测试编号442B：皮肤致敏：局部淋巴结检测：BrdU ELISA或FCM

OECD/OCDEYou are free to use this material subject to the terms and conditions available athttp://www.oecd.org/termsandconditions/OECD GUIDELINEFOR THETESTING OF CHEMICALSLocal lymph node assay: BRDU-ELISA or–FCMGENERAL INTRODUCTIONA skin sensitiser refers to a substance that will lead to an allergic response following repeated skin contactas defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNThere is general agreement regarding the key biological events underlying skin sensitisation. The currentknowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarised inthe form of an Adverse Outcome Pathway (AOP) (2), starting with the molecular initiating event throughintermediate events to the adverse effect, namely allergic contact dermatitis. This AOP focuses on chemicals thatreact with thiol (i.e. cysteine) and primary amines (i.e. lysine) such as organic chemicals. In this instance, themolecular initiating event (i.e. the first key event) is the covalent binding of electrophilic substances to nucleophiliccentres in skin proteins. The first key event can be addressed using the in chemico Direct Peptide Reactivity Assay(DPRA)TG 442C (3). The second key event in this AOP takes place in the keratinocytes and includes inflammatoryresponses as well as changes in gene expression associated with specific cell signalling pathways such as theantioxidant/electrophile response element(ARE)-dependent pathways. This key event can be addressed using thein vitro ARE-Nrf2 Luciferase Test Methods (KeratinoSensTM or LuSens) TG 442D (4). The third key event is theactivation of dendritic cells (DC), typically assessed by expression of specific cell surface markers, chemokines andcytokines, and can be addressed using either the in vitro Human Cell Line Activation Test (h-CLAT), the in vitroU937 Cell Line Activation Test (U-SENS™) or the Interleukin-9 Reporter Gene assay (IL-8 Luc assay) asdescribedin TG442E (5). The fourth key event is T-cell proliferation, which is indirectly assessed in the in vivo murine LocalThe first Test Guideline (TG) for the determination of skin sensitisation in the mouse, the LocalLymphNode Assay (LLNA; TG 429) was adopted in 2002, and has since then been revised (7). The details of the validationof the LLNA and a review of the associated work have been published (8) (9) (10) (11) (12) (13) (14) (15) (16). Inthe LLNA, radioisotopic thymidine or iodine is used to measure lymphocyte proliferation and therefore the assayhas limited use in regions where the acquisition, use, or disposal of radioactivity is problematic.This Test Guideline describes two non-radioactive modificationsto the LLNAtest method, which utilisenon-radiolabelled 5-bromo-2-deoxyuridine (BrdU) (Chemical Abstracts Service [CAS] No 59-14-3) in an ELISA[Enzyme-Linked Immunosorbent Assay]-or FCM[Flow Cytometry Method]-based test system to measureThe Local Lymph Node Assay: BrdU-ELISA (Appendix I), andThe Local Lymph Node Assay: BrdU-FCM (Appendix II).Similar to the LLNA, the LLNA: BrdU-ELISA and the LLNA: BrdU-FCM study the induction phase ofskin sensitisation and provide quantitative data suitable for dose-response assessment. Furthermore, an ability todetect skin sensitisers without the necessity for using a radiolabel for DNA eliminates the potential for occupationalexposure to radioactivity and waste disposal issues. This in turn may allow for the increased use of mice to detect ©OECD, (2024)GHS) (1).Lymph Node Assays (LLNA) (6).lymphocyte proliferation: 1.2.3.4.5. OECD/OCDE2skin sensitisers, which could further reduce the use of guinea pigs to test for skin sensitisation potential (i.e. TGThis Test Guideline is designed for assessing skin sensitisation potential of chemicals in animals. TG 406utilises guinea pig tests, notably the guinea pig maximisation test and the Buehler test (17). The LLNA (TG 429)(7) and the non-radioactive modifications, LLNA: BrdU-ELISA and FCM (TG 442 B) and LLNA: DA (TG 442 A)(18), all provide an advantage over the guinea pig tests in TG 406 (17) in terms of reduction and refinement of1)UN, 2017. Globally Harmonised Sysem of Classification and Labelling of Chemicals (GHS)edition.UnitedNations,https://www.unece.org/trans/danger/publi/ghs/ghs_rev07/07files_e0.html2)OECD (2012). The Adverse Outcome Pathway for Skin Sensitisation Initiated by CovalentBinding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No.168.Availableat:ENV/JM/MONO(2012)10/PART1OECD (2015), In Chemico Skin Sensitisation, Test Guideline No. 442C, Guidelines for Testingof Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]OECD (2015), In Vitro Skin Sensitisation, Test Guideline No. 442D, Guidelines forTesting ofChemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]5)OECD (2017), In Vitro Skin Sensitisation, Test Guideline No. 442E, Guidelines for Testing ofChemicals, OECD,