Thyroid method 1a: PATHHUNTER® BETA-ARRESTIN thyrotropin-releasing hormone (TRH) receptor activation (beta-galactosidase) measuring agonist and antagonist activities

Topic: Endocrine disruption

Test Method Number:

TM2019-02 (EU)

Short Name of TM:

TRH-CHOK1

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method is a chemiluminescent in vitro method which measures the agonist and antagonist activities of test items by assessing the thyrotropin-releasing hormone receptor (TRHR) activation. The method employs the commercially available Chinese hamster ovary cells (CHO-K1) engineered to stably express the tagged proteins TRHR and β-Arrestin (PathHunter® CHO-K1 TRHR β-Arrestin Cell Line). Following chemical-TRHR binding, the ProLink™ tagged GPCR is activated inducing the recruitment of Enzyme Acceptor tagged β-Arrestin, forcing complementation of the two enzyme fragments. The resulting functional enzyme hydrolyses the substrate to generate a chemiluminescent signal.

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, the test system was authenticated, screened for contaminants and information on genetic modification was gathered 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 'Eurofins - Discover X (United States of America)' and EU-NETVAL facility 'Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (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.
Mar 2023	Stopped	The method definition and description prior to experimental assessment could not be completed. Therefore data were not generated and the method could not be assessed.

Peer-review

Recommendation

Regulatory acceptance/Standards