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The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergone cell division during or after exposure to the test substance. This Test Guideline allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B. Cytochalasin B allows for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis, because such cells are binucleate. This Test Guideline also allows the use of protocols without cytokinesis block provided there is evidence that the cell population analysed has undergone mitosis.   

This method provides information on health hazard likely to arise from exposure to test substance (liquids, solids and aerosols) by application on the eye. This Test Guideline is intended preferably for use with albino rabbit. The test substance is applied in a single dose in the conjunctival sac of one eye of each animal. The other eye, which remains untreated, serves as a control. The initial test uses an animal; the dose level depends on the test substance nature. A confirmatory test should be made if a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals. It is recommended that it be conducted in a sequential manner in one animal at a time, rather than exposing the two additional animals simultaneously. The duration of the observation period should be sufficient to evaluate fully the magnitude and reversibility of the effects observed. The eyes should be examined at 1, 24, 48, and 72 hours after test substance application. The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. Use of topical anesthetics and systemic analgesics to avoid or minimize pain and distress in ocular safety testing procedures is described.

This Test Guideline describes an in vitro assay that may be used for identifying water soluble ocular corrosives and severe irritants as defined by the UN Globally Harmonized System of Classification and Labelling, Category 1. The assay is performed in a well where a confluent monolayer of Madin-Darby Canine Kidney (MDCK) is used as a separation between two chambers. It uses a fluorescein dye as marqueur. The test substance has the potential to impair the junctions of the MDCK cells and thus to increase the monolayer¡¯s permeability. Consequently the fluorescein passes through the monolayer and the fluorescein leakage (FL) increases. The FL is calculated as a percentage of leakage relative to both a blank control and a maximum leakage control. The concentration of test substance that causes 20% FL (FL20, in mg/mL) is calculated and used in the prediction model for identification of ocular corrosive and severe irritants. The cut-off value of FL20 to identify water soluble chemicals as ocular corrosives/severe irritants is ¡Ü 100mg/mL. The FL test method should be part of a tiered testing strategy.

This Test Guideline describes in vitro assays, which use Androgen Receptor TransActivation (ARTA) to detect Androgen Receptor Agonists and Antagonists. The ARTA assay methods are mechanistically and functionally similar test methods that provide information on the transcription and translation of a reporter gene following the binding of a chemical to the androgen receptor and subsequent transactivation. The cell lines used in these assays express AR and have been stably transfected with an AR-responsive luciferase reporter gene, and are used to identify chemicals that activate (i.e. act as agonist) or inhibit (i.e. act as antagonists) AR-dependent transcription. Some chemicals may, in a cell type-dependent manner, display both agonist and antagonist activity and are known as selective AR modulators. The AR is activated following ligand binding, after which the receptor-ligand complex binds to specific DNA responsive elements and transactivates the receptor gene, resulting in an increase cellular expression of the luciferase enzyme. The enzyme then transforms the substrate to a bioluminescent product that can be quantitatively measured with a luminometer. This Test Guideline includes ARTA assays using the AR-EcoScreenTM cell line, the AR-CALUX® cell line, and 22Rv1/MMTV_GR-KO cell line.

The present Key Event based Test Guideline (TG) addresses the human health hazard endpoint skin sensitisation, following exposure to a test chemical. More specifically, it addresses the activation of dendritic cells, which is one Key Event on the Adverse Outcome Pathway (AOP) for Skin Sensitisation. Skin sensitisation refers to an allergic response following skin contact with the tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). This TG provides three in vitro test methods addressing the same Key Event on the AOP: (i) the human cell Line Activation Test or h-CLAT method, (ii) the U937 Cell Line Activation Test or U-SENS and (iii) the Interleukin-8 Reporter Gene Assay or IL-8 Luc assay. All of them are used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with the UN GHS. Test methods described in this TG either quantify the change in the expression of cell surface marker(s) associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g. CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. In the h-CLAT and U-SENS assays, the changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. In the IL-8 Luc assay, the changes in IL-8 expression are measured indirectly via the activity of a luciferase gene under the control of the IL-8 promoter. The relative fluorescence or luminescence intensity of the treated cells compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.