Behr, A.-C.; Vettorazzi, A.; Streel, C.; Mertens, B.; Antonissen, R.; Guerreiro, B.; Ventura, C.; Vilela, R. S.; Novak, M.; Zegura, B.; Reith, F.; Oltmanns, L.; Prisyazhnoy, V.; Suessmuth, R.; Silva, M.; Louro, H.; Marko, D.

Alternaria toxins are naturally occurring food contaminants with limited and often inconsistent genotoxicity and mutagenicity data. Within the European Partnership for the Assessment of Risks from Chemicals (PARC), an OECD-aligned in vitro testing strategy was applied to fill existing data gaps and to characterize the genotoxic potential of major Alternaria toxins using high-purity test materials. Mutagenicity was assessed using bacterial reverse mutation test (OECD TG 471) and SOS/umu assay, while chromosomal damage was assessed using the in vitro micronucleus (MN) assay (OECD TG 487) in TK6 and HepG2 cells, complemented by fluorescence in situ hybridization (FISH) and {gamma}H2AX assay in HepaRG cells. Alternariol (AOH), alternariol monomethyl ether (AME), and altertoxin-I (ATX-I) showed clear mutagenicity in bacteria, whereas altenuene (ALT), tenuazonic acid (TeA), and tentoxin (TEN) were negative under the tested conditions. In mammalian cells, AOH, AME, and ATX-I induced MN formation in TK6 cells at concentrations [≥]5.5 M, [≥]2.5 M, and [≥]0.21 M, respectively, with FISH analysis supporting a clastogenic mode of action. In HepG2 cells, all tested toxins induced chromosomal damage, with effect threshold ranging from [≥]6.25 M (AOH) to [≥]50 M (TeA). {gamma}H2AX induction confirmed DNA damage for AOH and ATX-I, and at higher concentrations for TeA (1000 M). Overall, the data indicate clear in vitro genotoxic potential for AOH, AME, and ATX-I and provide evidence of chromosomal damage for ALT, TEN, and TeA, thereby reducing critical data gaps for hazard assessment.