Purpose: To investigate the toxic effects of benzalkonium chloride (BAC), a preservative commonly used in ophthalmic preparations, on DNA single-and double-strand breaks in immortalized human corneal epithelial cells (HCEs).Methods: HCEs were treated with BAC in concentrations ranging from 0.00005% to 0.001% for 30 min.Cells were examined immediately after BAC exposure and after 24 h recovery.Alkaline comet assay was used to detect DNA single-strand breaks (SSBs).Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (H2AX) foci indicated DNA double-strand breaks (DSBs).Cell viability was measured by the MTT test.Results: A significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with BAC exposure in HCEs at concentrations of 0.00005% and higher.Such BAC treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of H2AX foci.In addition,a significant change in the relative cell survival rate of HCEs was observed after exposure to 0.001% BAC for 30 min.Although the toxic effects of BAC could be partly repaired after 24 h of cell recovery, SSBs and DSBs in HCEs were still present after BAC removal.Conclusions:The results demonstrated that exposure to BAC in HCEs even at low concentrations could induce DNA strand breaks, which were present after BAC removal.Cell survival analysis indicated that BAC-induced DNA damage was correlated with the cytotoxic effects.