ToxTracker: Bringing Mechanistic Insight to Genotoxicity Testing of Medical Devices

ToxTracker (recently adopted as OECD 446A) is a mammalian cell-based reporter in vitro assay designed to provide mechanistic insight into the genotoxic mode of action of test substances in 2-3 weeks.

Rather than only indicating whether a compound is “positive” or “negative” for genotoxic effects, ToxTracker can help distinguish between direct and indirect genotoxic mechanisms as explicitly requested by the standard, such as indirect genotoxicity due to oxidative stress [1]. This is important, as regulatory frameworks for medical devices often differentiate between direct DNA-reactive substances that may trigger stricter safety evaluations and indirect genotoxic mechanisms, which can require additional mechanistic evidence to justify biocompatibility assessments.

ToxTracker: 6 Reporter Genes to Discriminate between Direct and Indirect Genotoxicity, and Cytotoxicity with High Accuracy

The mechanistic information provided by ToxTracker enables toxicologists to make more confident decisions and strengthen regulatory submissions with scientifically grounded explanations.

Here, we describe two use cases showing how ToxTracker can be used to support a weight-of-evidence approach or make faster and more informed decisions when preparing for regulatory submissions.

1: Cho E, et al. (2022). Environ Mol Mutagen, 63(3), 118–134. DOI: 10.1002/em.22479

Use Case 1: Resolving Unexpected Genotoxicity Results with Acrylate-based Medical Devices

In the development of medical devices, particularly those based on polymeric materials such as polyacrylates, safety assessment can sometimes generate conflicting genotoxicity data that complicates regulatory acceptance.

This first use case illustrates how a combination of the conventional bacterial and mammalian gene mutation assays and ToxTracker was used to resolve such uncertainty and support a clear regulatory conclusion.

The Problem – Unexpected outcomes with short- and long-term OECD 490

A manufacturer of polyacrylate-based medical devices conducted a standard genotoxicity test battery as part of ISO 10993 requirements for its regulatory submission. The bacterial Ames test (OECD 471) was negative, suggesting the material was not mutagenic in a prokaryotic system. However, the mammalian cell gene mutation assay (OECD 490) gave an unexpected outcome: no effect was observed after short-term exposure, but a positive signal was observed after long-term exposure conditions.

• Ames test (OECD 471): Negative
• Mouse lymphoma assay (OECD 490): Positive in long-term exposure, negative in short-term exposure.

This discrepancy created uncertainty with the sponsor and raised concerns about genotoxicity, which could trigger additional testing and delay the complex regulatory submission.

The sponsor proposed conducting an in vivo genotoxicity study; however, this approach was not accepted by the authorities, which requested a scientific rationale instead.

The Approach

To resolve this ambiguity, a weight-of-evidence strategy was applied.

First, with the support of a registered toxicologist, the sponsor conducted a literature review to evaluate known genotoxic mechanisms associated with acrylate-based materials.

Considering these findings, the time pressure and the need for mechanistic insight, the sponsor decided to contact us to perform ToxTracker.

Extracts from the medical device were prepared according to ISO 10993-12:2021, similar to what was done for the MLA assay, and were tested in the ToxTracker assay

Conclusions from the ToxTracker study:

• No indications for direct DNA reactivity
• Activation of the oxidative stress response, suggesting an indirect mechanism that could explain the positive genotoxicity result in the MLA.

The Outcome – Device approved for market

Extending the conventional genotoxicity testing strategy with mechanistic data from ToxTracker®, the overall weight of evidence indicated that the material was not directly genotoxic and that the positive OECD 490 result was likely a secondary effect related to oxidative stress.

As a result, no additional follow-up studies were required, and the medical device got market approval.

• A comprehensive weight-of-evidence package was submitted
• Regulatory authorities accepted the explanation
• No additional studies were required
• The device was approved for market

Use Case 2: Assessing Genotoxicity of Unknown Extractables from medical devices for Quick Decision-Making

In biomedical device development, chemical characterization of extractables is required under ISO standards to assess potential risks from substances released by a medical device.

The Problem – Unidentified extractable compounds in the chromatogram

During the final stage of medical device development, chemical analysis of extracts obtained from the device revealed unidentified extractable compounds in the chromatogram. According to regulatory guidance, unidentified substances must be treated as potentially mutagenic under a worst-case assumption. As such, those extractables did not pass the risk assessment as per ISO 10993-17. As a result, two additional OECD genotoxicity studies would need to be performed to exclude risk for mutagenicity. These assays are costly and time-consuming, with an estimated turnaround of six months.

The manufacturer was under high time and budget pressure and had a second R&D project whose initiation was directly dependent on the results of this late-stage project.

Challenges:

• Several extractable compounds from the medical device could not be identified
• ~6 months delay for additional OECD genotoxicity studies
• Limited budget and high time pressure
• A second R&D project dependent on the outcome

The Approach

To avoid unnecessary delay and cost escalation, the team decided to use ToxTracker instead of immediately proceeding with the required OECD tests. This approach aimed to estimate genotoxic potential early, before committing to full regulatory genotoxicity testing.

The Outcome

Within 10 days, extracts from the medical device were tested in the ToxTracker assay. ToxTracker was negative, suggesting that the device was unlikely to be genotoxic. This allowed informed decision-making: the team proceeded with confidence, launched the dependent R&D project, and later confirmed the prediction through the required OECD studies, which validated the non-genotoxic outcome.

Within 10 days:

• Results indicated no genotoxicity concern
• The team gained early confidence in the safety of the device
• The second R&D project proceeded without delay
• Subsequent OECD studies later confirmed the ToxTracker findings

Conclusion: Modernizing genotoxicity testing of medical devices

These examples highlight a reality in biomedical development where the uncertainty about genotoxicity and safety of medical devices that sometimes emerges when results are difficult to interpret can represent a major bottleneck for regulatory approval.

This is where ToxTracker can provide added value by helping bring clarity to unexpected equivocal or positive genotoxicity findings and providing mechanistic insight into what is driving a genotoxic response.

In this context, ToxTracker empowers teams to move beyond a “positive or negative” interpretation towards a deeper understanding of the underlying mechanism driving a positive call in the traditional genotoxicity tests and strengthening regulatory submissions.

As a result, ToxTracker supports more informed and confident decision-making by:

• Providing clarity on unexpected or equivocal genotoxicity results
• Delivering mechanistic evidence to strengthen regulatory arguments
• Reducing the need for other follow-up studies, saving both time and cost
• Keeping development timelines and downstream projects on track