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S.P. Epstein, M. Ahdoot, C. Koonapareddy, K. Yee, E. Marcus, P.A. Asbell; Toxicity of Ocular Preservatives and Buffering Agents on the Ocular Surface (Corneal and Conjunctival Epithelium) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4961.
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Nearly all eye drops contain preservatives to decrease contamination of multi–use bottles. Non–preservatives disodium–ethylene diamine tetra–acetate (EDTA) and phosphate buffered saline (PBS) are also added as buffering agents. These components can cause ocular surface disease. To evaluate the potential toxicity of these preservatives as well as each of these components to the ocular surface, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized.
Immortalized human conjunctival cells [Chang's Conjunctival Cells] and corneal epithelial cells (CEPI 17)] were seeded at 103 cells/well and kept at 37oC, 5% CO2. At confluency, medium was arbitrarily replaced with 100 uls of: varying concentrations of ophthalmic preservatives: benzalkonium chloride (BAK), methyl paraben (MP), sodium perborate (SOP), chlorobutanol (Cbl) and stabilized thimerosal (Thi); varying concentrations of ophthalmic buffering agents: EDTA and PBS; cell media (viable control); and formalin (dead control). After 1 hour, the testing solutions were 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 exhibited an average toxicity from approximately 89% (Chang: 86.71%±22.38, HCE: 91.98%±25.70) to 56% (Chang: 54.02%±25.99, HCE: 58.12%±31.56). In comparison, Cbl exhibited a range of toxicity from 86% (Chang: 83.65%±23.21, HCE: 87.48%±33.22) to 50% (Chang: 46.77%±27.39, HCE: 52.46%±35.71), MP from 76% (Chang: 73.92%±26.24, HCE: 77.30%±33.89) to 30% (Chang: 24.48%+23.24, HCE: 36.41%±33.95), SOP from 59% (Chang: 60.18%±28.04, HCE: 57.72%±32.65) to 23% (Chang: 27.00%±32.52, HCE: 19.75%±32.91) and Thi from 95% (Chang: 92.62%+20.08, HCE: 96.49%±21.59) to 70% (Chang: 68.76%±22.85, HCE: 70.24%±33.92). Na–EDTA and PBS were indistinguishable from SOP with minimal toxicity ranging from 59% (Chang: 60.07%±25.27, HCE: 57.86%±37.32) to 6% (Chang: 6.03%±24.74, HCE: 5.03%±39.53).
In general, the HCE cells were more sensitive than the conjunctival cells. The preservatives, in order of decreasing toxicity: Thi>BAK>Cbl>MP>SOP≈EDTA≈PBS. Even at low–concentration preservatives or buffering agents will cause some degree of cell damage to the ocular tissue as evaluated by corneal and conjunctival cells in tissue culture.
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