AimsUsing an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular-pressure-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015% and 0.005% BAC, respectively, and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison.MethodsThe 3D-HCEs were treated with solutions for 24 h followed or not by a 24 h recovery period. We used a modified MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) procedure to assess cell viability in the HCE. Frozen sections of HCE were analysed using fluorescence microscopy for the evaluation of apoptosis (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling), inflammation (ICAM-1) and proliferation (Ki67). Corneal epithelial tight junctions (occludin and tight junction protein 1 (zona occludens 1)) were also assessed by en face confocal microscopy in response to the different eye-drops.ResultsThe MTT test revealed that the cytotoxicity of antiglaucoma eye-drops was primarily related to the concentration of their common BAC preservative (0.02% BAC-latanoprost>0.015% BAC-travoprost>0.005% BAC-bimatoprost). PF-tafluprost did not induce any obvious cytotoxicity, showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions.ConclusionThe toxicological model of the 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared with the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration.