Phototoxic reactions are of particular concern for pharmaceuticals, cosmetics, and other consumer products that absorb UV-visible light, as they can result in skin damage and increased cancer risk. Current in vitro strategies, such as the 3T3 Neutral Red Uptake and reconstructed human epidermis assays, mainly address photocytotoxicity but fail to capture light-induced DNA damage (photogenotoxicity), a key endpoint for long-term health effects. To overcome these limitations, we developed an in vitro alkaline comet assay in HaCaT human keratinocytes, focusing on biomarkers of light-induced DNA damage (photogenotoxicity). Cytotoxicity was monitored through MTT and LDH assays, while DNA strand breaks served as a biomarker of genotoxic stress. Six reference chemicals (sodium dodecyl sulfate, chlorpromazine, benzophenone-3, 8-methoxypsoralen -8-MOP-, p-phenylenediamine, and 6-methylcoumarin – 6-MC-) were tested. Following UV-A irradiation (4 J/cm²), photocytotoxicity was quantified using Photo-Irritation Factor (PIF) and Mean Photo Effect (MPE) values, while genotoxic effects were determined by the comet assay at immediate and 24 h post-exposure. Phototoxicity was evident for benzophenone-3, 8-MOP, 6-MC and chlorpromazine, reflected in elevated PIF/MPE values. DNA damage, as detected by comet assay, was significant for benzophenone-3, p-phenylenediamine, and chlorpromazine, but absent for 8-MOP, consistent with its DNA crosslinking properties. Importantly, no persistence of DNA strand breaks was observed after 24 h, indicating DNA repair biomarkers of recovery. Altogether, cytotoxicity and genotoxicity biomarkers identified in this study highlight the alkaline comet assay as a relevant tool to detect photogenotoxic hazards in vitro.

Acknowledgement: Proyecto PID2020-113186RB-I00 fund by MCIN/AEI/10.13039/501100011033