Thyroid method 4a: Deiodinase 1 activity based on Sandell-Kolthoff reaction

Topic: Endocrine disruption

Test Method Number:

TM2019-10 (EU)

Short Name of TM:

DIO1-SKR

Year received:

2019

Responsible Organisation:

EURL ECVAM - European Union

General Comments:

Method Description

This method assesses the inhibition of thyroid hormones deiodination, the major pathway regulating thyroid hormones bioavailability in mammalian tissues. This method measures the activity of deiodinase enzyme D1 in human liver microsomes by measurement of free iodine with a spectrophotometric absorbance readout following the Sandell-Kolthoff reaction, during which the yellow cerium(IV) is reduced to the colourless cerium (III). 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)
Apr 2020	Finalised	Start of the EURL ECVAM coordinated validation study. Method developer 'Charité (Germany)' and EU-NETVAL facility 'BASF (Germany)' agree to assess the method.	EU-NETVAL
Sep 2020	Finalised	The method definition and description was finalised. Standard operating procedures were approved prior to experimental assessment.
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
Feb 2021	Finalised	PART 1: Assessment of method robustness, reliability and within laboratory reproducibility using few test items.	STANDARD OPERATING PROCEDURE for colorimetric assessment of deiodinases activity based on Sandell-Kolthoff reaction with human microsomes: DIO1-SK assay, version 1.0 STUDY REPORT for the colorimetric assessment of deiodinases activity based on Sandell-Kolthoff reaction with human microsomes: DIO1-SK assay – Part 1 A New Approach Method to Study Thyroid Hormone Disruption: Optimization and Standardization of an Assay to Assess the Inhibition of DIO1 Enzyme in Human Liver Microsomes
Mar 2022	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 colorimetric assessment of deiodinases activity based on Sandell-Kolthoff reaction with human microsomes: DIO1-SK assay, version 2.0 STANDARD OPERATING PROCEDURE for assessing specificity of DIO1 interaction using Alkaline phosphatase (ALP) testing as secondary readout, version 1.0 STUDY REPORT for the colorimetric assessment of deiodinases activity based on Sandell-Kolthoff reaction with human microsomes: DIO-SK assay - Part 2 Assessment of the Predictivity of DIO1-SK Assay to Investigate DIO1 Inhibition in Human Liver Microsomes
Feb 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 is ready for transfer to other laboratories to demonstrate between laboratory reproducibility.	Assessment reports by the OECD thyroid disruption methods expert group
Apr 2024	Initiated	PEPPER initiated a validation study, following the recommendations by the OECD TDM-EG. The method was accepted onto the OECD test guideline programme.	PEPPER website

Peer-review

Recommendation

Regulatory acceptance/Standards