ToxTracker

ToxTracker is a unique genotoxicity assay that combines multiple biomarkers to get mechanistic insight into the mode 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 are associated with increased cancer risk. The assay is part of the ToxTracker Suite and is currently being used by many of the top 10 pharma, chemistry and cosmetics companies.

Key features

• 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 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 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. Please read more on the tab validation.

Extensively validated

The assay is validated using the ECVAM suggested library (EU), a selection of compounds from the Toxcast library (USA) and carcinogens and non-carcinogens. The ToxTracker assay showed a very high sensitivity (94%) and specificity (95%) for the detection of genotoxic compounds. 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 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%.

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.

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 cell survival is plotted against the compound concentrations (figure 1). Secondly, the GFP induction is plotted against the compound concentrations for every compound and per reporter cell line.

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

The results for the compounds are summarised 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.

Application of ToxTracker

So far, we have tested over 1200 compounds in the ToxTracker assay, including various reference compound libraries suggested by ECVAM and in the US ToxCast program. We have also tested a large number of proprietary compounds with historical genotoxicity information available. ToxTracker has been extensively validated using small molecules, but other modalities have also been tested in the assay. We have experience in testing compounds from the following sectors:

• • • • • • Pharmaceuticals
          • Chemicals
          • Cosmetics ingredients
          • Food products
          • Nanomaterials
          • Pesticides/Herbicides
          • Heavy metals
          • UVCB’s

Thus far, we have found that the assay is compatible with many types of pharmaceuticals, chemicals, and nanomaterials. Should you wish to learn more about the compounds that we have tested or our experience with specific classes of compounds, please contact us at info@toxys.com.

Genotoxicity screening strategy

ToxTracker has been successfully applied as a fast and reliable screening assay, with an excellent correlation with the in vivo regulatory genotoxicity assays. Examples of the most commonly used applications for ToxTracker are as follows:

• Early non-regulatory preclinical development phase of pharmaceuticals. ToxTracker was applied as an early genotoxicity screen in parallel or as an alternative to AmesII/MPF and in vitro (flow-based) micronucleus assays. ToxTracker could predict with high accuracy the outcomes of the regulatory in vitro battery of genotoxicity tests (positive in mutation or chromosome damage tests).
• ToxTracker was applied as a follow-up of the regulatory in vitro battery of genotoxicity assays to provide insight into the MoA of genotoxic compounds. The MoA information was used in a weight of evidence approach during hazard assessment of novel compounds.
• ToxTracker was applied in screening or as a mechanistic follow-up of the regulatory in vitro genotoxicity tests for cosmetic ingredients and final products.
• Mechanistic studies and read-across approaches for retesting of marketed chemicals under REACH.
• ToxTracker was used as an in vitro indicator test for several non-genotoxic mechanisms of toxicity that are associated with increased carcinogenicity hazard.

ToxTracker in regulatory applications

We are frequently asked whether ToxTracker data has ever been included in safety dossiers to the regulatory authorities. While indeed, the data is included in these dossiers, this information is not always publicly available. We have put together an example of a regulatory dossier in which ToxTracker data has been applied in a weight of evidence strategy. 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. Please see Applications & Case Studies for more information. There is currently an international OECD validation for ToxTracker running. Publication of the results is expected to happen in 2021.

Timeline for the regulatory validation of ToxTracker

• In 2016 ToxTracker was submitted to ECVAM for validation
• In 2016, an SPSF application for regulatory validation of ToxTracker was submitted to the OECD.
• In 2017 an OECD validation of the ToxTracker was started. Seven expert labs installed the ToxTracker assay in their labs and tested a large collection of compounds.
• In 2020, most of the experimental work in the OECD validation project has been collected and data analysis was started
• Publication of the results from the validation project is expected in 2021

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 recognised by specialised sensor proteins that trigger a complex network of cellular signalling 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 were combined to 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

Click here to read more FAQs

ToxTracker services

The ToxTracker suite is available as service from our state-of-the-art laboratory in the Netherlands. You can then send your compounds for testing and receive a full report, in most cases within 2-3 weeks.

The following infographic depicts the typical workflow when doing a project with Toxys.

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. ToxTracker is also available via Labcorp in Harrogate(UK).

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 for these instances. 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. To be able to adhere to the strict timeline, it is essential that delivery of the test materials at Toxys is properly scheduled and that the relevant information about compound solubility and toxicity is shared before the start of the project.

Running ToxTracker in your labs

To accommodate the use of ToxTracker in your lab, Toxys now also 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 (each kit is sufficient to test 8 compounds +/- S9 in triplo) to allow flexible use and extensive insight into (geno)toxicity for your compounds.

Training and certification

We are very proud on 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 who 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 scientist who will perform a 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.