Multi-laboratory validation of a new animal product-free test guideline addition for the in vitro micronucleus test

Victoria will work with industry collaborators and international partners to undertake a multi-laboratory validation of six non-animal alternatives to replace rat S9 (metabolic enzymes derived from rat liver) and fetal bovine serum in an OECD Test Guideline for genotoxicity. The award follows on from previous NC3Rs funding in the animal-free in vitro CRACK IT Challenge and is intended to build confidence for regulatory acceptance of an animal-free protocol for the micronucleus test.

This award was made as part of the 2024 non-animal derived product validation grants supported with funding from the Department for Science, Innovation and Technology (DSIT).

Scope: This project aims to conduct a multi-laboratory validation of an animal-product free methodology adaptation to an established OECD test. Using this standard, widespread assay as a platform to build further confidence animal product-free alternatives and demonstrate that they are fit-for-purpose using a consortium of cross-sector industrial/academic partners to demonstrate applicability.

Objectives: The overall aim is to validate an animal product-free alternative protocol for the OECD TG487 mammalian in vitro micronucleus (MNvit) assay. This will be achieved by:

• conducting an interlaboratory comparison of an optimised animal product-free protocol (stemmed from CRACK IT Challenge 36 Animal Free In Vitro) using a panel of established reference compounds against the standard OECD protocol between three UK laboratories to demonstrate the performance, reliability and relevance of adapted protocol 
• generating a robust dataset for publication and consideration of datasets by OECD to support the inclusion of animal product-free approach to be incorporated into official test guidelines

Vision: The in vitro micronucleus assay is an internationally standardised test guideline for assessing the genotoxic potential of chemicals in humans. It is one of the principal tests run on all chemcials/pharmaceuticals to determine safety and is mandatory across a number of industries. It is our vision for the outputs of this project to be directly used as part of an application to the OECD to include an amendment to TG487 detailing an animal-product free
methodology.

Areas of focus: will be on standardisation, data integrity to get a well-designed and executed study of high quality that can be used in OECD evidence.

Significance: The non-animal derived products that this project will validate in the context of the performance of an adapted test guidance method are animal-free culture medium (to replace FBS) and non-animal derived enzymes (to replace induced rat liver fractions (S9)). Demonstrating the performance of these products will have a direct impact and applicability to <95% of in vitro culture cell culture assay methodologies available. Demonstrating the equivalent performance of animal product-free in vitro assays against standard assays will provide researchers with the tools and approaches to adapt
similar methodologies.

Success: This work is built on an established wide-reaching industrial-academic consortium (CRACK IT Challenge 36) with proven track record who have demonstrated functionality of the assay in a single laboratory and now seek to conduct reproducibility assessment of the assay with different laboratories and users, to build confidence in the approach and seek wider acceptance.