The Short Time Exposure (STE) test method is an in vitro method for assessing the eye irritation potential of chemicals and is used to classify the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Category 1 and No Category (NC). The method has been adopted by the Organisation for Economic Co-operation and Development (OECD) as test guideline (TG) 491 since 2015. While this method can be used to classify GHS NC, it is not suitable for testing highly volatile substances and solids other than surfactants. Here we evaluated highly volatile substances to expand the applicability domain. According to TG 491, acetone, ethanol, iso-propanol, and methyl acetate as highly volatile substances resulted in false negatives. Saline was selected as a solvent of these false negatives. In this study, mineral oil was used as the solvent, because these false negatives were amphiphilic. Based on this change, four highly volatile substances were correctly evaluated. The predictive performance for classifying GHS NC was then verified using a substance dataset constructed in reference to the Draize eye test Reference Database and STE Summary Review Document. The accuracy and false-negative rate were 86.6% (194/224) and 3.8% (3/80), respectively. Collectively, the applicability domain was expanded by changing the solvent to mineral oil for highly volatile substances, and the predictive performance for the new applicability domain including highly volatile substances was excellent. The STE test method is suitable to classify GHS NC, indicating its applicability as a test method in a bottom-up approach.

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The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.

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