Thyroid method 3a: Thyroxine-binding prealbumin (TTR) / thyroxine-binding globulin (TBG) binding using fluorescence displacement (ANSA)

Topic: Endocrine disruption

Test Method Number:

TM2019-08 (EU)

Short Name of TM:

ANSA-FD

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method investigates interference of test items with binding of thyroid hormones to human serum transport proteins 'thyroxine-binding prealbumin' (TTR) and 'thyroxine-binding globulin' (TBG). The binding of 8-anilino-1-naphtalenesulfonic acid (ANSA) to TTR or TBG produces fluorophore complexes with intrinsic fluorescence signal. In the presence of competitive ligands, displacement of ANSA occurs and the fluorescence signal detected decreases. Binding/displacement capacity of endocrine/thyroid disrupting chemicals can be assessed and compared to that of T3 and/or T4. 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

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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

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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 'Luxembourg Institute for Science and Technology (Luxembourg)' and EU-NETVAL facility 'EURL ECVAM (Italy)' agree to assess the method.	EU-NETVAL
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
Apr 2021	Finalised	Completion of method definition and description prior to experimental assessment.
Sep 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.
Oct 2022	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method. This study was performed on the quantitative High Throughput System.
May 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. Further work is needed on the criteria for interference with the fluorescence readout. Once the SOP has been updated, transferability to other laboratories is needed to confirm between laboratory reproducibility.	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards