试验编号442D：体外皮肤致敏：针对角质形成细胞激活不良结果途径关键事件的检测

OECD/OCDEYou are free to use this material subject to the terms and conditions available athttp://www.oecd.org/termsandconditions/1OECD GUIDELINEFOR THETESTING OF CHEMICALSIn vitroskin sensitisation assays addressing the Adverse Outcome Pathway Key Event onKeratinocyte activationKeratinocyte activation Key Event based Test GuidelineA skin sensitiser refers to a substance that will lead to an allergic response following repeatedskin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling ofChemicals (UNGHS) (1). There is general agreement on the key biological events underlying skinsensitisation. The current knowledge of the chemical and biological mechanisms associated with skinsensitisation has been summarised as an Adverse Outcome Pathway (AOP) (2), starting with the molecularinitiating event through intermediate events to the adverse effect, namely allergiccontact dermatitis. This AOPfocuses on chemicals that react with thiol (i.e. cysteine) and primary amines (i.e. lysine) such as organicchemicals. In this instance, the molecular initiating event (i.e. the first key event) is the covalent binding ofelectrophilic substances to nucleophilic centres in skin proteins. The second key event in this AOP takesplace in the keratinocytes and includes inflammatory responses as well as changes in gene expressionassociated with specific cell signalling pathways suchas the antioxidant/electrophile response element(ARE)-dependent pathways. The third key event is the activation of dendritic cells, typically assessed byexpression of specific cell surface markers, chemokines and cytokines. The fourth key event is T-cellThis Test Guideline describes in vitro assays that address mechanisms described under the secondKey Event of the AOP for skin sensitisation, namely keratinocyte activation (2). The Test Guideline comprisestest methods to be used for supporting the discrimination between skin sensitisers and non-sensitisers inaccordance with the UNGHS(1). The test methods currently described in this Test GuidelineincludetwoinvitroARE-Nrf2luciferasetestmethodsandatest method based on gene expressionquantification:•The ARE-Nrf2 luciferase KeratinoSens™test method (Appendix IA),•The ARE-Nrf2 luciferase LuSens test method (Appendix IB),•TheEpidermalSensitisationAssay–EpiSensA(AppendixIC).Thesethreetest methods have been considered scientifically valid. The KeratinoSens™test methodfirst underwent a validation study followed by an independent peer-review by EURL ECVAM ScientificAdvisory Committee (ESAC) and positive recommendations by EURL ECVAM, and is considered thevalidated reference method (VRM)with regards to ARE-Nrf2 luciferase test methods(3) (4) (5) (6). TheLuSens test method later underwent a Performance Standard-based validation study based on which it wasalso reviewed and received positive opinion by ESAC (7) (8) (9) (10).TheEpiSensA underwent validationstudies (11) followed by an independent peer review (12) conducted by the Japanese Center for the Validationof AlternativeMethods(JaCVAM).ItisconsideredaVRMwithregardstotestmethodsquantifying changes intheexpression of marker genes associated with keratinocyte activation (ATF3,IL-8,GCLM, andDNAJB4),using Reverse Transcription-quantitative PCR in reconstructed human epidermis (RhE) models. PerformanceStandards (13) are available for this type of methodtoo, to facilitate the validation and assessment of similar ©OECD, (2024)GENERAL INTRODUCTIONproliferation.and modified RhE-based test methods. 1.2.3. OECD/OCDE2Background and principles of the test methods included in the Key Event based TestThe assessment of skin sensitisation hashistoricallyinvolved the use of laboratory animals. Theclassical methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson andKligman and the Buehler Test (OECD TG 406) (14), assess both the induction and elicitation phases of skinsensitisation. The murine tests, the LLNA (OECD TG 429) (15) and its three non-radioactive modifications,LLNA: DA (OECD TG442A) (16) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG 442B) (17), allassess the induction response exclusively, and have gained acceptance sincethey provide an advantageover the guinea pig tests in terms of animal welfare together with an objective measurement of the inductionMechanistically-based in chemico and in vitro test methods addressing the first three key events ofthe skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisationhazard potential of chemicals: the OECD TG442C describes the Direct Peptide Reactivity Assay (18)addressing the first key event; the present Test Guideline assesses keratinocyte activation addressing thesecond key event and the OECD TG 442E addresses the activation of dendritic cells, the third key event ofthe skin sensitisation AOP (16). Finally, the fourth key event representing T-cell proliferation is indirectlyassessed in the murine Local Lymph No