IVM Institute for Environmental Studies. Screening for (potential) endocrine disrupting compounds

Transcription

1 Screening for (potential) endocrine disrupting compounds Strategy for the use of vitro bioassays in the environmental assessment of the (potential) endocrine disrupting properties of chemicals Timo Hamers Report R-16/04 30 September 2016

2 This report is released by: Prof. Dr. Jacob de Boer Department Head Chemistry & Biology This report was commissioned by The Netherlands Ministery for Infrastructure and the Environment (IenM). It was reviewed by the advisory board of the project, consisting of RIVM scientists Prof. Dr. Zhi-Chao Dang, Dr. Betty Hakkert, and Dr. Fleur van Broekhuizen. The views expressed in this publication reflect the views of the author. RIVM is not responsible for the content of this report. IVM Institute for Environmental Studies VU University Amsterdam De Boelelaan HV AMSTERDAM T F E info.ivm@vu.nl Ministry IenM Mrs. Heddy Lindeijer PO Box EX DEN HAAG T E heddy.lindemeijer@minienm.nl Copyright 2016, Institute for Environmental Studies All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the copyright holder

3 Screening for (potential) endocrine disrupting compounds Contents Executive Summary 5 1 Introduction 9 2 Inventory of bioassays 11 3 Test battery response to endocrine disrupting compounds Pilot inventory Selection of compounds and ED endpoints EAST profiling of nine industrial (potential) EDCs More environmentally focused inventory Selecting a set of environmentally relevant (potential) ED compounds Inventory of in vitro bioassay reports Inventory of the in vitro bioassay results EAST profiling of the twenty compounds EAST profiling - Conclusions In vivo validation of the in vitro EAS profile Predicting in vivo effects on Vtg based on in vitro EAS effects Interpretation of in vitro results similar as in GD Weight of in vitro evidence for in vivo ED effects In vivo validation - Conclusions 37 4 Outlook to a testing strategy Applicability of the existing bioassays Development of an in vitro testing strategy In vitro bioassay results in a weight of evidence approach EAST profiling with a pragmatic, minimum battery of four in vitro bioassays Testing strategy - Conclusions 44 5 Conclusions 47 References 49 Appendix A List of abbreviations 63 Appendix B Overview of the in vitro EAST results for the nine industrial potential EDCs (see 3.1), collected from open sources 65 Appendix C Data and scientific evidence on ED properties of the 9 industrial ED chemicals and the three steroid hormones from the BKH report [19] and the WRc-NSF report [20]. 73 Appendix D Overview of the in vitro EAST results for the twenty environmentally relevant potential EDCs (see 3.2) collected from open sources 79 Appendix E Determination of ultimate scores with their corresponding robustness for the EAST profiles in Figure 7 143

4 Screening for (potential) endocrine disrupting compounds Appendix F Lowest observed effect concentrations (LOEC; µm) for Vtg induction in fish according to inventory made by Dang et al. [28] 145 Appendix G Predictive capacity of five types of in vitro bioassays for Vtg induction in male fish 147

5 Executive Summary Screening for (potential) endocrine disrupting compounds 5 The possible risk of endocrine disrupting (ED) compounds for human and environmental health raises worldwide concern. Although ED compounds (EDCs) are widespread in the environment, current data requirements in EU regulations are unsufficient to detect compounds with ED properties. In the present study, the possibilities were explored to screen compounds for their (potential) environmental ED properties using in vitro bioassays, with a primary focus on estrogen, androgen, steroidogenesis, and thyroid (EAST) pathways. The goal of the current study was fourfold: 1. To make an inventory of in vitro EAST bioassays, for which test guidelines (TGs) have been adopted by the Organization for Economic Co-operation and Development (OECD) and the United States Environmental Protection Agency (US- EPA), or for which the development of such guidelines is supported by these bodies; 2. To determine to what extent this battery of in vitro bioassays would signal the endocrine disrupting properties of a selected set of (potential) EDCs; 3. To determine to what extent screening with human-oriented in vitro bioassays covers the environmental effects of (potential) EDCs; 4. To propose a strategy to include in vitro bioassays for endocrine disruption in regulatory frameworks for chemical hazard assessment. Throughout this study, EDCs have been defined according to the WHO definition [1] stating that an endocrine disruptor is 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. This definition inherently implies that in vitro bioassays per se are incapable of identifying EDCs, because they do not study intact organisms, let alone their progeny, or sub-populations. Consequently, in vitro bioassays can only contribute to the weight of evidence that a compound is a potential endocrine disruptor, which was defined by WHO [1] as an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub)populations. OECD and US-EPA have currently adopted seven TGs for in vitro EAST bioassays (Chapter 2), i.e. OECD TG-493 and US-EPA OPPTS for estrogen receptor (ER) binding, OECD TG-455 for the ER stably transfected transactivation assay (STTA), US- EPA OPPTS for androgen receptor (AR) binding, OECD TG-458 for the AR- STTA, US-EPA OPPTS for aromatase inhibition, and OECD TG-456 for steroidogenesis in the H295R adrenocortical cell line. For thyroid pathways, currently no TGs have been developed, but a recent OECD scoping document [2] characterized five in vitro bioassays as ready for validation in the short term, i.e. the thyroid peroxidase (TPO) inhibition assay, the transthyretin (TTR) binding assay, the thyroid hormone binding globulin (TBG) binding assay, the thyroid hormone (TH) membrane transport bioassay, and the thyroid hormone receptor (TR) STTA. For implementation of in vitro EAST profiling within regulatory frameworks (i.e. contributing to the weight of evidence for EDC identification), bioassays should be performed according to validated and standardized protocols. Because currently no TGs exist for thyroid endpoint, it is highly recommended to develop such TGs.

6 Screening for (potential) endocrine disrupting compounds 6 In Chapter 3, inventories were made of available in vitro bioassay results for two different sets of selected (potential) EDCs. A pilot inventory (3.1) was made for a list of nine industrial potential EDCs for which the in vitro bioassay data were easily obtained from a previous extensive evaluation. Still, results from the pilot study indicated that insufficient in vitro toxicity data were available from TG-like and alternative bioassays to estimate the capacity of in vitro bioassays to signal potential EDCs. Therefore, an alternative and longer list of (potential) EDCs was considered for further study. Given the environmental focus of the current project, a second set of (potential) EDCs with high environmental relevance was selected, including pesticides, plasticizers, detergents, and chemicals commonly found in consumer products and food packages. An extensive literature research was performed to obtain in vitro bioassay results for these twenty compounds. For both inventories, in vitro bioassay results obtained from literature were categorized per in vitro endpoint. In addition, distinction was made between results from TG-compliant bioassays (i.e. explicitly mentioned to be performed according to TG), TG-like bioassays (i.e. using the same biological material (cells, protein, origin of species) as prescribed by the TG, but not clear if performed according to TG), and non- TG bioassays (i.e. measuring similar endpoints but using different protocols). For the non-tg bioassays, further distinction was made in the second inventory between bioassays representing a human model or an environmental model. When considering all in vitro data available, the obtained EAST profile confirmed that all twenty environmentally relevant compounds might have ED properties. Good correspondence was observed when comparing in vitro EAST profiles from bioassays using human and environmental models to EAST profiles from bioassays using human models only. As far as data were available, good correspondence was also found when comparing in vitro EAS profiles from bioassays using human and environmental models to EAS profiles from TG-like bioassay only. Remarkably, TG-like in vitro bioassay results were obtained for only 32% of the cases. To determine to what extent the battery of TG-compliant in vitro bioassays would signal the endocrine disrupting properties of a selected set of (potential) EDCs, however, it is highly recommended that all selected compounds were tested in all TG-compliant bioassays. In addition, the exercise would become more reliable if the test set of compounds was extended with more compounds that were not used as positive reference chemicals for the validation of the in vitro performance based technical guideline (PBTG). The added value of in vitro EAST screening would be further confirmed if negative reference compounds were defined and tested for their lack of activity in the TG-compliant bioassays. In vitro EAST screening may overlook ED effects that occur in vivo. If such false negative results are due to the fact that the compound interferes with ED endpoints other than studied in the in vitro bioassay, they can only be avoided by expanding the test battery with additional bioassays covering these specific endpoints. False negative in vitro bioassay results may also be due to the fact that instead of the parent compound its metabolite(s) is (are) the active EDC(s). In such case, a biotransformation step is required to signal the ED properties in vitro. Not all bioassays have metabolizing capacity, but this may be overcome by using an alternative bioassay with inherent metabolic capacity, by transfecting metabolizing capacity into existing bioassays, or by extension of the bioassay with an (online or offline) metabolizing step. Methods covering these aspects should be developed, standardized, validated and being implemented in TGs for in vitro bioassays. In addition to the in vitro EAST profiling, an in vivo validation of the in vitro EAS profiles was performed with respect to their predictive capacity for vitellogenin (Vtg)

7 Screening for (potential) endocrine disrupting compounds 7 biomarker levels in fish (3.3). Given the limited number of compounds for which results from TG-like in vitro ER-STTA, AR-STTA, aromatase inhibition, and H295R steroidogenesis bioassays were available, the observed correspondence between in vitro bioassay results and increased Vtg biomarker responses in male fish cannot be generalized. Restricting the set of in vitro results to TG-like bioassays increased the number of correct and false positive predictions. False positives may be due to species differences and to metabolic differences between in vitro and in vivo test models, but also to non-specificity of the Vtg biomarker response and/or confounding factors. As false positives screening results lead to an overestimation of the ED properties, they should preferably be reduced to a minimum. From a signaling point of view, however, false positive results are less important for compound screening than false negative results, which lead to an underestimation of the ED properties. When interpreting the in vitro EAS profiles obtained in TG-like bioassays combined with the in vivo Vtg biomarker effects in male fish in a similar way as proposed in the OECD Guidance Document on standardized test guidelines for evaluating chemicals for endocrine disruption (GD-150;[3]), most firm possible conclusions were drawn for the four Vtg compounds in the test set causing Vtg induction in male fish. For these compounds, the in vitro data indicate a potential for (adverse) effects via multiple (EAS) mechanisms. For all other compounds, in vitro EAS effects were not confirmed by Vtg induction in vivo. Based on a very straightforward interpretation of Vtg induction in male fish as a consequence of feminization, demasculinization, or a combination of both, a weight of in vitro evidence score was calculated. For the TG-like bioassays, a minimum score was determined above which Vtg induction may take place. Similar to the other exercises, this result was steered by well-known ER-mediating compounds, which all had been used as positive reference compounds in the validation process of the underlying in vitro PBTGs. Because Vtg induction is generally not considered as an adverse effect, similar exercises focusing on more ecologically relevant effects on fecundity or reproduction are recommended with more compounds that are not used as positive reference compounds in PBTG development. Finally, in Chapter 4, the development of an in vitro testing strategy for ED was discussed. To cover the whole spectrum of EAST pathways as completely as possible, this strategy should preferably be performed with a battery of specific in vitro bioassays, each being indicative for a different mechanism of action. Although all seven TG-compliant EAS bioassays are human based, it was concluded that humanbased EAS profiles can be read-across to environmental EAS profiles. For the five in vitro thyroid bioassays categorized as ready for validation in the short term [2] TG development for the TPO inhibition assay, the TTR-binding assay, and the TH membrane transporter bioassay was prioritized above TG development for the TR transactivation bioassay and the TBG-binding assay. Read-across from human to environmental TH-disrupting properties seems appropriate for the TPO inhibition assay, but not for the TTR-binding and TH transmembrane transporter bioassays. For these latter two bioassays, it is recommended to develop TGs not only based on proteins or cells from human origin, but also from more environmentally relevant model species (amphibian, avian, fish). Further test strategy development should focus on additional relevant ED mechanisms of action in EAST pathways that could be turned into TG-compliant in vitro bioassays. Additional method standardization and validation should focus on the introduction of metabolic capacity in the in vitro bioassays, because many of the existing in vitro bioassays lack metabolic capacity, which is relevant for metabolism of the xenobiotic

8 Screening for (potential) endocrine disrupting compounds 8 compound and the endogenous hormone. Moreover, a testing battery that aims at covering the spectrum of ED pathways as completely as possible should not only include in vitro bioassays for EAST pathways, but also for other vertebrate and nonvertebrate hormonal pathways that can be disrupted by xenobiotic compounds such as AhR (aryl-hydrocarbon receptor), PPAR (peroxisome proliferator activated receptor), RXR (retinoid X receptor), PR (progesterone receptor), GR (glucocorticoid receptor), LXR (liver X receptor), ERR (estrogen related receptor), EcR (ecdysone receptor) and JHR (juvenile hormone receptor). From a pragmatic point of view, a minimum test battery has been proposed, which consists of four in vitro bioassays (representing E, A, S, and T). Three of these bioassays can be implemented immediately, as TGs are available for the ER STTA (TG- 455 [4]), the AR STTA (TG-458 [5]), and the H295R assay (TG-456 [6]) for testosterone and estradiol synthesis. For the TTR-binding assay, no TG is currently available, but its development is strongly encouraged. The EAST profile obtained with this minimum set of in vitro bioassays corresponded with the overall EAST profile obtained when considering all available in vitro bioassay results. In addition, this set possessed sufficient capacity to predict in vivo biomarker effects, albeit that this prediction was based on a limited set of compounds that had been used as reference compounds for in vitro PBTG development. For the moment, however, this minimum test battery is considered as the best available minimum selection to cover EAST pathways.

9 1 Introduction Screening for (potential) endocrine disrupting compounds 9 Nowadays, there is a worldwide concern about the possible risk of endocrine disrupting (ED) compounds for human and environmental health. In 2012, the World Health Organization (WHO) and the United Nations Environment Programme (UNEP) [7] demonstrated that ED compounds (EDCs) are widespread in the environment, in food and feed, and in human and animal bodies. Meanwhile, the Danish Centre on Endocrine Disruptors [8] concluded that information/data requirements in the EU regulations REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), PPPR (Plant Protection Products Regulation) and BPR (Biocidal Products Regulation) are not sufficient to adequately detect substances with endocrine disrupting properties. In January 2015, the Netherlands Ministry for Infrastructure and the Environment (IenM) commissioned the Institute for Environmental Studies (IVM) from VU University Amsterdam to perform a study on the possibilities to screen environmental effects of EDCs using in vitro bioassays. This study should primarily focus on estrogenic, androgenic, steroidogenesis, and thyroid (EAST) effects. Since in vitro EAST methods specifically developed for environmental model species are scarce, the study should also investigate to what extent existing human in vitro screening tests can cover environmental effects of EDCs. Compounds can act as EDCs in many different ways. They can interfere with central regulation, synthesis, transport, metabolism, cellular uptake and receptor activation of the hormones. Within each of these processes, different compounds may have different effects, for instance two compounds interfering with hormone transport bind to two different transporter proteins. In vitro bioassays may be developed for each single mechanism of action, ultimately leading to a large battery of bioassays each covering a different aspect of the spectrum of endocrine disruption. On short notice, however, it will be unrealistic to test compounds for their endocrine disrupting potencies in such a vast battery of bioassays. In addition, many of these bioassays have not (yet) been accepted, standardized, and/or validated at for instance OECD level to allow their application for chemical hazard assessment. Therefore, the goal of the current study was fourfold: 1. To make an inventory of in vitro EAST bioassays for which test guidelines (TGs) have been adopted by OECD and US-EPA, or for which the development of such guidelines is supported by these bodies (Chapter 2); 2. To determine to what extent this battery of in vitro bioassays would signal the endocrine disrupting properties of a selected set of (potential) EDCs (Sections 3.1and 3.2); 3. To determine to what extent screening with human-oriented in vitro bioassays covers the environmental effects of (potential) EDCs (Sections 3.3 and 4.1); 4. To propose a strategy to include in vitro bioassays for endocrine disruption in regulatory frameworks for chemical ED assessment (Chapter 4).

10

11 2 Inventory of bioassays Screening for (potential) endocrine disrupting compounds 11 The goal of this Chapter was to make an inventory of in vitro EAST bioassays for which test guidelines have been adopted by OECD and US-EPA, or for which the development of such guidelines is supported by these bodies. By studying the websites of the OECD Endocrine Disrupters Testing and Assessment Advisory Group (EDTA-AG) [9] and US-EPA [10] for ED testing, the correspondence by the OECD Validation Management Group Non-Animal testing (VMG-NA) forwarded by the commissioner, and the OECD 150 Guidance Document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption [3], a list of seven in vitro bioassays was composed (Table 1), for which test guidelines (TG) have been established. Out of these seven bioassay, three bioassays focus on estrogenicity, two on androgenicity, and two on steroidogenesis. So far, no TG have been adopted or drafted for in vitro bioassays measuring thyroid hormone disrupting activity. Nevertheless, the OECD 207 New Scoping Document on In Vitro and Ex Vivo Assays for the Identification of Modulators of Thyroid Hormone Signaling [2] suggested five additional in vitro thyroid bioassays for further development and inclusion in the OECD TG work plan (see Table 1).

12 Screening for (potential) endocrine disrupting compounds 12 Table 1: In vitro bioassays for testing of ED activity at different stages of adoption by OECD and US-EPA Body Reference Title Status Published Test Guidelines specifically developed or updated for the screening or testing of chemicals for endocrine disruption OECD TG 455 [4] Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists OECD TG 458 [5] Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals Adopted in 2009 Adjusted in 2012 Adjusted in 2015 a Adopted in 2016 b OECD TG 456 [6] H295R Steroidogenesis Assay Adopted in 2011 OECD TG 493 [11] Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrer) In Vitro Assays to Detect Chemicals with ER Binding Affinity Adopted in 2015 US-EPA OPPTS [12] Androgen Receptor Binding (Rat Prostate Cytosol) Adopted in 2009 US-EPA OPPTS [13] Aromatase (Human Recombinant) Adopted in 2009 US-EPA OPPTS [14] Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC) Adopted in 2009 In vitro Thyroid Assays with Level of Readiness A c for Inclusion in the OECD Test Guidelines (TG) Work plan according to Thyroid Scoping Document OECD 207 Paul et al. [15] TPO Inhibition Lans et al. [16] TTR binding Lans et al. [16] TBG binding Friesema et al. [17] TH Membrane Transporter Freitas et al. [18] TR Transactivation a : The reference test methods for TG-455 are the ERα-HeLa-9903 and the BG1Luc-4E2 cell lines. Although the ER- CALUX cell line has been approved as a me-too method in 2016, the online version of the PBTG has not been adjusted yet. Still, the ER-CALUX is considered in this report as a TG-455 compliant method. b : The reference test method for TG-458 is the AR-EcoScreen cell line. Although the AR-CALUX cell line has been approved as a me-too method in 2016, the online version of the PBTG has not been adjusted yet. Still, the AR-CALUX is considered in this report as a TG-458 compliant method. c : Readiness level A indicates ready for validation in the short term

13 Screening for (potential) endocrine disrupting compounds 13 Some of the twelve bioassays study similar endpoints (i.e. ER-binding in the ER-RUC and TG-493 bioassay), albeit with ER representing different species. Other bioassays allow the assessment of multiple endpoints (i.e. testosterone and estradiol measurements in TG-456). Altogether, the set of twelve selected bioassays altogether covered twelve different endpoints, i.e. 1. ER pathway: ER binding; 2. ER pathway: ER transcriptional activation/antagonism; 3. AR pathway: AR binding; 4. AR pathway: AR transcriptional activation/antagonism; 5. Steroidogenesis pathway: aromatase inhibition; 6. Steroidogenesis pathway: increased/decreased levels of testosterone; 7. Steroidogenesis pathway: increased/decreased levels of estradiol; 8. Thyroid pathway: inhibition of thyroid peroxidase (TPO); 9. Thyroid pathway: binding to plasma transporter protein transthyretin (TTR); 10. Thyroid pathway: binding to plasma transporter protein thyroid hormone binding globulin (TBG); 11. Thyroid pathway: increased/decreased transmembrane transport of TH; 12. Thyroid pathway: TR transcriptional activation/antagonism. The different endpoints and their corresponding bioassays have been classified according to the affected EAST pathway in Figure 1. Affected process Hormone synthesis Hormone binding Estrogen Androgen Steroidogenesis Thyroid Testosterone synthesis [TG 456] Estradiol synthesis [TG 456] Aromatase inhibition [OPPTS ] TPO inhibition TTR binding TBG binding Hormone transport Receptor binding Receptor activation ER binding [TG 493/ OPPTS ] ER (in)activation [TG 455] AR binding [OPPTS ] AR (in)activation [TG 458] TH membrane transporter TR (in)activation Figure 1: Schematic matrix-presentation of the endpoints represented by the different in vitro bioassays classified according to the hormone system (EAST) and their mechanism of action. In the next Chapters, in vitro bioassay results were collected, inventoried and analyzed that addressed these 12 pre-defined endpoints. In addition, many data were found from hormone-dependent cell-proliferation bioassays. Therefore, the list of endpoints was expanded with ER- AR-, and TR-transactivation dependent cell-proliferation assays.

14

15 Screening for (potential) endocrine disrupting compounds 15 3 Test battery response to endocrine disrupting compounds This Chapter addresses the second and third goal of the study, i.e. 2. To determine to what extent a battery of in vitro bioassays representing the 12 different endpoints distinguished in Chapter 2 would signal the endocrine disrupting properties of a selected set of xenobiotic compounds generally considered as EDCs; 3. To determine to what extent screening with human-oriented in vitro bioassays covers the environmental effects of EDCs. To reach these goals, it is important to know what is meant by an endocrine disruptor. In 2002, the World Health Organization (WHO) [1] defined an endocrine disruptor 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. It should be realized that this definition inherently implies that in vitro bioassays per se are incapable of identifying endocrine disruptors, since they do not study intact organisms, let alone their progeny, or sub-populations. Consequently, in vitro bioassays can only contribute to the weight of evidence that a compound is a potential endocrine disruptor, which was defined by WHO [1] as an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub)populations. A pilot inventory was made for a list of nine industrial potential EDC that were previously extensively evaluated (see 3.1). The in vitro bioassay data for these compounds were easily obtained from this evaluation, and were further extended with data obtained from a non-exhaustive literature search. Results from the pilot study indicated, however, that additional information was required to draw better conclusions regarding their EAST properties. Given the ultimate focus of this study on environmental effects of EDCs, the list of industrial chemicals was replaced by a second list of twenty potential EDCs, which was compiled in close consultation with the RIVM advisory board (see 3.2). Apart from industrial chemicals, this environmentally focused inventory included pesticides, plasticizers, detergents, and chemicals commonly found in consumer products and food packages. An extensive literature research was performed to obtain in vitro bioassay results for these twenty compounds. Results from this literature search were categorized as either representing a human model or an environmental model. EAST profiles were determined for each study in the pilot study and the more environmentally focused inventory. In addition, an in vivo validation was performed for the in vitro EAS profiles with respect to their predictive capacity regarding their effect on vitellogenin (Vtg) yolk protein levels in fish. Conclusions from the in vitro EAST profiling exercises and from the in vivo validation are given at the end of sections 3.2 and 3.3, respectively. In both inventories, three types of in vitro bioassay results could be distinguished, with respect to the underlying bioassay methods: 1. Results from bioassays that were explicitly mentioned to be performed according to the TGs inventoried in Figure 1. Such results are further referred to as obtained from TG-compliant bioassays. 2. Results from bioassays that were performed with the same biological material (cells, protein, origin of species) as prescribed by the TG, but for which it was further not clear if the test was performed according to the TG. This was

16 Screening for (potential) endocrine disrupting compounds 16 especially the case with studies that were performed before the TG was established. Results from such studies are further referred to as obtained from TG-like bioassays. 3. Results from bioassays that measure similar endpoints as the TG bioassays, but use different protocols (e.g. using transiently and not stably transfected transcription assays, recombinant receptors instead of receptors isolated from animals, different cell lines derived from different species) or use different read-out (hormone-dependent cell proliferation instead of hormone-receptor transactivation). Results from such studies are further referred to as obtained from non-tg bioassays. 3.1 Pilot inventory Selection of compounds and ED endpoints In 2000, BKH Consultancies published a study on EDCs, aiming at the establishment of a priority list of substances for further evaluation of their role in endocrine disruption [19]. From a total of 564 suspected EDCs, 147 were considered likely to be either persistent in the environment or produced at high volumes. For 66 of these 147, at least one study provided evidence of endocrine disruption in an intact organism (assigned Category 1). For another 52 of these 147, potential ED activity was indicated either by in vitro data or by in-vivo effects that may, or may not, be ED-mediated (Category 2). Out of the 118 Category 1 and 2 priority substances, a number of 109 substances had already been subject to bans or restrictions or was being addressed under existing Community legislation, although for reasons not necessarily related to ED. In 2002, WRc-NSF [20] published an in-depth evaluation of the remaining nine industrial chemicals, i.e. bisphenol-a-diglycidyl-ether (BADGE), carbon disulfide, 4- chloro-3-methyl-phenol, 2,4-dichlorophenol, 4-nitrotoluene, o-phenylphenol, resorcinol, 4-tert-octylphenol and 2,2,4,4 -tetrabromo diphenyl ether (BDE-47). The in vitro based evidence for the potential ED properties of the nine compounds has been collected from Chapters 3 and further of the WRc-NSF report [20]. For many compounds, however, limited or no in vitro bioassay results were available at the time of publication of the report (2002). Given the many developments in the field of in vitro toxicity testing over the last decade, an additional literature search was performed in Web of Science for the nine industrial chemicals in combination with search terms (vitro AND endocrine*) or with (H295R) EAST profiling of nine industrial (potential) EDCs Figure 2 provides an overview of the in vitro ED potencies towards the 12 in vitro endpoints selected in Chapter 2, expanded with ER-, AR-, and TR-transactivation dependent cell proliferation assays. All underlying data and references can be found in Appendix B. Two out of the fifteen endpoints have not been tested for any of the nine industrial compounds, i.e. microsomal aromatase inhibition and TH membrane transporter activity. Out of all endpoints considered, most compounds were tested in an ER-STTA, an AR-STTA, and a TR-transactivation dependent cell proliferation assay. Remarkably, in only 11 out of the 63 possible combinations (9 compounds x 7 endpoints), a compound was tested for an ED endpoint in a TG-like bioassay (indicated by bold font and bold bordered cells in Figure 2). BDE-47 was clearly best represented with five endpoints tested in TG-like bioassays. BDE-47 was also best represented in

17 Screening for (potential) endocrine disrupting compounds 17 the thyroid-related bioassays, in which it was tested for four out of five bioassays for which the thyroid scoping document proposed inclusion in the OECD TG work plan [2]. Substance BADGE* * BADGE is bisphenol-a-diglycidyl-ether, for which the official name is 2,2 bis(4-(2,3-epoxypropyl)phenyl) propane Figure 2: CAS number MW (g/mol) logkow ER-binding ER (in)activaton (reporter gene) 1 anta ER (in)activaton (cell proliferation) Overview of in vitro results for the nine industrial potential ED compounds, categorized per ED endpoint studied in vitro. Numbers and colors indicate level of ED potency, i.e. no potency (0; green), weak potency (1; yellow), moderate potency (2; orange), and strong potency (3). Potencies are based on qualification in WRC report or on EC50 value reported, with 1 (10<EC50 100µM), 2 (1<EC50 10µM), and 3 (0.1<EC50 1µM). White cells indicate that no data were available. Endpoints in grey cells refer to bioassays for which TG have been developed by OECD or US-EPA. Endpoints in italic refer to cell proliferation assays that were not selected in Chapter 2, but for which many data were available. The final columns refer to the ultimate conclusion for each compound as ED disruptor based on the in vitro EAST profile (weak-moderate potency in small characters and high potency in capital characters), the bkh classification, and the summarized conclusion of the WRC report regarding the ED properties of the compounds, based on in vitro and in vivo data. For carbon disulfide, no in vitro data were found for carbon disulfide at all. For 4- nitrotoluene, no responses were found for two endpoints studied in vitro. For the remaining seven industrial chemicals, in vitro EAST profiles could be determined (Figure 2) based on the positive responses that were found for at least one (ophenylphenol) to at most six (4-tert-octylphenol) of the fifteen endpoints considered. These seven compounds could be identified as weak (4-chloro-3-methyl-phenol and BDE-47) to moderate estrogens (4-t-octylphenol), as weak anti-estrogen (BADGE), as weak (BADGE, 4-chloro-3-methyl phenol, and resorcinol) to moderate anti-androgens (2,4-dichlorophenol, o-phenylphenol, 4-t-octylphenol, and BDE-47), as moderate inhibitor of estradiol synthesis (2,4-dichlorophenol), as moderate TPO-inhibitor (resorcinol), and as weak (BDE-47) or strong TTR-binder (2,4-dichlorophenol) (see Figure 2). Results from the TH-dependent cell proliferation bioassay (T-Screen) were not considered in the EAST profiling, because they were obtained from a single laboratory, which reported positive results for all phenolic and phthalate compounds AR-binding 2 AR (in)activation (reporter gene) 1 anta AR (in)activation (cell proliferation) Microsomal aromatase inhibition H295R steroidogenesis testosterone H295R steroidogenesis estradiol TPO inhibition TTR-binding TBG-binding TH membrane transporter activity TR (in)activation (reporter gene) TR (in)activation (cell proliferation) Conclusion in vitro EAST profile# BKH category WRc-NSF Human WRc-NSF Wildlife ea-- 2 +? -? Carbon disulfide ? 4-chloro-3-methylphenol ago 1 ago 2,4-dichlorophenol anta 2 anta 2 anta 1 ea ? ast 2 - +? 4-nitrotoluene ? +? o-phenylphenol Resorcinol tert-octylphenol ,2,4,4 -tetra BDE (BDE-47) ago 1 ago 2 ago anta 1 anta 2 anta 2 anta 0 -a ? 2 1 -a-t 1 + +? 2 ea ea-t 2 NA NA

18 Screening for (potential) endocrine disrupting compounds 18 tested [21]. This result was attribute to probable crosstalk between thyroid and estrogen signaling pathways in the GH3 rat pituitary cell line used in this assay, which is T3-dependent but E2 sensitive [22]. Therefore, these results were not considered as a true T endpoint. In conclusion, limited in vitro bioassay results for the 15 EAST endpoints were available for the nine industrial compounds as indicated by the white cells in Figure 2. Despite the existence of TGs adopted by OECD and/or US-EPA, the available information was derived from bioassays performed according to different protocols. For general acceptance of in vitro bioassay results in ED assessment, bioassays should be performed according to validated and standardized protocols. To assess the signaling capacity of in vitro bioassays for the potential ED properties of a compound, it is therefore highly recommended that the existing TGs are applied to a range of suspected EDCs. Despite the lack of data from TG-like and other bioassays, the in vitro EAST profiles (Figure 2) indicated potential ED activity for seven of the nine compounds tested. No conclusion could be drawn for the remaining two compounds due to lack of data. As such, the previous classification by BKH [19] was confirmed. On the other hand, the chronic in vivo data used in the in-depth evaluation by WRc-NSF [20] regarding reproductive, developmental or teratogenic effects did not unequivocally identify the nine compounds as EDCs (last two columns in Figure 2; see Appendix C). This was partly due to the fact that observed effects could not be related to an ED mechanism of action. Still, the in vitro EAST profiles confirmed the most firm conclusions by WRc-NSF regarding carbon disulfide (A), resorcinol (T), and 4-tert-octylphenol (E). In addition, the in vitro bioassay results confirmed the WRc-NSF conclusion that reproductive effects by o-phenylphenol on fish were evidently not ER mediated. The in vitro profile of BDE-47, which had the highest share of TG-like bioassay results, suggested that BDE-47 interferes with E, A, and T pathways. Unfortunately, WRc-NSF did not evaluate BDE-47 any further, because during the study this compound had entered the process of being banned. Later studies, as reviewed for instance by EFSA [23], have clearly indicated the ED potential of BDE-47, with the thyroid hormone system as main target. It should be stressed here that part of the in vivo ED activity of BDE-47 is attributed to its hydroxylated and methoxylated metabolites. For instance, the relatively low TTR-binding and TBG binding activity for BDE-47 reported in Figure 2 refers to the parent compound, which is not metabolized in the in vitro bioassay. Both underlying studies [24][25], however, have clearly demonstrated much higher TTR- and TBG-binding capacity for the hydroxylated metabolites of BDE-47. Although the underestimation of the thyroid hormone disrupting capacity of BDE-47 in the in vitro profiling seems to demonstrate a short-coming of in vitro ED screening, this may be overcome by using a bioassay with inherent metabolic capacity, by transfecting metabolizing capacity into the existing bioassay, or by extension of the bioassay with an (online or offline) metabolizing step [26], as was previously demonstrated for the TTR-binding assay [27]. Although the current inventory focused on EAST endpoints, it is worth mentioning that the literature search indicated that the nine industrial compounds were able to affect additional ED endpoints in in vitro bioassays. For seven out of the nine compounds, for instance, effects on transcriptional activation of the arylhydrocarbon receptor (AhR) were studied. Although true AhR agonism (i.e. dioxin-like activity) was not found for any of the test compounds, weak TCDD-potentiation (2,4-dichlorophenol and resorcinol) and AhR antagonism (4-chloro-3-methyl-phenol and BDE-47) were reported. Other endocrine-related endpoints that were reported to be affected by at least two of

19 Screening for (potential) endocrine disrupting compounds 19 the nine compounds included inhibition of hormone metabolizing sulfotransferase (SULT) enzymes (4-tert-octylphenol and BDE-47), increased adipogenesis (BADGE and BDE-47), effects on progesterone receptor (PR) transactivation (4-tert-octylphenol and BDE-47), and effects on the estrogen related receptor (ERR) transactivation (2,4- dichlorophenol and 4-tert-octylphenol). Given the fact that well-established bioassays are in place to test compounds for their capacity to interfere with some of these endpoints, expanding the inventory with these bioassays is worth to be considered. 3.2 More environmentally focused inventory In the pilot study a list of industrial compounds has been considered, which have clear human relevance with respect to occupational exposure and exposure through the use of consumer products. The study indicated that insufficient in vitro toxicity data were available from TG-like and alternative bioassays to estimate the capacity of in vitro bioassays to signal potential EDCs. Therefore, an alternative and longer list of (potential) EDCs was considered for further study Selecting a set of environmentally relevant (potential) ED compounds Given the environmental focus of the current project, the RIVM advisory board suggested to apply the approach followed in 3.1 to a set of potential EDCs with high environmental relevance. In addition, the set should contain compounds, for which the link between in vivo effects and underlying ED mechanism of action (MOA) was better established. Starting point for this selection was a set of 62 environmentally relevant compounds selected by Dang et al. [28], including typical agonists and antagonists of ERs and ARs, typical steroid metabolism modulators, chemicals that do not influence AR, ER and steroid metabolism, and chemicals with other MOAs (e.g. dopamine inhibitor) or uncertain MOAs. This set of compounds was then compared to a set of 189 compounds classified for fertility or developmental toxicity (H360) according to the EU Classification, Labeling and Packaging (CLP) regulation for fertility or developmental toxicity (H360). The list was further compared to the SIN (Substitute it Now!) list consisting of carcinogenic, mutagenic and reprotoxic (CMR) and ED compounds compiled by ChemSec (an NGO striving to a toxic free environment ), and to a list of 25 plant production products (PPP) that are currently under review by the Commission for an impact assessment. Based on the overlap between the different lists, a set of 20 compounds was selected (see Figure 3). For the selected twenty compounds, a literature search was performed in Web of Science, using the following search: ([compound name]) AND (vitro) AND ((endocrin*) OR (*estrogen*) OR (*androgen*) OR (*thyroid*) OR (steroidogen*) OR (H295R) OR (hela*) OR (bg1*)). If applicable, for [compound name], different names or spellings split by an OR argument were used. It should be stressed, that for some studies reporting in vitro EATS endpoints of the selected compounds, the exact identity of the tested compound could not be determined, as no CAS numbers were provided. This was especially the case for branched nonylphenol, which may have been confused with linear nonylphenol when only nonylphenol was mentioned, for di-n-butyl phthalate, which may have been confused with diisobutyl phthalate when only dibutyl phthalate was mentioned. Such subtle differences may affect the affinity of a compound for an EATS endpoint, as was

20 Screening for (potential) endocrine disrupting compounds 20 demonstrated by Simon et al. [29], who reported TTR-binding capacity for branched nonylphenol (technical mixture), but not for linear nonylphenol. Less importantly, most studies with tributyltin and triphenyltin did not mention the negative contra-ion, which may have been different than chloride. Figure 3: Venn diagram showing the overlapping compounds between the environmentally relevant set of compounds studied by Dang et al. [28] and the different lists of hazardous compounds considered. Similar as in the pilot study, all available in vitro bioassay results have been collected addressing the 12 pre-defined endpoints (Chapter 2) expanded with ER- AR-, and TRtransactivation dependent cell-proliferation. During the literature search, however, additional in vitro bioassay results for three more EAST endpoints were found, i.e. the potency to affect steroidogenesis of other hormones than testosterone and estradiol, to affect the expression and activity of enzymes involved in steroidogenesis, and to bind to the TR. Altogether, a list of eighteen EAST endpoints has been considered Inventory of in vitro bioassay reports Figure 4 indicates to what extent the eighteen selected endpoints have been tested for the twenty selected compounds. With respect to E and A, most compounds were tested for receptor (in)activation in stably or transiently transfected ER- or AR-reporter gene bioassays. For S, more reports were found on expression and activity of enzymes involved in steroidogenesis and on levels of (intermediates of) the mineralcorticoid (aldosterone) or glucocorticoid (cortisol) pathways than on levels of androgen (testosterone) or estrogen (estradiol) steroid hormones. Therefore, two additional endpoints were added to the inventory, i.e. steroidogenic effects on other hormones than testosterone and estradiol, and effects on expression/activity of steroidogenic enzymes. For T, most compounds were tested in the TTR-binding assay, followed by stably or transiently transfected TR-reporter gene bioassay and TR-binding assays. In

21 Screening for (potential) endocrine disrupting compounds 21 contrast to the EAS assays, most studies were performed on TTR or TR from nonhuman model species rather than from human model species. Given the focus of the current report on environmental ED screening, TR-binding was included as an endpoint, although it had not been labeled as ready for validation in the short term (readiness label A) in the scoping document (see Table 1). Compound characteristics Estrogenic Androgenic Steroidogenesis Endpoints tested ER-binding ER (in)activation (reporter gene) ER (in)activation (cell proliferation) AR-binding Chemical name CAS number MW (g/mol) log Kow Amitrole Benzophenone Bisphenol A p,p'-dde Hexachlorobenzene Ketoconazole Nonylphenol (branched) t-Octylphenol Pentachlorophenol Perfluorooctanesulfonate Butylbenzyl phthalate Di-n-butyl phthalate Dicyclohexyl phthalate Di-(2-ethylhexyl) phthalate Diethyl phthalate Dihexyl phthalate Dipentyl phthalate Tributyltin chloride Triphenyltin chloride Vinclozolin Number of compounds tested Number of compounds tested in TG-like bioassay 8 12 NA 1 12 NA NA NA NA NA NA NA NA NA NA 45 Figure 4: Overview of in vitro results of the selected bioassays for the 20 environmentally relevant potential EDCs. For each combination of a compound and an ED endpoint, the upper row (red) represents the numbers of studies reporting positive results and the lower row (green) the number of studies reporting negative results. For those endpoints for which OECD or US-EPA TG are available (indicated with grey), the left column (dark red/green colors) indicate the number of studies performed with TG-like bioassays, and the right column (light red/green colors) indicate the number of studies performed according to non-tg bioassays. Naturally, for endpoints for which no in vitro TG exists, number of positive or negative reports are also given by light red and light green numbers, respectively In 45 cases out of the 140 possible combinations (20 compounds x 7 endpoints [grey in Figure 4]), in vitro results were obtained from TG-like bioassays (i.e. 32%). This share is significantly higher than the 17% in the pilot assay (11 out of 63 cases), allowing further comparison between results from TG-like bioassays and results from non-tg AR (in)activation (reporter gene) AR (in)activation (cell proliferation) Microsomal aromatase inhibition Steroidogenesis testosterone Steroidogenesis estradiol Steroidogenesis other hormones Steroidogenesis enzyme expression and activity TPO inhibition TTR-binding TBG-binding TH membrane transporter activity TR binding TR (in)activation (reporter gene) TR (in)activation (cell proliferation) Nr of endpoints tested Number of endpoints for which TG protocols exist Nr of endpoints tested in TG-like bioassay