The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Evaluating endocrine disrupting chemicals: A perspective on the novel assessments in CLARITY-BPA.

BACKGROUND: The Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA) was a collaborative research effort to better link academic research with governmental guideline studies. This review explores the secondary goal of CLARITY-BPA: to identify endpoints or technologies from CLARITY-BPA and prior/concurrent literature from these laboratories that may enhance the capacity of rodent toxicity studies to detect endocrine disrupting chemicals (EDCs). METHODS: A systematic literature search was conducted with search terms for BPA and the CLARITY-BPA participants. Relevant studies employed a laboratory rodent model and reported results on 1 of the 10 organs/organ systems evaluated in CLARITY-BPA (brain and behavior, cardiac, immune, mammary gland, ovary, penile function, prostate gland and urethra, testis and epididymis, thyroid hormone and metabolism, and uterus). Study design and findings were summarized, and a risk-of-bias assessment was conducted. RESULTS: Several endpoints and methods were identified as potentially helpful to detect effects of EDCs. For example, molecular and quantitative morphological approaches were sensitive in detecting alterations in early postnatal development of the brain, ovary, and mammary glands. Hormone challenge studies mimicking human aging reported increased susceptibility of the prostate to disease following developmental BPA exposure. Statistical analyses for nonmonotonic dose responses, and computational approaches assessing multiple treatment-related outcomes concurrently in linked hormone-sensitive organ systems, reported effects at low BPA doses. CONCLUSIONS: This review provided an opportunity to evaluate the unique insights provided by nontraditional assessments in CLARITY-BPA to identify technologies and endpoints to enhance detection of EDCs in future studies.

Human and animal evidence of potential transgenerational inheritance of health effects: An evidence map and state-of-the-science evaluation.

BACKGROUND: An increasing number of reports suggest early life exposures result in adverse effects in offspring who were never directly exposed; this phenomenon is termed "transgenerational inheritance." Given concern for public health implications for potential effects of exposures transmitted to subsequent generations, it is critical to determine how widespread and robust this phenomenon is and to identify the range of exposures and possible outcomes. OBJECTIVES: This scoping report examines the evidence for transgenerational inheritance associated with exposure to a wide range of stressors in humans and animals to identify areas of consistency, uncertainty, data gaps, and to evaluate general risk of bias issues for the transgenerational study design. METHODS: A protocol was developed to collect and categorize the literature into a systematic evidence map for transgenerational inheritance by health effects, exposures, and evidence streams following the Office of Health Assessment and Translation (OHAT) approach for conducting literature-based health assessments. RESULTS: A PubMed search yielded 63,758 unique records from which 257 relevant studies were identified and categorized into a systematic evidence map by evidence streams (46 human and 211 animal), broad health effect categories, and exposures. Data extracted from the individual studies are available in the Health Assessment Workspace Collaborative (HAWC) program. There are relatively few bodies of evidence where multiple studies evaluated the same exposure and the same or similar outcomes. Studies evaluated for risk of bias generally had multiple issues in design or conduct. CONCLUSIONS: The evidence mapping illustrated that risk of bias, few studies, and heterogeneity in exposures and endpoints examined present serious limitations to available bodies of evidence for assessing transgenerational effects. Targeted research is suggested to addressed inconsistencies and risk of bias issues identified, and thereby establish more robust bodies of evidence to critically assess transgenerational effects - particularly by adding data on exposure-outcome pairs where there is some evidence (i.e., reproductive, metabolic, and neurological effects).

NIEHS/FDA CLARITY-BPA research program update.

Bisphenol A (BPA) is a chemical used in the production of numerous consumer products resulting in potential daily human exposure to this chemical. The FDA previously evaluated the body of BPA toxicology data and determined that BPA is safe at current exposure levels. Although consistent with the assessment of some other regulatory agencies around the world, this determination of BPA safety continues to be debated in scientific and popular publications, resulting in conflicting messages to the public. Thus, the National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S. Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively a variety of hypothesis-based research investigations and guideline-compliant safety testing with BPA. This collaboration is known as the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). This paper provides a detailed description of the conduct of the study and a midterm update on progress of the CLARITY-BPA research program.

A round robin approach to the analysis of bisphenol A (BPA) in human blood samples.

BACKGROUND: Human exposure to bisphenol A (BPA) is ubiquitous, yet there are concerns about whether BPA can be measured in human blood. This Round Robin was designed to address this concern through three goals: 1) to identify collection materials, reagents and detection apparatuses that do not contribute BPA to serum; 2) to identify sensitive and precise methods to accurately measure unconjugated BPA (uBPA) and BPA-glucuronide (BPA-G), a metabolite, in serum; and 3) to evaluate whether inadvertent hydrolysis of BPA-G occurs during sample handling and processing. METHODS: Four laboratories participated in this Round Robin. Laboratories screened materials to identify BPA contamination in collection and analysis materials. Serum was spiked with concentrations of uBPA and/or BPA-G ranging from 0.09-19.5 (uBPA) and 0.5-32 (BPA-G) ng/mL. Additional samples were preserved unspiked as 'environmental' samples. Blinded samples were provided to laboratories that used LC/MSMS to simultaneously quantify uBPA and BPA-G. To determine whether inadvertent hydrolysis of BPA metabolites occurred, samples spiked with only BPA-G were analyzed for the presence of uBPA. Finally, three laboratories compared direct and indirect methods of quantifying BPA-G. RESULTS: We identified collection materials and reagents that did not introduce BPA contamination. In the blinded spiked sample analysis, all laboratories were able to distinguish low from high values of uBPA and BPA-G, for the whole spiked sample range and for those samples spiked with the three lowest concentrations (0.5-3.1 ng/ml). By completion of the Round Robin, three laboratories had verified methods for the analysis of uBPA and two verified for the analysis of BPA-G (verification determined by: 4 of 5 samples within 20% of spiked concentrations). In the analysis of BPA-G only spiked samples, all laboratories reported BPA-G was the majority of BPA detected (92.2 - 100%). Finally, laboratories were more likely to be verified using direct methods than indirect ones using enzymatic hydrolysis. CONCLUSIONS: Sensitive and accurate methods for the direct quantification of uBPA and BPA-G were developed in multiple laboratories and can be used for the analysis of human serum samples. BPA contamination can be controlled during sample collection and inadvertent hydrolysis of BPA conjugates can be avoided during sample handling.

Persistently altered epigenetic marks in the mouse uterus after neonatal estrogen exposure.

Neonatal exposure to diethylstilbestrol (DES) causes permanent alterations in female reproductive tract gene expression, infertility, and uterine cancer in mice. To determine whether epigenetic mechanisms could explain these phenotypes, we first tested whether DES altered uterine expression of chromatin-modifying proteins. DES treatment significantly reduced expression of methylcytosine dioxygenase TET oncogene family, member 1 (TET1) on postnatal day 5; this decrease was correlated with a subtle decrease in DNA 5-hydroxymethylcytosine in adults. There were also significant reductions in histone methyltransferase enhancer of zeste homolog 2 (EZH2), histone lysine acetyltransferase 2A (KAT2A), and histone deacetylases HDAC1, HDAC2, and HDAC3. Uterine chromatin immunoprecipitation was used to analyze the locus-specific association of modified histones with 2 genes, lactoferrin (Ltf) and sine oculis homeobox 1 (Six1), which are permanently upregulated in adults after neonatal DES treatment. Three histone modifications associated with active transcription, histone H3 lysine 9 acetylation (H3K9ac), H3 lysine 4 trimethylation (H3K4me3), and H4 lysine 5 acetylation (H4K5ac) were enriched at specific Ltf promoter regions after DES treatment, but this enrichment was not maintained in adults. H3K9ac, H4K5ac, and H3K4me3 were enriched at Six1 exon 1 immediately after neonatal DES treatment. As adults, DES-treated mice had greater differences in H4K5ac and H3K4me3 occupancy at Six1 exon 1 and new differences in these histone marks at an upstream region. These findings indicate that neonatal DES exposure temporarily alters expression of multiple chromatin-modifying proteins and persistently alters epigenetic marks in the adult uterus at the Six1 locus, suggesting a mechanism for developmental exposures leading to altered reproductive function and increased cancer risk.

Expression of a dominant negative estrogen receptor alpha variant in transgenic mice accelerates uterine cancer induced by the potent estrogen diethylstilbestrol.

ERΔ3 transgenic mice expressing a dominant negative estrogen receptor α (ERα) variant lacking the second zinc finger in the DNA binding domain were developed to examine its potential to inhibit estrogen action in vivo. To investigate if ERΔ3 expression influences uterine carcinogenesis, ERΔ3 transgenic mice were exposed to diethylstilbestrol (DES) on post-natal days 1-5. Neonatal DES treatment induced uterine adenocarcinomas in 81% of 8-month-old ERΔ3 mice compared to 49% of wild-type females (p<0.016). ERΔ3 did not inhibit the expression of the estrogen-responsive progesterone receptor and lactoferrin genes in the presence of ERα or modify their expression in ERα knockout (αERKO) mice. Higher circulating 17ß-estradiol levels and non-classical signaling by ERΔ3 may be related to the earlier incidence of uterine cancer. These findings indicate that expression of this ERα variant can influence determining events in uterine cancer development and its natural occurrence in the human uterus would unlikely be protective.

Perinatal environmental exposures affect mammary development, function, and cancer risk in adulthood.

Puberty is an important transition that enables reproduction of mammalian species. Precocious puberty, specifically early thelarche (the appearance of breast "buds"), in girls of multiple ethnic backgrounds is a major health problem in the United States and other countries. The cause for a continued decrease in the age of breast development in girls is unknown, but environmental factors likely play a major role. Laboratory and epidemiological studies have identified several individual environmental factors that affect breast development, but further progress is needed. Current research needs include increased attention to and recording of prenatal and neonatal environmental exposures, testing of marketed chemicals for effects on the mammary gland, and understanding of the mammary gland-specific mechanisms that are altered by chemicals. Such research is required to halt the increasing trend toward puberty at earlier ages.

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay.

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-ß estradiol (E(2), 500µg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2).

Developmental exposure to endocrine-disrupting chemicals programs for reproductive tract alterations and obesity later in life.

Many chemicals in the environment, especially those with estrogenic activity, are able to disrupt the programming of endocrine signaling pathways established during development; these chemicals are referred to as endocrine-disrupting chemicals. Altered programming can result in numerous adverse consequences in estrogen-target tissues, some of which may not be apparent until later in life. For example, a wide variety of structural, functional, and cellular effects have been identified in reproductive tract tissues. In addition to well-documented reproductive changes, obesity and diabetes have joined the list of adverse effects that have been associated with developmental exposure to environmental estrogens and other endocrine-disrupting chemicals. Obesity is a significant public health problem reaching epidemic proportions worldwide. Experimental animal studies document an association of developmental exposure to environmental estrogens and obesity. For example, a murine model of perinatal exposure to diethylstilbestrol has proven useful in studying mechanisms involved in abnormal programming of differentiating estrogen-target tissues, including reproductive tract tissues and adipocytes. Other environmental estrogens, including the environmental contaminant bisphenol A, have also been linked to reproductive problems and obesity later in life. Epidemiology studies support similar findings in humans, as do studies of cells in culture. Together, these findings suggest new targets for abnormal programming by estrogenic chemicals and provide evidence supporting the scientific concept termed the developmental origins of adult disease. Furthermore, the association of environmental estrogens with obesity and diabetes expands the focus on these diseases from intervention or treatment to include prevention or avoidance of chemical modifiers, especially during critical windows of development.

Impact of environmental endocrine disrupting chemicals on the development of obesity.

Environmental chemicals with hormone-like activity can disrupt programming of endocrine signaling pathways during development and result in adverse effects, some of which may not be apparent until much later in life. Recent reports link exposure to environmental endocrine disrupting chemicals during development with adverse health consequences, including obesity and diabetes. These particular diseases are quickly becoming significant public health problems and are fast reaching epidemic proportions worldwide. This review summarizes data from experimental animals and humans which support an association of endocrine disrupting chemicals, such as diethylstilbestrol, bisphenol A, phytoestrogens, phthalates, and organotins, with the development of obesity. Potential mechanisms are summarized and future research needs are discussed.

Prenatal exposure to bisphenol a at environmentally relevant doses adversely affects the murine female reproductive tract later in life.

BACKGROUND: Exposure to endocrine-disrupting chemicals during critical developmental periods causes adverse consequences later in life; an example is prenatal exposure to the pharmaceutical diethylstilbestrol (DES). Bisphenol A (BPA), an environmental estrogen used in the synthesis of plastics, is of concern because its chemical structure resembles that of DES, and it is a "high-volume production" chemical with widespread human exposure. OBJECTIVES: In this study we investigated whether prenatal BPA causes long-term adverse effects in female reproductive tissues in an experimental animal model previously shown useful in studying effects of prenatal DES. METHODS: Timed pregnant CD-1 mice were treated on days 9-16 of gestation with BPA (0.1, 1, 10, 100, or 1,000 mug/kg/day). After delivery, pups were held for 18 months; reproductive tissues were then evaluated. RESULTS: Ovarian cysts were significantly increased in the 1-mug/kg BPA group; ovarian cyst-adenomas were seen in the other three BPA-treated groups but not in corn-oil controls. We observed increased progressive proliferative lesions of the oviduct after BPA treatment, similar to those described in response to DES. Further, although not statistically different from the controls, prominent mesonephric (Wolffian) remnants and squamous metaplasia of the uterus, as well as vaginal adenosis, were present in BPA-treated mice, similar to lesions reported following DES treatment. More severe pathologies observed in some BPA-treated animals included atypical hyperplasia and stromal polyps of the uterus; sarcoma of the uterine cervix; and mammary adenocarcinoma. We did not observe these lesions in controls. CONCLUSIONS: These data suggest that BPA causes long-term adverse reproductive and carcinogenic effects if exposure occurs during critical periods of differentiation.

Neonatal bisphenol-a exposure alters rat reproductive development and ovarian morphology without impairing activation of gonadotropin-releasing hormone neurons.

Developmental exposure to endocrine-disrupting compounds is hypothesized to adversely affect female reproductive physiology by interfering with the organization of the hypothalamic-pituitary-gonadal axis. Here, we compared the effects of neonatal exposure to two environmentally relevant doses of the plastics component bisphenol-A (BPA; 50 microg/kg and 50 mg/kg) with the ESR1 (formerly known as ERalpha)-selective agonist 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)trisphenol (PPT; 1 mg/kg) on the development of the female rat hypothalamus and ovary. An oil vehicle and estradiol benzoate (EB; 25 microg) were used as negative and positive controls. Exposure to EB, PPT, or the low dose of BPA advanced pubertal onset. A total of 67% of females exposed to the high BPA dose were acyclic by 15 wk after vaginal opening compared with 14% of those exposed to the low BPA dose, all of the EB- and PPT-treated females, and none of the control animals. Ovaries from the EB-treated females were undersized and showed no evidence of folliculogenesis, whereas ovaries from the PPT-treated females were characterized by large antral-like follicles, which did not appear to support ovulation. Severity of deficits within the BPA-treated groups increased with dose and included large antral-like follicles and lower numbers of corpora lutea. Sexual receptivity, examined after ovariectomy and hormone replacement, was normal in all groups except those neonatally exposed to EB. FOS induction in hypothalamic gonadotropic (GnRH) neurons after hormone priming was impaired in the EB- and PPT-treated groups but neither of the BPA-treated groups. Our data suggest that BPA disrupts ovarian development but not the ability of GnRH neurons to respond to steroid-positive feedback.

Environmental estrogens and obesity.

Many chemicals in the environment, in particular those with estrogenic activity, can disrupt the programming of endocrine signaling pathways that are established during development and result in adverse consequences that may not be apparent until much later in life. Most recently, obesity and diabetes join the growing list of adverse consequences that have been associated with developmental exposure to environmental estrogens during critical stages of differentiation. These diseases are quickly becoming significant public health issues and are fast reaching epidemic proportions worldwide. In this review, we summarize the literature from experimental animal studies documenting an association of environmental estrogens and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol (DES) that has proven useful in studying mechanisms involved in abnormal programming of various differentiating estrogen-target tissues. Other examples of environmental estrogens including the phytoestrogen genistein and the environmental contaminant Bisphenol A are also discussed. Together, these data suggest new targets (i.e., adipocyte differentiation and molecular mechanisms involved in weight homeostasis) for abnormal programming by estrogenic chemicals, and provide evidence that support the scientific hypothesis termed "the developmental origins of adult disease". The proposal of an association of environmental estrogens with obesity and diabetes expands the focus on the diseases from intervention/treatment to include prevention/avoidance of chemical modifiers especially during critical windows of development.

Few effects of multi-generational dietary exposure to genistein or nonylphenol on sodium solution intake in male and female Sprague-Dawley rats.

Previous work in our laboratory indicated that lifelong dietary exposure to estrogen-like endocrine disrupters increased sodium solution intake in adult male and female rats. Here, we sought to discern the critical periods necessary for this alteration as well as establish the effects of lower dietary concentrations of genistein and nonylphenol. Male and female Sprague-Dawley rats (F0) consumed phytoestrogen-free chow containing 0, 5, 100, or 500 ppm genistein (approximately equal to 0.0, 0.4, 8.0, and 40.0 mg/kg/day) or 0, 25, 200, or 750 ppm nonylphenol (approximately equal to 0.0, 2.0, 16.0, and 60.0 mg/kg/day). Rats were mated within treatment groups and offspring (F1) maintained on the same diets. Mating for the F1, F2, and F3 (genistein only) was within treatment groups. At postnatal day (PND) 21, the F3 generation began to consume unadulterated phytoestrogen-free chow such that genistein exposure occurred only in utero and preweaning. The F4 generation was never directly exposed to genistein. On PNDs 65-68, intake of regular water and a 3.0% sodium chloride solution was measured for F1-F4 generations (genistein portion) or F1-F2 (nonylphenol portion). Although body weights were decreased by the highest dietary concentrations of genistein and nonylphenol, there were only minimal effects of exposure on sodium solution intake. As expected, intake was highest in female rats. With previous data, these results indicate that the dietary concentrations necessary to increase adult sodium solution intake in rats are greater than 500 ppm genistein and 750 ppm nonylphenol and such effects do not appear to increase across generations.

Genistein and ethinyl estradiol dietary exposure in multigenerational and chronic studies induce similar proliferative lesions in mammary gland of male Sprague-Dawley rats.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and 2-yr chronic toxicity studies with different exposure durations across generations F(0) through F(4). Sprague-Dawley rats were exposed to genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb). Effects in the male mammary gland are described here. In the multigeneration studies, mammary hyperplasia was induced by both compounds; the chronic studies had a lower incidence, without proportionate neoplasia. Sexual dimorphism (predominant tubuloalveolar growth in females and lobuloalveolar in males) was retained without feminization in high dose genistein or EE(2). In the continuously exposed generations, mammary hyperplasia was sustained but not amplified, appeared morphologically similar across all generations, and was not carried over into unexposed offspring of previously exposed generations. The hyperplasia in male rats was similar whether induced by genistein or EE(2). Results substantiate and extend previous reports that mammary gland hyperplasia in the male rat is one of the most sensitive markers of estrogenic endocrine disruption.

Overlapping but distinct effects of genistein and ethinyl estradiol (EE(2)) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague-Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE(2) at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE(2) significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.