Bacterial Reverse Mutation Test. ISO 10993-3: 2014 -Biological Evaluation of Medical Devices, Part 3: Test for Genotoxicity, Carcinogenicity and Reproductive Toxicity, and OECD 471: 2020-Bacterial Reverse Mutation Test.

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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 devices. 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) but in which the determination of the number of colonies and its size with a view to covering both types of objectives (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.

The test described in the OECD 471 (Bacterial Reverse Mutation Test), uses strains of Salmonella typhimurium and Escherichia coli that require the supply of essential aminoacids for their growth, because these strains are unable to synthesize them by being mutants deficient in the metabolic pathways that must synthesize them. When there is a mutagenic compound (genotoxic), this compound can induce the reversion of the mutations, restoring the metabolic functional capacity of the bacteria to synthesize the essential amino acids. Reverted bacteria are detected by their ability to grow in the absence of the aminoacids previously required by the parental test strain. This test is faster and easier to perform than the tests performed with mammalian cells described in the OECD 476 standard. However, when using prokaryotic cells that differ from mammalian cells in several characteristics such as the uptake of molecules, metabolism of molecules, chromosomal structure and DNA repair process, the results obtained are not completely comparable to those that could be induced by the mutagenic substance in mammals.

Therefore, it is used as an initial screening method to detect possible genotoxic activity and in particular to detect point mutations. There is enough information that shows that many chemicals that show a mutagenic effect with this test, also exhibit mutagenic activity in other tests that use mammalian cells. In the same way, there are chemical substances that do not show a mutagenic effect with this test and that nevertheless can be evidenced by other tests. It has sometimes been suggested that this test may overestimate the mutagenic activity of some substances. This test is not useful for evaluating chemical substances with bactericidal activity, nor for those substances that are known to interfere specifically with the replication of mammalian cells. Nor is there an absolute correlation between compounds that demonstrate mutagenic capacity with this test and its carcinogenic capacity.

The performance of this test involves using suspensions of bacteria that are exposed to the substances under evaluation, in the presence or absence of an exogenous metabolic activation system. There are basically two methods of realization: a) plate incorporation method, in which the suspensions are mixed with coating agar and immediately sown in minimal medium; b) pre-incubation method in which the mixture of the bacterial suspension and the substance under test is incubated and then mixed with coating agar to be sown on a minimal medium. In both methods, after 2 to 3 days of incubation colonies should be counted, to detect reverted and compared with colonies reverted spontaneously into control plates. The suspensions of bacteria (5 strains: four of Salmonella spp. and one of Escherichia coli) must be used in the phase of initial exponential growth or initial stationary. The substance under test must be used in at least five concentrations and the corresponding solvent/vehicle controls of the substance under test must be included. Likewise, substances with and without mutagenic capacity must be tested in parallel. The most accepted criterion of mutagenesis is the increase in the detection of mutants depending on the concentration within the tested range and/or reproducible increase at one or more concentrations in the number of reverted colonies per plaque in at least one strain, with or without activation metabolic.

Test phases

The test consists of two phases:

Phase 1 - Previous toxicity test with a bacterium (Salmonella spp. CECT-880) to verify that the product (or that extracted from a medical device) is not toxic at the concentration recommended by the standard or indicated by the customer. If the product (or its extracted is toxic, the test cannot be completed and only this part of the test will be billed.

Phase 2- Bacterial reverse mutation test with five strains. For solid non-soluble medical devices, the extract of the product should be tested. To analyze liquid or solid medical devices or chemical products, with which dilutions can be prepared, 5 concentrations will be evaluated. See the total cost of the test depending on the type of product to be tested.

Test concentration

The recommended maximum concentration according to the standard for soluble non-cytotoxic substances is 5 mg/plate or 5 µl/plate. Test products that are cytotoxic already below 5 mg/plate or 5 µl/plate should be tested up to a cytotoxic concentration. Testing above the concentration of 5 mg/plate or 5 µl/plate may be considered when evaluating products containing substantial amounts of potentially mutagenic impurities.

The client must indicate if he wishes to perform the test at the maximum concentration indicated by the standard or at another concentration. From the maximum concentration requested by the client, the 4 lower test concentrations to be carried out will be determined, following the interval indicated by the OECD 471 Guideline. Note that if the test product is a non-soluble solid medical device, the test will be carried out only with the pure extract of the product.