Thyroid method 6b: CALUX human thyroid hormone receptor beta (TRβ) reporter gene transactivation measuring agonist and antagonist activities

Topic: Endocrine disruption

Test Method Number:

TM2019-15 (EU)

Short Name of TM:

TRβ-CALUX

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method assesses the activation of thyroid hormone receptor beta (TRβ). It is making use of proprietary TRβ CALUX® cells which originated from human osteoblastic osteosarcoma U2OS line. These cells express a functioning human thyroid (TRβ) receptor coupled with a luciferase reporter gene. Test items with both an agonist or antagonist effect on TR activity can be detected and an increase or decrease of thyroid hormone signalling, results in corresponding changes in the expression of luciferase activity in TRβ CALUX® cells that can be measured with a luminometer. 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 developer 'BioDetection Systems (BDS) B.V. (the Netherlands' and EU-NETVAL facility 'Arpae-Vitrox (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
Aug 2022	Finalised	Completion of method definition and description prior to experimental assessment.
Jul 2023	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.
Jul 2023	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method. The study was performed by the method developer BDS, using only a selection of 14 out of 30 chemicals to save resources.	STUDY REPORT for the thyroid hormone receptor transactivation (TRβ-CALUX®) assay, measuring agonist and antagonist activities – Part 1 & 2 - BDS
Mar 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. For agonism the assay looks specific. For antagonism further experimental data would be needed to conclude on the assay performance. More TR agonist and antagonist chemicals should be identified. In case there is regulatory need to further validate this method, between laboratory reproducibility will need to be demonstrated.	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards