New approach methods: What determines validated? A case study using the in vitro Bhas 42 cell transformation assay for carcinogenicity prediction

ESTIV Congress 2024 -- Tumour formation is a multi-stage process including an initiation stage (genomic level) and a promotion stage (cellular level). If required for regulatory submissions, the gold-standard method assay for carcinogenicity potential is the 2-year rodent carcinogenicity assay. Ex vivo (and subsequently in vitro) cell transformation assays (CTAs) were first developed nearly 60 years ago as a method for evaluating a substance's potential to drive cancer development. Of the two OECD-accepted methods (OECD Guidance Document 214 & 231), the in vitro Bhas 42 CTA uses a cell line that is already considered to be initiated (as a result of its selection process); therefore, this version of the CTA only needs to follow the promotor protocol. When the in vitro Bhas 42 CTA was being reviewed at OECD for a Test Guideline (in the early 2000s), its robustness/fit-for-purpose (validation) was scrutinised through review of the existing literature, resulting in more work being recommended and subsequently performed and a Guidance Document being published. More recently, an OECD workgroup has been active developing an Integrated Approach for Testing and Assessment (IATA) for non-genotoxic carcinogens (NGTxC) and recently published an overview. The in vitro CTA was also included in the Addendum to ICH S1(R2)12 released in March 2023. Laboratory proficiency testing for the in vitro Bhas 42 CTA involves testing two positive compounds and two negative compounds according to OECD GD 231. For our validation, we tested the published reference chemicals in triplicate experiments to provide robust data and indicate inter-assay variation. Additional experiments were performed using vehicle and positive controls in order to produce a robust historical control range (data not shown). These additional experiments were performed to provide confidence in the assay's performance and the compilation of the HCR was also performed to increase reviewers' confidence in the assay's consistency over time (robustness and demonstration of fit-for-purpose).