The Use of Cell Lysis as an Index of Ocular Irritation Potential

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Abstract
We report an in vitro method for assessing ocular irritation by measuring cell lysis of mouse connective tissue (strain L, Clone 929) and rabbit corneal cell lines (SIRC). Lysis of corneal epithelial cells in vivo leads to swelling and subsequent opacification of the underlying corneal stroma. In vitro lysis was determined by the measurement of changes in absorbance (360 nm) of cell suspensions over a 10-min exposure to test materials. These data were expressed as numbers of cells/ml using linear regression. The regression line obtained was linear and similar for both cell types. Loss of viability and spontaneous cell lysis were minimal over the assay interval. In a separate study, rabbit corneal cells were used to assess alteration of cell membrane integrity following 30-min exposures to test materials by measuring trypan blue dye exclusion in these cells. Activities of test materials were ranked according to the lowest concentration capable of producing statistically significant cell lysis as follows: triethanolamine lauryl sulfate (TLS) < triethanolamine (TEA) < propylene glycol (PG) (0.003, 0.1, and 10%, respectively, p < 0.01). The order was similar when dye exclusion ability was the index of activity except that benzalkonium chloride (BAK) was more active than the other materials. BAK and materials with cationic properties were not easily tested in the proposed suspension assay because of increased absorbance due to protein denaturation and subsequent masking of dissolution, if present. Six commercial formulations coded A through F were tested in the suspension assay as 1:3,000,1:1,000, and 1:300 dilutions and compared to similarly diluted standards to assess cell dissolution ability. A shampoo concentrate acted similarly to TLS and an amphoteric-surfactant-based shampoo acted similarly to TEA at dilutions of 1:3,000. Effects of other formulations were intermediate between these extremes (p < 0.05), but similar to one another. At 1:1,000 dilutions, the effects converged, with all formulations having the same activity, which was significantly less than that of TLS, but significantly greater than that of TEA (p < 0.05). Over the next interval, the effects paralleled that of TLS but were significantly less except for that of the concentrate (p < 0.05). Another formulation showed a plateau-like effect while the remaining one showed a decreased effect owing to its inherent cationic nature. Results were in general agreement with in vivo and in vitro observations as well as with known chemical activities of the materials. A method for estimating the relative ability of materials and formulations to cause cell lysis is proposed which may serve as a model for the assessment of a form of corneal opacity initiated by the loss of integrity of limiting corneal layers.