Assessment of Genotoxicity Prediction Using New Approach Methodologies (NAMs): A Comparative Study with In Vitro Micronucleus Assays on Small Molecule Pharmaceuticals (P408)

Genotoxicity evaluation to identify genetic changes that may lead to carcinogenic potential is critical in non-clinical safety assessment for small molecule drug development. According to the International Conference on Harmonisation S2(R1) regulatory guidance, genotoxicity is assessed using a battery of in vitro and in vivo assays. The micronucleus (MN) assay detects micronuclei formation, with Fluorescence In Situ Hybridization staining elucidating clastogenicity versus aneugenicity mechanisms. This study assessed the genotoxic potential of 22 anonymized small molecules in development using Litron MicroFlow® and MultiFlow® assays in human lymphoblastoid TK6 cells. Currently, the high content micronucleus (HC-MN) assay in Chinese Hamster Ovary cells serves as our screening assay, while the human peripheral blood lymphocytes micronucleus (HPBL-MN) assay is used for confirmatory testing. To evaluate the predictive value of the Litron assays as screening tools, we retrospectively analyzed the concordance between the Litron assays and the manual MN assay in HPBLs. Our findings indicate that Litron assays, with sensitivity ∼0.8 and specificity ∼0.7, outperform the standard HC-MN screening assay, with sensitivity ∼0.7 and specificity ∼0.2, in predicting HPBL-MN results for these small molecules. Additionally, the MultiFlow® assay provided mechanistic insights for MN-positive compounds, offering a comprehensive understanding of genetic damage mechanisms. We conclude that integrating New Approach Methodologies (NAMs) enhances mechanistic insights and predictive capabilities, refining genotoxicity screening processes and improving accuracy and reliability in toxicological evaluations. This approach shows promise in effectively categorizing compounds and delivering valuable insights to early-stage project teams for molecule optimization.