Genotoxicity Testing (ISO 10993-3 & ISO 10993-33)
Genotoxicity testing is a specialized suite of in vitro assays designed to identify whether a medical device contains leachable substances that can cause genetic mutations, chromosomal damage, or other permanent genetic alterations. Because DNA damage can lead to cancer or heritable defects, these tests are mandatory for all devices with long-term or permanent contact with the human body.
At EBI, we follow a tiered approach according to ISO 10993-3 and the more recent ISO 10993-33 guidance, ensuring that your device’s mutagenic potential is fully characterized for regulatory submissions (MDR, FDA, MHLW).
Our Genotoxicity Assays.
To cover all types of potential genetic damage, regulatory bodies typically require a battery of tests that look at both bacterial and mammalian cell models.
1. AMES Test (Bacterial Reverse Mutation Assay)
The Ames test is a primary screening assay for the detection of gene (point) mutations. It employs specific strains of Salmonella typhimurium and Escherichia coli that are unable to synthesize essential amino acids, such as histidine or tryptophan. Reversion of this deficiency following exposure to a test substance indicates mutagenic potential, and the assay is typically conducted both with and without metabolic activation (S9 mix).
- Methodology: The Ames test using the microplate method (MPA) involves exposing selected strains of Salmonella typhimurium and Escherichia coli to the test item. These strains carry mutations that prevent the synthesis of essential amino acids, and restoration of this ability indicates the occurrence of reverse mutations. The test enables the detection of point mutations, including base-pair substitutions and frameshift mutations. The assay is conducted in parallel with and without metabolic activation (S9), and following incubation, the growth of revertants is assessed in a microplate format.
- Analysis & Evaluation: After 48 hours of incubation, the number of positive wells (color change of the medium) is evaluated as an indicator of mutational reversions, in comparison with negative and positive controls, with and without metabolic activation (S9). A biologically relevant, typically at least two-fold increase in the number of positive responses compared to the negative control indicates the mutagenic potential of the test substance.
- Standard Turnaround Time: 6 weeks.
2. Mouse Lymphoma Assay (MLA)
The Mouse Lymphoma Assay (MLA) is an in vitro mammalian cell assay used to detect gene mutations at the thymidine kinase (TK) locus. Unlike bacterial assays, the MLA enables the assessment of a broad spectrum of genetic alterations, including gene mutations as well as large-scale genetic events of a chromosomal nature. The assay is conducted both with and without metabolic activation (S9 fraction).
- Methodology: The MLA test is performed using the L5178Y TK+/– mouse lymphoma cell line. Cells are exposed to the test item extract for 4 hours in the presence and absence of metabolic activation (S9) and for 24 hours without metabolic activation. Following the exposure and expression periods, cells are plated in selective medium containing trifluorothymidine (TFT). Only cells that have acquired mutations in the TK gene are able to survive and form colonies under selective conditions. The assay enables the detection of both point mutations and larger-scale genetic alterations.
- Analysis & Evaluation: Following incubation, mutant frequency is determined based on the number of TFT-resistant colonies. Colonies are classified according to their size: large colonies are typically associated with point mutations, whereas small colonies indicate deletions or other chromosomal-type genetic alterations. Results are compared with negative and positive controls, both with and without metabolic activation (S9). A statistically and biologically relevant increase in mutant frequency compared to the negative control is considered evidence of the mutagenic potential of the test item.
- Standard Turnaround Time: 8 weeks (ISO), 12 weeks (GLP).
Technical Specifications and Requirements
Genotoxicity assays require high concentrations of extracts to ensure sensitivity. Please refer to the sampling requirements below:
MDR standard:
| Test Type | Material Thickness | Surface Area Required | Weight / Volume | Minimum Quantity |
|---|---|---|---|---|
| AMES Assay | < 0.5 mm | 30 cm² | - | 4 items |
| ≥ 0.5 mm | 15 cm² | - | 4 items | |
| Liquid / Powder | - | 5 grams / 10 mL | - | |
| Mouse Lymphoma | < 0.5 mm |
pretest: 30 cm² testing: 420 cm² |
- |
pretest: 2 items testing: 1 item |
| ≥ 0.5 mm |
pretest: 15 cm² testing: 210 cm² |
- |
pretest: 2 items testing: 1 item |
|
| Liquid / Powder | - | 10 grams / 30 mL | - |
Note: For MHLW (Japanese market) submissions, specific volume requirements may apply (typically 20 ml or 8 grams). Please specify your target market during the inquiry.
GLP standard:
| Test Type | Material Thickness | Surface Area Required | Weight / Volume | Minimum Quantity |
|---|---|---|---|---|
| AMES Assay | < 0.5 mm | 30 cm² | - | 5 items |
| ≥ 0.5 mm | 15 cm² | - | 5 items | |
| Liquid / Powder | - | 5 grams / 20 mL | - | |
| Mouse Lymphoma | < 0.5 mm | 30 cm² | - | 5 items |
| ≥ 0.5 mm | 15 cm² | - | 5 items | |
| Liquid / Powder | - | 5 grams / 20 mL | - |
Compliance with Global Standards
Our protocols are designed to satisfy the requirements of global notified bodies and health authorities:
- ISO 10993-3: Tests for genotoxicity, carcinogenicity, and reproductive toxicity.
- ISO 10993-33: Guidance on tests to evaluate genotoxicity – Supplement to ISO 10993-3.
- OECD 471: Bacterial Reverse Mutation Test.
- OECD 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene.
How We Work
Discovery & Insight
We begin with an in-depth consultation to understand your needs, challenges, and objectives. This helps us define the right strategy and scientific approach for your project.
Design & Development
Our experts translate your vision into actionable plans — combining innovative research, data, and technology to build precise and reliable solutions.
Delivery & Support
We implement the solution with full transparency and ongoing communication. After delivery, we stay involved — supporting you with optimization, updates, and future improvements.
FAQ: Genotoxicity Strategies
Why do I need both the Ames and the MLA test?
Bacterial DNA (Ames) and mammalian DNA (MLA) react differently to chemical stressors. The Ames test is excellent for detecting simple mutations, while MLA can detect more complex damage to chromosomes. Using both provides a robust „weight-of-evidence” approach required by most regulators.
What is the role of the S9 metabolic activation?
Some chemicals are not mutagenic themselves but become mutagenic after being metabolized by the liver. We use S9 (a liver enzyme fraction) to simulate this biological process in the lab.
What happens if my device shows a positive result in a genotoxicity test?
A positive result does not necessarily mean your device is unsafe, but it does require a detailed toxicological risk assessment or follow-up in vivo testing (such as a Micronucleus test) to clarify the risk.
Why do I need both the Ames and the MLA test?
Bacterial DNA (Ames) and mammalian DNA (MLA) react differently to chemical stressors. The Ames test is excellent for detecting simple mutations, while MLA can detect more complex damage to chromosomes. Using both provides a robust „weight-of-evidence” approach required by most regulators.
What is the role of the S9 metabolic activation?
Some chemicals are not mutagenic themselves but become mutagenic after being metabolized by the liver. We use S9 (a liver enzyme fraction) to simulate this biological process in the lab.
What happens if my device shows a positive result in a genotoxicity test?
A positive result does not necessarily mean your device is unsafe, but it does require a detailed toxicological risk assessment or follow-up in vivo testing (such as a Micronucleus test) to clarify the risk.
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Location:
Europejski Instytut Biomedyczny sp. z o.o.
ul. Bartycka 63A lok. 6, 00-716 Warszawa
Mail:
contact@ebi.bio
Phone:
+48 22 780 06 32
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