Thyroid method 3b: Thyroxine-binding prealbumin (TTR) binding using fluorescence displacement (T4-FITC)

Topic: Endocrine disruption

Test Method Number:

TM2019-09 (EU)

Short Name of TM:

T4-FITC

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 protein 'thyroxine-binding prealbumin (TTR)'.
The principle of the method is that the addition of TTR-binding compounds to a mixture of TTR and a fluorescent conjugate FITC-T4 causes a decrease in fluorescence, because the TTR-binding compounds compete FITC-T4 out of the TTR binding-pocket and consequently lead to a decrease in the ratio of bound to unbound FITC-T4. The FITC-T4 conjugate exhibits higher fluorescence intensity when bound to TTR than in the unbound condition, probably due to the fact that the iodine atoms of the T4 present in the TTR binding pocket can no longer contribute to the intramolecular fluorescence quenching of the FITC-T4 conjugate.
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 'Vrije Universiteit Amsterdam (the Netherlands)' and EU-NETVAL facility 'Wageningen Food Safety Research - WUR (the Netherlands)' 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.
Mar 2021	Finalised	Completion of method definition and description prior to experimental assessment.
Jul 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.
Nov 2022	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method.
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. Some further work is needed to refine the criteria to measure interference with the fluorescence readout. Then the method is ready for transfer to other laboratories for between-reproducibility assessment.	Assessment reports by the OECD thyroid disruption methods expert group
Apr 2024	Initiated	PEPPER initiated a validation study, following the recommendations by the OECD TDM-EG. The method was accepted onto the OECD test guideline programme.	PEPPER website

Peer-review

Recommendation

Regulatory acceptance/Standards