Thyroid method 1b: Thyrotropin-stimulating hormone (TSH) receptor activation based on cAMP measurement

Topic: Endocrine disruption

Test Method Number:

TM2019-03 (EU)

Short Name of TM:

TSH-CHOK1

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method measures the production of cAMP after activation of the Thyroid Stimulating Hormone Receptor (TSHR). The production of intracellular adenosine 3’,5’ cyclic monophosphate (cAMP) is important for thyroid hormone physiology. cAMP is a critical component of a signal transduction pathway linking TSHR and its ligand TSH to the activation of adenylate cyclase and therefore cAMP production. The method measures the intracellular cAMP concentration in Chinese hamster ovary cells (CHO-K1) transfected with the gene of the human TSHR, using an Enzyme Linked Immuno Sorbent Assay. 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, 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 developers 'University of Pisa (Italy)' and EU-NETVAL facility 'National Institute for Public Health (Czech Republic)' 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
Nov 2021	Finalised	Completion of method definition and description prior to experimental assessment.
Nov 2023	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STANDARD OPERATING PROCEDURE for the thyrotropin-stimulating hormone (TSH) receptor activation assay based on cAMP measurement, version 1.0 STUDY REPORT for the thyrotropin-stimulating hormone (TSH) receptor activation assay based on cAMP measurement – Part 1
Feb 2024	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method.	STUDY REPORT for the thyrotropin-stimulating hormone (TSH) receptor activation assay based on cAMP measurement – Part 2 Advances in thyroid hormone system disruption testing – In vitro method for monitoring disrupted thyrotropin signaling through the human TSH receptor (hTSHR)
Apr 2024	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. There was insufficient information available to assess the reproducibility of the method. There are currently no chemicals identified for future validation activities with sufficient information.	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards