The 2025 update of ISO 10993-1 extended genotoxicity evaluation requirements to new categories of medical devices, namely those in prolonged contact with mucosal membranes or breached surfaces. Among other changes, the revised standard also places a stronger emphasis on prioritizing non-animal methods during the biocompatibility evaluation.
Several other ISO 10993 standards have since been amended or revised to apply the changes to individual biocompatibility tests.1 With regard to genotoxicity, the increased emphasis on non-animal methods is clear in ISO 10993-3:2026, which introduces chemical characterization and toxicological risk assessment as an explicit approach to addressing genotoxicity. The revised standard also removes in vivo tests from the follow-up evaluation when initial in vitro test results are positive.2
When is genotoxicity evaluation and testing required?
According to ISO 10993-1:2025, an evaluation of genotoxicity is required when medical devices come into prolonged (>24 h) or long-term (>30 d) contact with any tissues except intact skin, or if they contact circulating blood as implants or as part of an extracorporeal circuit for any duration. Evaluation may also be required if the device is made with novel materials or intended for sensitive patient populations, even if the contact criteria above are not met.3
The evaluation does not necessarily have to include genotoxicity tests; instead, chemical characterization and toxicological risk assessment (ISO 10993-18 and ISO 10993-17) may be sufficient. According to section 4.1 of ISO 10993-3:2026, further testing is not needed if the toxicological risk assessment concludes that exposure to extractable and leachable chemicals is within acceptable levels.
How is genotoxicity testing conducted?
If the toxicological risk assessment cannot address genotoxicity concerns adequately, testing is conducted using at least two genotoxicity test methods outlined in ISO 10993-3. A combination of tests is required, as no single test can detect all types of genotoxins.
The test battery should include:
A bacterial reverse mutation (Ames) test, conducted in vitro in accordance with OECD 471, and
One of the following in vitro tests:
A mouse lymphoma assay (MLA) in accordance with OECD 490,
A mammalian chromosomal aberration test in accordance with OECD 473, or
A mammalian cell micronucleus test in accordance with OECD 487
The most common approach is combining an Ames test with a mouse lymphoma assay, as the MLA captures the broadest range of genotoxic compounds and is therefore recommended by the U.S. Food and Drug Administration (FDA). However, ISO 10993-3 states that all three mammalian cell tests are “equally acceptable.”4
In vitro tests are sufficient to address genotoxicity in most cases. In vivo tests are only necessary in highly specific circumstances, for example if the device is suspected of causing accumulation of nanomaterials or other particulate matter in certain organs or tissues.5
Sample preparation considerations
Test samples for genotoxicity assays are typically extracted in accordance with ISO 10993-12, using exaggerated extraction conditions and two vehicles, one polar and one nonpolar, that do not dissolve or degrade the test material.6 The recommended polar vehicles are saline for the Ames test and cell culture medium without serum for the mammalian cell genetic toxicity tests (OECD 490, 473, and 487). Typical nonpolar vehicles include DMSO (recommended), PEG 400, and ethanol. In the mammalian cell tests, the final solvent concentration in the culture medium should not exceed 1% to prevent solvent cytotoxicity from influencing the results.
In the case of liquid or soluble devices, testing can also be performed on solutions, suspensions, or the device itself. Both extracts and solutions should be used immediately, or within 24 hours at the latest, to prevent compositional changes or adsorption onto the extraction container.
Typical genotoxicity test procedure with the Ames and MLA methods
While at least two different test methods must be used in a full medical device genotoxicity test battery, the Ames test (OECD 471) is sometimes performed first as a relatively fast and cost-effective screening method. This makes it possible to identify and address possible signs of genotoxicity before the more expensive mammalian cell test is performed. However, if the goal is to get the device to market as quickly as possible, both tests can be initiated simultaneously.
In a typical Ames test, four bacterial strains of Salmonella typhimurium and one strain of E. coli are exposed to the polar and nonpolar medical device extracts or solutions, both with and without a metabolic activation mix (S9). Triplicate test samples and controls (negative and positive) are incubated for 2 to 3 days at 37 °C, after which revertant colonies (i.e., colonies that have undergone reverse mutation) are counted. OECD 471 lists several criteria for identifying a positive result, including a reproducible increase in revertant colonies at one or more concentrations in at least one bacterial strain. If there is no such increase, the device is considered non-mutagenic in the Ames test.7
An in vitro mammalian cell genetic toxicity test, such as the MLA (OECD 490), is still required to screen for genotoxic effects in mammalian cells, including chromosomal damage that cannot be detected in the Ames test. The MLA is based on exposing L5178Y (TK+/-) mouse lymphoma cell cultures to polar and nonpolar medical device extracts. Test samples and positive and negative controls are tested under three different experimental conditions: 3–4-hour treatment with and without metabolic activation, and 24-hour treatment without metabolic activation. After a phenotypic expression period of 2 days, the cells are incubated in the presence of the selective agent trifluorothymidine (TFT), which inhibits the growth of non-mutant cells. The cultures are examined for mutant colonies, and the device is considered mutagenic if the induced mutant frequency exceeds the predefined global evaluation factor (GEF).8
What to do if the results are positive or inconclusive?
If the results from either in vitro genotoxicity test are positive or equivocal (i.e., elevated, but not to the extent of meeting the criteria for a positive result), ISO 10993-3:2026 suggests three alternative courses of action:9
Performing further chemical characterization to identify genotoxic constituents, allowing the related risks to be eliminated or managed.
Modifying the test protocol to address suspected confounding factors, such as cytotoxicity, pH, osmolality, or particulate formation during extraction.
Acknowledging and managing the genotoxicity risk.
In vivo tests are not recommended as a follow-up because they are usually not sensitive enough to detect genotoxic substances at the low concentrations typically present in medical devices or their extracts.
Our genotoxicity testing services
Measurlabs supports medical device manufacturers with the full genotoxicity evaluation required under the most recent version of ISO 10993-3. For more information on the individual tests in the recommended in vitro test battery, see the following:
We also offer testing with additional methods, including the mammalian cell micronucleus test (MNT) and the in vitro chromosomal aberration test. Chemical characterization (ISO 10993-18) and toxicological risk assessment (ISO 10993-17), which should be completed before genotoxicity tests are performed, are also available, along with a comprehensive range of other ISO 10993 biocompatibility tests.
Contact our experts through the form below for more information or a quote.
References:
1 Updates include ISO 10993-6:2026 and ISO 10993-7:2026. A new version of ISO 10993-4 is under development.
2 The main changes compared to the previous version are summarized in the Foreword to ISO 10993-3:2026.
3 Section 6.5.7: Genotoxicity of ISO 10993-1:2025.
4 See section F. Genotoxicity, in FDA industry guidance document Use of International Standard ISO 10993-1, "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process" and Section 5.2.2: In vitro genotoxic test battery in ISO 10993-3:2026.
5 Section 5.2.3: In vivo genotoxicity testing and Annex D of ISO 10993-3:2026.
6 Annex A: Test sample preparation method for genotoxicity testing in ISO 10993-3:2026. Exhaustive extraction is named as the ideal preparation method, but exaggerated conditions are typically used due to being more practical.
7 Result evaluation and interpretation are discussed in paragraphs 32 to 38 in OECD Test Guideline 471, Bacterial Reverse Mutation Test.
8 Result evaluation and interpretation are discussed in paragraphs 61 to 64 of OECD Test Guideline 490, In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene. The GEF is 90 × 10−6 if cells are incubated in soft agar and 126 × 10−6 if they are incubated in microwells.
9 Section 5.2.4: Follow-up evaluation of ISO 10993-3:2026.