Evidence for Bisphenol B Endocrine Properties: Scientific and Regulatory Perspectives

Vol. 127, No. 10 ReviewOpen AccessEvidence for Bisphenol B Endocrine Properties: Scientific and Regulatory Perspectives Hélène Serra, Claire Beausoleil, René Habert, Christophe Minier, Nicole Picard-Hagen, and Cécile Michel Hélène Serra Chemical Substances Assessment Unit, Risk Assessment Department, French Agency for Food, Environmental and Occupational Health Safety (ANSES), Maisons-Alfort, France Search for more papers by this author , Claire Beausoleil Chemical Substances Assessment Unit, Risk Assessment Department, French Agency for Food, Environmental and Occupational Health Safety (ANSES), Maisons-Alfort, France Search for more papers by this author , René Habert Unit of Genetic Stability, Stem Cells and Radiation, Laboratory of Development of the Gonads, University Paris Diderot, Institut national de la santé et de la recherche médicale (Inserm) U 967 – CEA, Fontenay-aux-Roses, France Search for more papers by this author , Christophe Minier UMR I-2 Laboratoire Stress Environnementaux et BIOsurveillance des milieux aquatique (SEBIO), Normandie University, Le Havre, France Search for more papers by this author , Nicole Picard-Hagen Toxalim, Institut National de la Recherche Agronomique (INRA), Toulouse University, Ecole Nationale Vétérinaire de Toulouse (ENVT), Ecole d’Ingénieurs de Purpan (EIP), Université Paul Sabatier (UPS), Toulouse, France Search for more papers by this author , and Cécile Michel Address correspondence to Cécile Michel, 14 rue Pierre & Marie Curie, F-94700 Maisons-Alfort, France. Email: E-mail Address: [email protected] Chemical Substances Assessment Unit, Risk Assessment Department, French Agency for Food, Environmental and Occupational Health Safety (ANSES), Maisons-Alfort, France Search for more papers by this author Published:16 October 2019CID: 106001https://doi.org/10.1289/EHP5200Cited by:27AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground:The substitution of bisphenol A (BPA) by bisphenol B (BPB), a very close structural analog, stresses the need to assess its potential endocrine properties.Objective:This analysis aimed to investigate whether BPB has endocrine disruptive properties in humans and in wildlife as defined by the World Health Organization (WHO) definition used in the regulatory field, that is, a) adverse effects, b) endocrine activity, and c) plausible mechanistic links between the observed endocrine activity and adverse effects.Methods:We conducted a systematic review to identify BPB adverse effects and endocrine activities by focusing on animal models and in vitro mechanistic studies. The results were grouped by modality (estrogenic, androgenic, thyroid hormone, steroidogenesis-related, or other endocrine activities). After critical analysis of results, lines of evidence were built using a weight-of-evidence approach to establish a biologically plausible link. In addition, the ratio of BPA to BPB potency was reported from studies investigating both bisphenols.Results:Among the 36 articles included in the analysis, 3 subchronic studies consistently reported effects of BPB on reproductive function. In rats, the 28-d and 48-week studies showed alteration of spermatogenesis associated with a lower height of the seminiferous tubules, the alteration of several sperm parameters, and a weight loss for the testis, epididymis, and seminal vesicles. In zebrafish, the results of a 21-d reproductive study demonstrated that exposed fish had a lower egg production and a lower hatching rate and viability. The in vitro and in vivo mechanistic data consistently demonstrated BPB’s capacity to decrease testosterone production and to exert an estrogenic-like activity similar to or greater than BPA’s, both pathways being potentially responsible for spermatogenesis impairment in rats and fish.Conclusion:The available in vivo, ex vivo, and in vitro data, although limited, coherently indicates that BPB meets the WHO definition of an endocrine disrupting chemical currently used in a regulatory context. https://doi.org/10.1289/EHP5200IntroductionSince the 1960s, bisphenol A (BPA) has been widely used in the production of a variety of polymers such as polycarbonate plastics, epoxy resins, or thermal papers and is therefore found in a wide range of consumer products, including plastics, receipts, and food packaging (ANSES 2011). Over the last decades, concerns on reproductive, metabolic, and developmental effects have led regulatory bodies worldwide to ban BPA from baby bottles (Government of Canada 2010; EC 2011). Further restrictions have been implemented for BPA’s use in food packaging (EC 2018) and in thermal papers (EC 2016a). In 2017, BPA was recognized as an endocrine disrupting chemical (EDC) and a substance of very high concern (SVHC) in the European Union (EU) for both human health (ECHA 2017a) and for the environment (ECHA 2017b), limiting its importation and use on the European market. To meet the regulatory agencies’ restrictions on BPA uses, the plastics industry has gradually replaced this substance with some structural analogs, although many voices have questioned whether these substitutes are indeed safer than BPA (Gao et al. 2015; Eladak et al. 2015; Kinch et al. 2015). Concern on some widely used substitutes have been substantiated, such as bisphenol S and bisphenol F (reviewed by Rochester and Bolden 2015), leading to further regulatory evaluation of their endocrine properties in the EU (ECHA 2018c). However, the health and environmental hazards of many other BPA analogs have not been addressed so far, albeit their endocrine activity might be similar to that of BPA (NTP 2017; Perez et al. 1998).Bisphenol B (BPB) shares a strong structural similarity with BPA. It differs from BPA only by an additional methyl group on the central carbon (Figure 1). BPB is identified in The Endocrine Disruptor Exchange list (TEDX 2018) of potential EDCs, and in vitro results of the U.S. EPA Endocrine Disruptor Screening Program (EDSP; U.S. EPA 2018) indicate an agonist activity toward the estrogen receptor (ER). BPB is currently registered by the U.S. Food and Drug Administration (FDA) as an indirect food additive used in food-contact resinous and polymeric coatings (FDA 2018) but not in the EU under the European regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH; ECHA 2018b). This means that BPB is produced or put on the European market at <1 ton/y. Therefore, no EU registrant has a legal obligation to produce toxicological or ecotoxicological data (EC 2006). Nevertheless, BPB has been detected in several European food products such as various canned foods (Cunha et al. 2011; Grumetto et al. 2008; Fattore et al. 2015; Alabi et al. 2014) and in commercial milk samples (Grumetto et al. 2013).Figure 1. Chemical structures of bisphenol A (BPA) and bisphenol B (BPB).Compared with BPA, there is limited data on human exposure levels to BPB. The biomonitoring data indicate that BPB was detected in the same order of magnitude to BPA in the urine of Portuguese volunteers (Cunha and Fernandes 2010) and in the serum of endometriotic women in Italy (Cobellis et al. 2009), although in a lower percentage of individuals screened. In contrast, BPB was not detected in urine samples of Australian pregnant women (Heffernan et al. 2016), nor of Norwegian mother–child pairs (Sakhi et al. 2018) or Chinese residents (Yang et al. 2014a). In the environment, BPB is one of the least investigated and detected bisphenols (reviewed by Noszczyńska and Piotrowska-Seget 2018). Mean total BPB concentrations of 2.5 ng/L and 8.46 ng/L were measured in municipal sewage treatment plants (STP) influents in India (Karthikraj and Kannan 2017) and in industrial STP effluents in Slovenia (Česen et al. 2018), respectively. BPB was quantified in 1 sediment sample in Korea at 10.6 ng/g of 172 samples collected in Japan, Korea, and the United States (Liao et al. 2012). BPB was not detected in surface water in Japan, Korea, or India (Yamazaki et al. 2015) or in different areas of China such as the Liaohe River basin (Jin and Zhu 2016), Beijing (Yang et al. 2014b), and the Jiuxiang river in Nanjing (Zheng et al. 2015). It was also not detected in the Taihu water source up to 2016 (Wang et al. 2017; Jin and Zhu 2016); however, two recent studies reported its quantification in almost all water and sediment samples of the same Chinese lake with mean concentrations in the low nanograms per liter (water) or nanograms per gram (sediment) (Yan et al. 2017; Liu et al. 2017). Information on BPB levels in European freshwater ecosystems is currently lacking.There are currently no mandatory regulatory requirements to assess the endocrine properties of industrial chemicals such as BPB. The challenge posed by BPA’s analogs lies in assessing their endocrine disrupting potential based on the available toxicological data. This is of particular importance to avoid industrial investment in unsafe substitutes and to prevent human and environmental health consequences. The World Health Organization (WHO) defines an EDC as “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations” (Damstra et al. 2002). This definition, which is also the basis of the EU criteria for EDC (EC 2016b; Slama et al. 2016), involves three elements that must be identified concomitantly: an adverse effect, a modulation of endocrine functions, and a plausible mechanistic link between the endocrine activity and the adverse effect. The relationship between these keystones necessary to identify an EDC has been long studied and debated in Europe (Munn and Goumenou 2013). In 2018, the European Commission published a guidance (EDC guidance; ECHA and EFSA 2018) based on the WHO and International Programme on Chemical Safety (WHO/IPCS) definition and the Organisation for Economic Co-operation and Development (OECD) conceptual framework for testing and assessment of endocrine disrupters (guidance document no. 150; OECD 2018). First developed to support the regulatory requirement to identify and regulate EDCs covered by the plant protection products and the biocidal products regulations, this EDC guidance provides a unique methodological approach to evaluate endocrine properties. Integrating these methodologic reflections, the objective of this work was to perform a systematic review of the existing scientific literature to assess BPB endocrine disruptive properties according to the WHO/IPCS definition, cconsidering both human health and wildlife.MethodsContextAs part of the French National Strategy on Endocrine disruptors (Ministries of Health and Ecological Transition 2014), BPB has been put on the list of compounds to be evaluated by the dedicated group of experts on EDC [i.e., the French Agency for Food, Environmental and Occupational Health Safety (ANSES) EDC working group]. This collective expert assessment undertaken at our agency enabled transparent and multidisciplinary discussions and debates on scientific data and regulatory decisions (see https://www.anses.fr/en/content/expert-committees-and-working-groups for more information). We conducted a systematic review on BPB endocrine disruptive properties by focusing on animal and in vitro mechanistic studies, but also on human epidemiological and case studies. However, biomonitoring and in silico data were not included in the review. This analysis was performed following the principles displayed in the EDC guidance developed by the European Chemical Agency (ECHA) and the European Food Safety Authority (EFSA), with the support of the Joint Research Centre (JRC), recently published to identify EDC under the plant protection products and the biocidal products regulations (ECHA and EFSA 2018). The EDC guidance provides a tiered approach to assess the adversity of chemicals on vertebrates, and to link it with an estrogenic (E), androgenic (A), thyroid hormone (T), or steroidogenesis-related (S) mode of action (the so-called EATS modalities). The evidence is first assembled by using a systematic review and weight-of-evidence approach. Then, the EATS-mediated adversity and the endocrine activity are assessed. If sufficient evidence is gathered, a mode of action is postulated and the plausible biological link discussed. The detailed methodology is presented in the following sections.Research QuestionA Population, Exposure, Comparator and Outcome (PECO) statement was developed to answer the question “Do the BPB endocrine properties meet the WHO definition of an endocrine disruptor?” (Table 1). The systematic review focused on studies investigating BPB effects for several levels of doses or concentrations, in in vitro, ex vivo, and experimental vertebrate models because they are relevant for human (mammals such as dogs, rodents, rabbits) and wildlife (e.g., fish, amphibians, birds, and reptiles), as well as human epidemiological and case studies, when available.Table 1 Population, Exposure, Comparator and Outcome (PECO) key information: definition and associated search terms.Table 1 has two columns. The first column lists the particulars PECO. The second column lists the corresponding definition of the particulars.DefinitionPopulationIn vitro, ex vivo, and experimental animal studies on vertebrates relevant for human health (e.g., mammals such as dogs, rodents, and rabbits) and wildlife (e.g., fish, amphibians, birds, reptiles) and human epidemiological and case studies [as defined in the EDC guidance (ECHA 2018c)]aExposureBisphenol B (CAS 77-40-7)ComparatorExposed groups vs. vehicle-treated controlsOutcomeChemically induced endocrine activity or adverse effects related to EATS modalities (e.g., testis weight, hormone levels), or not specific to EATS modality (e.g., fertility)Note: CAS, Chemical Abstracts Service; EATS, estrogenic, androgenic, thyroid hormone, and steroidogenesis-related; EDC, endocrine disrupting chemical.aHuman and wildlife biomonitoring studies and in silico data were not included.Search Design and Data CollectionThe systematic searches were performed on 5 September 2018 in PubMed and Scopus databases without limitations on year of publication. We applied a single concept strategy search to retrieve all relevant information on BPB by using its Chemical Abstracts Service Registry Number (CASRN; CAS 77-40-7), scientific chemical names, and common names (e.g., “bisphenol derivative” or “bisphenol substitute”), as recommended in the EDC guidance (ECHA and EFSA 2018). The literature search strategy is presented in Table S1.Studies were included in this systematic review when they met all of the following criteria: a) peer-reviewed research articles or primary reports of research findings that presented original data; b) exposure to one or various BPB doses; c) endocrine activity or adversity assessed in in vitro, ex vivo, or in vivo studies in vertebrate species (Table 1); and d) English-language articles. Accordingly, the exclusion criteria were as follows: a) no original data (e.g., review article) or abstract only, b) lack of exposure to BPB; c) lack of measurement of endocrine activity or adversity; d) in silico data, human or environmental biomonitoring studies; and e) full text not available in English. The relevance filtering was first based on title and abstract screening, and, second, on full-text screening. When checking title and abstract was insufficient to decide if the paper was relevant and should be included in the review, full-text screening was applied (e.g., BPB not explicitly mentioned in the abstract). Two reviewers (C.B. and H.S.) shared the two screening phases, and resolved any conflicts or discrepancies by complementary full-text screening and by discussion.In addition to the systematic literature search and screening, ToxCast (Chen et al. 2017) and EDSP (U.S. EPA 2018) databases were queried for BPB bioactivity results using the CASRN to identify high-throughput in vitro screening assays that measured endocrine activity. The endocrine activity of each assay was defined by modality, that is, estrogenic, androgenic, thyroid hormone, or steroidogenesis-related (i.e., EATS) or non-EATS (others) endocrine activity based on selected criteria presented in Figure 2. Cross references of peer-reviewed research articles and gray literature (e.g., reports by national agencies) were also included in the review.Figure 2. Estrogenic, androgenic, thyroid hormone, and steroidogenesis-related (EATS) and non-EATS (others) endocrine activity parameters selected for the systematic review analysis. The parameters were selected based on recommendations of the endocrine disrupting chemical (EDC) guidance (ECHA and EFSA 2018) and of the French Agency for Food, Environmental and Occupational Health Safety (ANSES) EDC working group.Initial Analysis of the ResultsThe following information was extracted from studies included in the review: author names, publication year, study design (biological model, type of treatment, exposure duration, range of concentrations tested) and the response observed. In addition, the ratios of BPA to BPB half maximal effective concentration (EC50) or half maximal inhibitory concentration (IC50) was reported from studies investigating both bisphenols to allow comparison of potency. Results on endocrine activity were grouped by EATS or non-EATS (others) modalities. In the next step, the data were grouped into three categories following OECD conceptual framework (OECD 2018) and EU EDC guidance (ECHA and EFSA 2018): a) in vitro mechanistic parameters (OECD Level 2); b) in vivo mechanistic parameters (OECD Level 3); and c) parameters providing information on adversity (OECD Levels 3, 4, and 5). OECD Level 2 and 3 data are mainly informative of endocrine activity, whereas Level 4 and 5 data provide information on adversity. All the results were combined by modality and parameter categories. Based on the adverse effects identified, results were further integrated into lines of evidence, defined as a “set of relevant information grouped to assess a hypothesis,” using a weight-of-evidence approach (ECHA and EFSA 2018).Assessment of the EvidenceEvaluation of study quality was performed using the Toxicological data Reliability Assessment Tool (ToxRTool) for all studies investigating adverse effects and for the mechanistic studies included in the lines of evidence (Schneider et al. 2009). The tool comprises 21 criteria for in vivo studies and 18 criteria for in vitro studies that cover information on the test substance, the test system, the study design, results, and plausibility of results. All criteria are answered either by 0 or by 1, and some selected criteria are deemed indispensable for evaluating the reliability of the study, namely: information on the identity and purity of the test substance, concentrations/doses tested, frequency and duration of exposure, time point of observation, species studied, inclusion of negative and positive controls, administration route, number of animals per group, and adequacy of the study design. The total number of criteria met enables the assignment of Klimisch Categories 1 (reliable without restrictions), 2 (reliable with restrictions), or 3 (not reliable) (Klimisch et al. 1997). The limitations of in vitro and in vivo studies identified were reported along with the results of the systematic review. All relevant studies were included and when the reliability was questionable (i.e., ToxR score of 3), the limitations were discussed as part of the weight-of-evidence approach. Teams of regulators and researchers of the EDC working group with relevant expertise in the field assessed in vivo studies investigating BPB adverse effects and discussed the biological link for the mode of action postulated (R.H., Ce.M., and C.B. for human health data, N.P.H., Ch.M., and H.S. for environmental data).ResultsThe systematic search and screening resulted in the identification of 494 unique documents that described studies of BPB in experimental animals, and in ex vivo or in vitro models as presented in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart (Figure 3). This included 484 articles from PubMed and Scopus databases and 10 documents identified by screening cross-references and gray literature. After title, abstract, and full-text screening, 36 articles fulfilled the inclusion criteria according to the PECO statement. The relevant studies are listed in Excel Table S1, and the detailed results from literature searches of all databases are provided in Excel Tables S2–S5. The majority of studies focused on BPB mechanistic effects, and only 3 studies investigated BPB adverse effects in intact organisms. An overview of the in vitro and ex vivo mechanistic information is presented in Figure 4. There were 801 assay results on BPB bioactivity available in the ToxCast database, among which, 132 met the definitions of EATS and non-EATS (others) endpoints (Figure 2; see also Excel Table S3). In addition, 33 bioactivity results retrieved from EDSP database on E, A, and T modalities were included in the analysis (see Excel Table S4).Figure 3. PRISMA flow diagram followed for studies selection. BPB, bisphenol B; EDC, endocrine disrupting chemical; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.Figure 4. Summary of in vitro and ex vivo endocrine activity results identified from (A) literature search and (B) database screening. The results are classified by modality [estrogenic, androgenic, thyroid hormone, and steroidogenesis-related (EATS), or others non-EATS] and end point category. (All the data are presented in Excel Tables S2–S4.) The total number of results refers to the number of experiments investigating a given end point (e.g., several end points may have been investigated within a given study). A, androgenic; E, estrogenic; H, hormone; S, steroidogenic; T: thyroidal; TA, transactivation assay; TPO, thyroid peroxydase; TR, thyroid hormone receptor.Adverse Effects of BPBThree recent in vivo studies identified adverse effects of BPB in vertebrates: two studies in rats from the same laboratory (Ullah et al. 2018a, 2018b) and one in zebrafish (Yang et al. 2017). The line of evidence for BPB adverse effect is presented in Table 2.Table 2 Line of evidence for BPB reproductive dysfunction in fish and male rats.Table 2 has seven columns, namely, end point; biological model; exposure; parameter; effect dose; ToxRscore; and reference.End pointBiological modelExposureParameterEffect doseToxR scoreaReferenceTestes histologyMale SD rats28 db50mg/kg BW/d: fewer secondary spermatocytes, tubules, and elongated spermatids in the lumen1Ullah et al. 2018aTestes histologyMale SD rats48 weeksLOAEL0.025mg/L: fewer spermatogonia, spermatocytes, and spermatids number3Ullah et al. 2018bTestes histologyMale zebrafish21 db1mg/L: alteration of testis tubules, decrease of mature spermatids1Yang et al. 2017Sperm parametersMale SD rats48 weeksLOAEL0.025mg/L: lower sperm number in the caput epididymis0.05mg/L: lower sperm motility, daily sperm production, sperm number in the cauda epididymisNo differences in the amount of viable sperm3Ullah et al. 2018bTestes histology, seminiferous tubulesMale SD rats28 dLOAEL50mg/kg BW/d: lower epithelial heightNo difference in the area of interstitium, nor on diameter of seminiferous tubule1Ullah et al. 2018aTestes histology, seminiferous tubulesMale SD rats48 weeksLOAEL0.05mg/L: lower epithelial heightNo difference in the area of interstitium, nor on diameter of seminiferous tubule3Ullah et al. 2018bGonado–somatic indexMale SD rats48 weeksLOAEC0.05mg/L: lower3Ullah et al. 2018bGonado–somatic indexMale zebrafish21 dLOEC1mg/L: lower1Yang et al. 2017Gonado–somatic indexFemale zebrafish21 dLOEC1mg/L: lower1Yang et al. 2017Hepato–somatic indexMale zebrafish21 dLOEC0.1mg/L: higher1Yang et al. 2017Hepato–somatic indexFemale zebrafish21 dLOEC0.1mg/L: higher1Yang et al. 2017FecundityAdult zebrafish21 dLOEC1mg/L: lower1Yang et al. 2017Hatching rate F1 generation)Adult zebrafish21 dLOEC1mg/L: lower1Yang et al. 2017Survival (F1 generation)Adult zebrafish21 dLOEC1mg/L: lower1Yang et al. 2017Note: BPB, bisphenol B; gonado–somatic index, [gonad weight/body weight]×100; hepatosomatic index, [liver weight/body weight]×100; LOAEC, lowest observed adverse effect concentration; LOAEL, lowest observed adverse effect level; LOEC, lowest observed effect concentration; SD, Sprague-Dawley.aToxR score refers to the study quality using Klimisch category (1, 2, or 3), which was assessed using the ToxRTool, which considers the test substance, test system, study design, results, and plausibility of results (Schneider et al. 2009).bQualitative assessment only, without statistical analyses reported.BPB and the male reproductive system in rodents. The two studies on male rats were conducted to compare the ability of several bisphenols (including BPA and BPB) to disturb male reproductive function (Ullah et al. 2018a, 2018b). In these papers, limitations in the description of the experimental procedure were identified. The modalities of the oral administration method performed was missing in the paper by Ullah et al. (2018a), and the histopathological evaluation was not sufficiently described. A low number of animals per group was used by Ullah et al. (2018b), and information on the sensitivity of hormonal assays and the number of replicates were lacking. Information on the CASRN and purity of the test chemical were not mentioned in either of the two papers by Ullah et al. (2018a, 2018b). Although to be taken with caution, these studies present a consistent set of data and were included in the weight-of-evidence approach.In the first study, male Sprague-Dawley rats (70–80 postnatal days [PND 70–80] of age) were orally exposed to BPB at 0, 5, 25, and 50mg/kg BW/d (7 animals/group) for 28 d (Ullah et al. 2018a). Effects on the testis morphology were evidenced with exposed rats exhibiting a statistically significant lower height of the seminiferous epithelium (−19%) compared with concurrent controls. The qualitative histological evaluation of the testis showed that exposed animals had fewer spermatids and sperm in the lumen of the seminiferous tubules compared with concurrent control animals, with only very few tubules and no elongated spermatids at the dose of 50mg/kg BW/d.In a follow-up study, Ullah et al. (2018b) reported a more documented analysis of the effects of a chronic exposure to low dose of BPB on testicular functions. PND-23 male Sprague-Dawley rats received drinking water containing 0, 5, 25, and 50μg/L BPB for 48 weeks. A daily intake of 0, 0.3, 1.5, and 3μg/kg BW/d can be roughly estimated from an average water daily intake of 6mL/d per 100g BW. However, it must be noted that the ingested BPB dose decreased over time given that the rats drink 10–14mL/d per 100g BW at PND 23 and 2–3mL/d per 100g BW at postnatal week 46 (Holdstock 1973). At the end of the treatment, rats exposed to 50μg/L BPB had a statistically significant lower relative weight of the testis, epididymis, and the seminal vesicle. Effects on sperm parameters were evidenced with a dose-dependent smaller daily sperm production statistically significant at 50μg/L (reduction of 9%) and a lower sperm number in the caput epididymis (statistically significant from 25μg/L onward) and in the cauda epididymis (statistically significant at 50μg/L). In the cauda epididymis of rats exposed at 50μg/L, sperm number and the motile sperm percentage were statistically significantly lower, whereas the viable sperm percentage remained similar to control levels. In this group, rats exhibited a statistically significantly lower height of seminal epithelium (−16%), without differences in the diameter and the relative area of seminiferous tubules. In addition, rats in the high-dose group had statistically significantly fewer spermatogonia, spermatocytes, and spermatids. Taken together, these data evidenced that a chronic exposure to BPB at low doses through drinking water altered the testis function of adult rat. Importantly, BPA response was assessed in the two papers by Ullah et al. (2018a, 2018b), and BPA and BPB had similar qualitative and quantitative effects.BPB adverse effects on fish reproduction. Yang et al. (2017) reported the results of a high quality and reliable fecundity study on zebrafish (Danio rerio) based on the OECD 229 and 230 technical guidelines with additional evaluation of endocrine parameters. Six 4-month-old male and six 4-month-old female zebrafish were exposed over 21 d to BPB at concentrations of 0, 0.001, 0.01, 0.1, and 1mg/L (nominal concentrations). The study showed that zebrafish exposed to BPB had a dose-dependently impaired reproductive function (i.e., male and female), evidenced by a lower number of eggs laid, and a smaller hatching rate and embryo survival, reaching statistical significance in the high-dose group (reduction of about 50% compared with concurrent control animals). Some malformations of the F1 generation (e.g., abnormal curvature of larvae) were also reported in this group. Exposed male and female zebrafish had a statistically significantly higher hepato-somatic index in the 0.1 and 1mg/L-exposure groups, and a statistically significantly lower gonado–somatic index in the 1mg/L-exposure group. At 0.1 and 1mg/L, the authors reported a histological testicular disorganization with the presence of an acellular area and a trend toward fewer mature spermatids, although not quantified. In the females, one fish exposed to 1mg/L lacked post-vitellogenic oocytes.BPB Endocrine ActivityAll 36 studies included in the review provided in vitro or ex viv