Development of the short time exposure (STE) test: An in vitro eye irritation test using SIRC cells

Abstract

Using SIRC (rabbit corneal cell line) cells, we developed an alternative eye irritation test: the short time exposure (STE) test. This STE test is a cytotoxicity test using physiological saline or mineral oil as the test solvent. Evaluation exposure time is short (5min), which is similar to actual exposure situations, and uses the cell viability (CV) at a constant concentration as the endpoint for irritation potential. First, in order to confirm the usefulness of this STE test in assessing eye irritation potential of chemicals, 51 raw materials were tested and the correlation between CV in the STE test and the eye irritation score in the Draize test was examined. For the undiluted raw materials tested in the Draize test, the 5% test concentration in the STE test gave irritation classes that correlated well with the irritation classes from the Draize test (accuracy: 89.6%). For those materials tested as a 10% solution in the Draize test, STE irritation classes with 0.05% test concentration corresponded well with the Draize irritation classes (accuracy: 80.0%). Next, using the cell viabilities at these two concentrations, the STE prediction model (PM) was developed. A score of 1 or 2 was given for the results from each tested concentration in the STE test and Draize test. The scores from each test were then summed to yield a 3-level (Rank 1: minimally irritant, Rank 2: moderate irritant, Rank 3: severe irritant) eye irritation potential classification. Rank classification in the STE test showed a good correlation mostly to that in the Draize test (irritation class correspondence rate: 70.2%, but after exclusion of data of alcoholic materials, the rate was 91.7%). In most cytotoxicity test, the cytotoxicity of acids and amines is generally underestimated due the use of medium as the solvent. This is the result of the buffering capacity of the media. On the other hand, the STE test could predict the eye irritation potential by evaluating the chemical with a 5% test concentration. Eleven water insoluble materials such as toluene, octanol, and hexanol could be evaluated by using mineral oil as test solvent in the STE test.The STE test demonstrated itself to be simple, promising, have great potential, be of value, and to be an easily standardized alternative eye irritation test.