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In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N -hydroxy-2-acetylaminofluorene (N -OH-2-AAF) and N -hydroxy-2-aminofluorene (N -OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N -OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N -OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N -OH-2-AF metabolite after multiple treatments over 72 h, whereas N -OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N -OH-2-AAF and N -OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay. [ABSTRACT FROM AUTHOR]