ToxTracker® is a state-of-the-art animal-free reporter assay that can readily identify the (geno)toxic and potentially carcinogenic properties of newly developed pharmaceuticals and chemicals in a single test. ToxTracker provides valuable information about the mode-of-action of toxic compounds which can be crucial for reliable chemical safety assessment. ToxTracker has been extensively validated and is currently under evaluation for official acceptance and inclusion of the test in the standard regulatory safety test strategy.
Technical background & rationale for development
Chemical exposure of cells may damage biomolecules, cellular structures and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. The ToxTracker assay is a mammalian stem cell based genotoxicity assay employing a panel of six green fluorescent protein reporters to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general p53-dependent cellular stress response.
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.
The ToxTracker assay consists 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 following the induction of promutagenic DNA lesions and subsequent DNA replication stress. The Rtkn-GFP genotoxicity reporter is associated with the formation of 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.
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
- Diving rapidly
Progenotoxic agents, such as Aflatoxin B1 and Benzo[a]pyrene, only become DNA reactive after metabolisation by detoxification enzymes. In vivo metabolism takes place in for example the liver, bone marrow or the lungs. In in vitro genotoxicity assays such as ToxTracker, metabolisation can be included by using S9 liver extract from rat or hamster.
In ToxTracker, rat S9 liver extracts derived from animals treated with Aroclor-1254 or a mixture of phenobarbital and beta-naphthoflavone can be applied. Both extracts are able to effectively metabolise compounds. For ToxTracker, the S9 metabolisation protocol has been optimised to allow the detection of various direct and indirect genotoxic properties of compounds following S9 metabolisation.
More information about the optimisation of in vitro metabolism using S9 can be found in our poster.
Validation against ECVAM and Toxcast libraries
The assay has been extensively validated by Toxys using the ECVAM suggested compound library (EU), a selection of compounds from the Toxcast program (USA) and of numerous well-established carcinogens and non-carcinogens. The ToxTracker assay showed a very high sensitivity (94%) and specificity (95%) for the detection of genotoxic compounds.
Limited Interlaboratory validation study
To Investigate the reliability and transferability of the ToxTracker assay, a limited interlaboratory validation study was performed. In total 27 compounds (24 compounds and 3 assay controls) were tested at least three times by two independent laboratories. The within-laboratory reproducibility (WLR) for the six different ToxTracker reporters was assessed per category of reporters (DNA damage, oxidative stress, protein damage) and for all reporters together. The overall WLR was on average 94% for both laboratories. Also the between-laboratory reproducibility (BLR) was evaluated for the different reporters with the highest concordance for the genotoxicity reporters (96,4%), followed by the reporters for p53 activation (92,6%), oxidative stress (88%) and protein damage (85,2%). This resulted in an overall BLR of 90,5%. The predictive capacity for genotoxicity was calculated for each laboratory and for the cumulative results of the two laboratories. The sensitivity for identification of genotoxic compounds was 77,8% (7 out of 9 genotoxic compounds tested positive in ToxTracker). The specificity was 94,1% (16 out of 17 non-genotoxic compounds tested negative in ToxTracker for both laboratories). In this study the overall concordance between ToxTracker and the standard battery of in vitro and in vivo genotoxicity tests was 85%.
International Validation Study for OECD application and ECVAM
Following the early interlaboratory validation study, in 2017 an official OECD validation study was started. In this validation study, 7 laboratories from various industries (Pharma, Chemical and CRO’s) will test a selection of 64 genotoxic and non-genotoxic compounds. The study is performed according to the OECD guidelines.
Correlation with regulatory genotoxicity assays
The results from the ToxTracker assay have been compared with the standard in vitro and in vivo regulatory genotoxicity assays for induction of gene mutation (Ames/MLA) and chromosomal damage (MN/CA).
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 negative ToxTracker results, in 30% of the cases the compound gave 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 cytotoxic conditions. However, the correlation with the in vivo MN/CA tests for non-genotoxic compounds was 93%.
ToxTracker extentions and applications
ToxTracker ACE for Aneugen and Clastogen evaluation
ToxTracker ACE is an extended version of the ToxTracker assay and includes cell cycle analyses and polyploidy detection to allow direct discrimination between an aneugenic and clastogenic mode-of-action of genotoxic compounds. ToxTracker ACE (Aneugen and Clastogen Evaluation) combines the differential response of the 6 fluorescent ToxTracker reporters with an extensive cell cycle analysis and aneuploidy detection. The differential activation of these endpoints is a unique and very powerful means to separate clastogens from aneugens, as well as the identification of tubulin poisons and cell cycle kinase inhibitors.
ToxTracker with ROS scavengers
High levels of oxidative stress and the production of reactive oxygen species (ROS) can indirectly lead to genotoxicity. To investigate the contribution of oxidative stress in the genotoxic profile of a compound, ToxTracker can be performed in presence of a ROS scavenger (NAC, GSH) and measure its impact on DNA damage, oxidative stress and cytotoxicity. This will allow the discrimination between direct DNA reactivity of a compound and indirect genotoxicity caused by oxidative stress.
For screening purposes, a selection of biomakers can be applied to test a large number of compounds for genotoxicity. Activation of the Bscl2-GFP biomarker for DNA damage has a very high correlation with the Ames test (figure 1). The Rtkn-GFP biomarker is activated by DNA strand breaks and strongly correlates with the regulatory clastogenicity assays (MN/CA) (figure 2). Combining these genotoxic markers will result in an assay which combines mutagenic and clastogenic endpoints in a fast format and requires little compound. The throughput of ToxTracker can be increased to analyse hundreds of compounds per week.
Data Analysis & ToxPlot software
For data analysis, the dedicated software tool ToxPlot has been developed. 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 an easy interpretable heatmap.
The software automatically calculates induction levels of various ToxTracker reporters as well as cytotoxicity of the compounds. ToxTracker activation at various levels of cytotoxicity are calculated, analysed and represented in easy interpretable heatmaps. GFP induction levels for each of the ToxTracker reporters are used for hierarchical clustering of the analyzed compounds based on similarity of reporter activation. Amongst others the IC50, IC10 and NOGEL are calculated. The software includes a feature to upload data into quantitative analysis tools such as PROAST for further analysis.
These are some of the typical questions we get asked about our technology platform. Should you not be able to find your question please feel free to contact us.
Yes, we can provide the cells and install the assay in your laboratory under license. Please contact us via the contact form or via email@example.com to find out more.
Depending on the size of the project it takes approximately 1-2 weeks before delivery of a final report. Please contact us in case you have specific requirements, for example to fast-track a report or for large size projects.
This depends on the compound and maximum concentration you wish to test. Usually 10 mg is sufficient for the test. This can be either dry powder material or as solution.
The type of solvent shall be agreed upon before start of the project. Typically DMSO or water is used, but other solvents are possible.
Usually it is possible to test auto-fluorescent materials. In our standard protocols we run along a non-GFP cell line to check for auto-fluorescence. Usually we can correct for the auto-fluorescence, however occasionally the auto-fluorescence is so strong that it interferes with the flow-cytometry measurements. If this is picked-up in the dose finding study we will revert to other technologies to measure reporter induction.
Yes, we can add S9 liver extract to our assays for drug metabolism.
The ToxTracker assay is highly specific and sensitive. Please see this publication for more information.
Example report is available, please contact us via the contact form or firstname.lastname@example.org for more information.
Yes, our scientist have extensive experience in genotoxicity and would like to design the optimal project for your needs. Please contact us via the contact form or email@example.com to find out more about custom projects.
We often get asked why we are not using human stem cells. There are various reasons why there is no direct advantage over our current cells:
- nearly all compounds that give rise to genotoxicity in humans will also do this in mouse cells – the predictivity of over 95% is already excellent and is not expected to increase a lot using human cells
- protocols to keep stem cells untransformed are not easily available for human stem cells
- the major difference between human cells and mouse cells is the metabolising system, however in human stem cells this is also not present
- the validation with over 400 compounds has been done in the current system and would need to be redone should we redevelop the system, this requires investment in lots of time and costs
- should we develop a human cell system it is expected that the assay will be a lot more expensive given the need for expensive media etc.
Based on all these reasons we have chosen to stick with our current system and not develop a human genotoxicity assay. Of course for other applications we will consider using human cell systems.
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 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.
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 vitro genotoxicity assays. Assessment of these non-genotoxic mechanisms can be very valuable in de-risking positive results from the conventional in vitro genotoxicity assays.
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.
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 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.
The genotoxicity reporters in ToxTracker show a high sensitivity and specificity for identification of in vivo genotoxic compounds. So far, we have not encountered a ToxTracker-unique positive compound.
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-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 followup 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.
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.