Thyroid method 7a: Measurement of intrafollicular thyroxine (T4) using zebrafish eleutheroembryos

Topic: Endocrine disruption

Test Method Number:

TM2019-16 (EU)

Short Name of TM:

ZETA

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

The Zebrafish Eleutheroembryo Thyroid Assay (ZETA) is used to detect direct thyroid gland function disruptors in the thyroid of 120 hours post- fertilisation (hpf) zebrafish embryos after their exposure to test items for 66 hours. The intrafollicular T4-content (IT4C) is determined by immunofluorescence signal. IT4C is a physiologically relevant integrative endpoint, as any potential disrupting mechanism of the thyroid gland function would result in increase or decrease of T4 production. This method was selected with other methods to assess modulating effects on the thyroid system (OECD, 2014). Information on these selected methods is available in TSAR as ‘Thyroid methods’.

Track Approval Status

Submission
Validation
Peer-review
Recommendation
Regulatory acceptance/Standards

Submission

Show status

Date	Stage	Comment	Documents/Links
Jun 2017	Assessment finalised	Selection of the test method for the Thyroid Validation Study based on the OECD scoping document on 'in vitro and ex vivo methods for the identification of modulators of thyroid hormone signalling' and guidance from the Endocrine Disrupters Testing and Assessment Advisory Group considering scientific advances since the issuing of the OECD scoping document, human relevance, possibility for High Throughput screening and availability of method elements (such as reagents and detection method) to a wide range of future users.	OECD scoping document (OECD, 2014)OECD detailed review paper (OECD, 2012)OECD Endocrine Disrupters Testing and Assessment Advisory Group

Validation

Show status

Date	Stage	Comment	Documents/Links
Sep 2019	Finalised	Preparatory work carried out by EURL ECVAM before the start of the validation study between November 2017 and September 2019. During this phase, intellectual property was addressed, legal agreements with method developer, test system supplier and EU-NETVAL laboratory were established and adequate training of the EU-NETVAL laboratory was ensured. An outline protocol describing the current status of the method, the quality controlled test system and reference and control items were made available to EU-NETVAL. OECD Good In Vitro Method Practices (GIVIMP, OECD, 2018) has been used as guidance.	OECD Good In Vitro Method Practices (GIVIMP, OECD, 2018)
Oct 2019	Finalised	Start of the EURL ECVAM coordinated validation study. Method developer 'Institute of Environmental Assessment and Water Research (Spain)' and EU-NETVAL facility 'Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (Italy)' agree to assess the method.	EU-NETVAL
Oct 2020	Finalised	The method definition and description was finalised. Standard operating procedures were approved prior to experimental assessment.
Feb 2021	Finalised	Final selection of the validation set chemicals to be used in the thyroid validation study PART 2. Selection was based on the advice provided by 15 experts in in vitro, in vivo and clinical thyroid disruption during an expert meeting in November 2019.	Chemical selection for the Thyroid Validation Study coordinated by EURL ECVAM and involving EU-NETVAL laboratories
Feb 2022	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STANDARD OPERATING PROCEDURE for the Zebrafish Eleutheroembryo Thyroid Assay (ZETA), version 1.0 STUDY REPORT for the Zebrafish Eleutheroembryo Thyroid Assay (ZETA) – Part 1
Jul 2023	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method.	STUDY REPORT Summary of results for all test items, assessed with the Zebrafish Eleutheroembryo Thyroid Assay (ZETA) – Part 2
Oct 2023	Finalised	The OECD Thyroid Disruption Methods Expert Group (TDM-EG) assessed the available data and study reports from EU-NETVAL, and provided its recommendation for further validation of the method. The conclusion of the assessment group was that the assay is promising but more work is needed on the experimental design and more chemicals should be tested	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards