None of the in vitro method are suitable for directly classifying of a substance as an eye irritant (category 2). They can classify substance as category 1 (serious eye damage) or as "no category" (not requiring classification). The aim of this study was to develop a new method for direct classification of a substance as category 2. Cytotoxicity Assay to Assess Eye Irritation (CEI) was performed on fibroblast - HDFn cell line with 36 substances. 5 concentrations of all substances and neat substances were applied directly to the cells. After 30 min, medium was added and cells were incubated at 37 °C. The next day, the cytotoxicity assay was performed (MTT assay in the first run and NRU assay in the second run). Based on viability and IC50 value (concentration with 50 % viability) a substance could be classified in category 2, category 1, and as "no category". The results obtained were referred to ECHA database. This new method had high sensitivity (53.8-88.9 %), specificity (73.9-100.0 %) and accuracy (69.4-88.9 %) in the classification to all categories. It effectively classifies not only substances in category 2 but also in category 1 and substances that do not require classification.
Keywords: Classification; Cytotoxicity; Eye irritation; IC50.
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