Genotoxicity
OECD 471 Bacterial Reverse Mutation Test (AMES Test)
Study Purpose and Introduction:
The bacterial reverse mutation test is a microbial assay which detects point mutations induced by chemicals causing base changes or frameshift mutations in the genome of amino-acid requiring strains of Salmonella typhimurium and Escherichia coli. The auxotrophic S. typhimurium and E. coli strains are unable to grow on minimal medium – containing inorganic salts and glucose as a carbon source – except for spontaneous revertants. But in the presence of a mutagenic agent, some of them can be converted to prototrophs after a reverse mutation to the wild type. These revertants can grow and form colonies on minimal medium.
An increased number of the revertant colonies indicates mutagenic activity of the test item. The tests are performed in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/b-naphthoflavone-induced rats (±S9). The standard testing procedures are plate incorporation and pre-incubation methods.
The bacterial mutagenicity test, along with other short-term assays, is used extensively to evaluate substances for mutagenic activity. For some mutagens, special cases that are poorly detected in the standard assays, modifications of standard procedures are required.
Modifications of standard procedure for special cases:
– „Micro Ames”: fluctuation method in microplate format;
– Prival modification for azo-dyes and diazo compounds;
– Treat and Wash procedure for peptides and amino acid containing materials;
– Procedure for investigation of glycosides;
– Enhanced Ames Test (EAT) Conditions for N-nitrosamines (EMA/409815/2020).
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 471, “Bacterial Reverse Mutation Test” (link: https://www.oecd-ilibrary.org/environment/test-no-471-bacterial-reverse-mutation-test_9789264071247-en)
– ICH S2 (R1)
– OPPTS 870.5100
OECD 471 Bacterial Reverse Mutation Test (AMES Test)
Study Purpose and Introduction:
The bacterial reverse mutation test is a microbial assay which detects point mutations induced by chemicals causing base changes or frameshift mutations in the genome of amino-acid requiring strains of Salmonella typhimurium and Escherichia coli. The auxotrophic S. typhimurium and E. coli strains are unable to grow on minimal medium – containing inorganic salts and glucose as a carbon source – except for spontaneous revertants. But in the presence of a mutagenic agent, some of them can be converted to prototrophs after a reverse mutation to the wild type. These revertants can grow and form colonies on minimal medium.
An increased number of the revertant colonies indicates mutagenic activity of the test item. The tests are performed in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/b-naphthoflavone-induced rats (±S9). The standard testing procedures are plate incorporation and pre-incubation methods.
The bacterial mutagenicity test, along with other short-term assays, is used extensively to evaluate substances for mutagenic activity. For some mutagens, special cases that are poorly detected in the standard assays, modifications of standard procedures are required.
Modifications of standard procedure for special cases:
– „Micro Ames”: fluctuation method in microplate format;
– Prival modification for azo-dyes and diazo compounds;
– Treat and Wash procedure for peptides and amino acid containing materials;
– Procedure for investigation of glycosides;
– Enhanced Ames Test (EAT) Conditions for N-nitrosamines (EMA/409815/2020).
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 471, “Bacterial Reverse Mutation Test” (link: https://www.oecd-ilibrary.org/environment/test-no-471-bacterial-reverse-mutation-test_9789264071247-en)
– ICH S2 (R1)
– OPPTS 870.5100
OECD 473 In vitro Mammalian Chromosome Aberration Test
Study Purpose and Introduction:
This in vitro test plays an important role in the evaluation of genotoxicity of a given item or agent. It is used to detect structural chromosome aberrations in somatic cells. Structural aberrations develop due to breaks in one or both DNA strands which can result in chromosome fragments (breaks, deletions). Faulty reunion of chromosome fragments results in formation of exchanges. These aberrations can be detected and quantified by light microscope. Extensive chromosome breaks usually cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are, however, not necessarily lethal and can be regarded as an indication of molecular events, which might lead to malignant transformation. The purpose of this study is to establish the potential of the test item to induce structural chromosome aberrations in cultured Chinese hamster cells.
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 473, “In vitro Mammalian Chromosome Aberration Test” (link: https://www.oecd-ilibrary.org/environment/test-no-473-in-vitro-mammalian-chromosome-aberration-test_9789264071261-en)
– OPPTS 870.5375
– EU Method B.10 (Mutagenicity – In Vitro Mammalian Chromosome Aberration Test)
OECD 474 Mammalian Erythrocyte Micronucleus Test
Study Purpose and Introduction:
The purpose of this study is to determine whether the test item causes genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated animals. 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 cells of animals, usually rodents. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.
Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 4, No. 474, “Mammalian Erythrocyte Micronucleus Test” (link: https://www.oecd-ilibrary.org/environment/test-no-474-mammalian-erythrocyte-micronucleus-test_9789264224292-en)
OPPTS 870.5395
EU Method B.12 (Mutagenicity – In Vivo Mammalian Erythrocyte Micronucleus Test)
OECD 475 Mammalian Bone Marrow Chromosomal Aberration Test
Study Purpose and Introduction:
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.
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 475, “Mammalian Bone Marrow Chromosomal Aberration Test” (link: https://www.oecd-ilibrary.org/environment/test-no-475-mammalian-bone-marrow-chromosomal-aberration-test_9789264264786-en)
– OPPTS 870.5385
– EU Method B.11 (In vivo mammalian bone-marrow cytogenetic test, chromosomal analysis)
OECD 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
Study Purpose and Introduction:
The objective of this study is to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Many mammalian cell gene mutation systems are available which may give a measure of the intrinsic response of the mammalian genome or its maintenance processes to mutagenes. The system used in this study has been extensively validated. The method is based on the detection of mutations (either induced or spontaneously generated) in the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located on the X chromosome. HPRT is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis. If a toxic base analog 6-thioguanine (6-TG) is present in the medium, then the analog will be phosphorylated via the HPRT pathway and incorporated into the nucleic acid. Thus, the cells die unless the enzyme is rendered inactive, by mutation.
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 476, “In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” (link: https://www.oecd-ilibrary.org/environment/test-no-476-in-vitro-mammalian-cell-gene-mutation-tests-using-the-hprt-and-xprt-genes_9789264264809-en)
– OPPTS 870.5300
– EU Method B.17 (In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes)
OECD 487 In Vitro Mammalian Cell Micronucleus Test
Study Purpose and Introduction:
The objective of this study is to evaluate the clastogenic and aneugenic potential of test item by its effects on the frequency of micronuclei in cultured mouse lymphoma L5178Y TK+/- 3.7.2 C or Chinese hamster V79 lung cell lines treated in the absence and presence of a rat liver metabolising system. The in vitro micronucleus assay is a genotoxicity test system used for the detection of micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The assay detects the activity of both clastogenic and aneugenic chemicals in mammalian cells that have undergone cell division during or after exposure.
Aneugenicity follow-up tests using fluorescence microscopy with pan-centromeric FISH probes enable centromeric staining and analysis to distinguish between centromere-negative (indicative of chromosome breakage) and centromere-positive (indicative of chromatid or chromosome loss) micronuclei
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 487, “In Vitro Mammalian Cell Micronucleus Test” (link: https://www.oecd-ilibrary.org/environment/test-no-487-in-vitro-mammalian-cell-micronucleus-test_9789264264861-en)
– EU Method B.49: In Vitro Mammalian Cell Micronucleus Test
OECD 489 In Vivo Mammalian Alkaline Comet Assay
Study Purpose and Introduction:
The purpose of the comet assay (single cell gel electrophoresis assay) is to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organ tissues. The comet assay is a method for measuring DNA strand breaks in the individual eukaryotic cells. It has gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. DNA fragments migrate from the “head” into the “tail” based on their size and the intensity of the comet tail relative to the total intensity (head plus tail) reflect the amount of DNA strand breaks. The corresponding measurements are performed by appropriate image analysis system.
Modifications of standard procedure for special cases:
Combined bone marrow micronucleus and comet assay on different target tissues
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 489, “In Vivo Mammalian Alkaline Comet Assay” (link: https://www.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264264885-en)
– EU Method B.62: Mutagenicity – In Vivo Mammalian Alkaline Comet Assay
OECD 490 In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene
Study Purpose and Introduction:
The purpose of the in vitro mammalian cell gene mutation tests is to detect gene mutations induced by chemicals. The cell line used in this test measure forward mutations in reporter genes, specifically the endogeneous thymidine kinase gene (TK).
The studies on mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (MLA) test the potential of test item to cause gene mutation and/or chromosome damage.
Cells in suspension are exposed to the test chemical, both with and without an exogenous source of metabolic activation (±S9), for a suitable period of time, and then sub-cultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection.
The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of the cell type, to allow near-optimal phenotypic expression of induced mutations. Following phenotypic expression, mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant colonies, and in medium without selective agent to determine the cloning efficiency (viability). Mutant frequency is calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.
Guidelines and literature:
– OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, “In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene” (link: https://www.oecd-ilibrary.org/environment/test-no-490-in-vitro-mammalian-cell-gene-mutation-tests-using-the-thymidine-kinase-gene_9789264264908-en)
– ICH S2 (R1)
– OPPTS 870.5300
– EC B.67 In Vitro mammalian cell gene mutation tests using the thymidine kinase gene