1 1 Detailed Review Paper on Retinoid Pathway Signalling 2 December 2020 3 2 4 Foreword 5 1. Project 4.97 to develop a Detailed Review Paper (DRP) on the Retinoid System 6 was added to the Test Guidelines Programme work plan in 2015. The project was 7 originally proposed by Sweden and the European Commission later joined the project as 8 a co-lead. In 2019, the OECD Secretariat was added to coordinate input from expert 9 consultants. The initial objectives of the project were to: 10 draft a review of the biology of retinoid signalling pathway, 11 describe retinoid-mediated effects on various organ systems, 12 identify relevant retinoid in vitro and ex vivo assays that measure mechanistic 13 effects of chemicals for development, and 14 Identify in vivo endpoints that could be added to existing test guidelines to 15 identify chemical effects on retinoid pathway signalling. 16 2. This DRP is intended to expand the recommendations for the retinoid pathway 17 included in the OECD Detailed Review Paper on the State of the Science on Novel In 18 vitro and In vivo Screening and Testing Methods and Endpoints for Evaluating 19 Endocrine Disruptors (DRP No 178). The retinoid signalling pathway was one of seven 20 endocrine pathways considered to be susceptible to environmental endocrine disruption 21 and for which relevant endpoints could be measured in new or existing OECD Test 22 Guidelines for evaluating endocrine disruption. Due to the complexity of retinoid 23 signalling across multiple organ systems, this effort was foreseen as a multi-step process. 24 This DRP is intended, in part, to be an initial scoping effort to identify retinoid signalling 25 pathway test methods, markers, and endpoints for consideration. Following the expert 26 group discussion of the technical aspects and regulatory context, the initial scoping effort 27 may lead to recommendations for identification of early screening assays that could be 28 included as a multi-tiered approach, identification and development of biomarkers to be 29 used in studies of humans and wildlife, and endpoints that could be added to enhance 30 exiting in vivo test guidelines. This effort was intended to be modelled after the thyroid 31 scoping effort (GD No 207). 32 3. The EU-Commission supported a first draft of DRP development through a 33 contract with Brunel University and the sub-contractors Technical University of 34 Denmark (DTU). Sections of the 2017 draft prepared for this project are included herein. 35 4. The Retinoid DRP project was discussed at two meetings of the Advisory Group 36 on Endocrine Testing and Assessment (EDTA) in 2017 and a meeting in 2018. The 37 EDTA recommended narrowing the scope of the original proposal to focus the DRP on 38 specific organ systems for which some information is known regarding the role of 39 retinoid signalling. 40 5. At the 2018 EDTA meeting, Sweden presented drafts of a section describing the 41 role of retinoids on female reproduction (Appendix A). A section reviewing the overall 42 biology of the retinoid pathway and additional information on the male reproductive 43 system (Appendix A) were added in 2019. 44 6. The retinoid pathway signalling was also discussed at a 2017 European 45 Commission workshop to identify gaps in current OECD Test Guidelines and prioritise 46 new assays to bridge the gaps. The meeting attendees identified the development of 3 47 retinoid pathway assays and endpoints for inclusion in OECD Test Guidelines as a high 48 priority. As a result, the European Commission supported the drafting of two additional 49 sections reviewing retinoid effects craniofacial/skeletal system development (Annex B) 50 and the central nervous system (CNS) (Annex C), also included in this DRP. These were 51 coordinated by the OECD Secretariat and prepared by subject matter expert consultants 52 noted below. In Q2 2019, OECD circulated a request for updated nominations to the 53 Expert Group on Retinoid Pathway Signalling. 54 7. The draft DRP was circulated for a WNT commenting round in Q3 2019 and 55 comments were discussed by the OECD Expert Group on Retinoid Pathway Signalling 56 in November 2019. The objectives of the meeting were to address any outstanding 57 comments received during the first commenting round and to collate recommendation 58 on possible next steps. The discussion of sections and recommendations are summarised 59 in Annex D. Following the meeting, the sections were revised by the lead experts. In 60 addition, the overview of retinoid biology, along with the annex on the male and female 61 reproduction, were previously published as a TemaNord Report (Retinoids in 62 Mammalian Reproduction, with an Initial Scoping Effort to Identify Regulatory 63 Methods, Nordic Council of Ministers, https://doi.org/10.6027/temanord2020-507, is 64 made available under the Creative Commons Attribution 4.0 International license (CC 65 BY 4.0).This material has been reformatted herein, but the content has not been changed. 66 8. Sections of this DRP were drafted by different experts and the content of some 67 sections was discussed extensively and revised several times prior to this draft, while 68 other sections were prepared more recently and with less initial review. The following 69 experts prepared the sections of this DRP indicated below: The body of the DRP and 70 Appendix A which was adopted from the TemaNord report (see paragraph 7) was 71 primarily authored by Charlotte Nilsson, Research Institutes of Sweden, in 72 collaboration with the Swedish Chemicals Agency, with financial support from the 73 Nordic Working Group for Chemicals and Health1 and the Swedish Chemicals 74 Agency2Agency1. The drafting of the body of the DRP and the section on male 75 reproduction built on the work performed by Brunel University and Technical University 76 of Denmark 2016-2017, supported by the EU Commission. In addition, a number of 77 experts outside the OECD retinoid expert group, made valuable contributions to the 78 Nordic Report, please see the acknowledgement section “List of contributors” in 79 https://doi.org/10.6027/temanord2020-507 for details. Appendix B was drafted by 80 Thomas B. Knudsen (Center for Computational Toxicology and Exposure (CCTE), 81 Office of Research and Development, U.S. Environmental Protection Agency) and 82 Nancy C. Baker (Leidos, Contractor to CCTE)2. Appendix C was drafted by Joshua F. 83 Robinson (University of California, San Francisco). Member of the OECD Expert Group 84 made contributions via written comments and discussion at the November 2019 meeting. 1 The contents of this working paper do not necessarily reflect the views, policies or recommendations of the Nordic Council of Ministers, or the Swedish Chemicals Agency 2 The views expressed herein are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 4 85 Abbreviations relevant to entire document ADH Alcohol dehydrogenase AhR Aryl hydrocarbon receptor ALDH Aldehyde dehydrogenase AMH Anti-müllerian hormone AO Adverse outcome AOP Adverse outcome pathway ARAT Acyl CoA:retinol acyltransferase ATRA All-trans retinoic acid BMP Bone morphogenic protein BTB Blood-testis barrier CAR Constitutive androstane receptor CF Conceptual framework CRABP Cellular retinoic acid-binding protein CRBP Cellular retinol-binding protein CYP Cytochrome P450 CYP17 Cytochrome P450 C17, 17,20-lyase, 17α-hydroxylase Cyp2b10 Cytochrome P450, family 2, subfamily b, polypeptide 10 CYP26 Cytochrome P450, Family 26 Dazl Deleted in azoospermia-like DEHP Diethyl hexyl phthalate DHRS3 Retinaldehyde reductase 3 DMRT1 Doublesex and mab-3 related transcription factor 1 dpc Days post coitum DRP Detailed Review Paper Foxl2 Forkhead box protein L2 FSH Follicle stimulating hormone FXR Farnesoid X receptor GD Gestational day GR Glucocorticoid receptor GW Gestational week HSD Hydroxysteroid dehydrogenase KE Key event LH Luteinizing hormone LRAT Lecithin:retinol acyltransferase LXR Liver X receptor MEHP Monoethyl hexyl phthalate MIE Molecular initiating event MMP Matrix metalloproteinases Nanos2 Nanos C2HC-Type Zinc Finger 2 5 NOAEL No observed adverse effect level OECD Organisation for Economic Co-operation and Development PCOS Polycystic ovarian syndrome PGC Primordial germ cells PND Post-natal day PPAR Peroxisome proliferator-activated receptor PXR Pregnane X receptor (Q)SAR (Quantitative) structure-activity relationship RA Retinoic acid RAL Retinaldehyde RALDH Retinaldehyde dehydrogenase RAMBA RA metabolism blocking agents RAR Retinoic acid receptor RARE Retinoic acid response element RBP Retinol binding protein RBP4 Retinol-binding protein 4 RDH10 Retinol dehydrogenase 10 RDH11 Retinol dehydrogenase 11 RE Retinyl esters Rec8 Meiotic recombination component gene Rec8 REH Retinyl ester hydrolase RIP140 Nuclear receptor interacting protein 1 ROH Retinol RXR Retinoid X receptor P450scc Cytochrome P450 cholesterol side-chain cleavage enzyme SDR Short-chain dehydrogenase/reductase Sox9 SRY -Box Transcription Factor 9 SRC-1; Ncoa1 Nuclear receptor coactivator 1 SREBP-1c Sterol regulatory element binding protein-1c Sry Sex-determining region Y protein Stra6 Stimulated by retinoic acid, gene 6 Stra8 Stimulated by retinoic acid, gene 8 TG Test guideline TTR Transthyretin VAD Vitamin A deficiency VDR Vitamin D receptor WEC Whole embryo culture Wnt4 Wnt Family Member 4 86 6 87 Table of Contents 88 Detailed Review