
Toxys offers the ToxTracker® assay, our state-of-the-art stem cell-based reporter assay that provides mechanistic insights into the mode-of-action (MOA) of genotoxic properties of pharmaceutical compounds and chemicals. ToxTracker can contribute to a mechanism-based, animal-free, cancer hazard assessment of chemicals and pharmaceuticals. ToxTracker can be particularly useful in Adverse Outcome Pathway (AOP) modelling for genotoxic and non-genotoxic carcinogens by revealing genotoxic modes-of-action and several non-genotoxic modes of action, such as oxidative stress and protein damage. ToxTracker is available as a contract service, as a kit or under a license, allowing you to perform ToxTracker in your own laboratories.
Key Features
- Mechanistic insight into (geno)toxicity
- Unsurpassed sensitivity of 95% and specificity of 94%
- Excellent predictor for regulatory in vivo genotoxicity assays
- A low amount of compound required
- Rapid and cost effective
- Extensively validated and applied in regulatory files
ToxTracker service
ToxTracker is routinely performed at our state-of-the-art laboratory. You can send your compounds and receive a full report in most cases within 2-3 weeks. The assay is available as a service directly from Toxys and is also available through the research service marketplaces Science Exchange and Scientist.com. ToxTracker is also available through Charles River, IIVS and Labcorp, where the assay is performed under GLP. Please read more on Service.
ToxTracker kit and license
ToxTracker can also be installed in your laboratory either under license or as a kit. We have performed an extensive inter-laboratory validation of ToxTracker that showed an excellent transferability of the assay. We provide full support for the integration of ToxTracker in your toxicology laboratory. Please read more on Kit & License.
ToxTracker is a unique genotoxicity assay that combines multiple biomarkers to get mechanistic insight into the MOA 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 are associated with increased cancer risk. Please read more about the assay’s panel of GFP-based reporters and the rationale for developing the assay here.
ToxTracker highlights
- Stem-cell-based in vitro assay for mechanistic toxicity testing
- A unique collection of biomarkers that discriminate between induction of DNA damage, oxidative stress, protein damage and general cellular stress
- Excellent correlation with in vivo regulatory assays
Typical applications
- Identification of genotoxic compounds with high sensitivity and specificity
- High throughput genotoxicity screening
- Investigations on mode-of-action of genotoxic compounds
The ToxTracker suite
Several extensions for ToxTracker are available, for example, to further identify and investigate aneugens and clastogens or whether genotoxicity is caused indirectly by oxidative stress. Please visit the ToxTracker Suite for more information on the extensions.
Meet the study director for ToxTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on ToxTracker in understanding where and how ToxTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
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Will you also take me through the data after a project in a TC or meeting?
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Dr. Inger Brandsma
Study director for ToxTracker suite
Inger Brandsma obtained her PhD from the Erasmus Medical Centre in Rotterdam, where she studied the role of changes in DNA double strand break repair in cancer cells relating to resistance to PARP inhibitors. Inger joined Toxys as a senior scientist, utilising her expertise in DNA damage responses, genome stability and cancer to develop novel mechanistic toxicity assays. Inger was pivotal in developing ToxTracker extensions for ToxTracker ACE, AO and TubulinTracker.
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Do you also have a question? Click here te send a question to Inger.
From sample to report
The ToxTracker project starts with a broad dose range-finding which is performed to select the compound concentrations. Next, the ToxTracker cells are exposed to 5 selected concentrations of the test compound in the presence and absence of S9 liver extract. GFP reporter induction is measured by flow cytometry. Finally, data analysis is performed using the ToxPlot software and our study directors create a report with the date and the expert interpretation for the project.
The 2-minute animated video below explains the relevant steps in the lab when we test compounds using the ToxTracker assay.
ToxTracker experimental design
Typical output generated from the ToxTracker assay includes:
- Broad cytotoxicity profile of each test compound
- Assessment of the induction profile of each biomarker
- Effect of metabolization on the compounds
- Hierarchical clustering of the analysed compounds
- Optionally we can compare the ToxTracker profile to the reference library of compounds
Cytotoxic profile and dose-finding
GFP inductions by the reporters per cell line are measured using flow cytometry.
On the left, the cell survival is plotted against the compound concentrations. On the right, the GFP induction is plotted against the compound concentrations for every compound and per reporter cell line.
To allow a comparison of induction levels of the ToxTracker reporter cell lines for a large number of compounds, Toxplot – a dedicated data analysis software package – was developed. Toxplot performs analysis of the data and automatically generates a visual report for a convenient interpretation of obtained test results. Read more about ToxPlot here.
Hierarchical clustering of the analysed compounds
ToxPlot calculates for each treatment the induction of the different ToxTracker reporters at a predefined level of cytotoxicity (typically 10%, 25% and 50%). Next, all tested compounds are clustered based on the differential induction of the reporters and test results are visualised in an easily interpretable heatmap (as shown above). After expert interpretation, the results for the compounds are summarised in a table (as shown below).
Project report
The project report contains the protocol, technical details, cytotoxicity testing/dose range finding results, biomarker expression with test compounds, morphological analysis with test compounds, and Lowest Observed Adverse Effect Level (LOAEL) concentrations of the compounds. We are happy to share a dummy report with you. Please click here to request a dummy report.
Dissemination meeting
Upon sharing the draft report, we will invite you for a dissemination meeting where the study director will go over the conclusions in the report. We will make sure you understand the results and provide our expertise on the results and potential next steps.
The ToxTracker assay is extensively validated
The assay is extensively validated – well over 1500 compounds have been tested, including various types of customer compounds as well as reference compounds listed by EU’s Reference Laboratory for alternatives to animal testing (ECVAM library) and by the 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.
Regulatory validation of ToxTracker
Currently, an international interlaboratory validation study for ToxTracker according to OECD guidelines is ongoing. Seven laboratories with expertise in the field have tested a large selection of compounds. The first results of the study have been shared in March 2022. A publication on the results is currently in preparation. Please contact us if you are interested in learning more.
ToxTracker in regulatory applications and screening strategies
ToxTracker results have been used in various regulatory files and screening strategies. For example, ToxTracker data has been included in various ECHA and EMA safety dossiers to the regulatory authorities as part of a weight-of-evidence (WOE) strategy or as supplemental data.
The latest revision of the SCCS notes of guidance for testing of cosmetic ingredients includes ToxTracker within the toolbox for further evaluation. The mechanistic insight provided by ToxTracker into the genotoxicity of chemicals is useful as an animal-free assessment and in a WOE evaluation. Please click here to find the SCCS guidelines.
In 2016, Luijten et al. proposed an integrative tiered test strategy, which includes the ToxTracker assay. This strategy could detect both genotoxic as well as non-genotoxic carcinogens and improve understanding of the underlying mode-of-action. You can read the publication on this strategy here.
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) assays.
Correlation with the AMES/MLA assays
The Bscl2-GFP reporter in the ToxTracker is directly associated with the 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 the 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 the case of negative ToxTracker results in 30% of the cases, the compound gave a positive test result in the in vitro micronucleus or chromosomal aberration assay, in line with the frequency of misleading positive test results for the MNvit under cytotoxic conditions. However, the correlation with the in vivo MN/CA tests for non-genotoxic compounds was 93%.
Flyers
Publications
Posters
ToxTracker – A Key to Early-Stage Molecule Genetox Testing
Evaluation of the ToxTracker® assay for DNA damage and pro-oxidative MoA using 10 coded chemicals
Optimisation of in vitro metabolism using S9 liver extract in ToxTracker
The ToxTracker assay as a tool for mechanism-based (geno)toxicity screening of nanoparticles
Genotoxicity assessment of Antimony compounds using the ToxTracker assay
The ToxTracker reporter assay detects indirect genotoxicity by high levels of oxidative stress
Interlaboratory validation of the ToxTracker genotoxicity reporter assay
Webinars
Case studies
In this section, you will find several case studies across various industries where ToxTracker was implemented in testing strategies successfully.
Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines
In this review, Karlsson et al. evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz). It was concluded that ToxTracker can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs.
New strategies for the safety assessment of new ingredients without testing in animals
The Safety and Environmental Assurance Centre (SEAC) of Unilever has included ToxTracker as part of their next-generation risk assessment (NGRA) and this video shows how it enables the delivery of safe products without animal testing.
Meet the study director for ToxTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on ToxTracker in understanding where and how ToxTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
Click for the answer
Will you also take me through the data after a project in a TC or meeting?
Click for the answer

Dr. Inger Brandsma
Study director for ToxTracker suite
Inger Brandsma obtained her PhD from the Erasmus Medical Centre in Rotterdam, where she studied the role of changes in DNA double strand break repair in cancer cells relating to resistance to PARP inhibitors. Inger joined Toxys as a senior scientist, utilising her expertise in DNA damage responses, genome stability and cancer to develop novel mechanistic toxicity assays. Inger was pivotal in developing ToxTracker extensions for ToxTracker ACE, AO and TubulinTracker.
Can you help with a study design for difficult to dissolve compounds?
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Do you also have a question? Click here te send a question to Inger.
Why are there 6 biomarkers included in ToxTracker?
ToxTracker combines biomarkers for direct genotoxicity with several 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 of in vitro genotoxicity 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 the 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 vitro genotoxicity assays. Assessment of these non-genotoxic mechanisms can be very valuable in de-risking positive results from conventional in vitro genotoxicity 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 the 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 vivo genotoxic 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 the induction of DNA double-strand breaks. Activation of the Rtkn reporter shows a very strong correlation with the in vivo micronucleus (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 vitro MN/CA aberration tests is 100% for ToxTracker-positive compounds, but only 62% for ToxTracker-negative compounds. The limited correlation between ToxTracker and the in vitro MN/CA assays is likely caused by the high frequency of misleading positive test results under highly cytotoxic conditions in the in vitro MN/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. The advantages of ToxTracker are its high-throughput format, short turn-around times, low amount of test material required and very good correlation with the in vivo regulatory genotoxicity assays. However, the ToxTracker assay can also be highly valuable as a follow-up of an in vitro genotoxicity 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.
Click here to read more FAQs
Meet the study director for ToxTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on ToxTracker in understanding where and how ToxTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
Click for the answer
Will you also take me through the data after a project in a TC or meeting?
Click for the answer

Dr. Inger Brandsma
Study director for ToxTracker suite
Inger Brandsma obtained her PhD from the Erasmus Medical Centre in Rotterdam, where she studied the role of changes in DNA double strand break repair in cancer cells relating to resistance to PARP inhibitors. Inger joined Toxys as a senior scientist, utilising her expertise in DNA damage responses, genome stability and cancer to develop novel mechanistic toxicity assays. Inger was pivotal in developing ToxTracker extensions for ToxTracker ACE, AO and TubulinTracker.
Can you help with a study design for difficult to dissolve compounds?
Click for the answer
Do you also have a question? Click here te send a question to Inger.
Service
We perform ToxTracker as a service for our clients. We work together on this basis with various of the top 10 pharma, chemical and cosmetics companies. We act as an extension of your R&D team by building a scientific case and thinking along with you so that you will receive a solution that goes beyond a “yes or no” answer. We advise you on what can be done to provide further evidence on the mode-of-action for your compound.
You can send your compounds and receive a full report within 2-3 weeks, or use our Xpress service and get your results within 3 days! It is also possible to request a ToxTracker test via scientist.com. Are you interested in receiving a quote or do you have any questions? Please reach out!
Next steps after sharing your interest in ToxTracker
Practical information
Compound requirement
- Pharmaceuticals: 5-10mg
- Chemicals: 50-100mg
Turn around time
- 2-3 weeks
Type of solvents compatible with the assay
- DMSO
- PBS
- Water
- Other solvents can be discussed with our Study Director
A standard ToxTracker report includes
- Extensive cytotoxicity profile
- Assessment of mechanism of toxicity
- EC 10/25/50 and LC 10/25/50
- Toxicity profile ± metabolising system
- Comparison of ToxTracker profile to reference compounds
ToxTracker in Japan via Eolas Biosciences
Toxys has partnered with Eolas Biosciences to include ToxTracker® in their portfolio of genetic toxicology services for the Japanese Market. Visit this page to learn more.
ToxTracker via research marketplaces Science Exchange and scientist.com
Toxys has made its ToxTracker assay available through the research service marketplaces Science Exchange and Scientist.com. 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 offered by other CROs
Toxys has partnered with Charles River to include ToxTracker® in their portfolio of genetic toxicology services for the North American Market. In mid-May of 2023, IIVS will become the first Contract Research Organization to offer ToxTracker at a US-based laboratory. ToxTracker is also available via Labcorp in Harrogate.
Meet the study director for ToxTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on ToxTracker in understanding where and how ToxTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
Click for the answer
Will you also take me through the data after a project in a TC or meeting?
Click for the answer

Dr. Inger Brandsma
Study director for ToxTracker suite
Inger Brandsma obtained her PhD from the Erasmus Medical Centre in Rotterdam, where she studied the role of changes in DNA double strand break repair in cancer cells relating to resistance to PARP inhibitors. Inger joined Toxys as a senior scientist, utilising her expertise in DNA damage responses, genome stability and cancer to develop novel mechanistic toxicity assays. Inger was pivotal in developing ToxTracker extensions for ToxTracker ACE, AO and TubulinTracker.
Can you help with a study design for difficult to dissolve compounds?
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Do you also have a question? Click here te send a question to Inger.
Running ToxTracker in your labs
To accommodate the use of ToxTracker in your lab, Toxys offers the assay as a kit or under a license. The lab interested in using the assay will first be trained and validated as ToxTracker certified laboratory. After certification, the license can be obtained or the kits can be purchased as required to allow flexible use and provide extensive insight into (geno)toxicity for your compounds.
Training and certification
We are very proud of the consistency and quality of the ToxTracker assays. To guarantee the same for our customers using the assay in-house we only offer the license and kits to labs that have had the training and have been certified (certification is included in the ToxTracker starter package). The training entails a 3-5 day on-site visit from one of our scientists who will perform hands-on training. You will gain experience with culturing the cells and performing the analysis. After the training you will be able to perform the assay yourself. All relevant documents, such as calculation sheets and protocols will be shared before the training. By testing a number of reference compounds we will assess whether results are in line with our expectations and quality criteria. When this is finalised you will get a ToxTracker certification allowing you to obtain the license or order further ToxTracker Kits.
Technical Requirements
To run ToxTracker in-house you will need a cell culturing laboratory and a flow cytometer.
Flow cytometer requirements to run ToxTracker assay
- Suitable for 96-well plates
- (Orbital) shaker or needle with mixing capacity for sample uptake
- Laser for GFP (488 nm)
How to order?
If you are interested in obtaining the ToxTracker license or kit please contact us via the button below of by sending an email to orders@toxys.com.
Meet the study director for ToxTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on ToxTracker in understanding where and how ToxTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
Click for the answer
Will you also take me through the data after a project in a TC or meeting?
Click for the answer

Dr. Inger Brandsma
Study director for ToxTracker suite
Inger Brandsma obtained her PhD from the Erasmus Medical Centre in Rotterdam, where she studied the role of changes in DNA double strand break repair in cancer cells relating to resistance to PARP inhibitors. Inger joined Toxys as a senior scientist, utilising her expertise in DNA damage responses, genome stability and cancer to develop novel mechanistic toxicity assays. Inger was pivotal in developing ToxTracker extensions for ToxTracker ACE, AO and TubulinTracker.
Can you help with a study design for difficult to dissolve compounds?
Click for the answer
Do you also have a question? Click here te send a question to Inger.