Influence of preservatives on conjunctival cells in vitro

Benzalkonium chloride (BAC), which is widely used in ophthalmic preparation s for its preservative properties, has been shown to cause conjunctival tox icity. The purpose of this study was to examine the effects of BAC on a con tinuous human conjunctival cell line (Wong Kilbourne-derived human conjunct iva). Cytotoxicity tests were assessed according to ECVAM recommendations u sing microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test), mitochondrial activity (rhod amine 123 test), and reactive oxygen species (ROS) production (dichlorofluo rescein diacetate test) were evaluated directly on living cells treated wit h different concentrations of BAC (0.00001-0.01%). Using the neutral red fl uorescence, we observed a decrease in membrane integrity even at a very low concentration of BAC (0.00001%) and after a short time (15 min). A stimula tion of ROS production was observed at this concentration, associated with chromatin condensation due to an apoptotic phenomenon. The apoptosis induce d by benzalkonium chloride on conjunctival cells in vitro can explain some of the ocular surface damage caused by preservatives.