The reproductive toxicology of ammonium perfluorooctanoate (APFO) in the rat
Introduction
Ammonium perfluorooctanoate (APFO; FC-143, C8, C7Fl5COO−NH4+, CAS Registry No. 3825-26-1) has been used as a processing aid in the production of fluoropolymers. Perfluorooctanoate (PFOA; C7Fl5COO−), the dissociation product of APFO, is not metabolized (Pastoor et al., 1987, Vanden Heuvel et al., 1991) and has been identified in blood samples from exposed workers and the general population (Taves et al., 1976, Hansen et al., 2001, Olsen et al., 2003a). The most extensive survey of population PFOA exposure comes from three studies involving children, adults, and the elderly residing in the United States (Olsen et al., 2002a, Olsen et al., 2002b, Olsen et al., 2003b, Olsen et al., 2004) showing geometric mean serum concentrations in all three age groups of 4–5 ng/ml (ppb). The sources and pathways of exposure resulting in the presence of PFOA in general population sera have not been specifically identified. These may include: (1) emission of PFOA and its salts from industrial processes; (2) environmental or metabolic degradation of certain fluorochemical product residuals to PFOA; or (3) the distribution of PFOA as a residual in various fluorochemical products. There are marked differences in excretion of PFOA between species and between sexes in some species (Vanden Heuvel et al., 1991, Hanhijarvi et al., 1988, Ohmori et al., 2003, Burris et al., 2002). Of the species for which data have been obtained, female rats have the highest rate of elimination with half-lives measured in hours. By comparison, male rats have elimination half-lives measured in days. In humans, PFOA appears to be poorly eliminated. The human serum elimination half-life for PFOA is currently estimated to be 4.4±3.5 years (Burris et al., 2002) although there are some uncertainties around this estimate. Considering the low elimination rate of PFOA in humans relative to rats and the small amount of PFOA found in sera of the general population, it is reasonable to assume that the magnitude of human exposure is quite small relative to doses typically used in toxicological studies.
A large database of experimental studies on the potential health hazards of PFOA is available, as are recent reviews (USEPA, 2002, Kennedy et al., 2004). In addition to toxicology studies in laboratory animals, the potential association of APFO exposure with health effects in fluorochemical production workers has been studied since 1976 through medical monitoring and epidemiological investigation (Ubel et al., 1980, Olsen et al., 1998, Olsen et al., 2000). No excess cause-specific mortality has been associated with APFO exposure in this workforce (Alexander, 2001), and clinical indications of liver and hormonal function (including estradiol, testosterone, and cholecystokinin) are normal (Olsen et al., 1998, Olsen et al., 2000). In response to toxicological findings that suggested PFOA might modulate endocrine activity in the male rat (Biegel et al., 1995, Biegel et al., 2001), serum levels of several hormones (estradiol, 17-hydroxyprogesterone, free and bound testosterone, dehydroepiandrosterone sulfate, follicle-stimulating hormone, luteinizing hormone, prolactin, and thyroid-stimulating hormone) of 3M Company’s Cottage Grove workers (predominantly male) have been analyzed. Mean estradiol levels were 10% greater among employees with the highest serum PFOA concentrations (30 ppm) (Olsen et al., 1998) but were confounded by body mass index, a factor known to be correlated positively with elevated estradiol in males (Schneider et al., 1979, Deslypere et al., 1985, Kley et al., 1980). It was concluded that there was a reasonable assurance of no significant hormonal changes associated with PFOA exposure in man (Olsen et al., 1998).
Numerous repeated-dose studies have been conducted with a variety of species (mice, rats, and monkeys), and these have recently been reviewed (Butenhoff et al., 2002a, Kennedy et al., 2004, USEPA, 2002). Together these demonstrate that liver is the primary target organ for PFOA toxicity, with liver-weight increases being observed at doses lower than those causing histological or biochemical indications of liver injury. The hepatotoxicity manifests as increased liver weights, hepatocellular hypertrophy, liver degeneration and necrosis, increases in plasma transaminases, and proliferation of smooth endoplasmic reticulum and peroxisomes in rodents. Hypolipidemia has also been reported in some rodent studies. The liver effects can be reversible on cessation of dosing. Other effects observed in one or more species include body weight decreases and increased kidney weights (without histological correlate).
Two cancer studies were conducted in rats (Riker Pharmaceuticals, 1983, Biegel et al., 2001), and increases in Leydig cell hyperplasia and benign Leydig cell tumors were observed in both studies at dietary dose of 300 ppm. In the study by Biegel et al. (2001) increases in benign liver tumors and benign pancreatic acinar cell tumors were also observed at 300 ppm.
The potential of APFO to produce developmental effects has been evaluated in rats by the oral (Gortner, 1981, Staples et al., 1984) and inhalation (Staples et al., 1984) routes, and in the rabbit by the oral route (Gortner, 1982). No evidence of embryo-fetal toxicity or gross developmental abnormalities was noted in these studies, even at doses that produced maternal toxicity as manifested by reduced body-weight gain and increased mortality. Neonatal effects did not occur, and pups were normal throughout lactation (Staples et al., 1984). Gortner (1981) originally reported a defect of the lens of the eye in the rat; however, this was later determined to be an artifact of methodology (Staples, 1985). Also, Gortner (1982) found an increase in a normal, stress-related structural variant in the rabbit, a 13th rib. This finding is not considered a significant structural malformation per se and is not likely to be relevant to humans (Christian et al., 1987). The current practice regarding this variant is to not even count it as a variant. These studies suggest that APFO is not toxic to the embryo and fetus of rats and rabbits and does not produce structural aberrations in fetuses of either species. The morphologic effects of APFO on reproductive organs have been evaluated in a number of bioassays in multiple laboratory animal species. Results indicated no abnormalities.
The reproductive system has not been the principal target of investigation of APFO toxicity in past studies; therefore, past studies have not looked at the functional capability of the reproductive systems. Two somewhat related compounds, perfluorooctanesulfonate (PFOS) and N-ethyl-N-(2-hydroxyethyl)-perfluorooctanesulfonamide (which converts metabolically to PFOS and the N-ethyl-N-(2-acetyl)-perfluorooctanesulfonamide), have been shown to cause an increase in perinatal mortality in reproduction studies (Lau et al., 2003; York, 1999a, York, 1999b; Butenhoff et al., 2002b). The extent to which the postnatal effects seen with PFOS and N-EtFOSE are transferable to another perfluorinated acid, such as PFOA, was not known. The purpose of the present study was to evaluate the potential effects of APFO on functional reproduction and postnatal development across two generations of offspring.
Section snippets
Animals
Crl:CD(SD)IGS BR VAF/Plus® (Sprague–Dawley) rats were supplied by Charles River Laboratory (Kingston, NY—male rats; and Raleigh, NC—female rats). Rats were housed individually except during mating and lactation. All cage sizes and housing conditions were in compliance with the “Guide for the Care and Use of Laboratory Animals”. Rats were maintained on a 12:12 h light–dark cycle and fed ad libitum Certified Rodent Diet® #5002 (PMI Nutrition International, St. Louis, MO). Water was provided ad
P generation—reproductive outcome
There were no changes in any of the reproductive performance and outcome parameters of either male or female rats exposed to up to 30 mg/kg APFO (Table 1). In male rats, fertility was normal as were all sperm parameters. Pairings of treated male and female rats resulted in normal fertility, pregnancy, and natural delivery including length of gestation. Estrous cycling in treated females prior to mating was normal. The only statistically significant difference was that the days in cohabitation
Discussion
This study evaluated the male and female reproductive systems of Sprague–Dawley (CD) rats exposed orally to APFO by gavage at either 0, 1, 3, 10, or 30 mg/kg for two generations, one litter per generation. Guidelines for the study design were USEPA OPPTS 870.3800 “Reproduction and Fertility Effects” testing guidelines and FIFRA/TSCA GLP standards (40 CFR Parts 160 and 792). Parameters evaluated were gonadal function, estrous cycling, mating behavior, conception, parturition, lactation, weaning,
Acknowledgements
The authors would like to thank Joseph W. Lech and W. Ray Brown for their technical assistance, and Jill Hogan and Rosamaria Maldonado for their help in manuscript preparation. This work was sponsored by 3M Company.
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