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Bacterial Reverse Mutation Test / OECD 471

ames test: The bacterial reverse mutation test uses amino-acid requiring at least five strains of Salmonella typhimurium and Escherichia coli to detect point mutations by base substitutions or frameshifts. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.

Suspensions of bacterial cells are exposed to the test substance (liquid or solid) in the presence and in the absence of an exogenous metabolic activation system. At least five different analysable concentrations of the test substance should be used. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 ml/plate. There are two methods: the plate incorporation method and the preincubation method. For both techniques, after two or three days of incubation at 37°C, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

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In Vitro Mammalian Chromosomal Aberration Test / OECD 473

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations may be of two types: chromosome or chromatid.

The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle lengths. At least three analysable concentrations of the test substance should be used. At each concentration duplicate cultures should normally be used. At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance, harvested, stained. Metaphase cells are analysed microscopically for the presence of chromosome aberrations.

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In vitro Mammalian Cell Gene Mutation Test / OECD 476

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. In the cell lines the most commonly-used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events.

Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation, for a suitable period of time. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. It is recommended to utilise at least 106cells. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.

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In Vitro Mammalian Cell Micronucleus Test / OECD 487
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.

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In Vitro Skin Irritation - Reconstructed Human Epidermis Test / OECD 439
This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2. It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test chemicals are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). This Test Guideline also includes a set of Performance Standards for the assessment of similar and modified RhE-based test methods. There are four validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

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In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method / OECD 431

The present Test Guideline addresses the human health hazard endpoint skin corrosion, following exposure to a test chemical. Skin corrosion is defined as the production of irreversible tissue damage, manifested as visible necrosis of the skin, according to the definition of the Globally Harmonised System for Classification and Labeling of Chemicals.

This Test Guideline describes an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures, based on three-dimensional human skin model which reliably reproduces histological, morphological, biochemical, and physiological properties of the upper layers of human skin, including a functional stratum corneum. The procedure on reconstituted human epidermis is based on the principle that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers. Two tissue replicates are used for each treatment (or exposure time), and for controls. Corrosive materials are identified by their ability to produce a decrease in cell viability below defined threshold levels at specified exposure time. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Corrosive substances are evidenced by their capacity to reduce cell viability below the defined threshold.

Several validated methods are referenced in the Test Guideline and follow the procedure described above. Some of the methods referenced allow sub-categorisation among corrosive chemicals.

This Test Guideline also includes a set of Performance Standards (PS) for the assessment of similar and modified TER-based test methods.

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In Vitro Membrane Barrier Test Method for Skin Corrosion / CORROSITEX / OECD 435

This Test Guideline is for an in vitro membrane barrier test method that can be used to identify corrosive substances.

The test method utilizes an artificial membrane designed to respond to corrosive substances in a manner similar to animal skin in situ. The in vitro membrane barrier test method may be used to test solids, liquids (aqueous substances with a pH in the range of 4.5 to 8.5 often do not qualify for testing) and emulsions. The test described in this Test Guideline allows the identification of corrosive chemical substances and mixtures and allows the subcategorisation of corrosive substances as permitted in the GHS. This classification is based on the substance penetration time through the membrane barrier. The test system is composed of two components, a synthetic macromolecular bio-barrier and a Chemical Detection System (which one detect the test substance). An appropriate number of replicates is prepared for each test substance and its corresponding controls. The times of applying the test substance to the membrane barrier are recorded and staggered. The time (in minutes) elapsed between application of the test substance to the membrane barrier and barrier penetration is used to predict the corrosivity of the test substance.

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