Thyroid method 8a: Measurement of proliferation of rat pituitary-derived cell line GH3

Topic: Endocrine disruption

Test Method Number:

TM2019-17 (EU)

Short Name of TM:

T-SCREEN

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method is based on thyroid hormone (TH) dependent cell proliferation of a rat pituitary tumour cell line (GH3) in serum-free medium. It can be used to study interference of test items with TH at the cellular level, thus bridging the gap between limitations of assays using either isolated molecules (enzymes, transport proteins) or complex in vivo experiments with all the complex feedback mechanisms present. GH3 cell growth, which is measured with the colorimetric AlamarBlue™ assay is increased in the presence of TH agonists, and decreased in the presence of TH antagonists.

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 'Luxembourg Institute of Science and Technology (Luxembourg)' and EU-NETVAL facility 'Nofer Institute of Occupational Medicine (Poland)' 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	Method definition and description prior to experimental assessment.
Nov 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STUDY REPORT for testing of six test items for TH receptor (TR)- mediated cell proliferation to assess the robustness and reliability of the T-screen method and confirm the reference and control items - Part 1
Feb 2023	Ongoing	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 by EURL ECVAM on the quantitative High Throughput System.	STANDARD OPERATING PROCEDURE T-Screen assay using a QUANTITATIVE HIGHTHROUGHPUT screening approach, version 1.0
Apr 2023	Planning	Assessment of the data and study reports + decision if further validation work is needed.

Peer-review

Recommendation

Regulatory acceptance/Standards