Genotoxicity - ISO 10993-3: 2014. Biological Evaluation of Medical Devices, Part 3: Test for Genotoxicity, Carcinogenicity and Reproductive Toxicity) and OECD 476: 2016 (In vitro mammalian cell gene mutation test using the Hprt and xprt genes).

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The ISO 10993-3: 2014 standard indicates strategies and tests to identify the risks of genotoxicity, carcinogenicity and reproductive toxicity applicable to assess the potential of medical devices to cause genotoxicity, carcinogenicity and reproductive toxicity. These tests can be performed using mammalian cells, bacteria, yeast or filamentous fungi to determine if the samples tested can induce gene mutations, alter the chromosomal structure or cause other changes in the DNA.

The ISO 10993-1: 2009/2010 standard indicates when these tests should be considered in the biological safety evaluation of a medical device. This standard states that genotoxicity tests are not necessary for medical devices and their components, when it is known that they do not have genotoxicity. The tests are indicated when the materials included in medical devices can have components that can interact with the genetic material, or when the chemical composition of the medical device is unknown.

Several types of in vitro tests can be used to perform the genotoxicity tests, either in 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 test, in vitro mutation of mammalian cell genes (In vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes), chemical mutations induced by chemicals 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). For the XPRT test (gpt gene) the AS52 cells derived from the CHO can be used, which contain the gpt transgene and lack the Hprt gene that has been deleted. These latter cells cannot be used for the HPRT test because they lack the hprt gene. These cell lines can develop a mutation in the hprt gene (Hypoxanthine-guanine phosphoribosyl transferase), as well as transgenic mutations of the gpt (Xanthine-guanine phosphoribosyl transferase) transgene. Mutation tests of the hprt and gpt genes detect different spectra of gene affectations. The autosomal localization of the gpt transgene allows detecting gene disorders (deletions) not detectable in the hprt locus of the X chromosome. Currently, the OECD 476 standard recommends the detection of mutations in the hprt and gpt genes.

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.

Cells with Hprt (hypoxanthine-guanine phosphoribosyl transferase gene) (in the HPRT test) or with gpt (guanine phosphoribosyl transferase transgene-gpt) (in the XPRT test), are sensitive to the action of 6-thioguanine -TG-, so they cannot develop in its presence. However, when mutated they are selected for their resistance to the nucleotide analog 6-thioguanine. Tests to detect Hprt and gpt mutants detect different genetic changes. The mutations detected by the HPRT test may correspond to nucleotide substitutions, frameshifts, small deletions or insertions located on the X chromosome. However, the autosomal localization (not on X chromosome) of the gpt transgene (in the case of XRTP), allows the detection of large deletions and possibly of mitotic recombinations not detected in the HRTP test since the Hprt gene is 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 four 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, the cells are washed and cultured to determine their survival in the presence of the selective agent (6-thioguanine -TG-) and allow the mutant phenotype to develop, if present. The measurement of the mutagenesis is determined according to the relative efficiency of cloning (survival) or the total relative growth of the cultures, seeding in a selective medium and in a non-selective medium. Each locus investigated has a minimum required time that is at least 7 to 9 days. The mutagenic capacity of a product depends on the mutation frequency related to the concentration or the reproducible increase in the mutation frequency. If it does not increase, the tested substance is not considered mutagenic.