Organisation de Coopération et de Développement Économiques
Organisation for Economic Co-operation and Development 21-Aug-2012 ___________________________________________________________________________________________
English - Or. English ENVIRONMENT DIRECTORATE
JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY
DETAILED REVIEW PAPER ON THE STATE OF THE SCIENCE ON NOVEL IN VITRO AND IN VIVO SCREENING AND TESTING METHODS AND ENDPOINTS FOR EVALUATING ENDOCRINE DISRUPTORS
Series on Testing & Assessment No. 178
JT03325419
Complete document available on OLIS in its original format
This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation of international frontiers and boundaries and to the name of any territory, city or area.
ENV/JM/MONO(2012)23Unclassified English - Or. En
OECD Environment, Health and Safety Publications Series on Testing and Assessment
No. 178
DETAILED REVIEW PAPER ON THE STATE OF THE SCIENCE ON NOVEL IN VITRO AND IN VIVO SCREENING AND TESTING METHODS AND ENDPOINTS FOR
EVALUATING ENDOCRINE DISRUPTORS
Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT Paris 2012
Also published in the Series on Testing and Assessment:
No. 1, Guidance Document for the Development of OECD Guidelines for Testing of Chemicals (1993; reformatted 1995, most recently revised 2009)
No. 2, Detailed Review Paper on Biodegradability Testing (1995)
No. 3, Guidance Document for Aquatic Effects Assessment (1995)
No. 4, Report of the OECD Workshop on Environmental Hazard/Risk Assessment (1995)
No. 5, Report of the SETAC/OECD Workshop on Avian Toxicity Testing (1996)
No. 6, Report of the Final Ring-test of the Daphnia magna Reproduction Test (1997)
No. 7, Guidance Document on Direct Phototransformation of Chemicals in Water (1997)
No. 8, Report of the OECD Workshop on Sharing Information about New Industrial Chemicals Assessment (1997)
No. 9, Guidance Document for the Conduct of Studies of Occupational Exposure to Pesticides during Agricultural Application (1997)
No. 10, Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (1998)
No. 11, Detailed Review Paper on Aquatic Testing Methods for Pesticides and industrial Chemicals (1998)
No. 12, Detailed Review Document on Classification Systems for Germ Cell Mutagenicity in OECD Member Countries (1998) No. 13, Detailed Review Document on Classification Systems for Sensitising Substances in OECD Member Countries 1998)
No. 14, Detailed Review Document on Classification Systems for Eye Irritation/Corrosion in OECD Member Countries (1998) No. 15, Detailed Review Document on Classification Systems for Reproductive Toxicity in OECD Member Countries (1998)
No. 16, Detailed Review Document on Classification Systems for Skin Irritation/Corrosion in OECD Member Countries (1998) No. 17, Environmental Exposure Assessment Strategies for Existing Industrial Chemicals in OECD Member Countries (1999) No. 18, Report of the OECD Workshop on Improving the Use of Monitoring Data in the Exposure Assessment of Industrial
Chemicals (2000)
No. 19, Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals used in Safety Evaluation (1999)
No. 20, Revised Draft Guidance Document for Neurotoxicity Testing (2004)
No. 21, Detailed Review Paper: Appraisal of Test Methods for Sex Hormone Disrupting Chemicals (2000)
No. 22, Guidance Document for the Performance of Out-door Monolith Lysimeter Studies (2000)
No. 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000)
No. 24, Guidance Document on Acute Oral Toxicity Testing (2001)
No. 25, Detailed Review Document on Hazard Classification Systems for Specifics Target Organ Systemic Toxicity Repeated Exposure in OECD Member Countries (2001)
No. 26, Revised Analysis of Responses Received from Member Countries to the Questionnaire on Regulatory Acute Toxicity Data Needs (2001)
No 27, Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment (2001)
No 28, Guidance Document for the Conduct of Skin Absorption Studies (2004)
No 29, Guidance Document on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (2001)
No 30, Detailed Review Document on Hazard Classification Systems for Mixtures (2001)
No 31, Detailed Review Paper on Non-Genotoxic Carcinogens Detection: The Performance of In-Vitro Cell Transformation Assays (2007)
No. 32, Guidance Notes for Analysis and Evaluation of Repeat- Dose Toxicity Studies (2000)
No. 33, Harmonised Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures (2001)
No. 34, Guidance Document on the Development, Validation and Regulatory Acceptance of New and Updated Internationally
Acceptable Test Methods in Hazard Assessment (2005)
No. 35, Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies (2002)
No. 36, Report of the OECD/UNEP Workshop on the Use of Multimedia Models for Estimating Overall Environmental Persistence and Long Range Transport in the Context of PBTS/POPS Assessment (2002)
No. 37, Detailed Review Document on Classification Systems for Substances which Pose an Aspiration Hazard (2002)
No. 38, Detailed Background Review of the Uterotrophic Assay Summary of the Available Literature in Support of the Project of the OECD Task Force on Endocrine Disrupters Testing and Assessment (EDTA) to Standardise and Validate the Uterotrophic Assay (2003) No. 39, Guidance Document on Acute Inhalation Toxicity Testing (2009)
No. 40, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures which Cause Respiratory Tract Irritation and Corrosion (2003)
No. 41, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures which in Contact with Water Release Toxic Gases (2003)
No. 42, Guidance Document on Reporting Summary Information on Environmental, Occupational and Consumer Exposure (2003)
No. 43, Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment (2008)
No. 44, Description of Selected Key Generic Terms Used in Chemical Hazard/Risk Assessment (2003)
No. 45, Guidance Document on the Use of Multimedia Models for Estimating Overall Environmental Persistence and Long-range Transport (2004)
No. 46, Detailed Review Paper on Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances (2004) No. 47, Detailed Review Paper on Fish Screening Assays for the Detection of Endocrine Active Substances (2004)
No. 48, New Chemical Assessment Comparisons and Implications for Work Sharing (2004)
No. 49, Report from the Expert Group on (Quantitative)
Structure-Activity Relationships [(Q)SARs] on the Principles for the Validation of (Q)SARs (2004)
No. 50, Report of the OECD/IPCS Workshop on Toxicogenomics (2005)
No. 51, Approaches to Exposure Assessment in OECD Member Countries: Report from the Policy Dialogue on Exposure Assessment in June 2005 (2006)
No. 52, Comparison of Emission Estimation Methods Used in Pollutant Release and Transfer Registers (PRTRs) and Emission Scenario Documents (ESDs): Case Study of Pulp and Paper and Textile Sectors (2006)
No. 53, Guidance Document on Simulated Freshwater Lentic Field Tests (Outdoor Microcosms and Mesocosms) (2006) No. 54, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (2006)
No. 55, Detailed Review Paper on Aquatic Arthropods in Life Cycle Toxicity Tests with an Emphasis on Developmental, Reproductive and Endocrine Disruptive Effects (2006) No. 56, Guidance Document on the Breakdown of Organic Matter in Litter Bags (2006)
No. 57, Detailed Review Paper on Thyroid Hormone Disruption Assays (2006)
No. 58, Report on the Regulatory Uses and Applications in OECD Member Countries of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models in the Assessment of New and Existing Chemicals (2006)
No. 59, Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats (2006)
No. 60, Report of the Initial Work towards the Validation of the 21-Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1A) (2006)
No. 61, Report of the Validation of the 21-Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1B) (2006)
No. 62, Final OECD Report of the Initial Work towards the Validation of the Rat Hershberger Assay: Phase-1, Androgenic Response to Testosterone Propionate, and Anti-Androgenic Effects of Flutamide (2006)
No. 63, Guidance Document on the Definition of Residue (2006) No. 64, Guidance Document on Overview of Residue Chemistry Studies (2006)
No. 65, OECD Report of the Initial Work towards the Validation of the Rodent Uterotrophic Assay - Phase 1 (2006)
No. 66, OECD Report of the Validation of the Rodent Uterotrophic Bioassay: Phase 2. Testing of Potent and Weak Oestrogen Agonists by Multiple Laboratories (2006)
No. 67, Additional Data Supporting the Test Guideline on the Uterotrophic Bioassay in rodents (2007)
No. 68, Summary Report of the Uterotrophic Bioassay Peer Review Panel, including Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow up of this Report (2006)
No. 69, Guidance Document on the Validation of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models (2007)
No. 70, Report on the Preparation of GHS Implementation by the OECD Countries (2007)
No. 71, Guidance Document on the Uterotrophic Bioassay - Procedure to Test for Antioestrogenicity (2007)
No. 72, Guidance Document on Pesticide Residue Analytical Methods (2007)
No. 73, Report of the Validation of the Rat Hershberger Assay:
Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists and Negative Reference Chemicals by Multiple Laboratories. Surgical Castrate Model Protocol (2007) No. 74, Detailed Review Paper for Avian Two-generation Toxicity Testing (2007)
No. 75, Guidance Document on the Honey Bee (Apis Mellifera L.) Brood test Under Semi-field Conditions (2007)
No. 76, Final Report of the Validation of the Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances: Phase 1 - Optimisation of the Test Protocol (2007) No. 77, Final Report of the Validation of the Amphibian Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study (2007)
No. 78, Final Report of the Validation of the 21-day Fish Screening Assay for the Detection of Endocrine Active Substances, Phase 2: Testing Negative Substances (2007)
No. 79, Validation Report of the Full Life-cycle Test with the Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007)
No. 80, Guidance on Grouping of Chemicals (2007)
No. 81, Summary Report of the Validation Peer Review for the Updated Test Guideline 407, and Agreement of the Working Group of National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007)
No. 82, Guidance Document on Amphibian Thyroid Histology (2007)
No. 83, Summary Report of the Peer Review Panel on the Stably Transfected Transcriptional Activation Assay for Detecting
Estrogenic Activity of Chemicals, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007)
No. 84, Report on the Workshop on the Application of the GHS Classification Criteria to HPV Chemicals, 5-6 July, Bern
Switzerland (2007)
No. 85, Report of the Validation Peer Review for the Hershberger Bioassay, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007)
No. 86, Report of the OECD Validation of the Rodent Hershberger Bioassay: Phase 2: Testing of Androgen Agonists, Androgen
Antagonists and a 5 α-Reductase Inhibitor in Dose Response Studies by Multiple Laboratories (2008)
No. 87, Report of the Ring Test and Statistical Analysis of Performance of the Guidance on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (Transformation/
Dissolution Protocol) (2008)
No. 88, Workshop on Integrated Approaches to Testing and Assessment (2008)
No. 89, Retrospective Performance Assessment of the Test Guideline 426 on Developmental Neurotoxicity (2008) No. 90, Background Review Document on the Rodent Hershberger Bioassay (2008)
No. 91, Report of the Validation of the Amphibian Metamorphosis Assay (Phase 3) (2008)
No. 92, Report of the Validation Peer Review for the Amphibian Metamorphosis Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2008)
No. 93, Report of the Validation of an Enhancement of OECD TG 211: Daphnia Magna Reproduction Test (2008)
No. 94, Report of the Validation Peer Review for the 21-Day Fish Endocrine Screening Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-Up of this Report (2008)
No. 95, Detailed Review Paper on Fish Life-Cycle Tests (2008) No. 96, Guidance Document on Magnitude of Pesticide Residues in Processed Commodities (2008)
No. 97, Detailed Review Paper on the use of Metabolising Systems for In Vitro Testing of Endocrine Disruptors (2008) No. 98, Considerations Regarding Applicability of the Guidance on Transformation/Dissolution of Metals Compounds in Aqueous Media (Transformation/Dissolution Protocol) (2008)
No. 99, Comparison between OECD Test Guidelines and ISO Standards in the Areas of Ecotoxicology and Health Effects (2008) No. 100, Report of the Second Survey on Available Omics Tools (2009)
No. 101, Report of the Workshop on Structural Alerts for the OECD (Q)SAR Application Toolbox, 15-16 May 2008, Utrecht, the Netherlands (2009)
No. 102, Guidance Document for using the OECD (Q)SAR Application Toolbox to Develop Chemical Categories According to the OECD Guidance on Grouping of Chemicals (2009)
No. 103, Detailed Review Paper on Transgenic Rodent Mutation Assays (2009)
No. 104, Performance Assessment: Comparsion of 403 and CxT Protocols via Simulation and for Selected Real Data Sets (2009) No. 105, Report on Biostatistical Performance Assessment of the draft TG 436: Acute Toxic Class Testing Method for Acute
Inhalation Toxicity (2009)
No. 106, Guidance Document for Histologic Evaluation of Endocrine and Reproductive Test in Rodents (2009)
No. 107, Preservative Treated Wood to the Environment for Wood Held in Storage after Treatment and for Wooden Commodities that are not Covered and are not in Contact with Ground. (2009)
No. 108, Report of the Validation of the Hershberger Bioassay (weanling model) (2009)
No. 109, Literature Review on the 21-Day Fish Assay and the Fish Short-Term Reproduction Assay (2009)
No. 110, Report of the Validation Peer Review for the Weanling Hershberger Bioassay and Agreement of the Working Group of National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2009)
No. 111, Report of the Expert Consultation to Evaluate an Estrogen Receptor Binding Affinity Model for Hazard Identification (2009)
No. 112, The 2007 OECD List of High Production Volume Chemicals (2009)
No. 113, Report of the Focus Session on Current and Forthcoming Approaches for Chemical Safety and Animal Welfare (2010)
No. 114, Performance Assessment of Different Cytotoxic and Cytostatic Measures for the In Vitro Micronucleus Test (MNVIT):
Summary of results in the collaborative trial (2010)
No. 115, Guidance Document on the Weanling Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties (2009)
No. 116, Guidance Document 116 on the Conduct and Design of Chronic Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 and 453 – 2nd Edition (2011)
No. 117, Guidance Document 117 on the Current Implementation of Internal Triggers in Test Guideline 443 for an Extended One Generation Reproductive Toxicity Study, in the United States and Canada (2011)
No. 118, Workshop Report on OECD Countries’ Activities Regarding Testing, Assessment and Management of Endocrine Disrupters Part I and Part II (2010)
No. 119, Classification and Labelling of chemicals according to the UN Globally Harmonized System: Outcome of the Analysis of Classification of Selected Chemicals Listed in Annex III of the Rotterdam Convention (2010)
No. 120, Part 1: Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism-based Structural Alerts for the Identification of DNA Binding Chemicals (2010)
No. 120, Part 2: Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism-based Structural Alerts for the Identification of DNA Binding Chemicals (2010)
No. 121, Detailed Review Paper (DRP) on Molluscs Life-cycle Toxicity Testing (2010)
No. 122, Guidance Document on the Determination of the Toxicity of a Test Chemical to the Dung Beetle Aphodius Constans (2010) No. 123, Guidance Document on the Diagnosis of Endocrine- related Histopathology in Fish Gonads (2010)
No. 124, Guidance for the Derivation of an Acute Reference Dose (2010)
No. 125, Guidance Document on Histopathology for Inhalation Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity:
28-Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010)
No. 126, Short Guidance on the Threshold Approach for Acute Fish Toxicity (2010)
No. 127, Peer Review Report of the Validation of the 21-Day Androgenised Female Stickleback Screening Assay (2010)
No. 128, Validation Report of the 21-Day Androgenised Female Stickleback Screening Assay (2010)
No. 129, Guidance Document on Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests(2010)
No. 131, Report of the Test Method Validation of Avian Acute Oral Toxicity Test (OECD Test Guideline 223) (2010)
No. 132, Report of the Multi-Laboratory Validation of the H295R Steroidogenesis Assay to Identify Modulators (2010)
No.133, Peer Review Report for the H295R Cell-Based Assay for Steroidogenesis (2010)
No.134, Report of the Validation of a Soil Bioaccumulation Test with Terrestrial Oligochaetes by an International ring test (2010) No.135, Detailed Review Paper on Environmental Endocrine Disruptor Screening: The use of Estrogen and Androgen Receptor Binding and Transactivation Assays in Fish (2010)
No. 136, Validation Report of the Chironomid Full Life-Cycle Toxicity Test (2010)
No. 137, Explanatory Background Document to the OECD Test Guideline on In Vitro Skin Irritation Testing (2010)
No. 138, Report of the Workshop on Using Mechanistic Information in Forming Chemical Categories (2011)
No. 139, Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism Based Structural Alerts for the Identification of Protein-binding Chemicals (2011)
No. 140, Report of the WHO/OECD/ILSI (Hesi) Workshop on Risk Assessment of Combined Exposures to Multiple Chemicals (2011) No. 141, Report of the Phase 1 of the Validation of the Fish Sexual Development Test for the Detection of Endocrine Active Substances (2011)
No. 142, Report of the Phase 2 of the Validation of the Fish Sexual Development Test for the Detection of Endocrine Active Substances (2011)
No. 143, Peer Review Report for the Validation of the Fish Sexual Development Test and Agreement of the Working Group of National Co-ordinators of the Test Guideline Programme on the Follow-up of the Peer Review (2011)
No. 144, Validation Report for the Acute Chironomid Assay (2011) No. 145, Transgenic Rodent Somatic and Germ Cell Gene
Mutation Assay: Retrospective Performance Assessment (2011) No. 146, Syrian Hamster Embryonic (SHE) Cell PH 6.7 Cell Transformation Assay Prevalidation Study Report (2012)
No. 147, Syrian Hamster Embryonic (SHE) Cell PH 7.0 Cell Transformation Assay Prevalidation Study Report (2012) No. 148, Guidance Document on the Androgenised Female Stickleback Screen (2011)
No. 149, Validation Report of the Balb/c 3T3 Cell Transformation Assay (2012)
No. 152, Case Study: Assessment of an Extended Chemical Category, the Short-chain Methacrylates, Targeted on Bioaccumulation (2011)
No. 153, Guidance Document for the Derivation of an Acute Reference Concentration (Arfc) (2011)
No. 154, Validation Report: Part 1 – Validation of Efficacy Methods for Antimicrobials used on Hard Surfaces (2011) No. 154, Validation Report: Part 2 – Validation of Efficacy Methods for Antimicrobials used on Hard Surfaces (2011) No. 155, Peer Review for the Validation of the Modified Skin Irritation Test Method using LabyCyte EPI-MODEL24; Additional Studies; and Agreement of the Working Group of National
Coordinators on the Follow-up to the Peer Review (2011) No. 156, Guidance Notes on Dermal Absorption (2011) No. 157, Validation Report Phase 1 for the Zebrafish Embryo Toxicity Test (2011)
No. 158, Report of Progress on the Interlaboratory Validation of the OECD Harpacticoid Copepod Development and Reproduction Test (2011)
No. 159, Validation Report for the Skin Irritation Test Method using Labcyte Epi-Model24 (2011)
No. 160, Guidance Document on the Bovine Corneal Opacity and Permeability (BCOP) and Isolated Chicken Eye (ICE) Test Methods:
Collection of Tissues for Histological Evaluation and Collection of Data on Non-Severe Irritants (2011)
No. 161, Peer Review Report for the Validation of the Stably Transfected Transcriptional Activation Assay for the Detection of Androgenic and Anti-Androgenic Activity of Chemicals (2011)
No. 162, Guidance Document 116 on The Conduct And Design Of Chronic Toxicity And Carcinogenicity Studies, Supporting Test Guidelines 451, 452 And 453 – 2nd Edition (2011)
No. 163, Assays and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up to the Peer Review (2012)
No. 164, Guidance Document on Crop Field Trials (2011) No. 165, Report of an OECD Workshop on Metals Specificities in Environmental Risk Assessment (2011)
No. 166, SIDS Initial Assessment Profiles agreed in the course of the OECD HPV Chemicals Programme from 1993 to 2013 (2012) No. 167, Crosswalk of Harmonized U.S. - Canada Industrial Function and Consumer and Commercial Product Categories with EU Chemical Product and Article Categories (2012)
No. 168, The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Binding to Proteins (2012)
No. 169, Use of the AOP to Develop Chemical Categories and Integrated Assessment and Testing Approaches (2012)
No. 170, Guidance Document for Demonstrating Efficacy of Pool and Spa Disinfectants and Field Testing (2012)
No. 171, Fish Toxicity Testing Framework (2012)
No. 172, Validity Report (Phase 2) for the Zebrafish Embryo Toxicity Test (2012)
No. 173, Performance Standards for Stably Transfected
Transactivation in vitro Assays to Detect Estrogen Agonists for TG 455 Guidance Document on Standardised Test (2012)
No. 174, Performance Standards for the BG1Luc ER TA Transactivation Method to Detect Estrogen Receptor Antagonists (2012)
No. 175, Validation Report of a Ring Test for the OECD 305 Dietary Exposure Bioaccumulation Fish Test (Part 1) with Additional Report Including Comparative Analysis of Trout and Carp Results (Part II) (2012)
No. 176, Report on the Retrospective Analysis of 2-Generation Reprotoxicity Data (2012)
No. 177, Guidance Document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (2012)
No. 178, Detailed Review Paper on the State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors (2012)
No. 179, Validation Report (Phase 2) for the Zebrafish Embryo Toxicity Test (2012)
© OECD 2012
Applications for permission to reproduce or translate all or part of this material should be made to: Head of Publications Service, RIGHTS@oecd.org. OECD, 2 rue André-Pascal, 75775 Paris Cedex 16, France
ABOUT THE OECD
The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental organisation in which representatives of 34 industrialised countries in North and South America, Europe and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and
harmonise policies, discuss issues of mutual concern, and work together to respond to international problems. Most of the OECD’s work is carried out by more than 200 specialised committees and working groups composed of member country delegates. Observers from several countries with special status at the OECD, and from interested international organisations, attend many of the OECD’s workshops and other meetings. Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is organised into directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in ten different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides and Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of Novel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission Scenario Documents; and Safety of Manufactured Nanomaterials. More information about the Environment, Health and Safety Programme and EHS publications is available on the OECD’s World Wide Web site (www.oecd.org/ehs/).
This publication was developed in the IOMC context. The contents do not necessarily reflect the views or stated policies of individual IOMC Participating Organisations.
The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was
established in 1995 following recommendations made by the 1992 UN Conference on Environment and Development to strengthen co-operation and increase international co-ordination in the field of chemical safety. The Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD. UNDP is an observer. The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by the Participating Organisations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment.
This publication is available electronically, at no charge.
For this and many other Environment, Health and Safety publications, consult the OECD’s
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OECD Environment Directorate, Environment, Health and Safety Division
2 rue André-Pascal 75775 Paris Cedex 16
France
Fax: (33-1) 44 30 61 80 E-mail: ehscont@oecd.org
FOREWORD
This Detailed Review Paper (DRP) was developed as a follow-up to the workshop on OECD countries’
activities regarding testing, assessment and management of endocrine disrupters, which was held in Copenhagen (Denmark) on 22-24 September 2010 (see document No. 118 published in the Series on Testing and Assessment).
In 2010, the project was included in the Test Guideline workplan. It was led by the US, with the support of the European Commission (EC) and the Secretariat – the EC and the Secretariat led the development of the chapter on Endocrine Disrupters and the Epigenome, included in the annex of this DRP. The document was developed by consultants in close cooperation with an advisory group on testing and assessment of endocrine disrupters (EDTA AG). The outline of the DRP was discussed at the meeting of the EDTA AG in April 2011. The 1st draft DRP was then sent to the Working Group of National Coordinators of the Test Guidelines Programme and the EDTA AG for comments in July 2011. The draft was revised by the consultants on the basis of the comments received and a 2nd draft was developed and sent for WNT comments in autumn 2011. The 2nd draft DRP was also reviewed and revised at a meeting of the EDTA AG in December 2011. The draft chapter on Endocrine Disrupters and the Epigenome was circulated once more to the WNT after the December EDTA AG meeting, as its section on Recommendations (section 8 of the chapter) had been revised in depth by the EDTA AG.
The draft Detailed Review Paper was approved by the WNT at its meeting held in April 2012. It was declassified by the Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology (hereafter Joint Meeting), on 7 August 2012. This document is published under the responsibility of the Joint Meeting.
Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors
Contractor:
RTI International 3040 Cornwallis Road P.O. Box 12194 Research Triangle Park, NC 27709 Draft #3 February 2012 Authors
Gerald A. LeBlanc, Ph.D.
Department of Environmental and Molecular Toxicology
North Carolina State University Raleigh, NC
David O. Norris, Ph.D.
Department of Integrative Physiology University of Colorado
Boulder, CO
Werner Kloas, Ph.D.
Department of Ecophysiology and Aquaculture IGB
Endocrinology at Humboldt University Germany
Seth W. Kullman, Ph.D.
Department of Environmental and Molecular Toxicology
North Carolina State University Raleigh, NC
William S. Baldwin, Ph.D.
Department of Biological Sciences and Environmental Toxicology
Clemson University Clemson, SC
John M. Greally, Ph.D.
Department of Genetics
Albert Einstein College of Medicine Bronx, NY
Table of Contents
ABOUT THE OECD ... 18 FOREWORD ... 20 ABSTRACT ... 32 INTRODUCTION ... 33 1.1 Relevance of this DRP to Diseases and Syndromes of Contemporary Concern ... 37 1.2 Relevance of this DRP to the Adverse Outcome Pathway approach ... 37 THE HYPOTHALAMUS:PITUITARY:ADRENOCORTICAL (HPA) AXIS ... 40 2.1 Overview ... 40 2.1.1 Corticotropin-Releasing Hormone (CRH) ... 41 2.1.2 Arginine Vasopressin (AVP)/Arginine Vasotocin (AVT) ... 42 2.1.3 Corticotropin (ACTH) ... 42 2.1.4 Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) ... 42 2.1.5 Glucocorticoids ... 42 2.1.5.1 Glucocorticoid Receptors (GRs)... 43 2.1.5.2 CRH and Glucocorticoid-Binding Protein (transcortinprotein and glucocorticoid-binding protein [transcortin]) ... 43 2.1.6. Neuroendocrine Regulation of the HPA Axis ... 43 2.2 Consequences of Disruption ... 44 2.3 Precedent Chemicals as Potential Disruptors of the HPA Axis... 44 2.3.1 Steroid Synthesis and Receptor Agonists and Antagonists ... 44 2.3.2 Metals ... 45 2.3.3 Neuroactive Chemicals ... 45 2.3.4 Vasopressin Receptor Agonists and Antagonists ... 45 2.3.5 CRH Receptor Antagonists ... 46 2.3.6 Pesticides ... 46 2.3.7 Arylhydrocarbon Receptor (AhR) Agonists ... 46 2.3.8 Estrogens and Androgens ... 46 2.4 In Vitro Assays ... 46 2.4.1 Transactivation Reporter Assays ... 46 2.4.2 Microarrays ... 47 2.4.3 Cell Culture Systems ... 47 2.4.3.1 Corticotropes ... 47 2.4.3.2 Adrenal Cortical Cells ... 47 2.4.3.3 Glucocorticoid Target Cells ... 47 2.5 In Vivo Assays ... 47 2.5.1 Mammals ... 48 2.5.2 Fish ... 48 2.5.2 Amphibians ... 48 2.6 Strengths, Challenges, and Limitations ... 48 2.6.1 Stress, the Adverse Outcome Pathway, and Assay Selection ... 49 HYPOTHALAMUS:PITUITARY:GONAD (HPG) AXIS ... 51 3.1 Overview ... 51 3.1.1 Structure of the HPG Axis ... 51 3.1.2 Structure and Actions of HPG Hormones ... 51
3.1.3 Function of the HPG Axis... 53 3.2 Consequences of Disruption ... 53 3.2.1 (Anti)estrogens ... 53 3.2.1.1 Reproduction ... 54 3.2.1.2 Metabolism and Growth ... 55 3.2.1.3 Immune System ... 55 3.2.2 (Anti)androgens ... 55 3.2.2.1 Reproduction ... 55 3.2.2.2 Growth ... 56 3.2.3 (Anti)gestagens ... 56 3.2.3.1 Reproduction ... 56 3.2.3.2 Immune System ... 57 3.3 Precedent Chemicals ... 57 3.3.1 (Anti)estrogens ... 57 3.3.1.1 Bisphenol A (BPA) ... 57 3.3.1.2 Phthalate Esters ... 58 3.3.1.3 Polychlorinated Biphenyls (PCBs) ... 58 3.3.2 (Anti)androgens ... 59 3.3.2.1 Di-(2-ethylhexyl)phthalate (DEHP) ... 59 3.3.2.2 Flutamide ... 59 3.3.3 (Anti)gestagens ... 59 3.3.3.1 Levonorgestrel (LNG)... 59 3.4 In Vitro Assays ... 60 3.4.1 (Anti)estrogens ... 60 3.4.1.1 ER Transactivation Assays ... 60 3.4.1.2 Membrane Receptor Binding ... 60 3.4.1.3 Cell-based Microarrays ... 60 3.4.2 (Anti)androgens ... 61 3.4.2.1 AR Transactivation Assay ... 61 3.4.3 (Anti)gestagens ... 61 3.4.3.1 PR Transactivation Assays ... 61 3.4.3.2 mPR Binding Assays ... 61 3.4.3.3 Cell-based Microarrays ... 61 3.5 In Vivo Assays ... 61 3.5.1 (Anti)estrogens ... 61 3.5.1.1 Microarrays ... 61 3.5.1.2 Disruption of Brain and Gonad Differentiation ... 62 3.5.2 (Anti)androgens ... 62 3.5.2.1 Behavioral Changes ... 62 3.5.3 (Anti)gestagens ... 62 3.5.3.1 Germinal Vesicle Breakdown (GVBD) ... 62 3.5.3.2 Sperm Motility... 62 3.6 Strengths, Challenges, and Limitations ... 63 3.6.1 (Anti)estrogens ... 63 3.6.2 (Anti)androgens ... 64 3.6.3 (Anti)gestagens ... 64 THE SOMATOTROPIC AXIS ... 68 4.1 Overview ... 68 4.2 Consequences of Disruption ... 70 4.3 Precedent Chemicals ... 70
4.3.1 Estrogenic Chemicals ... 70 4.3.2 Anti-thyroid Chemicals ... 70 4.3.3 Corticosteroid Stimulants ... 70 4.3.4 Chemicals that Directly Disrupt the Somatotropic Axis ... 71 4.4 In vitro Assays ... 71 4.5 In vivo Assays ... 72 4.6 Strengths, Challenges, and Limitations ... 72 THE RETINOID SIGNALING PATHWAY... 74 5.1 Overview ... 74 5.1.1 Retinoic Acid Receptor Signaling ... 74 5.1.2 The Retinoid X Receptor Signaling Network ... 74 5.1.2.1 Reproduction in Mammals ... 75 5.1.2.2 Development in Mammals ... 75 5.1.2.3 Lipid Homeostasis in Mammals ... 76 5.2 Consequences of Disruption ... 76 5.3 Precedent Chemicals ... 76 5.3.1 Reductions in Retinoid Levels ... 77 5.3.2 RAR Agonists ... 77 5.3.3 RXR Agonists/Antagonists ... 77 5.4 In Vitro Assays ... 78 5.4.1 AhR Transactivation Reporter Assay ... 78 5.4.2 RAR Transactivation Reporter Assay ... 79 5.4.3 RXR Transactivation Reporter Assay ... 79 5.4.4 Adipocyte Differentiation Assay ... 80 5.4.5 Cell-based Microarrays ... 81 5.5 In Vivo Assays ... 81 5.5.1 CYP 1A1 Induction ... 81 5.5.2 Alterations in Retinoid Levels and Metabolism ... 81 5.5.3 Alterations in Lipid Levels and Metabolism ... 82 5.6 Strengths, Challenges, and Limitations ... 82 THE HYPOTHALAMUS:PITUITARY:THYROID (HPT) AXIS ... 83 6.1 Overview ... 83 6.2 Consequences of Disruption ... 85 6.3 Precedent Chemicals ... 87 6.3.1 AhR and CAR Agonists ... 87 6.3.2 Deiodinase Inhibitors/Suppressors ... 88 6.3.3 Disruptors of TSH Signaling ... 88 6.3.4 Disruptors of TR Signaling ... 88 6.3.5 Disruptors of Iodine Uptake and Thyroid Hormone Synthesis ... 89 6.3.6 Disruptors of Plasma and Cross-Membrane Transport Proteins ... 89 6.4 In Vitro Assays ... 90 6.4.1 Transactivation Reporter Assays with TRα and TRβ ... 90 6.4.2 Two-hybrid Assays ... 91 6.4.3 DNA Binding Assays ... 92 6.4.4 Dendritic Arborization ... 92 6.4.5 Neurite Extension ... 93 6.4.6 Cell Proliferation Assay ... 93 6.4.7 Thyroid Peroxidase (TPO) Inhibition Assay ... 94 6.4.8 Iodide Uptake Assay ... 95
6.4.9 Thyroid Hormone Binding Protein Assays ... 95 6.5 In Vivo Assays ... 95 6.5.1 Modification of Long Term In Vivo Assays ... 95 6.5.2 Organ Culture ... 96 6.5.3 Additional In Vivo Models ... 97 6.5 Strengths, Challenges and Limitations ... 97 THE VITAMIN D SIGNALING PATHWAY ... 100 7.1 Overview ... 100 7.1.1 Synthesis ... 100 7.1.2 Catabolism ... 102 7.1.3 Calcium and Skeletal Maintenance ... 102 7.1.4 Immune System Function ... 102 7.1.5 Cancer ... 102 7.1.6 Neurodevelopment ... 103 7.1.7 Cardiac Function ... 103 7.1.8 Metabolism of Secondary Bile Acids ... 103 7.2 Consequences of Disruption ... 104 7.3 Precedent Chemicals ... 105 7.3.1 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ... 105 7.3.2 Polychlorinated Biphenyls (PCBs) ... 105 7.3.3 Ethanol ... 106 7.3.4 Lead ... 106 7.4 In Vitro Assays ... 106 7.4.1 Transactivation VDR Reporter Assays ... 106 7.4.2 AhR Transactivation Reporter Assays ... 106 7.5 In Vivo Assays ... 107 7.5.1 Serum Vitamin D Levels ... 107 7.5.2 Microarrays ... 107 7.5.3 Skeletal Morphology and Bone Densitometry ... 107 7.5.4 Histology ... 108 7.6 Current Challenges and Limitations ... 108 7.6.1 Limited Knowledge Regarding Non-mammalian Vertebrates ... 108 7.6.2 Broaden Focus beyond Skeletal Effects ... 109 THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR SIGNALING PATHWAY ... 110 8.1 Overview ... 110 8.1.1 PPARs in Non-Mammalian Species ... 111 8.2 Consequences of Disruption ... 111 8.2.1 PPARα ... 112 8.2.2 PPARβ/δ ... 113 8.2.3 PPARγ... 114 8.2.4 PPAR Disruption in Wildlife ... 115 8.3 Precedent Chemicals ... 116 8.3.1 PPAR Activators in Non-Mammalian Species ... 118 8.4 In Vitro Assays ... 118 8.4.1 Transactivation Reporter Assays ... 118 8.4.2 3T3-L1 Cell Differentiation Assay ... 119 8.5 In Vivo Assays ... 119 8.5.1 Peroxisome Proliferation ... 119 8.5.2 Lipid Accumulation ... 120
8.5.3 Microarrays ... 120 8.6 Strengths, Challenges, and Limitations ... 120 SUMMARY, CONCLUSIONS, AND CONSIDERATIONS ... 122 9.1 Summary and Conclusions ... 122 9.1.1 Cross Talk among Signaling Pathways ... 122 9.1.2 Assays ... 122 9.2 Next Steps for Further Consideration ... 125 9.2.1 Hypothalamus:Pituitary:Adrenocortical Axis ... 125 9.2.2 Hypothalamus:Pituitary:Gonadal Axis ... 126 9.2.3 Somatotropic Axis ... 126 9.2.4 Retinoid Signaling Pathway ... 127 9.2.5 Hypothalamus:Pituitary:Thyroid Axis ... 127 9.2.6 Vitamin D Signaling Pathway ... 127 9.2.7 Peroxisome Proliferator-Activated Receptor Signaling Pathway ... 128 9.2.8 Adverse Outcome Pathway approach and the Conceptual Framework... 128 ACKNOWLEDGEMENTS ... 129 REFERENCES ... 130 ANNEX 1: ENDOCRINE DISRUPTERS AND THE EPIGENOME ... 172 1.0 Introduction ... 172 2.0 Definitions ... 172 3.0 Potential effects ... 178 4.0 Evidence for endocrine disruption being mediated by epigenomic processes ... 178 5.0 Assay methods ... 181 6.0 Challenges ... 188 7.0 Conclusions and testing recommendations ... 189 8.0 Recommendations ... 197
Acronyms and Abbreviations
3β-HSD 3β-hydroxysteroid dehydrogenase 3,3’,5-triClBPA 3,3’,5-trichlorobisphenol A 17,20β-P 17,20β-dihydroxypregn-4-en-3-one 17,20β-S 17,20β,21-trihydroxypregn-4-en-3-one ACTH corticotropin
AhR aryl hydrocarbon receptor
AND androstenedione
ANSA 8-anilina-1-napthalenesulfonic acid
AOP adverse outcome pathway
AR androgen receptor
AR STTA AR transactivation assay
ARNT aryl hydrocarbon receptor nuclear translocator
ATPase adenosine triphosphatase
AVP arginine vasopressin
AVT argonine vastocine
BDE brominated diphenyl ether
BDE-47 2,2',4,4'-tetrabromodiphenylether
BDNF brain-derived neutrophic factor
BFR brominated flame retardant
BIAC Business and Industry Advisory Committee BKME bleached kraft mill effluent
BMI body mass index
BNF β-naphthoflavone
BNST bed nucleus of the stria terminalis
BPA bisphenol A
BTEB basic transcription element binding protein
cAMP cyclic adenosine monophosphate
CAR constitutive androstane receptor
CARLA co-activator-dependent receptor ligand assays
CAT chloramphenicol actyltransferase
CBG corticosteroid binding globulin CBP/p300 CREB binding protein
CCD charged couple device
CDCA chenodeoxycholic acid
CG chorionic gonadotropin
CHIP chromatin immunoprecipitation
CHO Chinese hamster ovary cells
CNS central nervous system
CoR co-repressors CPT1 carnitine palmitoyl transferase 1 CREB cAMP-responsive element binding
CRH corticotropin-releasing hormone
CRH-BP corticotropin-releasing hormone binding protein
CV coefficient of variation
DBD DNA binding domain
DBP di-n-butyl phthalate
DEHP di[2-ethylhexyl] phthalate
DES diethylstilbestrol DEX dexamethasone DHEA dehydroepiandrosterone
DHEAS DHEA sulfate
DHRA 9-cis-4-oxo-13,14-dihydroretinoic acid
DHT dihydrotestosterone
DIDP diisodecyl phthalate
DIT di-iodothyronine
DMBPA 3,3'-dimethylbisphenol A
DNHP di-n-hexyl phthalate
DnOP di-n-octyl phthalate
DPSA Differential Protease Sensitivity Assay
DRE dioxin response element
DRP detailed review paper
DUOX/ThOX dinucleotide phosphate oxidase
DXA dual-emission X-ray absorptiometry
EAT estrogen, androgen, and thyroid
EDC endocrine disrupting chemical
EDTA Endococrine Disrupter Testing and Assessment EE2 ethinylestradiol
EHA 2-ethylhexanoic acid
EMSA electrophoretic mobility shift assay ENCODE Encyclopedia of DNA Elements EPA U.S. Environmental Protection Agency
ER estrogen receptor
ERRγ estrogen-related receptor γ
FITC fluorescein isothiocyanate
FRET fluorescence resonance energy transfer
FSH follicle stimulating hormone
FXR farnesoid X receptor
GABAergic gamma-amino butyric acid
GHRH growth hormone releasing hormone
GH growth hormone
GHRH growth hormone releasing hormone
GnRH gonadotropin-releasing hormones
GPR30` G-protein-coupled receptor 30
GR glucocorticoid receptor
GR1 mineral corticoid receptor
GRE glucocorticoid response elements
GFP Green Fluorescent Protein
GST glutathione S-transferase
GSUα glycoprotein-hormone α-subunit
GVBD germinal vesicle breakdown
H3K9me 3histone H3 lysine 9 trimethylation HBCD 1,2,5,6,9,10-αHexabromocyclododecane HCBD hexabromocyclododecane HHPS hypothalamo-hypophysial portal system Hnpc human neural progenitor cell
HPA hypothalamus-pituitary-adrenocortical HPG hypothalamo-pituitary-gonadal HPI hypothalamic-pituitary-interrenal HPT hypothalamo-pituitary-thyroidal
hrTPO human recombinant TPO
IGF-1 insulin-like growth factor 1 IGF-2 insulin-like growth factor 2
IHEC International Human Epigenome Consortium IL interleukins
IP3 inositol trisphosphate
IPS induced pluripotent stem
JAK/STAT Janus Kinase/Signal Transducer and Activator of Transcription
K+ potassium ion
LBD ligand-binding domain
LC locus caeculeus
LCA lithocholic acid
LH luteinizing hormone
LICA ligand induced complex assay
LNG levonorgestrel
LUMA luminometric methylation analysis
LXR liver X receptor
MBP mono-n-butyl phthalate
MC2R melanocortin receptor
MCPA 2-methyl-4-chlorophenoxyacetic acid
MCT monocarboxylate transporter
MCT8 monocarboxylate transporter 8
ME malic enzyme
MEHP mono-2-ethylhexylphthalate MIT mono-iodothyronine
MMGT medaka multi-generation test
MMI methimazole
mPR membrane-G-protein-coupled gestagen receptor
MPS massively-parallel sequencing
MR mineralocorticoid receptor
MUFA monounsaturated fatty acid
Na+ sodium ion
NCoR nuclear receptor corepressor
NGF nerve growth factor
NIH National Institutes of Health
NIS sodium-iodide symporter
NR nuclear receptor
OATP organic ion transport proteins
OECD Organization for Economic Cooperative Development
P4 progesterone
PACAP pituitary adenylate cyclase-activating peptide PAH polycyclic aromatic hydrocarbon
PAX8 paired box gene 8
PBB polybrominated biphenyl
PBPK physiologically based pharmacokinetic
PCB polychlorinated biphenyl
PCDD polychlorinated dibenzo-p-dioxin
PCDF polychlorinated dibenzofuran
PFOA perfluorooctanoic acid
PFOS perfluorooctane sulfonate
PGJ2 prostaglandin J2
PKA phosphokinase A
PKC protein kinase C
PLC phospholipase C
PPAR peroxisome proliferator activated receptor
pQCT peripheral quantitative computed tomography
PR progesterone receptor
PRL prolactin
PTH parathyroid hormone
PTU propylthiouracil PUFA polyunsaturated fatty acid
PVN paravocellular nucleus
PXR pregnane X receptor
qPCR real-time polymerase chain reaction RAR retinoic acid receptor
RIA radioimmunoassay
RIC20 relative inhibitory concentration
RN raphe nucleus
RU486 mifepristone
RXR retinoid X receptor
SEXDAMAX Sexual differentiation and metamorphosis assaywith Xenopus
siRNA short interfering RNA
SMRT silencing mediator for retinoid and thyroid hormone receptors
SON supraoptic nucleus
SRIF somatotropin release inhibiting factor SRC-1 steroid receptor coactivator-1
SRC-2 steroid receptor coactivator-2
STAR steroidogenic acute regulatory protein
T3 thyroid hormone, triiodothyronine
T4 thyroid hormone, thyroxine
TBBPA 3,3’,5,5’-tetrabromobisphenol A
TBG Thyroid hormone-binding globulin
TBTO tributyltin oxide
TCBPA 3,30,5,50-tetrachlorobisphenol A TCDD tetrachlorodibenzo-p-dioxin
TCGA The Cancer Genome Atlas
TD thyroid disruptor
TDC thyroid disrupting compound
TDS testicular dysgenesis syndrome
TG test guideline
TH tyrosine hydroxylase
TIF2 transcriptional intermediary factor
TIQDT T4 immunofluorescence quantitative disruption test
TMBPA tetramethylbisphenol A
TMTU N,N,N’,N’-tetramethylthiourea
TPO thyroid peroxidase
TR thyroid hormone receptor
TRAP tartrate-resistant acid phosphatase
TRE thyroid response element
TRH thyrotropin releasing hormone
TRHR thyrotropin-releasing hormone receptor
TRIAC T3 signaling agonist
TSH thyrotropin stimulating hormone
TTF1 transcription termination factor 1 TTF2 transcription termination factor 2 TTR transthyretin Ucn Urocortin
V1aR vasopressin-1
VDBP vitamin D binding protein
VDR vitamin D receptor
VDRE vitamin D response element VTG vitellogenin
WHO World Health Organization
ZF zona fasciculata
ZG zona glomerulosa
ZR zona reticularis
ABSTRACT
Increasing incidents of disorders such as obesity/diabetes/metabolic syndrome, reproductive dysfunction, and neuro-developmental abnormalities in some human populations have raised concern that disruption of key endocrine-signaling pathways by exposure to environmental chemicals may be involved. This Detailed Review Paper describes some endocrine pathways that have been shown to be susceptible to environmental endocrine disruption and whose disruption could contribute to increasing incidents of some disorders in humans and wildlife populations. Assays and endpoints are described that could be used in new or existing Organization for Economic Cooperative Development (OECD) Test Guidelines for evaluating chemicals for endocrine-disrupting activity. Endocrine pathways evaluated were the hypothalamus:pituitary:adrenocortical (HPA) axis, the hypothalamus:pituitary:gonad (HPG) axis, the somatotropic axis, the retinoid signaling pathway, the hypothalamus:pituitary:thyroid (HPT) axis, the vitamin D signaling pathway, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In addition, the potential role of chemical-induced epigenetic modifications to endocrine signaling pathways, during sensitive windows of exposure, was evaluated as a mechanism of endocrine disruption, along with the examination of potential methods for assessing such disruption. This section is provided as an annex to the document (Annex 1). Potential targets of disruption along putative adverse outcome pathways associated with the signaling pathways were identified, along with assays that show promise in evaluating the target in a screening and testing program. Disruption of the HPA or retinoid X receptor signaling pathways could contribute to disorders of emerging concern, and adverse outcome pathways are well defined. However, assays for the assessment of disruption of these pathways are less well developed, and in some cases, are not specific to the pathway. Several new assays were described for the detection of disruption of the HPT axis. These assays may complement assays in the existing Test Guidelines and strengthen the adverse outcome pathway lineage. Assays for the detection of vitamin D signaling disruption and novel aspects of the HPG axis (membrane receptor signaling, progestin signaling) require further development and refinement prior to consideration for incorporation into Test Guidelines. Disruption of the somatotropic axis is likely to occur through disruption of other signaling pathways that cross-talk with the somatotropic axis. Disruption of the somatotropic axis may thus provide a more holistic view of the general integrity of the endocrine system. PPARs are involved in lipid and glucose homeostasis, inflammation, and aspects of development. The adverse outcome pathway for PPARγ is well established. Assays used to assess disruption of PPAR signaling are well developed, and many are suitable for incorporation into existing OECD Test Guidelines. In conclusion, OECD Test Guidelines could be modified to include new assays or the incorporation of novel endpoints into existing assays that would expand the repertoire of endocrine signaling pathways included in the screening and testing regimen.
INTRODUCTION
1. The endocrine system consists of an assemblage of ductless glands that secrete hormones directly into the blood or lymph, which regulate a wealth of biological processes.1 The endocrine system is comprised of multiple pathways, or axes, each consisting of different groupings of organs and hormones with distinct regulatory functions. These pathways are intricately involved in organizational, or programming, events during fetal development, as well as in the maintenance of homeostasis in the adult organism.
Mounting evidence has shown that aspects of the endocrine system are susceptible to perturbation by exogenous chemicals, resulting in the disruption of those processes under endocrine control. Evidence to date indicates that hormone nuclear receptors are a major target of endocrine disrupting chemicals (EDCs) because these receptors are designed to bind small, lipoidal molecules (i.e., steroid hormones), which can be mimicked by many environmental chemicals. These nuclear receptors, once activated by their ligand, regulate the transcription of target genes. Xenobiotics can disrupt normal nuclear receptor function by inappropriately activating the nuclear receptor (hormone receptor agonist) or by inhibiting the action of the nuclear receptor (hormone receptor antagonist). Some environmental chemicals also can disrupt normal endocrine function by altering circulating hormone levels. Accordingly, the World Health Organization (WHO) has defined an endocrine disruptor as an exogenous substance or mixture that alters functions(s) of the endocrine system and, consequently, causes adverse health effects in an intact organism, or its progeny, or (sub)populations.2 In this detailed review paper (DRP), an EDC is defined as a chemical substance that meets this definition of an endocrine disruptor.
2. At the request of member countries and its Business and Industry Advisory Committee, the Organization for Economic Cooperative Development (OECD) established a Special Activity on Endocrine Disrupter Testing and Assessment (EDTA) in 1996. The objective of the Special Activity was to coordinate the development of Test Guidelines to detect endocrine disruptors and to harmonize risk characterization approaches for such chemicals. As a result, several Test Guidelines have been developed or are presently in development. These guidelines have been integrated into a Conceptual Framework that can be used to evaluate chemicals for endocrine-disrupting activity. The Framework (http://www.oecd.org/document/58/0,3343,en_2649_34377_2348794_1_1_1_1,00.html) organizes tests into five levels of complexity dealing largely with the ability of chemicals to disrupt estrogen, androgen, and thyroid (EAT) signaling processes and steroidogenesis. Level 1 consists of the compilation of all existing test data, physical-chemical properties of the chemical, and various model predictions of activity.
Level 2 consists of in vitro screening assays that provide information on potential interactions between the chemical and specific endocrine target (e.g., receptors, enzymes). Level 3 consists of whole-organism screening assays that provide insight into chemical interactions with single selected endocrine mechanism(s) / signaling pathways. Level 4 consists of whole-organism assays that provide data on adverse effects on endocrine-relevant endpoints. These assays provide insight into chemical interactions with multiple endocrine signaling pathways or endpoints. Level 5 consists of whole-organism assays that are designed to provide more comprehensive data on adverse effects on endocrine-relevant endpoints over more extensive parts of the life cycle of the organism.
3. This Conceptual Framework provides a rational, step-wise approach to evaluating chemicals for their ability to disrupt signaling pathways, with emphasis on EAT endocrine pathways. However, the EAT pathways represent three of many endocrine pathways, and recent evidence indicates that other endocrine pathways also are susceptible to the disrupting effects of environmental chemicals. Accordingly, the OECD recognizes the need to have Guidance Documents in place that also would serve to evaluate the effects of chemicals on non-EAT endocrine pathways. This DRP describes assays that have been used to detect endocrine-disrupting effects of chemicals on non-EAT pathways, atypical EAT pathways (e.g., estrogen signaling via membrane receptors), and neuroendocrine pathways. In addition, new approaches to assessing chemical effects on EAT pathways are discussed. The neuro-endocrine pathways discussed may function upstream to regulate the production of hormones that interact with nuclear receptors, or may
function through the production of peptide hormones, which contribute directly to endocrine signaling.
(Note: The term neuro-endocrine is used in this document to denote both neuroendocrine and endocrine components to signaling pathways).
4. In 2007, the National Research Council published Toxicity Testing in the 21st Century: A Vision and a Strategy.3 This document served to redirect the standard toxicity testing paradigm, which consists of a patchwork of disparate tests performed largely with animals, to a more organized approach that makes extensive use of in vitro assays to identify and characterize toxicity pathways. The authors argue that the use of in vitro tests, coupled with modeling approaches (e.g., physiologically based pharmacokinetic [PBPK] modeling), could reduce the time and expense of chemical toxicity characterization and would relegate the use of whole-animal studies, mainly to the validation of toxicity predictions. Adverse outcome pathways (AOP) have been used as a tool to formulate pathway linkages among molecular events and toxicity. An AOP is a conceptual framework that integrates molecular events initiated by exposure to chemicals or other physiologic stressor to adverse biological outcomes at relevant levels of biological organization (Figure 1-1).4 In line with this emerging paradigm, assays described in this DRP are divided into in vitro screening assays designed to identify interactions of chemicals with specific components of toxicity pathways (OECD Conceptual Framework Level 2) and in vivo assays that would provide a more holistic evaluation of the chemical effects on endocrine signaling processes (OECD Conceptual Framework Levels 3–5). AOPs presented in the DRP are not meant to be definitive but are structured to define linkages between in vitro screening assays, which identify molecular initiating events, and in vivo toxicity tests that describe toxic events related to the initiating events. Readers are directed to Ankley et al.4 for the discussion and presentation of detailed AOPs.
5. Interaction of EDCs with nuclear receptors stands prominent among the molecular events that initiate adverse outcomes. The nuclear receptor family has 48 functionally distinct members in humans.5 In addition to the receptors involved in EAT signaling, hormone-activated nuclear receptors in vertebrates include the corticosteroid receptors (e.g., mineralocorticoid, glucocorticoid), retinoic acid receptor (RAR), retinoid X receptor (RXR), vitamin D receptor (VDR), and peroxisome proliferator activated receptor (PPAR). Ligands to some of these receptors (e.g., vitamin D binding to the VDR, retinoids binding to the RAR, fatty acids binding to PPAR) may not fit the conventional view of a hormone. Nonetheless, these ligands do fit the broad definition of a hormone as a substance, originating in one tissue and conveyed by the bloodstream to another to effect physiological activity,1 and this document will address the pathways to which these hormones and receptors contribute and their susceptibility to disruption by environmental chemicals.
6. Members of the nuclear receptor family all share a common domain structure (Figure 1-2). The A/B domains are highly variable among the nuclear receptors, but contain a transcriptional activation function (AF-1) that is vital to receptor activity. The C or DNA-binding domain (DBD) is highly conserved among the nuclear receptors, containing two zinc finger motifs that are responsible for recognition of specific DNA binding sites. The D domain functions as a hinge between the DBD and the ligand-binding domain (LBD). The LBD or E domain contains a hydrophobic ligand-binding pocket, which provides specific ligand recognition to the receptor. The E domain mediates dimerization and ligand-dependent transcriptional activation functions (AF-2). The F domain is not present on all nuclear receptors, and its function is not clear.
Figure 1-1 Adverse outcome pathway structure depicting the realms of in vitro and in vivo assays, site of initial interaction with toxicant (initiating event), and site of the typical initial
adverse outcome (Adapted from Ankley et al.4)
Figure 1-2. Domain structure of hormone nuclear receptors.
7. The susceptibility of peptide hormones, largely of neuroendocrine origin, to the action of EDCs has received relatively little attention. This may be because receptor proteins designed to recognize and bind peptide hormones are less likely to recognize typical environmental chemicals. However, precedent does exist for environmental chemicals modulating the secretion of peptide hormones (e.g., Fraites et al.6);
therefore, assays for the detection of such disruption will be described in this document. Endocrine signaling pathways for which evidence of endocrine disruption is limited to in vitro observations (e.g., MAP kinases) were not included in the DRP. Several endocrine pathways, that contribute to the regulation of apical processes relevant to this DRP (e.g., ghrelin and leptin signaling pathways), are not addressed since no data of endocrine disruption was revealed in our literature search. However, exclusion of such pathways likely indicates the absence of evaluation rather than the absence of disruption by environmental chemicals.
8. The intent of this DRP is to provide methods for both the mechanistic evaluation of the action of EDCs, as advocated in Toxicity Testing in the 21st Century: A Vision and a Strategy,3 and for the assessment of physiological consequences. This document is not all inclusive of neuro-endocrine pathways or the physiological processes regulated by the pathways. Rather, the document covers those neuro-endocrine pathways for which (a) significant evidence of susceptibility to disruption by environmental chemicals with potential for adverse outcome exists; and (b) assay procedures for the detection of environmental endocrine disruption are sufficiently developed for protocol standardization and validation. Chemicals that are known to disrupt each pathway are described in the respective sections.
These are not exhaustive lists of known EDCs, but rather are examples of chemicals that may serve as reference compounds in future standardization and validation of the assays. These pathways are diagrammed in Figure 1-3.
A/B C E
LBD AF-2
AF-1 DBD Hinge
F
Chemical properties
Receptor- ligand interactions Enzyme inhib.
Oxidative damage, etc.
Gene express.
Protein synth.
Altered signaling, etc.
Altered physiology, homeostasis, development, function
Lethality Impaired development, growth, reproduction
Altered structure, recruitment, Extinction
Toxicant Cellular
responses Macro-molecular
interactions Organ
responses Organism
responses Population responses
In vivo assays In vitro assays
Initiating event Apical event
Figure 1-3. Some examples of neuro-endocrine pathways that are affected by EDCs, resulting in symptoms of metabolic syndrome and disruptions in reproduction, growth, and development.
Black arrows denote contiguous pathways. Red arrows highlight examples of cross-talk between pathways.
9. The overall intent of this DRP is to provide guidance on testing approaches that can be used for assessing the actions and toxicity of environmental chemicals on neuro-endocrine pathways not addressed in current Test Guidelines. This DRP is not intended to introduce a new patchwork of disparate tests to add to the existing complement of testing procedures. Whenever possible, approaches for the integration of tests are described so that the greatest amount of information can be derived with the least investment of time, resources, and animals. Effort was made to minimize redundancy among assays; however, the assays are presented in the context of pathways, and pathways are typically branched, rather than linear, with various intersections among different pathways (Figure 1-3). Accordingly, some redundancy in assay descriptions was warranted to maintain the integrity of individual pathways. The DRP does not specifically address temporal aspects of susceptibility to EDCs such as effects in old animals exposed in utero (e.g. earlier menopause, reduced testosterone in old males) or hormonally induced cancers (e.g.
breast cancer, testicular cancer, prostate cancer), although such considerations are warranted in studies designed to identify “no effect” levels of the chemical. There exist many assays for the clinical evaluation of endocrine function. These assays are typically not addressed in this DRP unless they have been used to assess environmental endocrine disruption. However, such assays do hold promise for incorporation into testing schemes following evaluation for such application.
10. The OECD Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (ENV/JM/MONO(2005)14 describes eight criteria for test method validation. Assays recommended in this DRP were derived from the peer-reviewed research literature and generally do not formally meet criteria such as inter-laboratory reproducibility, extensive use of reference chemicals to determine assay performance, and assay performance under Good Laboratory Practices guidelines. However, assays recommended in the DRP do meet criteria such as existing rationale for the use of the assay, established relationship between the assay endpoint and the relevant biological response, and (reasonably) detailed assay protocols. These criteria are either evident in the descriptions of the assays or in the references provided.
metabolism reproduction growth development
fatty acids vitamin A retinoic acid
corticotropin
releasing hormone arginine vasopressin
ACTH
corticosteroids
thyroid releasing hormone
thyroid stimulating hormone
thyroid hormone vitamin D
somatostatin GHRH
growth hormone
IGF-1 gonadotropin releasing hormone
gonadotropin
testosterone estradiol