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Normal Reproductive Organ Development in CF-1 Mice Following Prenatal Exposure to Bisphenol A S. Z. Cagen, Shell Chemical Co., Houston, TX, USA J. M. Waechter, Jr, The Dow Chemical Co., Midland, MI, USA S. S. Dimond, General Electric Co., Pittsfield, MA, USA W. J .Breslin, MPI Research, Mattawan, MI, USA J .H. Butala, Consultant to Aristech, Pittsburgh, PA, USA F. W. Jekat, Bayer AG, Wuppertal, Germany R. L. Joiner, General Electric Co., Pittsfield, MA, USA R. N. Shiotsuka, Bayer Corp., Stilwel
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  Normal Reproductive Organ Developmentin CF-1 Mice Following Prenatal Exposure to Bisphenol A S. Z. Cagen, Shell Chemical Co., Houston, TX, USAJ. M. Waechter, Jr, The Dow Chemical Co., Midland, MI, USAS. S. Dimond, General Electric Co., Pittsfield, MA, USAW. J .Breslin, MPI Research, Mattawan, MI, USAJ .H. Butala, Consultant to Aristech, Pittsburgh, PA, USAF. W. Jekat, Bayer AG, Wuppertal, GermanyR. L. Joiner, General Electric Co., Pittsfield, MA, USAR. N. Shiotsuka, Bayer Corp., Stilwell, KS, USAG. E. Veenstra, Shell Chemicals Ltd., London, EnglandL. R. Harris, The Society of the Plastics Industry, Inc., Washington, DC, USA ABSTRACT 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 toevaluate the effects of BPA on male sexual development. This study, performed in CF-1mice, was limited to the measurement of sex organ weights, daily sperm production(DSP), epididymal sperm count, and testis histopathology in the offspring of female miceexposed to low doses of BPA (0, 0.2, 2, 20, or 200 µ g/kg/day) by deposition in the mouthon gestation days 11-17. Male sexual development determinations were made in offspringat 90 days of age. Since this study was conducted to investigate and clarify low-doseeffects 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 thoseinvestigators. An additional group was dosed orally with 0.2 µ g/kg/day of diethylstilbestrol (DES), which was selected based as the maternal dose reported to havethe maximum effect on the prostate of developing offspring (vom Saal et al  ., 1996;1997). No treatment-related effects on clinical observations, body weight, or foodconsumption were observed in adult females administered any dose of BPA or DES.Similarly, no treatment-related effects on growth or survival of offspring from damstreated with BPA or DES were observed. The total number of pups born per litter wasslightly lower in the 200 µ g/kg/day BPA group when compared to controls, but thischange was not considered treatment-related since the litter size was within the normalrange of historical controls. There were no treatment-related effects of BPA or DES ontestes histopathology, daily sperm production or sperm count, or on prostate, preputialgland, seminal vesicle, or epididymis weights at doses previously reported to affect theseorgans or at doses an order of magnitude higher or lower. In conclusion, under theconditions 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 notobserved. The absence of adverse findings in the offspring of dams treated orally withDES challenges the low-dose hypothesis of a special susceptibility of mammalsexposed perinatally to ultra low doses of even potent estrogenic chemicals. Based on thedata 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 reproductiveor developmental toxicant. INTRODUCTION Bisphenol A (BPA) is an important chemical used principally as a monomer in themanufacture of a multitude of chemical products, including epoxy resins and polycarbonate. BPA has been extensively evaluated for toxicity in a variety of tests inrodents, including developmental toxicity, reproductive toxicity, and carcinogenicity(Morrisey et al  ., 1987; Morrisey et al  ., 1989; NTP, 1982). Generally, BPA has shown alow order of toxicity, was not a selective developmental or reproductive toxicant, and wasnot 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 inspecialized protocols (Dodds and Lawson, 1936; Bitman and Cecil, 1970), recently therehas been renewed attention to the estrogenicity of BPA. In 1993, Krishnan and coworkersdiscovered that BPA leaching from autoclaved polycarbonate flasks was confoundingstudies to determine if S. cerevisiae  produced estrogens. More recently, Gaido et al  .(1997) confirmed the weak estrogenicity of BPA in vitro , showing BPA to beapproximately 15,000 times less potent than 17 β -estradiol, and Kuiper et al  . (1997)demonstrated that BPA could interact with both the α - and β -estrogen receptors.The in vivo  estrogenic potential of BPA was srcinally demonstrated in short-termuterotrophic assays in rodents using parenteral administration (Dodds and Lawson, 1936;Bitman and Cecil, 1970). However, recent studies have demonstrated a clear route-dependency in the magnitude of the uterotrophic response to BPA. Significantly larger oral doses were required to produce an estrogenic response than those requiredsubcutaneously (Laws and Carey, 1997; Twomey, 1998a; Twomey; 1998b). Nouterotrophic responses to BPA were observed at oral doses ranging from 2 to 100,000 µ g/kg/day, whereas the no observed effect levels (NOELs) following subcutaneousadministration ranged from 2 to 1,000 µ g/kg/day (Twomey, 1998a; Twomey, 1998b).This route-dependency in uterotrophic response is consistent with the more extensivemetabolic clearance of BPA via the oral route as significantly lower BPA concentrationsin blood were present in orally exposed rats when compared to the concentrations in ratsdosed intraperitoneally or subcutaneously (Pottenger et al  ., 1996). These data suggest thatthe estrogenic properties of BPA have not been manifest in previously conducted oraltoxicity studies because of the relatively rapid metabolism and elimination of orallyadministered 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 efficiencyof sperm production in their male offspring. These results were not consistent with the   previously reported absence of reproductive or developmental effects following oral BPAexposures.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 groupin a rigorously controlled environment. MATERIALS AND METHODSStudy 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 proceduresdetailed 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 hencesensitivity of the study, 2) four BPA doses instead of two were used, 3) two methods wereused for determination of sperm count, 4) male offspring were sacrificed at 90 daysinstead 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 Saaland 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 maleshaving significantly larger organ weights than subordinate males (Bartos and Brain,1993). In addition, food consumption and growth in pregnant dams and their offspringwere 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 TheDow 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 toachieve the desired concentrations. Fresh solutions were prepared weekly for eachconcentration and stored in glass containers. Based on the expected body weight of 40grams for pregnant CF-1 mice at the midpoint of dosing (GD14), 0.75mL/kg wasadministered to each mouse. The dose solutions from each weekly preparation wereanalyzed 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-dayacclimation period, all mice were weighed on GD 0 and GD 10, observed daily for anyclinical 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-exposureinterval 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 µ g/kg/day) or DES (0.2 µ g/kg/day) in a tocopherol-stripped corn oil vehicle by deposition into themouth using a micropipetter as reported by Nagel et al  . (1997). All doses were adjusteddaily, based on body weight, to provide constant dose levels (Table 1).Throughout the study, all mice were kept in an environmentally controlled room withtemperature 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-volumemusic was played in the animal rooms to provide background noise (vom Saal andThayer, 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 themanufacturer. The same lots of diet were provided to animals from all groups at the sametime during the course of the study to control across groups for possible variation in thecontent of the diet. Water was available via glass bottles with Teflon seals during theexposure period.Females were housed individually throughout the study except during lactation when theywere housed with their litters. Adult females and weanling males were individuallyhoused in polypropylene plastic tubs with stainless steel lids and corncob bedding. Malesto 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 eachmouse was performed once daily and weekly after treatment stopped. Each weaned pupwas given a detailed clinical examination on the day of weaning, daily for the 4consecutive days following weaning, and weekly thereafter until study termination.Food consumption in time-mated females was recorded during the intervals of GD 0-7, 7-10, 10-11, and 11-17. Following selection of females to be placed on study, foodconsumption was measured during GD 11-17. After parturition, food consumption wasrecorded twice during the first and second weeks of lactation and at 2- to 3-day intervalsduring the last week of lactation. For post-weaning males that were retained until 90 daysof age, food consumption was recorded weekly at the time each body weight wasrecorded.
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