A statement on the developmental immunotoxicity of bisphenol A (BPA): answer to the question from the Dutch Ministry of Health, Welfare and Sport
2016; Wiley; Volume: 14; Issue: 10 Linguagem: Inglês
10.2903/j.efsa.2016.4580
ISSN1831-4732
AutoresVittorio Silano, Claudia Bolognesi, Laurence Castle, Jean‐Pierre Cravedi, Karl‐Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer Gürtler, Sirpa Kärenlampi, Wim Mennes, Maria Rosaria Milana, André Penninks, Andrew T. Smith, Maria de Fátima Tavares Poças, Christina Tlustos, Detlef Wölfle, Holger Zorn, Corina‐Aurelia Zugravu, Stacey E. Anderson, Dori R. Germolec, Raymond Pieters, Anna Federica Castoldi, Trine Husøy,
Tópico(s)Agricultural safety and regulations
ResumoEFSA JournalVolume 14, Issue 10 e04580 Scientific OpinionOpen Access A statement on the developmental immunotoxicity of bisphenol A (BPA): answer to the question from the Dutch Ministry of Health, Welfare and Sport Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul Fowler, Paul FowlerSearch for more papers by this authorRoland Franz, Roland FranzSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorSirpa Kärenlampi, Sirpa KärenlampiSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorAndré Penninks, André PenninksSearch for more papers by this authorAndrew Smith, Andrew SmithSearch for more papers by this authorMaria de Fátima Tavares Poças, Maria de Fátima Tavares PoçasSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorCorina-Aurelia Zugravu, Corina-Aurelia ZugravuSearch for more papers by this authorStacey Anderson, Stacey AndersonSearch for more papers by this authorDori Germolec, Dori GermolecSearch for more papers by this authorRaymond Pieters, Raymond PietersSearch for more papers by this authorAnna F Castoldi, Anna F CastoldiSearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this author Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul Fowler, Paul FowlerSearch for more papers by this authorRoland Franz, Roland FranzSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorSirpa Kärenlampi, Sirpa KärenlampiSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorAndré Penninks, André PenninksSearch for more papers by this authorAndrew Smith, Andrew SmithSearch for more papers by this authorMaria de Fátima Tavares Poças, Maria de Fátima Tavares PoçasSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorCorina-Aurelia Zugravu, Corina-Aurelia ZugravuSearch for more papers by this authorStacey Anderson, Stacey AndersonSearch for more papers by this authorDori Germolec, Dori GermolecSearch for more papers by this authorRaymond Pieters, Raymond PietersSearch for more papers by this authorAnna F Castoldi, Anna F CastoldiSearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this author First published: 13 October 2016 https://doi.org/10.2903/j.efsa.2016.4580Citations: 5 Correspondence: fip@efsa.europa.eu Requestor: Ministry of Health, Welfare and Sport, The Netherlands Question number: EFSA-Q-2016-00301 Panel members: Claudia Bolognesi, Laurence Castle, Jean-Pierre Cravedi, Karl-Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer Gürtler, Trine Husøy, Sirpa Kärenlampi, Wim Mennes, Maria Rosaria Milana, André Penninks, Vittorio Silano, Andrew Smith, Maria de Fátima Tavares Poças, Christina Tlustos, Detlef Wölfle, Holger Zorn and Corina-Aurelia Zugravu Acknowledgements: The Panel wishes to thank EFSA staff member Cristina Croera for the support provided to this scientific opinion. Adopted: 14 September 2016 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract This statement addresses a request to EFSA from the Dutch Ministry of Health, Welfare and Sport to assess the impact of recent evidence underlying the conclusions of the 2016 RIVM report on the temporary tolerable daily intake (t-TDI) for BPA of 4 μg/kg bw per day set by EFSA in 2015. The CEF Panel has then evaluated the results of two studies published by Ménard et al. in 2014, suggesting food intolerance and impaired immune response to parasitic infection in rats exposed perinatally to BPA doses in the μg/kg bw per day range. The same appraisal criteria and weight-of-evidence analysis used for the 2015 EFSA opinion on BPA were applied to these studies. This new evidence adds to the indications of immunotoxicity of BPA in animals reported in previous reviews. For the only endpoint for which multiple BPA doses were tested (immunoglobulin G (IgG) levels), a benchmark dose analysis of the dose–response data was carried out. Due to the high interanimal variability within the treatment groups resulting in wide confidence intervals and the limited dose–response, the CEF Panel concluded that these data on anti-ovalbumin IgG antibodies are not suitable to derive a reference point for BPA on immunotoxicity. Furthermore, the limitations identified in both the Ménard et al. studies confound the interpretation of the results and prevent the assessment of the relevance to human health. The CEF Panel overall considers that the results from the two Ménard et al. studies are not sufficient to call for a revision of the EFSA t-TDI for BPA. EFSA will start a review of all the scientific evidence published after 2012 and relevant for BPA hazard assessment (including immunotoxicity) in 2017. The results of immunological studies, such as the two evaluated here, would form a useful contribution to this evaluation provided that the limitations identified herein were addressed. 1 Introduction 1.1 Background and Terms of Reference as provided by the Dutch Ministry of Health, Welfare and Sport The Background and Terms of reference for this statement is provided in a letter sent to EFSA from the Dutch Ministry of Health, Welfare and Sport on 19 April 2016 and stating the following: 'Please find enclosed the report "Bisphenol A, Part 2 Recommendations for Risk Management" drawn up by the Dutch National Institute for Public Health and the Environment (RIVM). In this report, the RIVM has made an assessment of environmental and health risks of bisphenol A (BPA) and possible risk management measures. BPA is in many different products and has an effect on the endocrine system. In 2014 and 2015 European standards for safe exposure to BPA for workers and consumers are strengthened. Recent scientific research shows that BPA can damage the immune system of the fetus or young children at lower exposure levels than to which the current standards for BPA are based. This lower level of exposure is at approximately the same level as the daily exposure of consumers and workers to BPA. As a result of this exposure people have possibly more chance to develop food intolerances and they can be more susceptible to infectious diseases. The RIVM concludes that these new findings constitute sufficient reason to consider further tightening of standards and suggests to take additional measures at the short term, to further reduce exposure to BPA. I would kindly but urgently request you to carefully examine the results of the RIVM study and take appropriate actions. The content of this report is an important addition to the existing knowledge about BPA'. 1.2 Interpretation of the Terms of Reference This statement only addresses the urgent request of the Dutch Ministry of Health, Welfare and Sport 'to carefully examine the results of the RIVM study' (RIVM, 2016). Accordingly, the current evaluation focuses only on the two studies by Ménard et al. (2014a,b) on BPA immunotoxicity underlying the conclusions of the RIVM report, leading RIVM to suggest a reconsideration of the EFSA t-TDI. 2 Data and methodologies 2.1 Data Ménard et al. (2014a) paper and original data Ménard et al. (2014b) paper 2.2 Methodologies The methodology used including the criteria and principles set for reviewing the experimental studies and the weight of evidence (WoE) approach applied to hazard identification, is the same as that used in the EFSA opinion on BPA of 2015 (EFSA CEF Panel, 2015). This is described in detail in Appendix A. 3 Assessment 3.1 Review of the two studies by Ménard et al. (2014a,b) 3.1.1 Ménard et al. (2014a) Food intolerance at adulthood after perinatal exposure to the endocrine disruptor bisphenol A. The FASEB Journal, 28, 4893–4900 Ménard et al. (2014a) studied multiple immune parameters to address the effects of exposure to BPA on the response to dietary antigens. In the first study, pregnant and lactating Wistar rats were treated orally (most likely gavage but not further specified by the authors) with BPA (0.5, 5 or 50 μg/kg body weight (bw) per day) or vehicle (4% ethanol in corn oil) for approximately 30 days from gestation day 15 until pup weaning at postnatal day (PND) 21. To evaluate the immune response to the food antigen ovalbumin (OVA), adult female offspring from these dams were fed 20 mg OVA (tolerised) or bicarbonate buffer vehicle control (immunised) via oral gavage on PND 45 (~ 6.5 weeks). All rats were given a subcutaneous (s.c.) injection of 100 μg OVA on PND 52 (plus Complete Freund Adjuvant) and PND 66, and euthanised on PND 73 (10.5 weeks). Following this treatment, serum OVA-specific antibody levels were examined along with splenic ex vivo proliferation and cytokine production following OVA stimulation. BPA treatment at all doses significantly increased anti-OVA immunoglobulin G (IgG) antibodies both in OVA-immunised and OVA-tolerised animals as compared with animals without BPA exposure. Irrespective of the BPA treatment, the tolerised rats had lower levels of OVA-specific IgG than their immunised counterparts. The authors claimed a non-monotonic BPA dose–response relationship with the highest antibody titres being observed for the 5 μg group (for both tolerised and immunised animals). The isotype of IgG was not indicated and the authors stated that no increases in immunoglobulin E (IgE) were observed. Additional endpoints were evaluated in adult females following perinatal exposure to 5 μg BPA/kg bw per day only. Ex vivo assays were conducted to assess multiple measures of cell responsiveness and activation responses in splenic lymphocytes obtained from control and OVA-immunised/tolerised animals exposed to BPA and following restimulation with OVA in vitro. For the OVA-specific splenocyte proliferation, a significant increase in cell proliferation was observed for the BPA/tolerised rats as compared to the non-BPA-exposed counterpart. There were no changes in proliferation between the BPA-treated and -untreated rats for the immunised group. A significant increase in interferon gamma (IFNγ) production was observed for the BPA/immunised rats when compared to the non-exposed immunised rats. This change was not observed for the tolerised rats. No changes in interleukin (IL)-10 were observed for any group. Immune phenotyping was conducted on splenocytes from PND 45 rats (these spleens were from rats in a separate study that were not exposed to OVA). A significant increase in activated splenocytes was observed along with no change in regulatory T-cells following exposure to BPA when compared to vehicle controls. This analysis was not conducted on OVA-tolerised/immunised rats. A long-term OVA oral challenge was also conducted to explore physiological consequences of developmental BPA exposure on food intolerance. Adult female offspring from dams treated with 5 μg BPA/kg bw per day (from gestational day 15 to PND 21 as described above) were fed 20 mg OVA (tolerised) or bicarbonate buffer vehicle control (immunised control) via oral gavage on PND 45, immunised on PND 52 (s.c. injection of 100 μg OVA + Complete Freund Adjuvant), and received an oral challenge via gavage with 50 mg OVA on PND 59, 61, 63, 65 and 67. The animals were euthanised on PND 67 (9.5 weeks). Colon samples were evaluated for cytokine production and myeloperoxidase (MPO) activity. Antibody levels were not evaluated to confirm tolerance. OVA-tolerised rats perinatally exposed to BPA had increased MPO activity and elevated concentrations of the cytokines IL-10 and IFNγ in colonic tissues as compared to the unexposed counterpart. No changes were identified in the immunised rats. Levels of transforming growth factor beta (TGFβ) were significantly decreased in both OVA-tolerised and OVA-immunised rats. It was indicated that 7–26 female offspring/group were included for the studies described above. In summary, the authors conclude that low-dose exposure to BPA induced the failure of oral tolerance in adult life and colonic inflammation following oral challenge. The strengths and weaknesses identified by the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) in this study are listed in Table 1. Comments from the CEF Panel The authors measured the critical cell populations, cytokines, immunoglobulins and MPO that provide a mechanistic framework underlying the immune-specific response to food allergens and the inflammatory response in the intestine using relevant models. There was some internal consistency in this study supporting the biological plausibility of the findings. The enhancement of OVA-specific IgG in the plasma of BPA-treated offspring may suggest a dysregulation of antigen-specific tolerance in the gastrointestinal (GI) tract. However, looking at the individual animal data, a high level of variability in the data set was observed. The study would benefit from additional controls including non-tolerised/non-immunised BPA controls and tolerised/non-immunised controls. In the measurement of OVA-specific IgG where three doses were used, the authors claim a non-monotonic dose–response curve, but no statistical support for this conclusion was provided. No power calculation was presented and the number of animals per experimental group varied substantially (n = 11–26 female offspring/group). The authors do not report on the allocation of the pups to the experimental groups and whether litter effects were controlled for. Neither was this evident from the raw data. Also, the number of dams was not reported, and given the number of pups used per group that of dams may have been as low as two or three per group. Only spleen cells were evaluated for in vitro OVA restimulation and proliferation (information from mesenteric lymph node (MLN) would be beneficial). Cell proliferation was evaluated following 6 days of OVA stimulation. However, cell viability was not reported. The above findings may be supported by the observed increase in OVA-specific cell proliferation in the BPA-exposed OVA-tolerised rats and the corresponding increase in the number of activated T-lymphocytes in the spleens from these animals. However, a lack of the expected response in immunised animals raises some doubts about the reliability of the increased cell proliferation reported for the BPA-exposed groups. The authors evaluated specific endpoints in the GI tract including cytokine and MPO production that suggest alterations in inflammatory responses in that organ, but no confirmatory pathology was provided. It is a significant limitation that the authors conducted the majority of their studies with only one dose of BPA (5 μg/kg bw per day). The study would have been considerably strengthened if all of the endpoints had been tested at multiple doses. BPA measurements in biological samples were not performed. Overall, the paper lacks details in the study design and reporting. Assessment of only one gender and use of an outbred strain (not stated by the authors but normally the Wistar strain is outbred unless differently specified) limits interpretation of the findings. Some of the data (cytokines and MPO) were inconsistent within the study. The lack of standard toxicological parameters (body and organ weights and histology of the spleen, thymus and intestine) is an additional limitation of the study. Methods and statistics conducted for the flow cytometry study were unclear, i.e. the number of gated events and cells were not reported and it is unclear if the percentages reported reflect the total number of cells or CD4+ cells). Immune phenotyping was not evaluated on OVA-tolerised/immunised animals and this was only conducted on spleens (information from MLN would be beneficial). Although the Panel noted significant limitations in this study, the reported alterations in endpoints associated with food allergy and intolerance suggest there may be some potential immunotoxic effects in rats associated with perinatal exposure to 5 μg BPA/kg bw per day. 3.1.2 Ménard et al. (2014b). Perinatal exposure to a low dose of bisphenol A impaired systemic cellular immune response and predisposes young rats to intestinal parasitic infection. PLOS One, 9, e112752 Pregnant and lactating Wistar rats were treated orally (most likely gavage but not further specified by the authors) with BPA (5 μg/kg bw per day) or vehicle (4% ethanol in corn oil) for approximately 30 days from gestation day 15 until weaning on PND 21. To evaluate the immune response to dietary antigens in juvenile animals, female offspring from these dams were fed by gavage 20 mg OVA (tolerised) or control bicarbonate buffer (immunised) on PND 25 (~ 3.5 weeks). All rats were challenged on PND 32 (100 μg OVA s.c. injection + Complete Freund Adjuvant) and PND 46 (100 μg OVA s.c.), and euthanised on PND 53 (7.5 weeks). After this treatment, serum OVA-specific antibody levels were examined along with splenic and MLN cytokine production following OVA stimulation. Irrespective of BPA treatment, OVA-specific IgG titres were lower in tolerised than in immunised rats. Perinatal exposure to 5 μg BPA/kg bw per day did not affect the anti-OVA IgG antibodies either in immunised or tolerised animals. The isotype of IgG was not indicated and the authors stated that no increases in IgE were observed. Ex vivo assays were conducted to assess multiple measures of cell responsiveness and activation responses in spleen and MLN cells obtained from control and OVA-immunised/tolerised animals exposed to BPA and following in vitro OVA restimulation. It was indicated that for these studies 12–17 female offspring were included per group. Splenocytes from the BPA/immunised and BPA/tolerised rats produced reduced amounts of IFNγ as compared to cells obtained from the non-BPA-exposed counterparts. A similar BPA-induced decrease in IFNγ production was observed only in MLN cells obtained from OVA-immunised animals, and not in cells from OVA-tolerised animals. No changes in IL-10 were observed in the spleen or mesenteric lymph node. Immune phenotyping was conducted on splenocytes from PND 25 rats (these spleens were from rats that were not exposed to OVA). A significant decrease in regulatory T-cells, T-helper cells (which also were composed of T-regs) and dendritic cells was observed following exposure to BPA in the spleen and MLN compared to vehicle controls. This analysis was not conducted on OVA-tolerised/immunised rats. It was indicated that 12 female offspring were included per group for the studies described above. Host resistance to the helminthic parasite Nippostrongylus brasiliensis following developmental exposure (from gestational day 15 to PND 21 as described above) to 5 μg BPA/kg bw per day was also evaluated. At PND 25, female offspring were infected with 1,000 infective stage larvae N. brasiliensis subcutaneously and euthanised 1 week later (PND 32). Colon samples were evaluated for cytokine production, MPO activity and living larvae. While there was an increase in IgE following infection, there was no difference in response to BPA exposure. Rats perinatally exposed to BPA had elevated levels of living larvae in their faecal material as compared to controls. This was accompanied by a significant decrease in MPO activity and elevated levels of cytokines (IL-4 and IL-13 (TH2), IL-10 (anti-inflammatory) and growth-regulated oncogene/keratinocyte chemoattractant (GRO/KC), and IFNγ (pro-inflammatory)) in the small intestine as compared to the non-BPA-exposed infected animals. It was indicated that 7–8 female offspring were included per group for the studies described above. In summary, the authors conclude that in juvenile rats, low-dose perinatal exposure to BPA resulted in normal responses to food antigen but failed to induce a proper cellular immune response following systemic immunisation and suggest an immunosuppressive effect. In addition, they report a decreased host resistance in juvenile rats following perinatal BPA exposure. The strengths and weaknesses identified by the CEF Panel in this study are listed in Table 1. Comments from the CEF Panel The authors measured the cell populations, cytokines, immunoglobulins and MPO that provide a mechanistic framework underlying the immune-specific response to an antigen and parasitic infections using relevant models. There was some internal consistency in this study supporting the biological plausibility of the findings. Lack of enhancement of OVA-specific IgG in the plasma suggests no effect on antigen-specific tolerance in the GI tract, but in the disease-resistant model, the authors report an increase in the number of larvae in faeces and significant decrease in MPO following infection in BPA-exposed female offspring. It is a significant limitation that the authors conducted these experiments using only one dose of BPA (5 μg/kg bw per day). The findings would have been considerably strengthened if all of the endpoints had been tested at multiple doses. The authors do not report on the allocation of the pups to the experimental groups and whether litter effects were controlled for. Also, the number of dams was not reported, and given the number of pups used per group, that of dams may have been as low as two or three per group. The study overall lacks details in the study design and report. Assessment of only one gender and use of outbred strain (not stated by the authors but normally the Wistar strain is outbred unless differently specified) limits interpretation of the findings. The lack of standard toxicological parameters (body and organ weights and histology of the spleen, thymus and intestine) is a limitation of the study. Methods and statistics conducted for the flow cytometry study are unclear (i.e. number of gated events not reported, number of cells not reported and if the percentages reported reflect total number of cells or CD4+ cells). Given the small increase in the living larvae and the large variation in these data for the host resistance model, the biological significance of this result is considered questionable. 3.2 Weight of evidence of the immune effects of BPA In the 2015 EFSA opinion, a WoE analysis was performed for each toxicological endpoint including the immune effects. In particular, whether BPA induces immune effects was considered using a tabular format for weighing different lines of evidence. The overall outcome of this WoE evaluation is presented in the conclusions on the immune effects taken from the 2015 EFSA opinion and reported below. 'Based on recent human studies, there are indications that BPA may be linked to immunological outcomes in humans, although these studies had limitations and confounding factors may have been present. A causal link between BPA exposure during pregnancy or in childhood and the immune effects in humans cannot be established.Studies in animals lend support to the possibility of immunological effects of BPA. Most of these studies suffered from shortcomings in experimental design and reporting. Although dose-responses could not be confidently established in most studies, a dose-related effect was observed in allergic lung inflammation.Using a WoE approach, the CEF Panel assigned a likelihood level of "-as likely as not- to likely" to immunotoxic effects of BPA. Since the likelihood level for this endpoint is less than "likely" (see Appendix A of EFSA CEF Panel, 2015), this endpoint was not taken forward for assessing the toxicological reference point, but was taken into account in the evaluation of uncertainty for hazard characterisation and risk characterisation (Section 4.3 of EFSA CEF Panel, 2015).' The CEF Panel had already included the study by Bauer et al. (2012) in its 2015 WoE analysis of animal studies and reviewed it as follows: 'The CEF Panel notes that also the study by Bauer et al. (2012) indicated enhancement of ovalbumin-induced allergic responses, notably inflammation, by oral exposure to BPA, and that a dose-dependence was evident. The CEF Panel also noted that in this latter study the inflammation noted was seen in females but not males. It should be mentioned that elevated immune responses in female humans as well as female animals have been reported previously, including innate responses, cytokine responses and vaccine responses (Klein et al., 2010; McLelland and Smith, 2011; Hochstenbach et al.,2012).' For the present statement, the CEF Panel took the 2015 WoE evaluation carried out on animal studies on the immune effects (EFSA CEF Panel, 2015) as the starting point and assessed whether the two Ménard et al. studies from 2014 have an impact on the overall outcome of the WoE analysis (see Table 1). No additional literature has been searched for in the public domain,1 since a review of all the scientific evidence published after 2012 and relevant for BPA hazard assessment (including BPA immunotoxicity) will start next year. This will follow a protocol currently under development which will define a priori the strategy for collecting, appraising, analysing and integrating the relevant evidence. The strengths and weaknesses identified by the Panel in the two new Ménard et al. studies are listed in the left hand side column of the WoE table (Table 1). The second column reports the (positive) answer to the question 'Is BPA immunotoxic in animals?' as reported by the study authors. Taking into consideration all the strengths and weaknesses of each study, the Panel assigned to the new evidence a low score for reliability (third column) and a limited influence (•/↑, see Table A.4 in Appendix A for an explanation of the symbols used for expressing the weight of each new line of evidence) on the likelihood of a positive answer to the question (fourth column). After considering the individual influences of the two new lines of evidence and the starting point the Panel concluded that evidence from the new studies adds to the indications of immunotoxicity of BPA in animals reported in previous reviews. However, uncertainties in the dose–response, the study conduct and design, along with a high variability in the observed responses, lower the confidence in the data as presented. Overall, the CEF Panel reconfirmed the overall conclusion already expressed in 2015 of a likelihood level of 'from -as likely as not (ALAN)- to likely' (see Table A.2 for standard terms used for expressing the overall likelihood in the WoE tables) for BPA immunotoxic effects in animals. Table 1. Assessment of convincing associations between BPA exposure and immunotoxic effects in animals Question 1: Is BPA immunotoxic in animals? Answer to the question as reported by the study authors (positive, negative or uncertain) Reliability of evidence (low, medium or high) Influence on likelihood (see Table A.4 for key to symbols) Starting point based on previous assessments (EFSA CEF Panel, 2015): Evidence from the new studies adds to the indications of immunotoxicity of BPA in animals reported in previous reviews. Some positive ALAN to Likely a Line of Evidence: Ménard et al., 2014a Positive Low •/↑ Strengths Relevant models are used Three dose levels tested (only for IgG) Phytoestrogen-free diet Use of non-polycarbonate cages and non-polycarbonate water bottles and negligible oestrogenicity for cages/water/bedding at E-screen Weaknesses Single dose-level study for all endpoints aside from antibody titres No positive control Tests performed in female offspring only Insufficient study reporting (information not given on: animal body weight, BPA source, BPA oral administration mode, number of dams/pups, endotoxin levels in OVA (this may drive food tolerance response
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