Toxys offers the ToxTracker® assay, our state-of-the-art stem cell-based reporter assay that provides mechanistic insights into the genotoxic properties of pharmaceutical compounds and chemicals. ToxTracker can contribute to a mechanism-based, animal-free, cancer hazard and risk assessment of chemicals. ToxTracker can be particularly useful in an Adverse Outcome Pathway (AOP) approach for both genotoxic and non-genotoxic carcinogens by revealing genotoxic modes of action and a number of non-genotoxic modes of action, such as oxidative stress and protein damage.

Key Features

  • mechanistic insight into (geno)toxicity
  • unsurpassed sensitivity and specificity of 95%
  • excellent predictor for regulatory in vitro and in vivo assays
  • low amount of compound required
  • rapid and cost effective
  • metabolic activation using S9 liver extract
  • extensively validated

ToxTracker is an unique genotoxicity assay that combines multiple biomarkers to get mechanistic insight into the of action of genotoxic compounds. The assay not only includes markers for DNA damage as predictors for the standard genotoxicity assays, but also includes markers for non-genotoxic MOA, including oxidative stress, protein misfolding and general cellular stress. All these types of cellular damage have been associated with increased cancer risk. The assay is currently being used by many of the top 10 pharma, chemistry and cosmetics companies.

Key featurestoxtracker

  • Unique genotoxicity screening platform
  • stem-cell based in vitro assay for mechanistic toxicity testing
  • Unsurpassed sensitivity and specificity
  • Excellent correlation with regulatory assays

Unique biomarkers

The ToxTracker assays consist of 6 reporter cell lines which are developed for unique biomarkers that discriminate between induction of DNA damage, oxidative stress, protein damage and general cellular stress. Please read more on the biomarkers on the tab principles.

Extensively validated – high correlation with regulatory assays

The assay is extensively validated, over 450 compounds have been tested, including various types of customer compound as well as reference compounds listed by EU’s Reference Laboratory for alternatives to animal testing (ECVAM library) and by US’ Environmental Protection Agency (EPA; Toxcast library). The ToxTracker assay showed a very high sensitivity (94%) and specificity (95%) for the detection of genotoxic compounds and has a strong correlation with the regulatory assays. Please read more on the tab validation.

Extensively validated

The assay is validated using the ECVAM suggested library (EU) and a selection of compounds from the Toxcast library (USA) and of carcinogens and non-carcinogens. The ToxTracker assay showed a very high sensitivity (94%) and specificity (95%) for the detection of genotoxic compounds. Please read this article for more information on the validation. Currently over 450 compounds have been tested in the assay, and we are routinely screening for customers.


Correlation with regulatory genotoxicity assays

We have assessed the results from the ToxTracker assays with the standard in vitro and in vivo regulatory genotoxicity assays and found an excellent correlation with the gene mutation (Ames/ MLA) and chromosomal damage (MN/CA) test.

Correlation with the AMES/MLA assays
The Bscl2-GFP reporter in the ToxTracker is directly associated with induction of promutagenic DNA lesions. In a comparative study using the ECVAM-suggested library of mutagenic and non-mutagenic compounds (Kirkland et al 2016) we found that the Bscl2-GFP reporter was highly predictive for the bacterial Ames and mammalian MLA mutation assays. We found that if our biomarker is positive, 93% of these compounds will also be positive in a regulatory mutation test. When our reporter is negative 95% of the compounds will test negative in the Ames and/or MLA assay.

Correlation with the micronucleus assay
The Rtkn-GFP genotoxicity reporter is activated upon induction of DNA double strand breaks. Using the ECVAM library of genotoxic and non-genotoxic compounds (Kirkland et al 2016) we have found that this reporter was highly predictive for the in vitro micronucleus (MN) and chromosome aberration (CA) assays in case of genotoxic compounds. We found that if the Rtkn reporter is positive, the compounds will test 100% positive in the in vitro micronucleus or chromosomal aberration assay. However in case of a negative ToxTracker results, in 30% of the cases the compound gave a positive test results in the in vitro micronucleus or chromosomal aberration assay, in line with the frequency of misleading positive test results for the MNvit under cytotoxicxic conditions. However, the correlation with the in vivo MN/CA tests for non-genotoxic compounds was 93%.


Rationale for developing the assay

Interaction of newly developed materials, chemicals and drugs with biomolecules may disrupt cellular homeostasis and can ultimately lead to severe tissue damage or induction of cancer. Inflicted cellular damage is recognized by specialized sensor proteins that trigger a complex network of cellular signaling pathways resulting in activation or inactivation of specific enzymes and altered expression of distinct gene networks. Therefore, visualization of damage specific cellular stress response pathways that are activated upon exposure to chemicals or xenobiotics provides insight into the type and extent of cellular damage that has been induced and thus the biological (re)activity of compounds.

Identification of the genes that specifically represent certain types of damage

We have identified a panel of genes that are preferentially activated upon exposure to different classes of carcinogens following extensive whole-genome transcription profiling of mouse embryonic stem (mES) cells after exposure to over 40 different carcinogenic chemicals.


From the genes that were activated following exposure to the chemicals we have identified the genes that specifically represent DNA damage, oxidative stress and the unfolded protein response, being the major biological damages associated with carcinogenesis. We have identified 6 genes representing these damages very specifically for which we have created green fluorescent mES reporter cell lines, which combined form the ToxTracker assay.

Unique biomarkers

The ToxTracker assays consist of 6 reporter cell lines which are developed for unique biomarkers that discriminate between induction of DNA damage, oxidative stress, protein damage and general cellular stress. Genotoxicity is detected by the Bscl2-GFP reporter that is activated by promutagenic DNA lesions and DNA replication stress and the Rtkn-GFP reporter that is associated with DNA double strand breaks. The Srxn1-GFP and Blvrb-GFP reporters indicate activation of the Nrf2 and Hmox1 antioxidant responses. The Ddit3-GFP reporter is directly associated with the unfolded protein response and Btg2-GFP is activated as part of a p53-mediated stress response.


Cell system

ToxTracker consists of a panel of GFP-based reporters in mouse embryonic stem (mES) cells. mES cells are genetically stable and proficient in all cellular pathways required for accurate detection of potentially carcinogenic properties of compounds, in contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing.

Advantage of mouse embryonic stem cells

            • Untransformed, non-cancerous mammalian cell line
            • Infinite lifespan
            • Proficient in all major DNA damage signalling and cell cycle regulation pathways
            • Dividing rapidly

Please also see FAQ for questions on the cells used.

We aim to provide you with in-depth toxicity information on your compounds. We will inform you whether compounds are toxic, what mechanism of toxicity is involved and by using our proprietary ToxPlot software we can compare the toxic profiles of your compounds to a database of reference compounds for further interpretation of data (part of ToxTracker Plus). A standard report contains the following information:

            • Extensive cytotoxic profile
            • Assessment of DNA Damage, oxidative stress and protein damage at various concentrations
            • EC 10/25/50 and LC 10/25/50 calculations
            • Toxicity profile in absence or presence of metabolising system

ToxPlot software for data analysis

For data analysis, we have developed a dedicated software tool, ToxPlot. Automated data analysis and graphical representation of the test results allow clear and rapid assessment of the reactive properties of novel compounds. ToxPlot visualises induction of the ToxTracker reporters in a easy interpretable heatmap.

ToxPlot key features:

            • ToxPlot©, custom-made software (windows, macOS, linux)
            • Automated data integration for GFP reporter activation and toxicity
            • Statistical analysis, data clustering and heatmap creation

Read more on ToxPlot 

Example output

GFP inductions by our reporters is measured in 5000 cells using a flow-cytometer per reporter cell line. First the survival is plotted against the compound dosis (figure 1). Secondly the GFP induction is plotted against the compound dosis for every compound and per reporter cell line.

To generate easy to read heatmaps we plot GFP induction against a set cytotoxicity level (survival). Standard we plot at 10%, 25% and 50%cytotoxicity, but these can be changed according to customer specifications.

The results for the compounds are summarized in a table and explained later in the document. We state whether the compounds activate any of the markers, and if so at what levels of cytotoxicity and compare this to induction by the controls.


Step by step

The ToxTracker assay is a stem cell-based assay in which 6 GFP reporter cell lines are exposed to various concentrations of test compound.  The 2-minute animated video below explains the relevant steps in the lab when we test compounds using the ToxTracker assay.

From sample to report

Reception of your test compound at the Toxys lab is where our ToxTracker service starts.  Below is a visualisation of the sequence of events between receiving your test compound,  performing the ToxTracker assay and discussing the final results.

Application by type of compound

So far we have tested over 1000 compounds in the assay, including various reference compound libraries (ECVAM and ToxCast library), but we have been testing increasing numbers and types of customer compounds. We have experience testing compounds from the following sectors:

            • Pharmaceuticals
            • Chemicals
            • Cosmetics ingredients
            • Food products
            • nanomaterials
            • pesticides/herbicides
            • heavy metals
            • UVCB’s

So far we have demonstrated that the assay is compatible with many types of pharmaceuticals, chemicals and nanomaterials. Should you wish to learn more on which compound we have tested, or whether we have experience with your class of compounds please contact us via

Application in screening strategy

Our customers apply the assay in several variations in the screening strategy, which also varies widely over the different industries. Here we will explain the most common use strategies for the assay.

1) early non-regulatory preclinical development phase of pharmaceuticals. ToxTracker can be applied as early genotoxicity screen in parallel or as alternative to AmesII/MPF, GreenScreen HC, flow micronucleus. ToxTracker can predict with high accuracy the outcomes the regulatory in vitro battery of genotoxicity tests (positive in mutation or chromosome damage tests).
2) ToxTracker can be applied as follow-up of the regulatory in vitro battery of genotoxicity assays to provide insight into MOA of genotoxic compounds. This information can be used in a weight of evidence approach during hazard assessment of novel compounds.
3) As prescreen or as mechanistic follow up of the regulatory in vitro genotoxicity tests for cosmetic ingredients and final products.
4) Mechanistic studies and read across approaches for retesting of marketed chemicals under REACH and novel substances.
5) In vitro indicator test for a number of non-genotoxic mechanisms of toxicity that are associated with increased carcinogenicity hazard.

Use Case – ToxTracker in a regulatory file

We are frequently asked whether ToxTracker data have ever been included in safety dossiers to the regulatory authorities. The answer is yes, but often this information is not publicly available. And in many cases we simply don’t know. But now there is an example available of a regulatory dossier in which ToxTracker data have been applied in a weight of evidence strategie. In this case, the standard regulatory battery of genotoxicity assay  gave equivocal results and ToxTracker was used to understand the mode-of-action of the substance and argue that the compound was not genotoxic.
Click here for the use case:

Why are there 6 biomarkers included in ToxTracker?

ToxTracker combines biomarkers for direct genotoxicity with a number of markers for non-genotoxic effects that can indirectly cause DNA damage or that have been known to cause misleading positive results in the standard battery if in vitrogenotoxicity assays. ToxTracker consists of genotoxicity reporters for direct DNA binding (Bscl2) and induction of DNA double strand breaks (Rtkn), but also markers for oxidative stress (Srxn1 and Blvrb), cytotoxicity/apoptosis and p53 activation (Btg2) and protein damage (Ddit3). Collectively these 6 biomarkers address a broad spectrum of genotoxic and non-genotoxic mechanisms of toxicity that are associated with increased cancer risk.

Why would you include markers for non-genotoxic endpoints in a genotoxicity assay?

Induction of oxidative stress can lead to oxidative DNA lesions. Protein damage is a strong trigger for apoptosis. Therefore, these non-genotoxic mechanisms of toxicity can indirectly cause genotoxicity. However, compounds that cause oxidative stress or protein unfolding are generally not mutagenic. The ability to identify the mode-of-action of a compound and to discriminate between DNA binding and indirect genotoxicity can be crucial for hazard and risk assessment of a compound.
Furthermore, oxidative stress and protein damage have been described as potential causes for misleading positive test results in the in vitrogenotoxicity assays. Assessment of these non-genotoxic mechanisms can be very valuable in de-risking a positive results from the conventionalin vitrogenotoxicity assays.

What does a positive result for a non-genotoxic endpoint mean for genotoxicity classification?

A positive result for a non-genotoxic endpoint in absence of a negative genotoxic endpoint will not implicate a test compound as genotoxic. However, a compound tested as positively genotoxic as well as non-genotoxic offers support for positive results due to indirect genotoxicity. Dose response information from ToxTracker plays a crucial role in hazard assessment of a compound.

What is the sensitivity and specificity of ToxTracker?

The sensitivity of the ToxTracker assay is defined as the ability to correctly identify in vivogenotoxic compounds. The specificity indicates the accuracy of correctly classifying compounds as non-genotoxic. Activation of the Bscl2-GFP reporter in ToxTracker indicates induction of bulky, pro-mutagenic DNA lesions and DNA replication inhibition. Activation of the Bscl2 reporter shows a very strong correlation with the bacterial (Ames) and/or mammalian (MLA) mutation assays, with a sensitivity of 93% and a specificity of 95%. The Rtkn-GFP reporter in ToxTracker indicates induction of DNA double strand breaks. Activation of the Rtkn reporter shows a very strong correlation with the in vivomicronucleus (MN) and/or chromosome aberration (CA) assays, with a sensitivity of 92% and specificity of 93%. The correlation of the Rtkn reporter with the in vitroMN/CA aberration tests is 100% for ToxTracker-positive compounds, but only 62% for ToxTracker-negative compounds. The limited correlation between ToxTracker and the in vitroMN/CA assays is likely caused by the high frequency of misleading positive test results under highly cytotoxic conditions in the in vitroMN/CA tests.

Where is ToxTracker typically positioned in a genetox strategy?

The ToxTracker can be adopted during the early genotoxicity screening of compounds (Tier-1) as well as in a Tier-2 strategy. Advantages of ToxTracker are its high-throughput format, short turn-turn-around times, low amount of test material required and very good correlation with the in vivoregulatory genotoxicity assays. However, the ToxTracker assay can also be highly valuable as followup of an in vitrogenotoxicity assay to de-risk positive results. The mode-of-action information about a compound can be used to determine if it is possible to continue the development of a compound. The MoA information can also be used in a regulatory dossier under a weight of evidence approach.

Can ToxTracker results be included in registration dossiers for submission to regulatory agencies?

ToxTracker results have been accepted as part of regulatory dossiers submitted to various regulatory agencies. Using information from ToxTracker as weight of evidence for hazard and risk assessment for a compound is fully in line with the ICH S2(R1) and M7 guidelines.

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We can perform the assay for you at our state-of-the-art laboratory. You can then send your compounds and receive a full report, in most cases within 2 weeks. Most of our customers ask us to perform the assay as a fee-for-service project as we have all tools and equipment in house and are experienced in the data interpretation. We work together on this basis with various of the top 10 pharma, chemical and cosmetics companies

Technology access

The assay can also be installed in your laboratory either under license or as a kit. We have performed an extensive inter-laboratory validation of the ToxTracker that showed an excellent transferability of the assay. We provide full support for integration of the ToxTracker in your toxicology laboratory. Please contact us via the contact form below or via email:

ToxTracker in North America via Charles River

Toxys has partnered with Charles River to include ToxTracker® in their portfolio of genetic toxicology services for the North American Market. Visit this page to learn more.

ToxTracker via research marketplaces Science Exchange and

Toxys has made its ToxTracker assay available through the research service marketplaces Science Exchange and These platforms are used worldwide by large and small companies to source their testing services. Organisations listed as buyers can now make use of the standardised legal agreements of these platforms and efficiently order ToxTracker services.

ToxTracker Service Plan

Toxys offers a prepaid ToxTracker service plan for easy testing at lower cost with a lower administrative burden. Service plans are available in packages of 25, 50 or 100 tests. Once a package is purchased, tests can be ordered in a series of projects within the duration of a year. ToxTracker results are delivered according to our usual turnaround time of 2 to 3 weeks. Click to read more

ToxTracker Xpress

Do you need your genetic toxicology data as soon as possible to enable fast and informed decision making? We offer the ToxTracker Xpress service. In this package we will fast-track ToxTracker analysis for your compounds. Receive a full report within 3 working days for a maximum of 4 compounds. Click to read more

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Why use ToxTracker?

This video gives you insight into why you could use ToxTracker

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ToxTracker protocol

This video gives you insight into how the ToxTracker assay is performed in the lab

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More information

If you have any questions on the assay please contact us via or complete the contact form. Please also see:

Unraveling the toxic mode of action further: ToxTracker MoA Extensions

The standard ToxTracker identifies genotoxicity after 24 hours exposure to the compound. We can then discriminate between mutagenic DNA lesions and chromosome damage and indicate whether compounds induce oxidative stress and protein misfolding. Sometimes these results prompt further research into the toxic mode of action. We can extend the standard ToxTracker assay with additional protocols and assays aimed at further unraveling the genotoxic mode of action.

ToxTracker MoA allows:

  • Discriminate between a clastogenic and aneugenic mode-of-action
  • Identification of indirect genotoxicity by kinase inhibitors
  • further investigating oxidative stress (ROS production, mitochondrial damage)

Read more on ToxTracker MoA Extensions

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