Sun, XiaowenDertinger, Stephen DElhajouji, AzeddineRoberts, Daniel Jvan Benthem, JanSchuler, MaikRoy, ShambhuTaraboletti, AlexandraChen, Connie L2025-08-132025-08-132025-07-184068229710.1002/em.70023https://rivm.openrepository.com/handle/10029/655560The genotoxic potential of chemicals must be evaluated in regulatory safety assessment settings, including but not limited to, the development of new pharmaceuticals, industrial chemicals, food and cosmetic ingredients, and agrochemicals. Initial assessment of the chromosome-damaging potential of chemicals is often conducted in mammalian cells using the micronucleus (MN) assay, a method capable of detecting both aneugenicity and clastogenicity. When differentiation between these modes of action (MOAs) is necessary, microscopy-based analyses using fluorescent In Situ Hybridization (FISH) or CREST staining have traditionally been employed. More recently, semi-automated in vitro new approach methods (NAMs), which leverage technologies like flow cytometry and high-content imaging, have increasingly been used across sectors due to their higher throughput and faster turnaround times. A SWOT (strengths, weaknesses, opportunities, and threats) analysis was conducted to systematically evaluate the merits and limitations of widely used NAMs in industry, with a focus on the pharmaceutical sector. Data from cultured mammalian cells exposed to reference aneugens (colchicine, taxol, and AMG900) and DNA-reactive clastogens (mitomycin C and methyl methanesulfonate) across methodologies are presented to illustrate the process of distinguishing aneugens from clastogens for the different techniques described herein. Collectively, these analyses highlight the capabilities of NAMs to distinguish aneugens from clastogens. The newer, high information content, semi-automated approaches were considered preferable to traditional microscopy-based FISH and CREST techniques as they provide insight into molecular mechanisms of aneugenicity and help optimize the design of future in vivo genotoxicity studies to facilitate deriving points of departure which may contribute to margin of exposure estimates.en© 2025 The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.CRESTFISHaneugenflow cytometrygenotoxicityimage analysismicronucleus positivemitotic kinase inhibitiontubulin bindingJournal ArticleEnviron Mol Mutagen 2025; online ahead of print