Publications & Documents

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.

This Test guideline describes studies on phototransformation in water to determine the potential effects of solar irradiation on chemicals in surface water, considering direct photolysis only. It is designed as a tiered approach. The Tier 1 is based on a theoretical screen. The rate of decline of a test chemical in a direct photolysis study is generally assumed to follow pseudo first-order kinetics. If the maximum possible losses is estimated to be superior or equal to 50% of the initial concentration over a 30-day period, an experimental study is proceeded in Tier 2. The direct photolysis rate constants for test chemicals in the laboratory is determined using preferably a filtered xenon arc lamp capable of simulating natural sunlight in the 290 to 800 nm, or sunlight irradiation, and extrapolated to natural water. If estimated losses are superior or equal to 20%, the transformation pathway and the identities, concentrations, and rate of formation and decline of major transformation products are identified. An optional task is the additional determination of the quantum yield for various types of water bodies, seasons, and latitudes of interest. The test chemical should be directly dissolved in the aqueous media saturated in air at a concentration which should not exceed half its solubility. For linear and non-linear regressions on the test chemical data in definitive or upper tier tests, the minimum number of samples collected should be 5 and 7 respectively. The exact number of samples and the timing of their collection is determined by a preliminary range-finding. Replicates (at least 2) of each experimental determination of kinetic parameters are recommended to determine variability and reduce uncertainty in their determination.

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 Test Guideline describes an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line, used for the assay, expresses genes that encode for all the key enzymes for steroidogenesis. After an acclimation period of 24 h in multi-well plates, cells are exposed for 48 h to seven concentrations of the test chemical in at least triplicate. Solvent and a known inhibitor and inducer of hormone production are run at a fixed concentration as negative and positive controls. At the end of the exposure period, cell viability in each well is analyzed. Concentrations of hormones in the medium can be measured using a variety of methods including commercially available hormone measurement kits and/or instrumental techniques such as liquid chromatography-mass spectrometry. Data are expressed as fold change relative to the solvent control and the Lowest-Observed-Effect-Concentration. If the assay is negative, the highest concentration tested is reported as the No-Observed-Effect-Concentration.

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 (TG) describes the IL-2 Luc Assay test method to evaluate the potential immunotoxic effects of chemicals on T lymphoblastic cell line. This cell line allows quantitative measurement of luciferase gene induction by detecting luminescence from well-established light producing luciferase substrates as indicators of the activity of IL-2, IFN-γ and GAPDH in cells following exposure to immunotoxic chemicals. The method is intended to be used as a part of a battery to determine immunotoxic potential of chemicals.

This Test Guideline (TG) describes a method to determine the hydrophobicity index (Hy) of nanomaterials (NMs), through an affinity measurement. Hydrophobicity is defined as 'the association of non-polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non-polar molecules'. By measuring their binding rate to different engineered surfaces (collectors), Hy expresses the tendency of the NMs to favour the binding to a non-polar (hydrophobic) surface because of its low affinity for water. The method applies to NMs dispersed in an aqueous solution or to NM powders after their dispersions in aqueous solutions, with or without a surfactant, using a recommended protocol.

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.

This Test Guideline describes a cytotoxicity-based in vitro assay that is performed on a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, cultured on a 96-well polycarbonate microplate. After five-minute exposure to a test chemical, the cytotoxicity is quantitatively measured as the relative viability of SIRC cells using the MTT assay. Decreased cell viability is used to predict potential adverse effects leading to ocular damage. Cell viability is assessed by the quantitative measurement, after extraction from the cells, of blue formazan salt produced by the living cells by enzymatic conversion of the vital dye MTT, also known as Thiazolyl Blue Tetrazolium Bromide. The obtained cell viability is compared to the solvent control (relative viability) and used to estimate the potential eye hazard of the test chemical. A test chemical is classified as UN GHS Category 1 when both the 5% and 0.05% concentrations result in a cell viability smaller than or equal to (≤) 70%. Conversely, a chemical is predicted as UN GHS No Category when both 5% and 0.05% concentrations result in a cell viability higher than (>) 70%.

This Test Guideline describes the Medaka Extended One Generation Test (MEOGRT), which exposes fish over multiple generations to give data relevant to ecological hazard and risk assessment of chemicals, including suspected endocrine disrupting chemicals (EDCs).  Exposure in the MEOGRT starts with spawning fish (P or F0 generation) and continues until hatching (until two weeks post fertilization, wpf) in the second (F2) generation. This Test Guideline measures several biological endpoints.  Primary emphasis is given to potential adverse effects on population relevant parameters including survival, gross development, growth and reproduction (fecundity).  Secondarily, in order to provide mechanistic information and provide linkage between results from other kinds of field and laboratory studies, where there is a posteriori evidence for a chemical having potential endocrine disrupter activity (e.g. androgenic or oestrogenic activity in other tests and assays) then other useful information is obtained by measuring vitellogenin (vtg) mRNA (or vitellogenin protein, VTG), phenotypic secondary sex characteristics (SSC) as related to genetic sex, and evaluating histopathology.