity (Py and Fy) was observed at all dose levels (1,3, 10, and 30 mg/kg) and minimal matemal toxicity was observed at 30 mg/kg. While several possible reproductive/developmental effects were observed (i.., decreased pup weights, increased pup mortality, and delayed sexual maturation in Fy offspring), the reproductive success of the rats was not compromised. Notably, the overall results of the first and second generation appear tobe similar in that there was no apparent increase in adverse outcome(s) in the second generation. The effects that were observed could be suggestive of reproductive and/or developmental effects or they could be due to general delays in growth. Unknown mechanisms may be contributing to the effects that were observed at 30 mg/kg. At dosages of< 10 mg/kg, no reproductiveordevelopmental parameters were affected, while parental males showed clear signs of toxicity. The no-observed-adverse-effectlevel (NOAEL) for reproduction in the two-generation reproduction study was 10 mg/kg, while the NOAEL for general toxicity would be < 1mg/kgfor the male parental animals and 10 mg/kg, for the female parental animals. The effects that were observed with PFOA in the twogeneration reproduction study are consistent with those observed in studies with other peroxisome-proliferating compounds (Fitzgerald er al., 1987; Gibson ef al, 1981; Malinverno er al., 1996). Human Experience with Respect to Development and Reproduction An episodes-of-care study (Olsen er al., 2001b) at the 3M Decatur plant site examined. reproductive outcomes associated with fluorochemical exposure (which includes potential PFOA exposure). Regarding pregnancy and its potential complications, there were 40 episodes of care. reported in 13 female employees in the fluorochemical plant (44.7 expected) compared to 23 episodes of care (26.3 expected) reported in eight female employees in the film plant (a nonfluorochemical plant at the same site as the Decatur fluorochemical plant) between 1993 and 1998. This resulted in an episodes of care risk ratio of 1.0 (95% CI 0.6-1.8). The total number of female employees was 122 and 101 in the chemical and film plants, respectively. The episodes-of-care risk ratios for congenital anomalies (1.0, 95% C1 0.6-1.8) as well as perinatal disorders (0.2, 95% C1 0.0 - 2.4) were also comparable between employees in the fluorochemical 4 and film plants. There is no evidence from this study to suggest increases in reproductive and developmental effects associated with exposure to fluorochemicals including PFOA. Hormones `The association of PFOA serum and hormone concentrations in workers has been studied at three. production facilities (Cottage Grove, Decatur and Antwerp). The episodes-of-care study conducted only at the Decatur facility also allowed observation of episodes of care that may relate to hormonal status. Two cross-sectional studies of 111 and 80 Cottage Grove male. fluorochemical production workers were conducted and measured their serum PFOA cFoSnHc,enDtrHaEtAioSn,s iTnSrHe,lactoirotnistooltahendcosnecxenhtorramtoinoens-boifnsdeivnegragllohbourlmino)ne(sOl(steensteotstale.r,on1e9,98e)st.raPdiFolO,ALH, serum concentration was not associated with changes in hormone concentrations. Although a 10% increase in mean estradiol level was observed among employees who had the highest levels of serum PFOA, this association was confounded by body mass index and was likely not due to PFOA exposure. Further, an analysis of thyroid hormone levels in 3M Antwerp and Decatur workers did not show substantial changes in TSH, T4, free T4, T3 or thyroid hormone binding globulin associated with serum PFOA concentrations (Olsen et al., 2003b). The risk ratio for disorders of the thyroid in the episodes-of care study was comparable between Decatur fluorochemical and film plant workers (1.1, 95% C1 0.6-1.8) (Olsen ef al, 2001b). In addition to these human observations, a six-month oral toxicity study in male cynomolgus monkeys did not produce significant changes in either sex hormones or thyroid hormones (Butenhoff ef a, 2002). Therefore, there is no observed association of PFOA exposure with changes in hormone levels in man or monkeys. Genotoxicity `The weight of evidence from studies evaluating the genotoxicity of PFOA indicates that PFOA is not genotoxic. These studies include evaluation of mutagenicity, clastogenicity and cell transformation. PFOA has not shown a potential to effect DNA point mutations or recombinations. PFOA has shown a lackofactivity in bacterial reverse mutation assays including Salmonella typhimurium and Escherechia coli strains and in yeast recombination assays (Saccharomyces cerevisiae) in the absence and the presence of metabolic activation (Litton, 1978; Hazleton, 1995, 19962). Similarly, in the Chinese hamster ovary (CHO) forward mutation assay, PFOA did not induce a statistically 2002). significant increase in the number of mutant colonies in the treated cells (Toxicon, Chromosomal aberrations were assessed in human lymphocytes and CHO cells. PFOA did not induce significant increases in the numbersof chromosomal aberrations in human lymphocytes (Hazleton, 1996b; NOTOX, 2000). When tested in CHO cells, significant cytotoxicity was observed at the highest doses tested, and these doses also increased chromosomal aberrations. 5 (Hazleton, 1996, 19964). In view of the high toxicity, the biological significance of this positive response is questionable. PFOA did not induce a significant increase in bone marrow polychromatic erythrocytes after oral administration to mice (Hazleton, 1995b, 1996e). There was no evidence of cell transformation using the C3H 10T1/2cell line observed at any of the dose levels tested (Stone, 1981). The `genotoxicity profile for PFOA indicates a lack of activity in a range of test systems and endpoints. Peroxisome Proliferation PFOA is aperoxisome proliferator (PP) in numerous studies and belongs to a widening group of substances including plasticizers and hypolipidemic drugs that are known to be PPs (Ikeda ef al., 1985;Just et al., 1989; Pastooref al, 1987;Cooketal., 1992, 1994; Biegeletal., 1995,2001). The liver is a primary target organ for both short-term and chronic effects of PFOA in rats (Griffith & Long, 1980; Olson & Anderssen, 1983; Kennedy, 1985; Pastoor ef al, 1987) and cynomolgus monkeys (Butenhoff ef al, 2002). The increased liver weight does not appear to be a result of hepatocellular hyperplasia (no increase in nuclear DNA) and has been variously a1t9t8r5i;buPtaedsttoooirnectreala,ses19i8n7p;eBruotxeinshoomfefs,efeanld,op2l0a0s2m;iBcerretthiicauulmume a&ndWamliltaocceh,on2d0r0i2a; (BIikeegdealeeffaall,, 2001). PFOA has been shown to activate the PPAR receptor (Maloney & Waxman, 1999). Higher doses lead to liver degeneration andnecrosis and the appearance in the serumof enzymes reflecting liver damage. Treatment of rodents with Ps initiates a characteristic sequence of morphological and biochemical events in the liver and to a lesser extent the kidney. These events include marked hepatocellular hypertrophy due to an increase in number and size of peroxisomes, large increases in peroxisomal fatty acid B-oxidation, an obvious swelling and proliferation of the mitochondria `and endoplasmic reticulum, increased cytochrome P-450-mediated a-hydroxylation of lauric acid, and various changes in lipid metabolism (Ikeda ef al, 1985; Pastoor ef al, 1987; Berthiaume & Wallace, 2002). This response is initiated by the activation of the nuclear receptor, PPARc. (Green, 1995; Ashby ef al, 1994; Lake, 1995). PPAR:is a steroid hormone receptor able to increase the transcription rate of responsive genes and is the major mediator of PP in rodent liver. The critical role of PPARct in PP in mice has recently been clearly established. PPARo-null mice do not show the typical PP-mediated responses or signs of hepatic h1y9p9e7r;plWaasriadoerf nael,opl1a99s8i)a.(aLdoenngo-mtaesrmorexcpaorscuirneomoafs)roidnencthsrotnoiPc sstucdhiaersacwtietrhisPtPcsall(yPerteesrusltestianl.,an increased incidence of liver tumors (Doull ef al, 1999; IARC, 1995). There are differences in the effects exerted by different PPs. Pronounced species differences have been reported following treatment of animals with PPs in vivo and have been observed in hepatocyte cultures in vitro (Ashby et al., 1994; IARC, 1995; Bentley ef al. 1993; Elcombe et al. 1997; Lake, 1995; Maloney & Waxman, 1999). Rats and micearehighly, perhaps uniquely, responsive to the effects of PPS; whereas, Syrian hamsters exhibit an intermediate response and guinea pigs seem to be practically nonresponsive6, as are primates - including both Old World and New World (c.g., marmoset) species, and humans (Bentley er al., 1993; Pugh ef al, 2000; Butenhoff er al. 2002; Tucker & Orton, 1993; Graham ef al., 1994). A large number of humans have been treated for relatively long periods of time with hypolipidemic drugs that are potent PPs in rodents. No significant changes in the peroxisome number or volume occur in humans taking substantial doses of these drugs for extended periods of time (up to 3 years) (Ashby et al., 1994). Two human epidemiology studies showed no indicationofan increase in cancer associated with long-term human exposure (ranging up to cight years) to hypolipidemic drugs (Ashby ef al, 1994). Rodents are poor models for human risk assessment with respect to livereffects observed with PPS. The reason for the non-responsiveness of humans to PPs is not yet fully understood; although, research shows differences in amount and expression of PPAR. between humans and rodents (Cattley et al., 1998; Palmer ef al, 1998). Induction of liver, testicular Leydig cell and pancreatic acinar cell tumors is a common finding for PPs (Cook et al, 1999). In chronic bioassays in rats, Cook et al. (1999) reported that 7 out of 11 PPs induced all three tumor types (Cook ef al.,1999), and 10 of the 11 PPs produced liver and Leydig cell tumors (Cook et al., 1999). Cancer `The oncogenicity of PFOA has been investigated in two separate two-year feeding studies in rats. PFOA was found to increase the incidence of three tumor types (liver, Leydig cell, and pancreatic acinar cell tumors-Riker,1983, Biegel et al., 2001). In the following discussion, cach tumor type will be discussed in tum. Hepatocellular Adenoma Ina chronic dietary study conducted with 156 male Sprague Dawley rats fed diets containing 300 ppm PFOA for two years (Biegel ef al, 2001), histopathological evaluation revealed PFOArelated increases in hepatocellular adenoma. Hepatocellular adenoma occurred at an incidence of 13 % (10/76) as compared to 3 % (2/80) and 1% (1/79) inadlibitum and pair-fed controls, respectively. These liver tumors are believed to have resulted from peroxisome proliferation. Evidence for this comes from the measurement of hepatocellular peroxisome proliferation at three-month intervals during the study. Increased liver weights and hepatic B-oxidation activity were observed in the PFOA-treated rats at all time points; however, PFOA did not significantly increase hepatic cell proliferation. It is generally agreed that liver tumors in rats produced by PPs are unlikely to be relevant to humans. Human Experience with Regard to PFOA and Liver Toxicity Several worker studies investigated the possible association between either liver cancer or liverrelated disease with PFOA exposure and have shown no association. Exposures to PFOA in 7 these workers, as measured by serum PFOA concentration starting in 1993, ranged from less than 110 routinely 114 ppm (Olsen et al, 2000, prior to 1993 becauseatotal 2001a, 2001c, 2003a, 2003). PFOA was not organofluorine method was used. Past serum measured PFOA `concentrations in workers may have been higher. Epidemiological assessments of liver cancer deaths among 3M workers with potential exposure 10 PFOA have not shown significantly increased Standardized Mortality Ratios (SMRs) for liver cancer; although, very few deaths fromliver cancer were expected. Among 182 workers identified with definite PFOA exposure at 3M's Cottage Grove plant, there were no deaths rreelsapteecdtitvoelliyv)er(Aclaenxcaenrdeorr,ci2r0r0h1oas)i.s oAfmtohenlgiv1e,r4d9u1riwnogrkae5r0s-wyietahr tpirmoebapbelreioPdF(O0.A3 eaxnpdos1u.r2ee,xtpheecrteed, was one liver cancer death auributable to cirrhosis of compared to the liver (6.4 2.0 expected (SMR = expected, SMR = 1.0, 0.5, 95% CI 0.0 95% C1 0.4-2.1) 2.0) and 6 deaths AU3M's Decatur plant, PFOA has been used as an emulsifier in fluoropolymer production and phraosdaulcstoiboneednidanroetsiodcucaulrbyun-tpirlotdhuectlaotfe p1e9r9f0l'su.orEomocptlaonyeesuelsfoenryulmfPluForOiAdecpornocdeuncttriaotni.onPsFhOavAe ranged up to 13 ppm in sampling conducted in 1998 and 2000. In this population, there were. 3tw8o-yleivaerr csatnucdeyrpdeeraitohds(o1b9s6e1r-v1e9d98c)omopfa1r,e0d65t0wo0r.7keerxspe(cAtleedxa(nSdeMrR, 2=030.11b,)9. 5I%tiCs1un0l.i4k-e1l1y.1t)hadturing these observations represent a response to PFOA. Analysis of episodes of care (health claims data)over a six-year interval (1993-1998) of a subset of these Decatur workers (n = 652) did not show differences in reported disorders of the liver (ecxiprorshoesdiswoarnkdfhoerpcaetit(iDse)cabteutrwefielnmtphliasnDteecmaptluoryfeleuso)ro(cOhlesmeincaelt awl.o,r2k0f0o1rbc)e.anTdhearceowmapsaraison nonbniolnisairgyntirfaicctanrtelpyoritnecdrienas1e3d irnisdkivriadtuiaol(s1.i6n,t9he5%flCuoIr0o.c8h-e2m.i9c)oalfpelpainsto(dNes=of6c5a2)r.e Tohfidsiscopridseordseosfotfhe care risk ratio increas1e0d 2.6 (95% CI 1.2-5.5) when restricted to the 211 fluorochemical workers with > 10 years work experience (based on eight individuals' health claims data). An episodes of care study has not been done for Cottage Grove or Antwerp fluorochemical production workers Hepatic clinical chemistry test results have been reported in a series of cross-sectional assessments of medical surveillance examinations for both the Cottage Grove and Decatur employee populations as well as the fluorochemical production workforce located in Antwerp (Gilliland & Mandel, 1996; Olsen et al, 2000; 2003b). None of these study populations have had changes in hepatic enzyme assays or bilirubin analyses that could be associated with b`moedays-umraesdsseinrduemxPaFndOAalccoonhcoelnctornastuiomnpstiaofnte.r aSdejrusutminPgFfOorApoctoenncteinatlrcatoinofnosunindi3nMg fAancttowresripncluding workers were 2001a, 2001c, approximahtalefloyf 2003b). those measured in the Decatur workforce (Olsen ef al, Liver Tumor Summary 8 In summary, the lackof indications of increased risk of liver disease in 3M workers with exposure to PFOA suggests that the exposures encountered by non-occupationally exposed individuals should presenta low risk of liver disease and, by extension, liver cancer. The lack of `genotoxicity observed in genotoxicity assays and the increase in peroxisome proliferation observed in the lifetime dietary study in rats suggests a potential mechanism for the increase in hepatocellular adenoma in rats. If peroxisome proliferation is involved in the etiology of the hepatocellular adenoma observed in rats, the risk of hepatocellular adenoma developing in exposed humansis expected tobe quite low due to the much lower-degree of response 0 PPARG. agonists in human liver. Leydig Cell Tumors Two chronic studies in Sprague Dawley rats have shown increases in hyperplasia and benign tumors (adenoma) of testicular Leydig cells. In the first study (Riker, 1983), the incidence of Leydig cell adenomas was 0/50, 3/50, and 7/50 at dosages of 0, 30, and 300 ppm PFOA, respectively. A second study by DuPont included numerous mechanistic endpoints (i.c., cell proliferation, hepatic enzyme measurements, hormone measurements) and was specifically designed to evaluate the mechanism of Leydig cell tumor induction (Biegel et al. 2001). In this study, PFOA was administered at 0, 0-pair-fed, or 300 ppm PFOA to male rats. There was a increase in the incidence of Leydig cell hyperplasia and adenomas, with adenoma incidences of 0/80, 2/78, and 8/76 in the 0, 0-pair-fed, or 300 ppm PFOA group, respectively (Biegel ef al, 2001). Experimental evidence for the mechanism of PFOA-induced Leydig cell tumor formation, while not conclusive, tends to support the hypothesis that a sustained increase in estradiol within the testes may be responsible for the increased incidence of Leydig cell tumors in male Sprague Dawley rats ( Cook ef al, 1992; Biegel ef al, 1995; Liu ef al, 1996a, 1996b). The extent to which this effect may be linked to PPARa activation is not clear. Other PPs (DEHP and clofibrate) have been shown to increase serum estradiol concentrations in male rats (Eagon ef al. 1994; Rao ef a. 1984), and several PPs (c.g, clofibrate, DEP, gemfibrozil, dibutyl phthalate, and Wyeth 14,643) have been showntoreduce estradiol metabolism, resulting in an increase in circulating levelsof estradiol (Corton e a, 1997; Eagon ef al, 1994; Fan ef al, 1998; Rao ef al, 1984). This pattern of hormonal alteration has also been observed in vitro, where 10 of 11 peroxisome proliferators evaluated increased estradiol levels, and 11 of these PPs decreased testosterone levels (Liu ef al., 1996a, 1996b). While most PPs may increase estradiol in rats, the direct association of elevated estradiol with the production of Leydig cell tumors remains to be demonstrated. There are seven proposed mechanisms for Leydig cell tumorigenesis in rodents, all of which disrupt the hormonal milieu within the testes (Clegg et al., 1997; Cook et al., 1999). `The attribution of sustained estradiol increase as part of the response to PPARa activation and as the operative mechanism for PFOA-induced Leydig cell tumors as well as the relevance of these tumors to humans will require additional research. Human Experience with Testicular Tumors Testicular cancer is most commonly diagnosed under the age of 40 in humans (Schottenfeld, 1996). Ninety-five percent of neoplasms of the testes arise from germinal cells and are divided 9 Ncloinn-igcaelrlmyiinnatlo tnheeospleamsimnsomcoansatnidtuateva5r%ietoyfotfesptuicruelaarntdummiorxsedwittyhpeaspopfronxoinmsaetmeilynohmaalftooufsthteusmeors. being histologically classified as Leydig cell tumors. Mortality data do not adequately explain `occupational risk for testicular cancer because of the high five-year survivability rates for ateusrtiibcuutlaabrlceatnocetres(t>ic9ul5a%r csaunrcvievrala)m.onThgutsh,eit3iMs nCootttuangeexpGercotveed ftlhautortohcehreemhiacsalbepernodouncltyioonnewodrekaetrhs (0:4 expected) during a 50-year study period (Alexander ef al., 2001a) and no deaths due to testicular cancer observed among the Decatur occupational population (0.2 expected) in a 38 year study period (Alexander et al, 20010). Analysis of episodes of care among the Decatur population from 1993-1998 did find two individuals with health claims data coded to testicular cancer (0.6 expected) (Olsen ef al, 2001b). One of these two workers had > 10 years of work experience in the fluorochemical plant. hAosrmnootneeds.preAvi1ou0s%lyi,ntchreeraesearien nmoeadnireecsttraasdsiooclilaetvieolnsobosfePrvFeOdAamexopnogsuermeplwoiytheecshawnhgoeshiandstehxe highest levels of serum PFOA was confounded by body mass index and likely was not due to PFOA exposure (Olsen et al, 1998). Testicular Tumor Summary tAhlitshtouumgohrLteyypdeiignchelulmtaunmsorissrharaev.e Tbheeerneiobssceurrvreedntilnytnwooecvaindceenrcesttuhdaitesairnelraattsi,ontshheipocbceutrwreeencne of cPhFaOngAesextphoatsumraeyanbde mineccrheaansiesdtitceasltliycurlealractaendcteortreisstkiceuxliasrtcsainncheurmhaanvse.beIennadodbisteironv,ednoinhmoormnokneayls or humans with PFOA exposure. Pancreatic Acinar Cell Tumors 2M0a0l1e),SpexrhaigbuieteDdaawnleinycrraetassefeidndpiaentcsrceoantitcaianciinngar30ce0llpapdmePnoFmOaAafnodrctowmobiyenaerds p(aBnicergeealtiecf aa.c,inar acdellliabdietnuommfae/dcacrocntirnoolmsa,.anAdcpianiarr-fceedllcoandteronlosm,areisnpceictdievnecley.waAs 9pr%io,r0t%w,o-y1e%arindiPeFtOaAry-tbrieoaatsesadyraitns, male and female Sprague Dawley rats at 30 and 300 ppm PFOA did not result in an increase in pancreatic tumors (Riker, 1983); although, a subsequent pathology peer review has noted the presence of hyperplastic foci. ePxanpcorseuarteicofacriondaerntceslltotoutmhoerrsPP(sR.edTdhye&meRcaoh,an1i9s7m7)bayrewhoiftcehnPoFbsOeArvaenddfsololmoewiontghecrhrPoPnsicinduce these tumors is not well understood. The development of these tumors is known to be modified cahnodl/eocrymsetdokiiantiend(bCyCsKe)vearanldfdaicettoarrsysfuacth(aOsbstoeuroiedf ahlo,rm1o99n7e).levBelise,geglroewttahl,fa(c2t0o0rs1)suhcahveas rperloepaosseetdhatth,aitnPtFumO,Acaonudldotlheeard ePvPesntcuoaullldyitnocrheyapseerptlhaesfiaaticnonttheenptanincrtehaetigcutacainndarstcielmlusl.atIet mCuCsKt be concluded that, at the present time, this is a speculative mechanism that is not supported by teoxpheurmiamennstaisluenvciedretnacien (foGravPiFnOeAf a(lB,ie1g9e9l6,et1a9l9,7;20C0a1t;tlBeuyteefnahlo,,ff19ef98a;l,Pa2n0d0o2l),a1n9d98i)t.s aPpapnlcirceaabtiilicty 0 acinar cell adenomas are rare in humans (Anderson ef al, 1996) and when considering the relevance of this rat tumor data with regard to human health risk, the non-genotoxic mechanism (with a likely threshold), and the relatively low exposure in humans shouldbetaken into account Human Experience with Pancreatic Disease `The pancreatic acinar cell tumors observed in PFOA-treated rats (Biegel et al., 2001) are not commonly diagnosed in humans. Among the Cottage Grove workforce with definite PFOA e(xSpMosRur=e1(.n3,=9158%2)C,1th0e.r0e7w.a4)s (oAnleexdeaantdherreeptoarl,t,ed20f0o1rap).ancErmeaptliocyceaensce(rnc=om1p,4a9r1e)ddetofi0n.e8dewxiptehcted eprxopbeacbtleed P(FSMORA =ex1p.o4s,u9r5e%haCdIs0i.x5d-ea2t.7h)s.atTthreisbuetapbalnecrteoaptainccrceaantciecrscawnecreerlciokmelpyatroehdatvoe4b.8een of ductular origin rather than acinar. At the 3M Decatur manufacturing site there were no deaths attributable to pancreatic cancer among the 1,065 employees with one expected (Alexander er al., 2001b). One episode of care for pancreatic cancer has been reported (Olsen ef al, 2001b). Although the episodesofcare risk ratio for acute pancreatitis was increased (2.6, 95% CI 0.615.8) among the fluorochemical production workforce, this effect is difficult to interpret because itis based on six health claims from just one employee. Because asustained elevation of CCK has been suggested as a potential mechanism for pancreatic cancer, plasma CCK levels were assayed in 74 Cottage Grove PFOA production wvaolrukeesrs(mpearatnic2i8p.a5tipngg/imnl,meSdDica17l.1s,urmveedililaannc2e2.e7xapmgi/nmalt,iroannsgien81.98-9876.(7Olpsge/nmlet) aalp,pr20o0x0i)m.atCedCtKhe assay' reference range (upto 80 pg/ml) and were negatively, not positively, associated with employees' serum PFOA concentrations. Pancreatic Tumor Summary sPeFpaOrAatweatswoa-syseoacriabtieodaswsiatyhs.anTihnicsretausmeoirntaycpienairs rcealrletiunmhourmsaonfs,thaenpdantcherreeasisinnoraetpsiidnemoinoeloogfitcwaol evidence for a relationship between PFOA exposure and pancreatic cancer. The relevance of acinar cell tumors of the pancreas in rats to human pancreatic cancer risk is uncertain. Mammary Gland Tumors Ifnibtrhoea3dMe-ncoamnacseorfstthuedmyawmitmhaPryFOgAlanidn SapppraargeunetlDyawwlaesyinractrsea(sReidkeirn,f1e9m8a3l)e, rtahtesi(n2c2i%d,en4c2e%o,f and 48% at 0, 30, and 300 ppm in diet, respectively). There was no apparent difference in incidence over a ten-fold dose range. The authors of this study concluded that the mammary tumor data did not reflect an effect of PFOA. Tcohnetrloalbodraattaobraysec.onHdouwcetvinegr,thuentsrteuadyt,edRcioknetrroPlharramtsac(esuatmiecasltsr,aidniadnndotsuhpapvlieear)n fardoeqmua13techhrisotnoircical toxicityloncogenicity studies conducted at Haskell Laboratory from 1984-87 provided 947 n control rats, which were on test for at least one year (scheduled sacrifice at two years). Charles River, the supplier, also maintains a control database. Statistical evaluation of the incidence of fibroadenomas in the PFOA-treated groups versus the Haskell Laboratory historical controls was not significant (p = 0.3). The incidence of fibroadenomas in the 13 reference Haskell laboratory studies ranged from 24 to 54% with a mean of 37%. In the PFOA study, the control group incidence lies just below and the test group incidences lie near the top of the control range. The incidences in the PFOA-treated groups (42 and 48%) are similar to the average of the Haskell Laboratory historical control groups (37%). Historical control data posted on the Charles River Laboratories Web-Site, gives the average fibroadenoma incidence of 41% with a range among 24 studies of 13 - 61%. These data further support the study authors' conclusion that the distribution of fibroadenomas in the PFOA study were a reflection of background incidence and were not related to PFOA treatment. `When all mammary tumorsofepithelial origin in this study are combined, there is no statistically significant increase in total tumors. Mammary tumors in rats present as a continuum from benign to malignant. In composition. They range from tumors of primarily epithelialcellsto various degrees of connective tissue involvement. Froma biological perspective, both adenomas and fibroadenomas are classified as benign fibroepithelial tumors, and, when combined for the PFOA study is not statistically increased. Similarly, there is no biological difference between the terms adenocarcinoma and carcinoma. The data for total malignant tumors shows a lower incidence of malignant tumors in the high-dose compared to the control animals (17, 31, 11% in the 0, 30, 300 pp groups). Human Experience with Breast Cancer `The available human data do not suggest an increased breast cancer risk. There have been no breast cancer deaths observed among Cottage Grove workers identified with definite PFOA exposure (0.2 expected) and two breast cancer deaths observed among those with probable PFOA exposure (3.6 expected, SMR = 0.6, 95% CI 0.1 - 2.0) (Alexander, 2001a). There have been no breast cancer deaths in the Decatur fluorochemical production workforce (0.9 expected) (Alexander, 2001b). There were two episodes of care for breast cancer (3.5 expected) among a subset of the Decatur fluorochemical production workforce compared to zero episodes of care in the comparison film plant employee population (4.0 expected) (Olsen ef al. 2001b). One of these: individuals had worke>d 10 years. As for benign neoplasms of the breast, the risk ratio was 1.1 (i0n.d4i-v2i.d8u)albaespeidsoodnesnionfecairndeiivnidtuhaelfeiplimspoldaenst.ofNcoanr-emainlitghneaDnetcadtiusrorfdleurosroofchtehmeibcraelasptlawnetraensdlitgehntly higher among Decatur fluorochemical female employees as the episodes of care risk ratio was 1.6 (95% CI10.9-2.9) based on 28 individual episodes of care in the chemical plant and 19 individual episodes of care in the film plant. The majority of these episodes of care were. identified as fibrocystic disease. Mammary Tumor Summary 2 In summary, the tumors seen in the mammary glands of rats fed PFOA reflect background incidence. Prostate Tumors An epidemiological investigation of the Cottage Grove chemical division workforce associated prostate cancer mortality with employment duration in perfluorochemical production activities (Gilliland, 1992; Gillila&nd Mandel, 1993). Specifically > 10 years of employment was associated with a 3.3 fold increase (95% CI 1.0 -10.6) in prostate cancer mortality relative to workers not employed in the chemical division. A major limitation of ths investigation, with regard to evaluating the potential effects of PFOA exposure, was the lack ofjob and department specificity in the duration of employment analyses. Only one Cottage Grove employee had worked directly in the PFOA production building (Olsen ef al, 1998). Alexander (2001a) addressed this limitation by computerizing all work history records of Cottage Grove employees with at least one year of cumulative employment and constructing a calendar year, job- and department- specific exposure matrix from this computerized database. Alexander (20012) did not find prostate cancer mortality associated with duration of employment among those Cottage: Grove employees with definite or possible exposure to PFOA (cases observed/expected): 0 - < 1 year (0/0.1),1 - <5 years (2/14),5 - < 10 years (0/9.8) and > 10 years (4/2.9). The SMR was 1.4 (95% C1 0.4 - 3.5) for prostate cancer in the > 10 year duration category. The Alexander (20012) investigation improved upon the methods used for exposure assessment, nevertheless, some misclassification of exposure is likely. Maintenance and other mobile workers not specifically identified as definitely PFOA exposed workers may have routinely entered the areas of high exposure (drying and packaging). The extent to which this misclassification occurred and the effects on the study results is unknown. Among the Decatur fluorochemical production workforce, there have been no prostate cancer deaths (1.0 expected) (Alexander, 2001b). In the episodes of care investigation of this same workforce with 10or more years of experience, however,a isk ratio of 8.2 (0.8-399) was reported for prostate malignant neoplasms based on 4 episodesof care among fluorochemical workers (1.5 expected) compared to 1 episode of care among the comparison film plant workers (3.1 expected) (Olsen ef al, 2001b). On the other hand, there was no evidence of prostatic hypertrophy as the episodesofcare risk ratio was 1.0 (95% CI 0.6-1.5) based on 24 individual episodes of care in the Decatur fluorochemical plant and 52 episodes of care in the film plant. Conclusions At the exposure levels encountered in either the workplace or the environment, PFOA does not appear to present a human health risk. The chemical is not genotoxic in assays measuring various endpoints and utilizing test systems ranging from bacteria to mammals. The developing fetus is not uniquely sensitive to the effects of PFOA. Indications oaf fetal response are seen only under dosing/exposure conditions in which the adult animal is also responding. No evidence of structural abnormalities produced by in utero exposure to PFOA exists from animal tests. Clearly, the effects observed in the two-generation reproduction study (decreased pup weights, increased pup mortality, and sexual maturation delays only at the 30 mg/kg dose) did not compromise the reproductive success (i.., mating and fertility) of PFOA-exposed rats. With 1B respect to the human experience there is no evidence of increases in episodes of medical care. related to either developmental or reproductive health matters. In addition, evaluation of the hormonal status of 3M workers from the Cottage Grove, MN plant did not reveal any changes in sex hormones associated with PFOA exposure, In the long-term studies with PFOA iin rats, the incidence of tumors of the liver, pancreas, and testes was increased. An apparent increase in mammary fibroadenomas, seen in the PFOA treated females, was the result ofan unusually low incidence of fibroadenomas in this particular control group. The incidence of mammary tumors in all test groups was within the range expected for this strainofratbasedon historical control data `The tumors whose incidence is increased in rats treated with PFOA (liver, testes and pancreas) arefrequently observed in rats treated with Ps. It is generally recognized that rats have a heightened response to peroxisome proliferators relative to other species, including man, due in part due to their higher level of expression of the nuclear receptor PPARa. Because of the increased sensitivity of rats to PPs, the human significance of these three tumor types is not clear. `With respect to the liver, tumors observed in rats result from PPAR activation and are unlikely 10 be relevant to humans. The relevance to humans of pancreatic acinar cell tumors and Leydig cell tumors is also questionable. In addition, available data for humans who have had long-term treatment with hypolipidemic drugs (which are potent peroxisome proliferators in rats) show no increase in these three cancers associated with their long-term use. Studies of workers, believed to be the highest exposed human population, have not shown an increased cancer risk. Mortality studies show no increase in any cancer that could be associated with PFOA exposure. In addition, the episodes-of-care study and clinical studies of workers do not reveal any indications of PFOA-related response of liver, testes, and pancreas. 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