What Is Genotoxicity Testing?

Genotoxicity testing, as outlined in ISO 10993-3:2023, is a fundamental component of the biological evaluation of medical devices intended to identify materials that may induce genetic damage, either directly to DNA or indirectly through cellular processes. This standard emphasizes that such testing is necessary when there is a potential for prolonged or repeated exposure to leachables that may interact with genetic material. Genotoxicity is assessed using a combination of in vitro and in vivo assays, each targeting different mechanisms of genetic toxicity.

The three primary types of tests include: (1) the Ames test (bacterial reverse mutation assay), which detects point mutations in the DNA of Salmonella typhimurium or E. coli strains; (2) the HPRT assay (hypoxanthine-guanine phosphoribosyltransferase mutation assay), an in vitro mammalian cell gene mutation test that measures forward mutations at the HPRT locus; and (3) the comet assay (single-cell gel electrophoresis), which detects DNA strand breaks and alkali-labile sites in individual eukaryotic cells, providing insight into primary DNA damage. Together, these assays offer a comprehensive evaluation of mutagenic and clastogenic potential, informing risk assessment and ensuring the genetic safety of device materials.

Types of Genotoxicity Testing

 The Ames Assay

The Ames test, formally known as the Bacterial Reverse Mutation Assay, is a cornerstone in the genotoxicity assessment of medical device materials as prescribed by ISO 10993-3:2022, which governs the biological evaluation of medical devices for genotoxic, carcinogenic, and reproductive toxicity. The test evaluates a material’s potential to induce point mutations in the DNA of Salmonella typhimurium or Escherichia coli strains that carry specific mutations preventing histidine or tryptophan biosynthesis.

Upon exposure to a mutagenic extract, these bacteria may undergo reverse mutations restoring their ability to synthesize the essential amino acid, enabling colony growth on minimal media. The number of such revertant colonies provides a direct measure of mutagenic potential.

In practice, the Ames test is performed using multiple bacterial strains with different mutation spectra to capture a range of genotoxic mechanisms, both in the presence and absence of a metabolic activation system (typically rat liver S9 mix). For medical device testing, extracts are prepared under controlled conditions as defined by ISO 10993-12, simulating clinical exposure scenarios. These extracts are tested at various concentrations alongside appropriate controls. A positive mutagenic response is indicated by a reproducible, dose-dependent, and statistically significant increase in revertant colonies compared to negative controls.

The test requires careful attention to strain selection, cytotoxicity assessment, validation of metabolic activation, and adherence to OECD TG 471 guidelines, all within a robust quality system to ensure regulatory acceptability and biological relevance.

The HPRT Assay

The HPRT gene mutation assay, a key component of the in vitro genotoxicity test battery recommended by ISO 10993-3:2022, is designed to detect gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in mammalian cells, typically using Chinese hamster ovary (CHO) or Chinese hamster lung (V79) fibroblast cell lines. The HPRT enzyme is involved in the purine salvage pathway, and mutations at this locus result in resistance to toxic purine analogs such as 6-thioguanine (6-TG). When cells acquire such mutations—either spontaneously or as a result of exposure to a genotoxic agent—they survive and form colonies in 6-TG–containing media, allowing for quantitative detection of induced gene mutations.

In the context of medical device evaluation, this assay is applied to extracts prepared from device materials, following the ISO 10993-12 sample preparation standard to reflect worst-case clinical exposure. The test is typically performed with and without metabolic activation (S9 mix) to detect both direct-acting and metabolically activated mutagens. The exposure is followed by an expression period (typically 7–9 days) to allow mutant phenotype expression, after which the treated cultures are plated in selective and non-selective media to determine mutation frequency.

Expert execution of this assay requires not only proficiency in mammalian cell culture techniques and stringent cytotoxicity controls but also validated protocols for expression, selection, and statistical interpretation of results. Its strength lies in the detection of forward mutations in mammalian cells, complementing the bacterial-based Ames assay and providing a more biologically relevant perspective for potential human health hazards.

The Comet assay

The Comet assay, also known as the Single Cell Gel Electrophoresis (SCGE) assay, is an advanced in vitro genotoxicity method recommended under ISO 10993-3:2022 for detecting primary DNA strand breaks at the single-cell level. Unlike mutation-based assays such as Ames or HPRT, the Comet assay provides direct evidence of DNA damage, including strand breaks, alkali-labile sites, and oxidative DNA lesions, which are early indicators of genotoxic insult. Cells exposed to test substances (often extracts of medical device materials, prepared per ISO 10993-12) are embedded in agarose on microscope slides, lysed to remove membranes and proteins, and subjected to alkaline electrophoresis. Damaged DNA migrates toward the anode, forming a “comet tail” when stained and visualized under fluorescence microscopy—the extent of which correlates with the degree of DNA damage.

From an expert standpoint, the Comet assay offers high sensitivity and broad applicability, especially for detecting genotoxic effects of low-extractable substances or complex mixtures often found in medical device materials. The assay can be applied to a variety of mammalian cell types (e.g., human lymphocytes or cultured cell lines), and testing is conducted both with and without metabolic activation (S9 mix) to capture pro-mutagenic compounds. Proper execution demands stringent control of electrophoresis parameters, cell viability, scoring criteria (tail length, tail moment, % DNA in tail), and statistical robustness.

Given its mechanistic relevance and ability to detect a range of genotoxic effects—including those missed by mutation assays—the Comet assay is particularly valuable as a complementary test within a weight-of-evidence approach in biological risk assessment of medical devices.

Why Choose International Nikopharmad Laboratory for Genotoxicity Testing?

ILAC Accreditation and ISO/IEC 17025 Certification

International Nikopharmad Laboratory is fully accredited under ISO/IEC 17025 and recognized by ILAC (International Laboratory Accreditation Cooperation)—a testament to our technical competence, methodological rigor, and compliance with globally accepted quality assurance systems. These accreditations ensure that our genotoxicity testing services meet the highest international benchmarks, delivering data that is both reliable and regulatory-ready for submission to health authorities worldwide, including the FDA, EMA, and PMDA.

Comprehensive Genotoxicity Testing Portfolio

Our laboratory offers a full suite of genotoxicity assays in alignment with ISO 10993-3, OECD Test Guidelines, and ICH S2(R1) recommendations. Our validated test systems include the Ames test (bacterial reverse mutation assay), in vitro HPRT gene mutation assay, and the Comet assay (single-cell gel electrophoresis)—each designed to detect specific mechanisms of DNA damage, gene mutation, and chromosomal instability. Whether your product requires basic screening or an in-depth multi-tiered risk assessment, we provide scientifically sound and regulation-compliant testing solutions.

Expertise, Infrastructure, and Technical Excellence

With deep-rooted experience in medical device and pharmaceutical safety evaluation, Nikopharmad is equipped with state-of-the-art platforms for both bacterial and mammalian cell-based assays. Our team of highly trained molecular biologists and toxicologists apply industry best practices and advanced statistical tools to ensure analytical sensitivity, biological relevance, and regulatory robustness of your test results. All procedures are performed under strict quality control systems, using fully validated SOPs and GLP-aligned workflows.

Timely Turnaround and Project Agility

We recognize the critical importance of time-to-market in product development. Our streamlined workflows, dedicated project management, and automation-integrated infrastructure enable rapid turnaround without compromising data integrity or scientific validity—allowing your development program to proceed without regulatory delay.

Confidentiality and Data Integrity

At Nikopharmad, we treat your proprietary data with the highest degree of confidentiality and security. We operate under strict non-disclosure agreements and implement robust data integrity protocols to protect your intellectual property throughout the testing lifecycle.

Partner with Nikopharmad for Your Genotoxicity Testing Needs

By partnering with International Nikopharmad Laboratory, you gain access to a world-class team, internationally accredited methods, and a service philosophy centered on scientific excellence, operational efficiency, and regulatory compliance. Trust us to deliver the genotoxicity data you need to bring safe, effective, and compliant medical and pharmaceutical products to the global market.

 To request testing or a complimentary consultation contact Nikopharmad

The timeline for genotoxicity testing

The HPRT assay typically takes 9 days, the Ames test approximately 14 days, and the Comet assay requires around 30 days to complete.

Reference:mddionline