Genotoxicity Evaluation of Pharmaceuticals

Abstract

This chapter provides a comprehensive overview of genomic safety considerations for both small molecules and cell and gene therapies. For small chemical entities, testing is essential to identify a possible genotoxic potential early in drug development. Mutations in somatic and germline cells can lead to cancer or heritable diseases, necessitating robust testing strategies. Standard assays include in vitro assays such as bacterial reverse mutation tests (e.g., Ames test), mammalian cell-based gene mutation, and chromosomal damage assays, and in vivo models. Emerging technologies (e.g., error-corrected sequencing) enhance sensitivity for detecting ultra-rare mutations and provide opportunities for testing in cells and tissues under conditions otherwise unavailable. Regulatory frameworks such as ICH S2(R1) guide preclinical testing, whereas exploratory and screening strategies help interpret ambiguous results and support early candidate selection. Impurity testing, especially for mutagenic contaminants (e.g., N-nitrosamines), employs in silico tools and enhanced Ames protocols. In addition, the chapter addresses the genotoxicity risks associated with gene and cell therapies, particularly those involving designer nucleases (e.g., CRISPR/Cas9) and viral vectors. It explores mechanisms such as off-target editing and insertional mutagenesis and presents a multi-tiered genotoxicity assessment strategies combining in silico prediction (for off-target editing), genome-wide experimental methods, and the evaluation of functional consequences. Regulatory guidance emphasizes case-by-case risk assessment and supports the use of innovative in vitro transformation assays. Several such assays are reviewed in this chapter, including SACF, GILA, IVIM, and SAGA, which demonstrate suitability for assessing the oncogenic potential across different therapeutic modalities. These approaches also align with the 3Rs principles by reducing reliance on animal testing. Overall, the chapter explains the need for modality-specific, mechanistically informed safety strategies to ensure genomic integrity across therapeutic platforms.