Study of the toxic effect of benzalkonium chloride on epithelial cells of the lacrimal canaliculi

Purpose The study aimed to investigate the cytotoxic effect of benzalkonium chloride on human lacrimal canalicular epithelial cells under both constant and dynamically decreasing concentrations, which more accurately reflects real ocular surface conditions after eye drop instillation.Methods This in vitro study was conducted at the Krasnov Research Institute of Eye Diseases. Primary cell cultures were established from human lacrimal canaliculi tissue and characterized as exhibiting a partial epithelial – mesenchymal transition (EMT) phenotype, confirmed by spindle-shaped morphology and immunocytochemical expression of cytokeratin-19. Cytotoxicity was assessed using live/dead staining and MTT assay following exposure to benzalkonium chloride (BAC) at constant concentrations (2.5–100 µg/mL) and dynamically decreasing concentrations simulating physiological tear dilution.Results The half-lethal dose (LD50) of benzalkonium chloride at constant concentrations was 21.02 µg/mL (range: 16.45–26.87 µg/mL). When dynamically decreasing concentrations were used (mimicking physiological dilution), the cytotoxic effect was significantly reduced compared to constant concentration exposure at equivalent time points (p < 0.05, two-way ANOVA). Cell viability measurements demonstrated that decreasing benzalkonium chloride concentration over time substantially preserved epithelial cell metabolic activity and membrane integrity.Conclusions The dynamically decreasing concentration model, which better reflects in vivo conditions, demonstrates that physiological dilution of BAC in tear fluid significantly reduces its cytotoxic effect on lacrimal canalicular cells undergoing partial EMT. These findings support the hypothesis that dry eye syndrome and impaired tear drainage – conditions that prolong BAC exposure and may promote EMT – increase the risk of secondary lacrimal drainage obstruction in patients receiving long-term BAC-preserved therapy.