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The purpose of the Dominant lethal (DL) test is to investigate whether chemical agents produce mutations resulting from chromosomal aberrations in germ cells. In addition, the dominant lethal test is relevant to assessing genotoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA-repair processes are active and contribute to the response. Induction of a DL mutation after exposure to a test chemical indicates that the chemical has affected germinal tissue of the test animal. This modified version of the Test Guideline reflects more than thirty years of experience with this test and the potential for integrating or combining this test with other toxicity tests such as developmental, reproductive toxicity, or genotoxicity studies; however due to its limitations and the use of a large number of animals this assay is not intended for use as a primary method, but rather as a supplemental test method which can only be used when there is no alternative for regulatory requirements.

This screening Test Guideline describes the effects of a test chemical on male and female reproductive performance. It has been updated with endocrine disruptor endpoints, in particular measure of anogenital distance and male nipple retention in pups and thyroid examination. The test substance is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks. Females should be dosed throughout the study, so approximately 63 days. Matings 'one male to one female' should normally be used in this study. This Test Guideline is designed for use with the rat. It is recommended that each group be started with at least 10 animals of each sex. Generally, at least three test groups and a control group should be used. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. The test substance is administered orally and daily. The results of this study include clinical observations, body weight and food/water consumption, oestrous cycle monitoring, offspring parameters observation/measurement, thyroid hormone measurement, as well as gross necropsy and histopathology. The findings of this toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. Because of the short period of treatment of the male, the histopathology of the testis and epididymus should be considered along with the fertility data, when assessing male reproductive effects.

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.

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. Genetic events detected using the tk locus include both gene mutations and chromosomal 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 micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents (mice or rats). The purpose of the micronucleus test is to identify substances (liquid or solid) that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. Animals are exposed to the test substance by an appropriate route (usually by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection). Bone marrow and/or blood cells are collected, prepared and stained. Preparations are analyzed for the presence of micronuclei. Each treated and control group must include at least 5 analysable animals per sex. Administration of the treatments consists of a single dose of test substance or two daily doses (or more). 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.

This Test Guideline describes a method to estimate the developmental toxicity of a test chemical to the dung dwelling life stages of dung-dependent dipteran species. Two test species can be used. The test chemical is mixed with bovine faeces, to which either 10 eggs of Scathophaga stercoraria or 10 larvae of Musca autumnalis are added. The test will be terminated 5 days after emergence of the last adult in the control (> 18 days for S. stercoraria, >13 days for M. autumnalis). Then the possible impacts of the test chemical on the following measurement endpoints are assessed under controlled conditions: sex and total number of emerged adult flies, retardation of emergence indicated by the developmental rate and morphological change. Depending on the experimental design, the No Observed Effect Concentration (NOEC) or the Effect concentration for x percent effect (ECx) can be determined. This Guideline can be used for water soluble or insoluble substances, but is not applicable to volatile substances. If the toxicity of the chemical is unknown, five nominal test concentrations should be conducted. A positive control should be tested periodically. The test is considered valid if in the controls hatching of larvae is superior or equal to 70% of the number of introduced eggs, emergence of adults is superior or equal to 70% and superior or equal to 50% of the respectively hatched and introduced larvae and if the emergence of adult flies starts after 18 +- 2 days (S. stercoraria) or after 13 +- 2 days(M. autumnalis).

This Test Guideline describes procedures designed to estimate the acute oral toxicity of substances to birds, and it provides three testing options: (1) limit dose test, (2) LD50-slope test, and (3) LD50-only test. The LD50-slope and LD50-only options are sequential testing procedures. The test method selected will depend on whether or not a definitive median dose (LD50) and slope of the dose-response curve are both needed. The limit dose test is the preferred test when toxicity is expected to be low and lethality is unlikely at the limit dose. The limit dose should be adequate for assessment purposes, and it is usually 2000 mg/kg-bwt. Five or ten birds are tested at the limit dose in addition to a control group. The LD50-slope test is the preferred test when regulatory or other requirements determine that the slope of the dose-response curve and/or the confidence interval is required in addition to an estimate of the LD50. This is a 3- or 4-stage test with 24 or 34 birds in addition to a control group. The LD50-only test is the preferred test when regulatory or other requirements determine that only the median lethal dose is required but neither the slope of the dose response curve or the confidence interval for the LD50 is required. This may be the appropriate test to estimate a percentile of a species sensitivity distribution of LD50s and to provide information for product labelling purposes. This test has two stages, with 14 birds in addition to a control group. Software to be used with TG 223. Click here. Software not part of the Mutual Acceptance of Data.