Bisphenol A (BPA, CAS #80-05-7) is a high-production-volume industrial chemical used as a monomer for polycarbonate plastics and epoxy resins that have broad applications in consumer products, including storage containers for foods and beverages and medical devices. The potential toxicity resulting from chronic exposure to BPA as an indirect food additive is the concern addressed in this study.

This study is part of the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA), a research program between the National Institute of Environmental Health Sciences (NIEHS) and the National Center for Toxicological Research (NCTR) of the Food and Drug Administration (FDA), developed to bridge guideline-compliant research conducted at the FDA with hypothesis-based research investigations conducted by academia on the toxicity of BPA. The CLARITY-BPA research program has two components: 1) A “core” guideline-compliant chronic study conducted at NCTR according to FDA Good Laboratory Practice (GLP) regulations and 2) studies of various endpoints, conducted by NIEHS-funded researchers at academic institutions using animals born to the same exposed pregnant rats as the core GLP study. The purpose of this research program was to evaluate chronic exposure to BPA over a broad dose range using traditional and non-traditional endpoints. It aimed to determine if non-traditional endpoints reveal toxicity not detected by traditional guideline study endpoints and provide mechanistic support for observations made in the guideline study. The current research report covers only data from the “core” guideline-compliant chronic study.

The toxicity of BPA administered by oral gavage from gestation day (GD) 6 through the start of labor and then by oral gavage to pups from postnatal day (PND) 1 (day of birth = PND 0) until termination at one year or two years was examined in Sprague-Dawley rats from the NCTR breeding colony (Sprague-Dawley/CD23/NctrBR). BPA doses were 2.5, 25, 250, 2,500, and 25,000 μg/kg body weight (bw)/day. A vehicle (0.3% carboxymethylcellulose (CMC)) control group was also included. In addition to animals that were dosed daily throughout the study, a stop-dose study arm was included with animals dosed daily until PND 21 and then held without further treatment until termination to assess any effects that were due to early exposure only. Because many of the effects of BPA reported in the literature are associated with estrogen signaling pathways, two doses (0.05 and 0.5 μg/kg bw/day) of the orally active estrogen ethinyl estradiol (EE₂) were also included in the continuous-dose arm to assess the sensitivity of the test system to low doses of an estrogen. Reference estrogen groups were not included in the stop-dose study arm of the core study due to resource constraints, primarily lack of animal facility space. Rats were obtained as weanlings from the NCTR breeding colony and placed under study conditions (soy- and alfalfa-free diet (5K96, LabDiet, Purina Mills), polysulfone cages, hardwood chip bedding, glass water bottles, and food-grade silicone stoppers) until mating. Study materials were monitored for background BPA levels; the only material with detectable levels of BPA was the diet, which had less than 3 ppb BPA. Prior to mating to males that were not siblings or first cousins, female rats were stratified by body weight and were randomized to treatment groups to give approximately equivalent mean starting body weights in each group. Each morning after pairing, females were examined for evidence of mating (presence of an in situ vaginal plug or sperm-positive vaginal smear). Upon evidence of mating, the females were separated from the males and individually housed; this day was considered GD 0. On GD 6, daily dosing of the dam with BPA, EE₂, or vehicle began and was based on the body weight measured immediately prior to the administration of these compounds. Direct gavage dosing of the pups was started on PND 1, with the same dose and agent that was administered to their dams. At weaning on PND 21, no more than one animal per sex per litter was assigned to the following study arms: 1) continuous dosing to sacrifice at two years (terminal sacrifice, 46–50 animals per sex per vehicle control or BPA treatment group and 26 animals per sex per EE₂ group); 2) continuous dosing to sacrifice at one year (interim sacrifice, 20–26 animals per sex for all groups); 3) no further treatment after PND 21 until sacrifice at two years (stop-dose terminal sacrifice, 46–50 animals per sex per preweaning vehicle control or BPA group); and 4) no further treatment after PND 21 until sacrifice at one year (stop-dose interim sacrifice, 20–26 animals per sex for preweaning vehicle control and BPA groups). The stop-dose study arms for which gavage dosing was not continued beyond weaning were included to assess the potential of permanent effects induced by exposure to hormonally active compounds during developmental stages. The interim (one-year) sacrifice group was included to allow evaluation of long-term exposure effects with less confounding due to background lesions of aging than would be expected at two years, and to allow assessment of any precursors of any treatment-related lesions observed at two years.

Data collected included body weights, litter parameters, age at vaginal opening, vaginal cytology, clinical pathology (interim sacrifice only), sperm parameters (interim sacrifice only), organ weights (interim sacrifice only), and histopathology (both interim and terminal sacrifices). Vaginal cytology data were collected for 14 consecutive days at approximately 16 weeks of age from the same subset of females in the terminal sacrifice arm that had been monitored for vaginal opening; these same animals were then monitored for five consecutive days monthly to estimate the time at which they began having aberrant estrous cycles. In addition to the summary tables provided in this report and appendices, all individual animal data are available online (https://doi.org/10.22427/NTP-DATA-018-00015-0001-000-6).

Table 1 lists all non-histopathology endpoints analyzed and associated statistical findings. For histopathology data, Table 1 only lists the endpoints where a statistically significant difference was found by the primary statistical tests applied (Cochran-Armitage/Fisher’s Exact Test for interim sacrifice animals; survival-adjusted Poly-3 test for terminal sacrifice animals). Results from all statistical tests applied to the histopathology data, which further included Jonckheere-Terpstra/Shirley-Williams (JT/SW) and relative treatment effect (RTE) tests for non-neoplastic lesions assigned severity scores, are included in the text of this abstract and in the report text and tables. Statistically significant results are indicated regardless of biological significance.

There were few significant effects of BPA treatment in the in-life data collected. In the late stages of the study (weeks 96–104), mean female body weights in the 250 μg BPA/kg bw/day continuous-dose group were significantly higher than the mean vehicle control body weights. For clinical pathology endpoints and organ weights, some statistically significant effects of continuous- or stop-dose BPA treatments were observed. These effects were of questionable relevance to BPA toxicity given that they were seen only in single-dose groups, in several cases differed from the vehicle control by less than 10%, and, in the case of organ weights, were not significant when adjusted for body weight.

In the stop-dose BPA study arm at two years, there was a statistically significant increase in the incidence of female mammary gland adenocarcinoma (22% versus 6%; p = 0.016) and the combination of adenoma and adenocarcinoma (24% versus 8%; p = 0.018) in the 2.5 μg BPA/kg bw/day dose group. No increase in female mammary gland neoplasms was observed in the continuous BPA dose arm at two years. There were no significant treatment-related non-neoplastic lesions in the mammary gland of interim or terminal sacrifice stop-dose BPA groups. In the interim and terminal BPA continuous dosing arm, there was an increase, significant by the secondary RTE test only, in female mammary gland atypical foci at 2.5 μg BPA/kg bw/day (14% versus 0% and 15% versus 4% for the interim and terminal dose group animals, respectively). Increased adenoma/adenocarcinoma incidence observed only in the stop-dose animals, lack of a dose response, absence of non-neoplastic lesions in interim or terminal sacrifice stop-dose animals, and comparison to limited historical control data for this strain of rats in experiments conducted at NCTR bring into question the biological plausibility of this lesion as a BPA treatment-related effect. In addition to mammary gland neoplasms, a significant trend (p = 0.037) for uterine stromal polyps in the interim sacrifice animals in the continuous BPA dose arm was observed; this was not observed in the terminal sacrifice animals.

In the histopathological evaluations, there were many non-neoplastic lesions associated with aging in this strain of rats in both males and females that were variable across control and BPA treatment levels. In the interim stop-dose sacrifice BPA females, there was a significant dose trend with a significant increase in follicular cysts in the ovary at 25,000 μg BPA/kg bw/day dose group. The secondary statistical tests, which incorporated both incidence and severity scores, indicated an increase in cystic endometrial hyperplasia and squamous metaplasia in the uterus at 25,000 μg BPA/kg bw/day in the interim stop-dose females. In the terminal stop-dose animals, secondary tests indicated an increase in cystic endometrial hyperplasia at 2,500 and 25,000 μg BPA/kg bw/day, although severity was similar in the vehicle control and the BPA-treated groups. Cardiomyopathy was increased in the terminal stop-dose females at 2.5, 250, 2,500, and 25,000 μg BPA/kg bw/day, as assessed by statistical tests that incorporated incidence and severity scores, although background incidence was high at this age and severity score differences across dose groups were similar. In interim continuous-dose females, uterine apoptosis and vaginal epithelial hyperplasia were elevated at 25,000 μg BPA/kg bw/day. Vaginal epithelial hyperplasia was also increased in terminal continuous-dose animals at doses from 25 to 25,000 μg BPA/kg bw/day, with a similar response across each of those dose levels.

There were no significant differences between treatment groups and vehicle controls in the incidences of neoplastic lesions in stop-dose or continuous-dose interim or terminal sacrifice males. There were also no apparent treatment-related non-neoplastic effects in interim stop-dose males; in terminal stop-dose BPA males, an increase of hyperplasia in the pars distalis of the pituitary at 25,000 μg BPA/kg bw/day was noted. In interim, but not terminal, continuous-dose males there was an increase in exfoliated germ cells and an increase in lymphocyte infiltration in the epididymis at 25,000 μg BPA/kg bw/day. No significant effects on sperm parameters or testicular histopathology were noted in the BPA dose groups. In the terminal continuous-dose males, hyperplasia of the pars distalis of the pituitary was increased at 25 and 25,000 μg BPA/kg bw/day. Increases in dorsal/lateral prostate inflammation in most BPA dose groups were variable across a high background in both interim and terminal sacrifice animals.

In the EE₂ reference estrogen dose groups, there were multiple significant treatment-related effects at the 0.5 μg/kg bw/day exposure level in females. At the time of estrous cycle evaluation at 16 weeks, more than 90% of the females in the 0.5 μg EE₂/kg bw/day dose group were exhibiting prolonged estrus. At the interim sacrifice, mean weights of the adrenal glands, heart, kidney, liver, and pituitary gland were higher in the 0.5 μg EE₂/kg bw/day dose group than the vehicle control means. Ovarian/parametrial fat pad and ovary weights were significantly lower than mean vehicle control weights in the high EE₂ dose group. At the interim sacrifice, lobular hyperplasia and ductal dilatation were elevated in the mammary glands of the 0.5 μg EE₂/kg bw/day dose group. Increases in apoptosis, cystic endometrial hyperplasia, and squamous metaplasia were observed in the uterus of the interim high dose EE₂ females. Atrophy and cystic follicles were increased in the ovaries, the incidence of vaginal hyperplasia was increased, and increases in hyperplasia of the pars distalis and angiectasis were observed in the pituitary at 0.5 μg EE₂/kg bw/day. The incidences of cardiomyopathy and nephropathy were also increased in the high dose EE₂ females at one year. At terminal sacrifice, there were significant increases in the incidence of mammary gland adenocarcinomas and combined adenomas/carcinomas of the pituitary pars distalis in the 0.5 μg EE₂/kg bw/day dose group. There was a trend toward increasing uterine metaplasia at two years, and the incidence of nephropathy was increased in both the 0.05 and 0.5 μg EE₂/kg bw/day dose groups.

Few statistically significant effects of EE₂ in males were observed. In the high dose EE₂ group, there was an elevated incidence of lymphocyte infiltration observed in the epididymis in interim sacrifice animals and an increase in hyperplasia in the pars distalis of the pituitary at two years.

In conclusion, in the CLARITY-BPA core study, statistical differences between BPA treatment groups, particularly below 25,000 μg/kg bw/day, and the vehicle control group detected by the low-stringency statistical tests applied to histopathology lesions, were not dose responsive, sometimes occurring in only one low or intermediate dose group, and did not demonstrate a clear pattern of consistent responses within or across organs within the stop- and continuous-dose arms and sacrifice times. In contrast, the high EE₂-dose elicited several estrogenic effects in females in a clearly interpretable and biologically plausible manner. Several observations at 25,000 μg BPA/kg bw/day may be treatment related, including effects mentioned above in the female reproductive tract (ovary, uterus, and vagina) and in the male pituitary.