Genotoxicity. In vitro Mammalian Cell Gene Mutation Test using the Hprt gene – OECD 476: 2016.

Information 10-01-2017.

            Several types of in vitro tests can be used to perform the genotoxicity tests, either a series of two tests or three tests. When three tests are chosen, they will include: a test for gene mutations using bacteria (OECD 471), a test for gene mutations using mammalian cells (OECD 476), and a clastogenicity test using mammalian cells (OECD 473). When two tests are chosen, a test for gene mutations using bacteria (OECD 471) and a test for gene mutations using mammalian cells (OECD 476) will be required, but in which the determination of the number of colonies is made and its size with a view to covering both types of targets (gene mutations and clastogenicity). If the results of in vitro tests are negative, it is usually not indicated to perform tests on animals.

            The primary function of genotoxicity tests is to investigate, using cells or organisms, the potential of the products tested to induce genetic changes in humans that can be transmitted to future generations. The scientific data generally support the hypothesis that damage to the DNA of somatic cells is critical for the onset of cancer, so these tests can identify chemical substances with carcinogenic potential. As far as we know, there is no international agreement on the best combination of tests for a specific purpose. The most recommended methods are those described by the OECD 471 and OECD 476 standards.

            In the OECD 476: 2016, In vitro Mammalian Cell Gene Mutation Test using the Hprt gene, chemical mutations induced by chemical substances are detected using eukaryotic cells. For the HPRT test, various types of cell lines can be used such as CHO, CHL and V79 cells of hamster cells (Chinese hamster cells), L5178Y cells of mouse lymphoma, or TK6 cells (human lymphoblastoids).

            En la prueba OECD 476: 2016, in vitro de mutación de genes celulares de mamíferos (In vitro Mammalian Cell Gene Mutation Test using the Hprt gene), se detectan mutaciones génicas inducidas por sustancias químicas utilizando células eucariotas. Para la prueba HPRT, pueden utilizarse varios tipos de líneas celulares como las células CHO, CHL y V79 de células de hámster (Chinese hámster cells), las células L5178Y de linfoma de ratón, o las células TK6 (linfoblastoides humanas).

            In vitro tests of gene mutations of mammalian cell genes use established cell lines. These cells have been selected based on their growth capacity in culture and their gene stability in terms of the frequency of spontaneous mutations. In vitro tests may require the use of an exogenous metabolic activator, although this cannot completely simulate in vivo conditions. Conditions that may not reflect the intrinsic mutagenesis of the cells, such as variations in pH, osmolarity, interactions with components of the culture medium and cytotoxicity due to high concentrations of the tested substance, should be avoided. Many compounds for which a mutagenic potential is detected with this test are carcinogenic to mammals, but there is no perfect correlation between these results and carcinogenicity.

            The cells with Hprt (hypoxanthine-guanine phosphoribosyl transferase gene) (in the HPRT test), are sensitive to the action of 6-thioguanine -TG-, so they cannot develop in their presence. However, when mutated they are selected for their resistance to the nucleotide analog 6-thioguanine. The mutations detected by the HPRT test can correspond to nucleotide substitutions, frameshifts, small deletions or insertions located on the X chromosome.

            To perform the test, cell suspension or monolayer cultures, depending on the type of cells used, are exposed to the test substance, with and without metabolic activation, for an appropriate period of time and are subcultured to determine if they have been inhibited. by incorporating the nucleotide analogs or if on the contrary they are not inhibited by having mutated in the presence of the analog used. Cytotoxicity is determined by measuring the relative efficiency of cloning (survival) or the relative total growth of the cells of a culture after the exposure period. The treated cells are maintained in growth medium for a sufficient period of time, characteristic for each locus and cell type, to allow the phenotypic expression of the induced mutations.

            The frequency of mutants is determined by seeding a known number of cells in medium with the selective agent to determine the presence of the mutated cells, as well as in a culture medium without the selective agent to determine the cloning efficiency (viability). After a period of time the colonies are counted and the mutation frequency is obtained from the number of mutant colonies in selective medium and the number of colonies in non-selective medium. The test must use at least 4 concentrations that cover a maximum range with little or no toxicity if there is cytotoxicity, as well as positive controls with known substances capable of inducing mutations, either in the absence or in the presence of an exogenous metabolic activator. Exposures should be made between 3 and 6 hours in duplicate, including negative controls in duplicate.

            After exposure to the test product, the cells are cultured and incubated for a period of 7 to 9 days in cell growth medium, for the time to grow the cultures to proliferate or not depending on the genotoxicity of the test substance. Once that time has elapsed, to add the selective agent (6-thioguanine), the cells are cultured for a period of 9 to 12 days to allow the development of the mutant phenotype, if present.