Thyroid method 4b: Inhibition of thyroid hormones (THs) glucuronidation using liquid chromatography/mass spectrometry (LC/MS)

Topic: Endocrine disruption

Test Method Number:

TM2019-11 (EU)

Short Name of TM:

GLUC-LCMS

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method assesses the potential of test items to inhibit the metabolism and excretion of thyroid hormones. Glucuronidation is one of the main pathways in the metabolism of thyroid hormones (T3 and T4). The method consists in the incubation of either T4 or T3 with human liver microsomes that contain UDP-glucuronosyltransferases (UGT), the enzyme involved in the glucuronidation. Following separation and purification the mixture is analysed by liquid chromatography/mass spectrometry (LC/MS). Acyl- and Phenolic- Glucuronides are separated and peaks identities confirmed. 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. EU-NETVAL facility 'Accelera Srl (Italy)' agreed to develop one method from available published methods and 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
Jun 2021	Finalised	Completion of method definition and description prior to experimental assessment.
Oct 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STANDARD OPERATING PROCEDURE for determination of the potential inhibition of thyroid hormones glucuronidation in human liver microsomes, version 1.0 STUDY REPORT Inhibition of thyroid hormones (THs) glucuronidation using liquid chromatography/mass spectrometry (LC/MS-MS) – Part 1
Feb 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 using only a selection of 8 out of 30 chemicals to save resources.	STANDARD OPERATING PROCEDURE for Inhibition of thyroid hormones (THs) glucuronidation using liquid chromatography/mass spectrometry (LC/MS-MS), version February 2023 STUDY REPORT Inhibition of thyroid hormones (THs) glucuronidation using liquid chromatography/mass spectrometry (LC/MS-MS) – Part 2
Aug 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. The method gives reproducible results. In case of further validation testing of more negative chemicals is necessary to confirm its specificity and the between laboratory reproducibility will need to be confirmed.	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards