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S. P. Epstein, Y. Wei, P. A. Asbell; Toxicity of Low-Concentration Benzalkonium Chloride on the Ocular Surface (Corneal and Conjunctival Epithelium). Invest. Ophthalmol. Vis. Sci. 2009;50(13):5525.
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Most eye drops contain preservatives to decrease contamination of multi-use bottles, the most common of which is benzalkonium chloride (BAK), used in concentrations from 0.004% to 0.05% whose degradation byproduct, H2O2, is an ophthalmic irritant. However, low doses of BAK have been considered to decrease potential effects. Therefore in this study, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized to evaluate BAK at concentrations below those commonly used in pharmaceutical preparations.
Immortalized human conjunctival cells (CCC: P61 HCK) and corneal epithelial (HCE: CEPI 17) at confluency were incubated with 100 µL of: BAK from 0.00001 to 0.001% at 37oC, 5% CO2 for 5, 10 and 15 mins. Pure cell media (viable control); and formalin (dead control) were used as controls. The testing solutions were then replaced with 150 uls of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide) and incubated at 37oC for 4 hours. After decanting, 100 uls of acid isopropanol was added and the absorbance of each determined at 572 nm. Absorbance, relative to the controls, was equated with "viability", and conversely, "toxicity".
BAK 0.001% incubated for 15 mins exhibited an average toxicity of approx 34% (CCC: 29.60%±4.34, HCE: 37.75%±3.06), 29% for 10 mins (CCC: 25.04%±2.08, HCE: 33.37%±4.96) and 24% for 5mins (CCC: 20.22%±2.01, HCE: 26.98%±3.81). BAK 0.0005% incubated for 15 mins exhibited an average toxicity of approx 24% (CCC: 22.04%±4.10, HCE: 25.44%±4.04), 21% for 10 mins (CCC: 17.96%±2.91, HCE: 23.32%±4.50) and 16% for 5mins (CCC: 13.76%±3.30, HCE: 18.59%±4.10). BAK 0.0001% incubated for 15 mins exhibited an average toxicity of approx 15% (CCC: 15.07%±4.15, HCE: 14.98%±5.53), 11% for 10 mins (CCC: 10.79%±2.85; HCE: 11.16%±4.87) and 8% for 5mins (CCC: 8.97%±4.60, HCE: 6.75%±3.83). BAK 0.00005 and 0.00001% induced only non-statistically significant levels of toxicity.
Even low doses of BAK have toxic effects on sample cells. In general, the human corneal cells were more sensitive than the conjunctival cells. The concentration of BAK has much more of an impact on toxicity than the exposure time. BAK concentrations as low as 0.0001% for as little as 5 mins will evoke some degree of cell damage to the ocular tissue as evaluated by corneal and conjunctival cells in tissue culture.
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