Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells

Abstract

Purpose: Nearly all eye drops contain preservatives to decrease contamination. Nonpreservatives such as disodium-ethylene diamine tetra-acetate (EDTA) and phosphate-buffered saline are also regularly added as buffering agents. These components can add to the toxicity of eye drops and cause ocular surface disease. To evaluate the potential toxicity of these common components and their comparative effects on the ocular surface, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized.

Methods: Immortalized human conjunctival and corneal epithelial cells were grown. At confluency, medium was replaced with 100 microL of varying concentrations of preservatives: benzalkonium chloride (BAK), methyl paraben (MP), sodium perborate (SP), chlorobutanol (Cbl), and stabilized thimerosal (Thi); varying concentrations of buffer: EDTA; media (viable control); and formalin (dead control). After 1 h, solutions were replaced with 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide). After 4 h, solutions decanted, 100 microL of acid isopropanol added, and the optical density determined at 572 nm to evaluate cell viability.

Results: Conjunctival and corneal cell toxicity was seen with all preservatives. Depending upon concentration, BAK exhibited from 56% to 89% toxicity. In comparison, Cbl exhibited from 50% to 86%, MP from 30% to 76%, SP from 23% to 59%, and Thi from 70% to 95%. EDTA with minimal toxicity (from 6% to 59%) was indistinguishable from SP.

Conclusions: Generally, the order of decreasing toxicity at the most commonly used concentrations: Thi (0.0025%) > BAK (0.025%) > Cbl (0.25%) > MP (0.01%) > SP (0.0025%) approximately EDTA (0.01%). Even at low concentration, these agents will cause some degree of ocular tissue damage.

FIG. 1.

Graphic representation of overall relative toxicities of human corneal (gray bars; 10.014 pRSV-T) and conjunctival epithelial cells (black; Wong–Kilbourne derivative of conjunctiva, clone 1-5c-4) after being put into contact with the most commonly used concentration of each of the experimental testing solutions (10% formalin [dead control], stabilized thimerosal [Thi: 0.0025%], benzalkonium chloride [BAK: 0.025%], chlorobutanol [Cbl: 0.25%], methyl paraben [MP: 0.01%], sodium perborate [SP: 0.0025%], disodium-ethylene diamine tetra-acetate [EDTA: 0.01%], and media alone [total viable control]) for 1 h.