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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.

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 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.

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 is designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp. First instar chironomid larvae are exposed to at least five concentrations of the test chemical in sediment-water systems. The test starts by placing first instar larvae into the test beakers containing the sediment-water system and subsequently spiking the test substance into the water. Chironomid emergence and development rate is measured at the end of the test. The maximum exposure duration is 28 days for C. riparius, C. yoshimatsui, and 65 days for C. tentans. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). The study report should include the development time and the total number of fully emerged midges (sex and number are recorded daily), the observation of any abnormal behaviour, the number of visible pupae that have failed to emerge and any egg masses deposition. The data are analysed either by using a regression model in order to estimate the concentration that would cause x % reduction in emergence, larvae survival or growth, or by using statistical hypothesis testing to determine a NOEC/LOEC.

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 in vitro macromolecular test method is a biochemical in vitro test method that can be used to identify chemicals (substances and mixtures) that have the potential to induce serious eye damage as well as chemicals not requiring classification for eye irritation or serious eye damage. The in vitro macromolecular test method contains a macromolecular reagent composed of a mixture of proteins, glycoproteins, carbohydrates, lipids and low molecular weight components, that when rehydrated forms a complex macromolecular matrix which mimics the highly ordered structure of the transparent cornea. Corneal opacity is described as the most important driver for classification of eye hazard. Test chemicals producing protein denaturation, unfolding and changes in conformation will lead to the disruption and disaggregation of the highly organised macromolecular reagent matrix, and produce turbidity of the macromolecular reagent. Such phenomena is quantified, by measuring the changes in light scattering (at a wavelength of 405 nm using a spectrometer), which is compared to the standard curve established in parallel by measuring the increase in OD produced by a set of calibration substances.