Normal Reproductive Organ Development

Bisphenol A (BPA) is a monomer used in the manufacture of a multitude of chemical products, including epoxy resins and polycarbonate. The objective of this study was to evaluate the effects of BPA on male sexual development. This study, performed in CF-1 mice, was limited to the measurement of sex organ weights, daily sperm production (DSP), epididymal sperm count, and testis histopathology in the offspring of female mice exposed to low doses of BPA (0, 0.2, 2, 20, or 200 μg/kg/day) by deposition in the mouth on gestation days 11-17. Male sexual development determinations were made in offspring at 90 days of age. Since this study was conducted to investigate and clarify low-dose effects reported by Nagel et al. (1997) and vom Saal et al. (1998), the study protocol purposely duplicated the referenced studies for all factors indicated as critical by those investigators. An additional group was dosed orally with 0.2 μg/kg/day of diethylstilbestrol (DES), which was selected based as the maternal dose reported to have the maximum effect on the prostate of developing offspring (vom Saal et al., 1996; 1997). No treatment-related effects on clinical observations, body weight, or food consumption were observed in adult females administered any dose of BPA or DES. Similarly, no treatment-related effects on growth or survival of offspring from dams treated with BPA or DES were observed. The total number of pups born per litter was slightly lower in the 200 μg/kg/day BPA group when compared to controls, but this change was not considered treatment-related since the litter size was within the normal range of historical controls. There were no treatment-related effects of BPA or DES on testes histopathology, daily sperm production or sperm count, or on prostate, preputial gland, seminal vesicle, or epididymis weights at doses previously reported to affect these organs or at doses an order of magnitude higher or lower. In conclusion, under the conditions of this study, the effects of low doses of BPA reported by Nagel et al. (1997) and vom Saal et al. (1998) or of DES reported by vom Saal et al. (1997) were not observed. The absence of adverse findings in the offspring of dams treated orally with DES challenges the "low-dose hypothesis" of a special susceptibility of mammals exposed perinatally to ultra low doses of even potent estrogenic chemicals. Based on the data in the present study and the considerable body of literature on effects of BPA at similar and much higher doses, BPA should not be considered as a selective reproductive or developmental toxicant. INTRODUCTION Bisphenol A (BPA) is an important chemical used principally as a monomer in the manufacture of a multitude of chemical products, including epoxy resins and polycarbonate. BPA has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental toxicity, reproductive toxicity, and carcinogenicity (Morrisey et al., 1987; Morrisey et al., 1989; NTP, 1982). Generally, BPA has shown a low order of toxicity, was not a selective developmental or reproductive toxicant, and was not carcinogenic to rats or mice. Several in vitro and in vivo genotoxicity studies have been conducted, all with negative results (BIBRA, 1989). Although it has been known for several decades that BPA is weakly estrogenic in specialized protocols (Dodds and Lawson, 1936; Bitman and Cecil, 1970), recently there has been renewed attention to the estrogenicity of BPA. In 1993, Krishnan and coworkers discovered that BPA leaching from autoclaved polycarbonate flasks was confounding studies to determine if S. cerevisiae produced estrogens. More recently, Gaido et al. (1997) confirmed the weak estrogenicity of BPA in vitro, showing BPA to be approximately 15,000 times less potent than 17β-estradiol, and Kuiper et al. (1997) demonstrated that BPA could interact with both the αand β-estrogen receptors. The in vivo estrogenic potential of BPA was originally demonstrated in short-term uterotrophic assays in rodents using parenteral administration (Dodds and Lawson, 1936; Bitman and Cecil, 1970). However, recent studies have demonstrated a clear routedependency in the magnitude of the uterotrophic response to BPA. Significantly larger oral doses were required to produce an estrogenic response than those required subcutaneously (Laws and Carey, 1997; Twomey, 1998a; Twomey; 1998b). No uterotrophic responses to BPA were observed at oral doses ranging from 2 to 100,000 μg/kg/day, whereas the no observed effect levels (NOELs) following subcutaneous administration ranged from 2 to 1,000 μg/kg/day (Twomey, 1998a; Twomey, 1998b). This route-dependency in uterotrophic response is consistent with the more extensive metabolic clearance of BPA via the oral route as significantly lower BPA concentrations in blood were present in orally exposed rats when compared to the concentrations in rats dosed intraperitoneally or subcutaneously (Pottenger et al., 1996). These data suggest that the estrogenic properties of BPA have not been manifest in previously conducted oral toxicity studies because of the relatively rapid metabolism and elimination of orally administered BPA and the low estrogenic potency of BPA. Recently, however, experiments by Nagel et al. (1997) and vom Saal et al. (1998) reported that administration of low oral doses of BPA to pregnant female mice (n = 5-7) on gestation days (GD) 11-17 produced statistically significant increases in the weights of the prostate and preputial glands and a decrease in epididymis weights and the efficiency of sperm production in their male offspring. These results were not consistent with the previously reported absence of reproductive or developmental effects following oral BPA exposures. Therefore, the objective of the present study was to repeat the experiments of Nagel et al. (1997) and vom Saal et al. (1998) using a larger number of animals per treatment group in a rigorously controlled environment. MATERIALS AND METHODS Study Design Since the present study was specifically undertaken to repeat the experiments of Nagel et al. (1997) and vom Saal et al. (1998), the study was designed to duplicate the procedures detailed in those reports as closely as possible with the following exceptions: 1) larger numbers of mice were used in all groups to increase the statistical power and hence sensitivity of the study, 2) four BPA doses instead of two were used, 3) two methods were used for determination of sperm count, 4) male offspring were sacrificed at 90 days instead of 180 days because effects on male sex accessory organs were reportedly driven by the in utero BPA exposure and sacrifice time after puberty was not critical (vom Saal and Thayer, 1997), and 5) males were individually housed from the time of weaning because group housing of males (in triads) is known to significantly affect the weight of sex accessory organs, including the prostate and preputial glands with dominant males having significantly larger organ weights than subordinate males (Bartos and Brain, 1993). In addition, food consumption and growth in pregnant dams and their offspring were measured throughout the study, as well as delivery and litter data. Test Materials Bisphenol A (BPA) used in this study, with a purity of >99%, was obtained from The Dow Chemical Company (Midland, MI). Diethylstilbestrol (DES), with a purity of 99%, was obtained from Sigma Chemical Co. (St. Louis, MO). Tocopherol-stripped corn oil (ICN Biomedicals Inc., Aurora, OH) served as the vehicle and control substance. Dose Preparation and Analysis Appropriate amounts of BPA and DES were mixed with tocopherol-stripped corn oil to achieve the desired concentrations. Fresh solutions were prepared weekly for each concentration and stored in glass containers. Based on the expected body weight of 40 grams for pregnant CF-1 mice at the midpoint of dosing (GD14), 0.75mL/kg was administered to each mouse. The dose solutions from each weekly preparation were analyzed prior to dosing to determine that the BPA and DES concentrations were within ∀10% of targeted concentrations. Animals and Treatment Time-mated CF-1 mice were obtained from Charles River Laboratories (Portage, MI). Upon receipt at the laboratory, the mice were housed individually. During the 11-day acclimation period, all mice were weighed on GD 0 and GD 10, observed daily for any clinical signs of disease, and given a detailed physical examination prior to the start of the study. Animals gaining >4.5 grams in body weight during the GD 0-10 pre-exposure interval were randomized into seven groups on GD 10, using a stratified (by weight) block randomization procedure, until 28 mice/treatment group were assigned (Table 1). The weight gain criterion reduced placement of non-pregnant females on the study. On GD 11-17, the mice were dosed orally with BPA (0, 0.2, 2, 20, or 200 μg/kg/day) or DES (0.2 μg/kg/day) in a tocopherol-stripped corn oil vehicle by deposition into the mouth using a micropipetter as reported by Nagel et al. (1997). All doses were adjusted daily, based on body weight, to provide constant dose levels (Table 1). Throughout the study, all mice were kept in an environmentally controlled room with temperature and relative humidity maintained between 69°-75°F (21°-24°C) and 43-65%, respectively. Fluorescent lighting provided illumination 12 hr/day via an automatic timer and lighting levels were maintained below 18 ft-candles (measured 1 meter off the floor and approximately 1-6 inches in front of the cages on each side of the rack). Low-volume music was played in the animal rooms to provide background noise (vom Saal and Thayer, 1997). Diet (Certified Rodent Chow #5002, PMI Feeds, Inc., St. Louis, MO) and drinking water were available ad libitum. Certification analysis of each lot of diet was performed by the manufacturer. The same lots of diet were provided to animals from all groups at the same time during the course of the study to control across groups for possible variation in the content of the diet. Water was available via glass bottles with Teflon seals during the exposure period. Females were housed individually throughout the study except during lactation when they were housed with their litters. Adult females and weanling males were individually housed in polypropylene plastic tubs with stainless steel lids and corncob bedding. Males to be retained to 90 days were individually housed following weaning in suspended, stainless steel, wire-mesh cages. In-Life Observations All mice were observed at least twice a day, seven days a week, for morbidity, mortality, and signs of injury. During treatment (GD 11-17), a detailed clinical examination of each mouse was performed once daily and weekly after treatment stopped. Each weaned pup was given a detailed clinical examination on the day of weaning, daily for the 4 consecutive days following weaning, and weekly thereafter until study termination. Food consumption in time-mated females was recorded during the intervals of GD 0-7, 710, 10-11, and 11-17. Following selection of females to be placed on study, food consumption was measured during GD 11-17. After parturition, food consumption was recorded twice during the first and second weeks of lactation and at 2to 3-day intervals during the last week of lactation. For post-weaning males that were retained until 90 days of age, food consumption was recorded weekly at the time each body weight was recorded. Time-mated females were weighed on GD 0, 10, and 11-18. Females that delivered litters were weighed on postnatal days (PND) 1, 4, 7, 14, and 21. Male and female pups were weighed individually on PND 1, 4, and 22. Each male pup to be maintained to 90 days of age was weighed on PND 22 and weekly thereafter. Table 1. Assignment to Study Number of Animals Group Dose (μg/kg/day) Dams Selected F1 Males