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This revised Test Guideline 413 (TG 413) has been designed to fully characterize test article toxicity by the inhalation route following repeated exposure for a period of 90 days, and to provide data for quantitative inhalation risk assessments.  It was updated in 2017 to enable the testing and characterisation of effects of nanomaterials tested. Groups of at least 10 male and 10 female rodents are exposed 6 hours per day for 90 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. The results of the study include measurement and daily and detailed observations (haematology and clinical chemistry), as well as ophthalmology, gross pathology, organ weights, and histopathology. This Test Guideline allows the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

This revised Test Guideline 412 (TG 412) has been designed to fully characterize test article toxicity by the inhalation route following repeated exposure for a limited period of time (28 days), and to provide data for quantitative inhalation risk assessments.  It was updated in 2017 to enable the testing and characterisation of effects of nanomaterials tested. Groups of at least 5 male and 5 female rodents are exposed 6 hours per day for 28 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test article. This guideline allows the study director the flexibility to include satellite (reversibility) groups, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

This Test Guideline describes a chronic oral toxicity test on adult worker honey bees under laboratory conditions over an exposure period of 10 days. Young bees (max. 2 days old) are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical by continuous and ad libitum feeding over a period of 10 days. Mortality and behavioural abnormalities are observed and recorded daily during the 10 day test period. The chronic effects of the test chemical are evaluated by comparing the results of the test chemical treated group to those of the respective control group. The test is designed for the determination of the following endpoints  LC50 (median Lethal Concentration) and the LDD50 (median Lethal Dietary Dose) values after 10 days of exposure, and NOEC (No Observed Effect Concentration) and NOEDD (No Observed Effect Dietary Dose).  

This test guideline describes a test procedure to gain information on dispersion stability of manufactured nanomaterials in simulated environmental media. The main purpose of this guideline is to assess the ability of a nanomaterial to attain a colloidal dispersion and to conserve this dispersion under environmentally relevant conditions. The test procedure involves a dispersion of the nanomaterial with the aid of a calibrated sonication procedure and the determination of the mass concentration of the nanomaterial in a set of test vials while the particles undergo homoagglomeration and settling in environments of different hydrochemistry

This test guideline is a laboratory test method, designed to assess the acute oral toxicity of pesticides and other chemicals to adult worker bumblebees. Adult worker bumblebees are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical. The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.

This test guideline is a laboratory test method, designed to assess the acute contact toxicity of pesticides and other chemicals to adult worker bumblebees. Adult worker bumblebees are exposed to the test chemical dissolved in an appropriate carrier, by direct application to the dorsal thorax (droplet). The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.

This Test Guideline describes a method to assess effects of a test chemical on the phagocytotic activity of activated sludge containing protozoan organisms under defined conditions in the presence of different concentrations of the test chemical. The principle of biological sewage-treatment plants (STP) is to transform the organic matter of incoming waste-water in microbial biomass, which in turn is separated from the liquid yielding a purified effluent. The purpose of the test is to provide a means to record effects of test chemicals on ciliated protozoa in sewage treatment plants, which due to their grazing on bacteria considerably contribute to the functioning of STPs.

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.

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. In this test, the used genetic endpoints measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT), and at a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The 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. 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.

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents (rats, mice and Chinese hamsters). Structural chromosome aberrations may be of two types: chromosome or chromatid. Animals are exposed to the test substance (liquid or solid) by an appropriate route of exposure (usually by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection) and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting agent. Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosome aberrations. Each treated and control group must include at least 5 analysable animals per sex. The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.