Test No. 492B: Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard Identification
OECD
Abstract
2a synopsis of the important elements of the test method, as well as flowcharts providing guidance for specific situations. 5.The purpose of this TG is to describe the procedure used to evaluate the eye hazard potential of a test chemical based on its ability to induce cytotoxicity in a RhCE tissue construct, as measured by reduction of a vital dye (MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS RN 298-93-1]) hereafter designated as the tetrazolium dye (TD) (15) (see paragraph 23).The viability of the RhCE tissue following exposure to a test chemical is determined in comparison to tissues treated with the negative control substance (%viability) for the two or three exposure times, and is then used to predict the eye hazard potential of the test chemical. 6.Definitions are provided in Annex I. INITIAL CONSIDERATIONS AND LIMITATIONS 7.The SkinEthic™ HCE tissue construct is a three-dimensional model produced using cells from the species of interest which mimics the in vivo corneal epithelium(17).The test method directly measures cytotoxicity resulting from penetration of the chemical through the corneal epithelium and production of cell and tissue damage following chemical exposure, which is used to predict the eye hazard identification of a test chemical.Cell damage can occur by several modes of action (see paragraph 16), but cytotoxicity plays an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye irritation response of a chemical, manifested in vivo mainly by corneal opacity, iritis, conjunctival redness and/or conjunctival chemosis, regardless of the physicochemical processes underlying tissue damage. 8.A total of 151 chemicals covering a variety of chemical types, chemical classes, chemical structures, as well as molecular weights, LogP and other physical-chemical properties have been tested in the validation study underlying this TG.The validation database covered 134 different organic functional groups (11)(12)(13) and all key in vivo drivers of classification (26)(27).The majority of these chemicals represented monoconstituent substances (a total of 151 substances, of which 16 were tested in dilution), but several multi-constituent substances (including surfactants or polymers) were also included in the study.In terms of physical state and according to UN GHS Categories (1), the 151 tested chemicals were distributed as follows: 70 liquids, comprising 21 Cat. 1, 25 Cat. 2 (incl.16 Cat.2A and 9 Cat.2B) and 24 No Cat, and 81 solids, comprising 29 Cat. 1, 19 Cat.2, and 33 No Cat.(11)(12)(13). 9.The SkinEthic™ HCE TTT is not intended to discriminate between UN GHS Category 2A (eye irritation, effects fully reversible within 21 days) and UN GHS Category 2B (mild eye irritation, effects fully reversible within 7 days).This differentiation needs to be addressed by other methods or approaches, if discrimination is deemed necessary (3). 10.This TG is applicable to substances and mixtures, to solids, liquids, semi-solids and waxes.The liquids may be aqueous or non-aqueous, solids may be soluble or insoluble in water.Caution should be used when testing solid chemicals with poor water solubility (< 0.014 mg/mL) as they were frequently underpredicted by SkinEthic TM HCE TTS in the validation study (e.g., 5 out of 9 Cat.1 chemicals were underpredicted to be Cat.2).Gases and aerosols have not been assessed in a validation study.While it is conceivable that these can be tested using RhCE technology, the current TG does not allow testing of gases and aerosols.Limited information is currently available on the applicability of the test method to multi-constituent substances/mixtures (11).The test method is nevertheless technically applicable to the testing of multi-constituent substances and mixtures.In cases where evidence demonstrates the non-applicability of the TG to specific formulations, the TG should not be used for those formulations.When considering testing of difficult-to-test OECD/