Benzalkonium Chloride Homologs in Ocular Surface Cells in vitro and in vivo

Abstract

Purpose: : Ocular surface toxicity of commercial prostaglandin analogues seems to be related to the concentration of benzalkonium chloride (BAC) used as a preservative. BAC is a mixture of straight chain homologs and the proportions of these homologs in the mixture determine its effectiveness. The cytotoxic effects of different BAC homologs (BAC-C₁₂, BAC-C₁₄, BAC-C₁₆) and BAC mixture (containing 64.1% C₁₂, 33.8% C₁₄, 2.1% C₁₆) were studied in vitro in corneal and conjunctival epithelial cell cultures. In vivo in rabbit eyes, the distribution of BAC homologs in ocular surface tissues was examined.

Methods: : Human corneal epithelial (HCE) and human conjunctival epithelial (IOBA-NHC) cell cultures were exposed to BAC homologs or mixture for one hour. Cytotoxicity was assessed with the WST-1 assay for cellular growth and viability. BAC mixture as 0.02% (v/v) aqueous solution was applied into rabbit eyes once a day for 14 days and homologs in corneal and conjunctival tissues were analyzed using liquid chromatography-tandem mass spectrometry.

Results: : In vitro, conjunctival cells appeared to be more sensitive to BAC exposure than corneal cells. In corneal cells, the cytotoxicity for tested preservatives did not make much difference, with the EC₅₀ values 0.00130% for C₁₆, 0.00127% for BAC mixture, 0.00101% for C₁₂, and 0.00097% for C₁₄. In conjunctival cells, the EC₅₀ values were 0.00065% for C₁₆, 0.00047% for BAC mixture, 0.00041% for C₁₄, and 0.00038% for C₁₂. In vivo, the amounts of C₁₂, C₁₄ and C₁₆ in corneal and conjunctival tissues were respectively (mean ± SEM, n=5): 0.37 ± 0.08 and 2.64 ± 0.27 ng/mg, 0.42 ± 0.07 and 4.77 ± 0.43 ng/mg, 0.04 ± 0.01 and 0.54 ± 0.05 ng/mg.

Conclusions: : Besides different potency of toxicity, BAC homologs possess different absorption kinetics in ocular surface tissues. The greater sensitivity of conjunctival cells than corneal cells in vitro may be explained by higher absorption of BAC homologs into the conjunctiva in vivo.

Keywords: ocular irritancy/toxicity testing • cornea: epithelium • conjunctiva