An in vitro alternative to the Draize eye-irritation test: Evaluation of the crystal violet staining method

To evaluate a simple in vitro method as an alternative to the Draize eye-irritation test, 12 surfactants were tested by both of the above methods, and the correlation between the results was examined. The in vitro method used was the crystal violet staining method. HeLa cells or SIRC cells from rabbit cornea were inoculated into each well of 96-well microplates which contained serially diluted test chemicals. They were cultured for 72 hr, and then fixed and stained with crystal violet. After spectrophotometric measurement, the concentrations of test materials that inhibited the absorbance to 50% of the control level (IC(50)) were determined. The in vivo data used for comparison were the maximum corneal score and the maximum total score obtained by the eye test when the substances were tested at a concentration of 10%. The correlation coefficients between the maximum corneal scores of the 12 surfactants and their IC(50)s in HeLa cells and in SIRC cells were -0.821 and -0.816, respectively, while those between the maximum total scores and IC(50)s in both cell lines were -0.855 and -0.863, respectively. All of these values are statistically highly significant, indicating that the crystal violet staining method may have value in predicting the eye irritancy of surfactants. Further interlaboratory validation will, however, be needed.