Thyroid method 2a: Thyroperoxidase (TPO) inhibition based on oxidation of Amplex UltraRed

Topic: Endocrine disruption

Test Method Number:

TM2019-04 (EU)

Short Name of TM:

AUR-TPO

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method evaluates the activity of the thyroperoxidase (TPO), a key enzyme for the synthesis of thyroid hormones. The method allows to identify TPO-inhibiting test items by assessing the enzyme activity in prepared extracts from FTC-238 cells transfected with human recombinant TPO. TPO activity is measured through the oxidation of a commercially-available peroxidase substrate (Amplex UltraRed, AUR, ® LifeTech) and its conversion to a fluorescent product.

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 ' US-EPA (United States of America)' and EU-NETVAL facility 'RISE (Sweden)' 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	Completion of method definition and description prior to experimental assessment.
Sep 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STANDARD OPERATING PROCEDURE for thyroperoxidase activity assay with Amplex UltraRed (AUR-TPO), version 1.0 STANDARD OPERATING PROCEDURE for culturing FTC-238 and FTC-238/hrTPO cells used in the AUR-TPO assay, version 3.0 STANDARD OPERATING PROCEDURE for TPO extract preparation, used in the AUR-TPO assay, version 1.0 STANDARD OPERATING PROCEDURE for solubility testing, used in the AUR-TPO assay, version 2.0 STUDY REPORT for the thyroperoxidase activity assay with Amplex UltraRed (AUR-TPO) – Part 1
Jan 2023	Finalised	PART 2: Assessment of the method's mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method.	STANDARD OPERATING PROCEDURE for thyroperoxidase activity assay with Amplex UltraRed (AUR-TPO), version 2.0 STUDY REPORT for the thyroperoxidase activity assay with Amplex UltraRed (AUR-TPO) – Part 2
Jun 2024	Finalised	The OECD Thyroid Disruption Methods Expert Group (TDM-EG) assessed the available data and study reports from EU-NETVAL, and asked some clarification on the data, before giving its final recommendation on further validation of the method. There was high within laboratory variability found of especially the inactive chemicals. The cause would need to be identified and the SOP optimised before continuing further validation.	Assessment reports by the OECD thyroid disruption methods expert group

Peer-review

Recommendation

Regulatory acceptance/Standards