The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids was requested to evaluate the genotoxic potential of one flavouring substance from subgroup 1.1.1(b) of FGE.19 in the Flavouring Group Evaluation 226 (FGE.226). The flavour industry provided genotoxicity studies for the substance 4,5-epoxydec-2(trans)-enal [FL-no: 16.071]. Based on these data, the Panel concluded in FGE.226 that 4,5-epoxydec-2(trans)-enal did not induce gene mutations in bacterial cells but was positive in an in vitro micronucleus assay, so, 4,5-epoxydec-2(trans)-enal is considered an in vitro genotoxic agent. The negative results obtained in an in vivo micronucleus assay cannot overrule the positive results of the in vitro micronucleus assay with and without S9-mix due to the lack of demonstration of bone marrow exposure. Following this, the flavour industry has provided plasma analysis of a satellite group of rats treated with 4,5-epoxydec-2(trans)-enal in order to investigate the systemic exposure of animals in the in vivo micronucleus assay. However, the plasma analysis did not provide enough evidence of target tissue exposure. An in vivo Comet assay in rodents was recommended in FGE.226, in order to investigate possible genotoxic effects at the first site of contact (e.g. stomach/duodenum cells) and in the liver. An in vivo Comet assay in liver and duodenum was provided that suggests that 4,5-epoxydec-2(trans)-enal [FL-no: 16.071] did not induce DNA damage in the duodenum of rats. However, the genotoxic effect observed in vitro was confirmed in the in vivo Comet assay in the liver of rats. The Panel concluded that 4,5-epoxydec-2(trans)-enal [FL-no: 16.071] does raise a safety concern with respect to genotoxicity and, therefore, it cannot be evaluated according to the Procedure.