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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 in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells. Each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified. Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.

The Potamopyrgus antopodarumon reproduction test is designed to assess potential effects of prolonged exposure to chemicals on reproduction and survival of parthenogenetic lineages of the freshwater mudsnail Potamopyrgus antipodarum. Adult female P. antipodarum are exposed to a concentration range of the test chemical. The test chemical is dispersed into the reconstituted dilution water, added to test beakers, and adult snails are subsequently introduced into the test beakers. When testing 'difficult chemicals' (i.e. volatile, unstable, readily biodegradable and adsorbing chemicals) the test can be conducted under flow-through conditions as an alternative to the semi-static design with fixed renewal periods of the medium (see paragraph 29). P. antipodarum survival over the 28 days exposure period and reproduction at the end of the test after 28 days are examined. Reproduction is evaluated by counting the number of embryo in the brood pouch (without distinction of developmental stages) at the end of 28 days exposure. The toxic effect of the test chemical on embryo numbers is expressed as ECX by fitting an appropriate regression model in order to estimate the concentration that would cause x % reduction in embryo numbers or alternatively as the No Observed Effect Concentration and Lowest Observed Effect Concentration (NOEC/LOEC) value (2).

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 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 Test Guideline is designed to assess effects of prolonged exposure to chemicals on the reproduction and survival of the hermaphrodite freshwater snail Lymnaea stagnalis (the Great Pond Snail). Reproducing adults of L. stagnalis are exposed to a concentration range of the test chemical and monitored for 28 days for their survival and reproduction (number of egg clutches). As additional information, the number of eggs per clutch may also be determined. Adult shell length increase may also be measured. The toxic effect of the test chemical on the cumulated number of clutches produced per individual-day is expressed as ECx by fitting an appropriate regression model to the data in order to estimate the concentration that would cause x% reduction in the reproductive output. Alternatively, the toxic effect of the test chemical can be expressed as the No Observed Effect Concentration and Lowest Observed Effect Concentration (NOEC/LOEC) values. Both ECx and NOEC/LOEC can be determined from a single study.

This test measures structural chromosomal aberrations (both chromosome- and chromatid-type) in dividing spermatogonial germ cells and is, therefore, expected to be predictive of induction of heritable mutations in these germ cells. The purpose of the in vivo mammalian spermatogonial chromosomal aberration test is to identify those chemicals that cause structural chromosomal aberrations in mammalian spermatogonial cells (1) (2) (3). In addition, this test is relevant to assessing genetoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA-repair processes are active and contribute to the response. The original Test Guideline 483 was adopted in 1997. This modified version of the Test Guideline reflects many years of experience with this assay and the potential for integrating or combining this test with other toxicity or genotoxicity studies.

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.

This Test Guideline describes a method to assess the effects of chemical substances in soil on the reproductive output of the soil mite species Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae). It can be used for water soluble or insoluble substances, but not with volatile substances. Adult females of similar age are exposed to a range of concentrations of the test substance mixed into 20 g dry mass of artificial soil 28-35 days after the start of the egg laying period. Depending on the endpoint (ECx, NOEC or both), five to twelve concentrations should be tested. At least two to four replicates for each test concentrations and six to eight control replicates, of 10 animals each, are recommended. At 20¡ãC, the test lasts 14 days after introducing the females, which usually allows the control offspring to reach the deutonymph stage. The number of surviving females (mortality ¡Ü 20% for a valid test) and the number of juveniles per test vessel (at least 50 for a valid test) are determined. The fecundity of the mites exposed to the test substance is compared to that of controls in order to determine the ECx (e.g. EC10, EC50) or the No Observed Effect Concentration (NOEC). Any observed differences between the behaviour and the morphology of the mites in the control and the treated vessels should be recorded.