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Challenge 46


Launched Awarded Completed

This Challenge aims to develop an assay using non-protected life-stages of fish to detect thyroid-active chemicals that is designed to meet OECD principles for use in a regulatory context.

Challenges briefing webinar

Find out more about this Challenge in the webinar recording and summary of the Q and A session with the Sponsors below. 


The application deadline for this Challenge has now closed. 


Challenge launched

Sponsored by BASF SE and Bayer AG, and co-funded by Bayer AG, this Challenge aims to develop an assay using non-protected life-stages of fish to detect thyroid-active chemicals that is designed to meet OECD principles for use in a regulatory context.

Read the full Challenge Brief here


Environmental Risk Assessment (ERA) evaluates the likelihood that organisms in the environment are impacted due to exposure to chemicals. ERA mainly relies on the relationship between the exposure of natural ecosystems to a chemical (e.g. emissions resulting from consumer use or from industrial processes into water bodies) and the inherent hazard of that chemical (e.g. potential for it to cause harm to relevant species). Traditionally, ERA relies on animal testing to assess toxicological properties of chemicals, including the evaluation of potential endocrine disruption (ED) through interaction with endocrine-related receptors such as the oestrogen, androgen, and thyroid receptors (1). Current regulatory ED assessment for aquatic species relies mostly on data from chronic in vivo studies in fish in accordance with US Environmental Protection Agency (EPA) and OECD Test Guidelines (TG). The assessment of thyroid-mediated endocrine-disrupting properties of chemicals is unique in that amphibian rather than fish are almost exclusively used, as the current fish tests do not include endpoints relevant for thyroid disruption. These studies often use large numbers of animals.

Global research efforts into the development and application of New Approach Methodologies (NAMs) aim to provide information for chemical hazard and risk assessment and reduce the reliance on in vivo studies. An amphibian eleutheroembryonic  assay – the XETA assay – (2) has been developed as a NAM to evaluate endocrine activity mediated via the thyroid axis, but it does not currently cover all thyroid-mediated mechanisms, such as thyroperoxidase (TPO) and sodium-iodide symporter (NIS) inhibition. Therefore, testing using amphibians and fish (for other endocrine modes of action) is still conducted to cover these and to address adverse effects on growth and development to fulfil data requirements for regulatory ERA.

Ongoing research initiatives such as the European Programme Horizon2020 ERGO project (3) are working on the development and incorporation of thyroid-specific endpoints into existing OECD in vivo fish TGs 210, 234 (4). These developments hold promise in terms of reduction of animal testing, as fish TGs that include thyroid-specific endpoints could avoid the need for amphibian studies. However, a NAM based thyroid-specific test using fish eleutheroembryos, that covers an increased range of thyroid-mediated mechanisms and builds on other work in the area (5) has the potential to replace the use of both fish and amphibian protected life-stages for assessing thyroid-mediated ED.

The aim of this Challenge is to develop an assay using fish eleutheroembryos to specifically detect thyroid activity for use in ED and ERA.


3Rs benefits

Thousands of animal tests are conducted worldwide each year for ERA purposes. Implementing a thyroid-specific fish eleutheroembryo test to identify chemical classes with potential thyroid-mediated endocrine activity has the potential to replace some of these studies. In the short-term, a reliable thyroid-specific fish eleutheroembryo test would preclude the requirement for studies in amphibians. In the longer term, extending the scope and coverage of NAMs for ED and wider ERA could lead to a suite of NAMs that can begin to fully replace the use of fish in regulatory testing.

Current European Chemicals Agency (ECHA) and European Food Safety Authority ED guidance for the identification of endocrine disruptors (6,7) includes an annex where the specific conditions for the use of the NAM-based XETA assay are defined. It is expected that this guidance will evolve to include newly developed eleutheroembryo assays for endocrine modes-of-action other than the thyroid such as the OECD EASZY (8) and RADAR assays (9) over the next few years. This provides a significant opportunity for a successful FET4Thyroid Challenge to be included. Draft ECHA guidance supporting the new regulation (10) on the classification, labelling and packaging of substances and mixtures also includes recommendations on the tests to be conducted for ED assessment, presenting additional opportunity for the inclusion of new NAMs.


  1. European Chemicals Agency (2022) Hot topics: Endocrine disruptors
  2. OECD (2019), Test No. 248: Xenopus Eleutheroembryonic Thyroid Assay (XETA), OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris. doi: 10.1787/a13f80ee-en
  3. [Accessed 27 June 2023]
  4. Kraft M et al. (2023) Developmental exposure to triclosan and benzophenone-2 causes morphological alterations in zebrafish (Danio rerio) thyroid follicles and eyes. Environ Sci Pollut Res 30, 33711–24. doi: 10.1007/s11356-022-24531-2
  5. Project 2.64; Inclusion of thyroid endpoints in OECD fish Test Guidelines Work plan for the Test Guidelines Programme (TGP) [Accessed 4 July 2023]
  6. Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC
  7. Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products
  8. OECD (2021), Test No. 250: EASZY assay - Detection of Endocrine Active Substances, acting through estrogen receptors, using transgenic tg(cyp19a1b:GFP) Zebrafish embrYos, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris,
  9. OECD (2022), Test No. 251: Rapid Androgen Disruption Activity Reporter (RADAR) assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris. doi :10.1787/da264d82-en
  10. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (Text with EEA relevance)
  11. OECD (2018), Test No. 150: Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Parisdoi: 10.1787/9789264304741-en



Dr Fiona Marston (Chair)Independent consultant
Dr Laurent Lagadic (Sponsor)Bayer AG
Prof Dr Lennart Weltje (Sponsor)BASF SE
Dr Alice BaynesBrunel University London
Prof Thomas DickmeisKarlsruhe Institute of Technology
Dr Elke EilebrechtFraunhofer Institute for Molecular Biology and Applied Ecology IME
Prof Awadhesh JhaUniversity of Plymouth
Dr Zahid LatifShionogi Europe 
Dr Scott LynnUS Environmental Protection Agency