In vitro metabolomics approaches to decipher the biochemical and cellular response to genotoxic compounds.

New approach methodologies (NAMs) are gaining increasing attention within the scientific community as strategies to reduce the use of animal testing. Their integration into regulatory frameworks for chemical hazard and risk assessment in food and feed would directly support the goals of the European Food Safety Authority (EFSA). Within the framework of the EU-FORA fellowship programme, this study investigated the use of metabolomic approaches to evaluate cellular responses to genotoxic agents, serving as a proof of concept for the development, standardisation and future regulatory application of metabolomics in genotoxicity testing. Human liver-derived cells (HepRG) were exposed to two well-characterised genotoxicants, and both intracellular (endometabolome) and extracellular (exometabolome) metabolites were extracted and analysed using gas chromatography-mass spectrometry (GC-MS). Metabolites associated with energy metabolism, sugar metabolism and antioxidant capacity were identified, and both multivariate and univariate statistical analyses were applied to characterise metabolic profiles. Results obtained under the experimental conditions tested in this pilot study, do not highlight significant differences between exposed and control cultures. Nevertheless, the findings demonstrate the potential of metabolomics to capture biologically relevant pathways, supporting its future application in genotoxicity assessment under alternative experimental conditions and designs.