试验492B：眼睛危害识别的重建人角膜样上皮（RHCE）试验方法

OECD/OCDEYou are free to use this material subject to the terms and conditions available athttp://www.oecd.org/termsandconditions/1OECD GUIDELINE FORTHETESTING OF CHEMICALSReconstructed Human Cornea-like Epithelium (RHCE) Test Methodfor Eye Hazard IdentificationSerious eye damage refers to the production of tissue damage in the eye, orserious physical decay of vision, which is not fully reversible, occurring after exposure of theeye to a test chemical, as defined by the United Nations Globally Harmonized SystemofClassification and Labelling of Chemicals (UNGHS) (1). Alsoaccording to UN GHS, eyeirritation refers to the production of changes in the eye, which are fully reversible, occurringafter exposure of the eye to a test chemical. Test chemicals inducingserious eye damageare classified as UN GHS Category 1, while those inducing eye irritation are classified asUN GHS Category 2. Test chemicals not classified for eye irritation or serious eye damageare defined as those that do not meet the requirementsfor classification as UN GHSCategory 1 or 2 (2A or 2B) i.e., they are referred to as UN GHS No Category (No Cat).The assessment of serious eye damage/eye irritation has initially involved the useof laboratory animals (OECD Test Guideline (TG) 405; adopted in 1981 and revised in 1987,2002, 2012 and 2017) (2).In vitroorex vivotest methods have been adopted as OECD TestGuidelines (TGs) 437(4), 438 (5), 460 (6), 491 (7), 492 (8),494 (9)and 496 (10)to identifyeitherchemicals for serious eye damage potential and/or to identify chemicals not requiringclassification for eye hazard potential.The choice of the most appropriate test method to beused should be considered in the context of the OECD Guidance Document on an IntegratedApproaches on Testing and Assessment (IATA) for Serious Eye Damage and EyeirritationThis TG describes anin vitroprocedure allowing the identification on its own ofchemicals(substances and mixtures)not requiring classification(No Cat),requiringclassification for eye irritation (Cat 2) and requiring classification for serious eye damage(Cat 1) according to the UN GHS ocular hazard categories (1).This TG describes avalidated test method, namely the SkinEthic™Human CornealEpithelium (HCE) Time-to-Toxicity (TTT) testusing a commercially available reconstructedHumanCornea-like Epithelium(HCE).The RhCE is designed to closely mimic thehistological, morphological, biochemical and physiological properties of the human cornealepithelium.A validation study forassessing the three UN GHS ocular hazard categories hasbeen conducted (11)(12)(13) on this method, referred to in the following text as the ValidatedReference Method (VRM). From the validation study and its independent peer review (14) itwas concludedthatthe SkinEthic™HCE TTT is able to correctly identify chemicals (bothsubstances and mixtures) by discriminating the three UN GHS categories for serious eyedamage/eye irritation, i.e. UN GHS Cat. 1, Cat. 2 and No Cat chemicals (1),andthe testmethodwas recommended as a full replacement to thein vivoDraize acute eye irritationtest for classification of chemicals.It is recognized that the use of this test guideline is subjecttonational and international regulatory considerations and conditions.The GuidanceDocument No. 263 onIATAshould be consulted for further testing with other adequateinvitrotests in a weight-of-evidence approach, if deemed necessary (3).AnnexesII-V provide ©OECD, (2023)INTRODUCTION1.2.(3).3.4. OECD/OCDE2a synopsis of the important elements of the test method, as well as flowcharts providingguidance for specific situations.The purpose of this TG is to describe the procedure used to evaluate the eyehazard potential of a test chemical based on its ability to induce cytotoxicity in a RhCE tissueconstruct, as measured by reduction of a vital dye (MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;Thiazolyl blue tetrazolium bromide;CAS RN 298-93-1])hereafter designated as the tetrazolium dye (TD)(15) (see paragraph 23).The viability ofthe RhCE tissue following exposure to a test chemical is determined in comparison to tissuestreated 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.Definitions are provided in Annex I.INITIAL CONSIDERATIONS AND LIMITATIONSThe SkinEthic™HCE tissue construct is athree-dimensional model producedusing cells from the species of interestwhich mimics thein vivocorneal epithelium(17).Thetest method directly measures cytotoxicity resulting from penetration of the chemical throughthe corneal epithelium and production of cell and tissue damage following chemicalexposure, whichis used to predict the eye hazard identificationof a test chemical.Celldamage can occur by several modes of action (see paragraph16), but cytotoxicity plays animportant, if not the primary, mechanistic rolein determining the overall serious eyedamage/eye irritation response of a