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 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 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.
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.
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.
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 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 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 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.
This Test Guideline describes an in vivo erythrocyte Pig-a gene mutation assay (hereafter called the Pig-a assay) which uses an endogenous mammalian gene, the phosphatidylinositol glycan class A gene (Pig-a), as a reporter of somatic-cell gene mutation. In vivo gene mutation tests, such as the Pig-a assay, are especially relevant for assessing mutagenicity because physiological factors, such as absorption of the test chemical from the site of exposure, distribution of the test chemical throughout the test system via systemic circulation, and in vivo metabolism and DNA repair processes, all contribute to the mutagenic responses. The Pig-a assay can be performed with commonly used strains of rats or mice and the test can be conducted without euthanizing the animals. These properties facilitate integration of the Pig-a assay into many in vivo rodent testing protocols.
This Test Guideline proposes defined approaches (DA) combining data generated in vitro methods, with information sources such as physicochemical properties. The prediction from a DA may be used alone to determine eye hazard potential according to the hazard classes of the UN GHS (Categories 1, 2, or not classified). A DA consists of a fixed data interpretation procedure (DIP) (i.e. a mathematical model, a rule-based approach) applied to data (e.g in silico predictions, in chemico, in vitro data) generated with a defined set of information sources to derive a prediction without the need for expert judgment. The DAs use method combinations intended to overcome some of the limitations of the individual, stand-alone methods in order to provide increased confidence in the overall obtained result.
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 describes in vitro assays, which use Androgen Receptor TransActivation (ARTA) to detect Androgen Receptor Agonists and Antagonists. The ARTA assay methods are mechanistically and functionally similar test methods that provide information on the transcription and translation of a reporter gene following the binding of a chemical to the androgen receptor and subsequent transactivation. The cell lines used in these assays express AR and have been stably transfected with an AR-responsive luciferase reporter gene, and are used to identify chemicals that activate (i.e. act as agonist) or inhibit (i.e. act as antagonists) AR-dependent transcription. Some chemicals may, in a cell type-dependent manner, display both agonist and antagonist activity and are known as selective AR modulators. The AR is activated following ligand binding, after which the receptor-ligand complex binds to specific DNA responsive elements and transactivates the receptor gene, resulting in an increase cellular expression of the luciferase enzyme. The enzyme then transforms the substrate to a bioluminescent product that can be quantitatively measured with a luminometer. This Test Guideline includes ARTA assays using the AR-EcoScreenTM cell line, the AR-CALUX® cell line, and 22Rv1/MMTV_GR-KO cell line.
Test No. 457: BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists
Information: Following the OECD Council decision, the Test Guideline 457 was deleted on 29 January 2018.
Those who would like to obtain it should contact [email protected]
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, which provides mechanistical information, and can be used for screening and prioritization purposes. The test system utilises the hERalpha-HeLa-9903 cell line derived from a human cervical tumor and stably transfected. This cell line can measure the ability of a test chemical to induce hERalpha-mediated transactivation of luciferase gene expression. The cells are exposed to 7 non-cytotoxic concentrations of the test chemical for 20-24 hours to induce the reporter gene products. Four reference chemicals should be included in each experiment: a strong estrogen (17beta-estradiol), a weak estrogen (17alpha-estradiol), a very weak estrogen (17alpha-methyltestosterone) and a negative control (corticosterone). The activity of the luciferase enzyme is measured in a luminometer. A test chemical is considered to be positive if the maximum response induced is equal to or exceeds 10% of the response of the positive control (1 nM 17alpha-estradiol) in at least two of two or two of three runs.
Software to be used with TG 425, 432, 455. Click here. Software not part of the Mutual Acceptance of Data.
This Performance-Based Test Guideline (PBTG) describes in vitro assays, which provide the methodology of Stably Transfected Transactivation to detect Estrogen Receptor Agonists and Antagonists (ER TA assays). It comprises mechanistically and functionally similar test methods for the identification of estrogen receptor agonists and antagonists and should facilitate the development of new similar or modified test methods. The two reference test methods that provide the basis for this PBTG are: the Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line, derived from a human cervical tumor, and the BG1Luc ER TA assay using the BG1Luc-4E2 cell line, derived from a human ovarian adenocarcinoma. The cell lines used in these assays express ER and have been stably transfected with an ER responsive luciferase reporter gene. The assays are used to identify chemicals that activate (i.e. act as agonists) and also suppress (i.e. act as antagonists) ER- dependent transcription. ER are activated following ligand binding, after which the receptor-ligand complex binds to specific DNA response elements and transactivates the reporter gene, resulting in increased cellular expression of a marker enzyme (e.g. luciferase in luciferase based systems). The enzyme then transforms the substrate to a bioluminescent product that can be quantitatively measured with a luminometer. These test methods are being proposed for screening and prioritisation purposes, but also provide mechanistic information that can be used in a weight of evidence approach.
This Performance-Based Test Guideline (PBTG) describes in vitro assays, which provide the methodology of Stably Transfected Transactivation to detect Estrogen Receptor Agonists and Antagonists (ER TA assays). It comprises mechanistically and functionally similar test methods for the identification of estrogen receptor agonists and antagonists and should facilitate the development of new similar or modified test methods. The two reference test methods that provide the basis for this PBTG are: the Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line, derived from a human cervical tumor, and the BG1Luc ER TA assay using the BG1Luc-4E2 cell line, derived from a human ovarian adenocarcinoma. The cell lines used in these assays express ER and have been stably transfected with an ER responsive luciferase reporter gene. The assays are used to identify chemicals that activate (i.e. act as agonists) and also suppress (i.e. act as antagonists) ER- dependent transcription. ER are activated following ligand binding, after which the receptor-ligand complex binds to specific DNA response elements and transactivates the reporter gene, resulting in increased cellular expression of a marker enzyme (e.g. luciferase in luciferase based systems). The enzyme then transforms the substrate to a bioluminescent product that can be quantitatively measured with a luminometer. These test methods are being proposed for screening and prioritisation purposes, but also provide mechanistic information that can be used in a weight of evidence approach.
This Performance-Based Test Guideline (PBTG) describes in vitro assays, which provides the methodology of Stably Transfected Transactivation to detect Estrogen Receptor Agonists (ER TAs). It comprises mechanistically and functionally similar test methods for the identification of estrogen receptor agonists and should facilitate the development of new similar or modified test methods. The two reference test methods that provide the basis for this PBTG are: the Stably Transfected TA (STTA) assay using the (h) ERá-HeLa-9903 cell line, derived from a human cervical tumor, and the BG1Luc ER TA assay using the BG1Luc-4E2 cell line, derived from a human ovarian adenocarcinoma. The cell lines used in these assays express ER and have been stably transfected with an ER responsive luciferase reporter gene. The assays are used to identify chemicals that activate the ER following ligand binding, after which the receptor-ligand complex binds to specific DNA response elements and transactivates the reporter gene, resulting in increased cellular expression of a marker enzyme (e.g. luciferase in luciferase based systems). The enzyme then transforms the substrate to a bioluminescent product that can be quantitatively measured with a luminometer.These test methods are being proposed for screening and prioritisation purposes, but also provide mechanistic information that can be used in a weight of evidence approach.
The objective of a combined chronic toxicity/carcinogenicity study is to identify carcinogenic and the majority of chronic effects, and to determine dose-response relationships following prolonged and repeated exposure.
The rat is typically used for this study. For rodents, each dose group and concurrent control group intended for the carcinogenicity phase of the study should contain at least 50 animals of each sex, while for the chronic toxicity phase of the study should contain at least 10 animals of each sex. At least three dose levels should be used, in addition to the concurrent control group for both the chronic toxicity phase and the carcinogenicity phase of the study. The three main routes of administration are oral, dermal, and inhalation. The Test Guideline focuses on the oral route of administration.
The period of dosing and duration of the study is normally 12 months for the chronic phase, and 24 months for the carcinogenicity phase. The study report should include: measurements (weighing) and regular detailed observations (haematological examination, urinalysis, clinical chemistry), as well as necropsy procedures and histopathology. All these observations permit the detection of neoplastic effects and a determination of carcinogenic potential as well as the general toxicity.