Food and feed can be contaminated by fungal strains producing mycotoxins, such as the emerging enniatins and Alternaria toxins. Since no manufacturer is responsible for providing hazard data for these mycotoxins, important data gaps exist. In the project P.5.1.1.a_Y1_Natural toxins, toxicological studies are conducted to collect data for several human health-related endpoints, including genotoxicity. Based on a literature review and practical considerations like availability and price, several enniatins and Alternaria toxins were selected for genotoxicity testing including enniatin (ENN) B, B1, A and A1, beauvericin (BEA), altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME), altertoxin-I (ATX-I), tentoxin (TEN) and tenuazonic acid (TeA). These mycotoxins were tested in the in vitro cytokinesis-block micronucleus (MN) assay in mammalian TK6 cells, both with and without metabolic activation  (OECD test guidelines No 487). After 24h exposure without metabolic activation, only AOH, ATX-I, AME and TeA induced an increase in MN formation, indicating the induction of structural and/or numerical chromosome damage. However, the effect of TeA was considered inconclusive since the increased MN formation occurred at highly cytotoxic levels. After 3h exposure with metabolic activation, only AOH and AME showed a positive effect. To investigate the genotoxic mode of action of AME, AOH, and ATX-I, the MN assay was followed by a fluorescence in situ hybridization (FISH) staining using centromere probes. This technique allows to discriminate between aneugenic and clastogenic genotoxicants. Preliminary results identified ATX-I and AOH as clastogens. The clastogenicity of AOH aligns with the literature data, while, to our knowledge, no similar experiments have been conducted so far for ATX-I and AME. In the next step, these results will be combined with data from other genotoxicity assays, such as the Ames test, and new approach methodologies like γH2Ax/pH3 and/or GENOMARK, a transcriptomics-based biomarker for genotoxicity.