ToxTracker AO

The ToxTracker assay is a state-of-the-art stem cell-based reporter assay that provides mechanistic insight into genotoxic properties of compounds. ToxTracker AO (Antioxidant) is an extended version of the ToxTracker assay and includes discrimination between direct DNA reactivity of a compound and indirect genotoxicity caused by high levels of oxidative stress and the production of reactive oxygen species (ROS). ToxTracker AO combines the power of 6 fluorescent reporter cell lines and ROS scavengers NAC and GSH for the detection of DNA damage response, oxidative stress and protein damage. This unique combination gives unparalleled mechanistic insight into the hazardous properties of chemicals and pharmaceuticals in a single assay. The information generated is particularly useful in Mode-of-Action (MoA) and Adverse-Outcome Pathway (AOP) approaches.

A pipette drawing medium

Key Features

  • assesses production of ROS and oxidative stress inducing properties
  • discriminates between direct and indirect genotoxicity
  • 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

ToxTracker AO is an extended version of the ToxTracker assay. In ToxTracker AO, the reporter assay is performed in the absence and presence of two ROS scavengers, n-acetyl cysteine (NAC) and reduced glutathione (GSH). This protocol can be used to assess the contribution of oxidative stress to the genotoxic profile of a compound. The combination of differential activation of these endpoints is a unique and very powerful means to identify the MOA of genotoxicity and the dose response relationship between oxidative stress and genotoxic effects.

Unique combination of endpoints

DNA damage vs. oxidative stress

We compared potassium bromate (KBrO3) and methyl methanesulfate (MMS)  in the absence and presence of a ROS scavenger.

The graphs in this visual compared the effect of potassium bromate (KBrO3) on the ToxTracker cells in the absence and presence of a ROS scavenger. Oxidative stress and DNA damage was measured.The graphs in this visual compared the effect of MMS on the ToxTracker cells in the absence and presence of a ROS scavenger. Oxidative stress and DNA damage was measured.

In this example, KBrO3 and MMS were tested in the ToxTracker assay. Both compounds demonstrate DNA damage (genotoxicity) and oxidative stress activity (2-fold induction defines a positive ToxTracker result). Introduction of NAC reduces oxidative stress as determined using ToxTracker for both compounds; however DNA damage for KBrO3 only. Apparently, DNA lesions caused by MMS are ROS-independent while DNA lesions caused by KBrO3 are partially ROS-dependent.

This example illustrates one possible approach to investigate the role of oxidative stress for a given test compound. Evidently, we can and have performed variations on this approach, tailored to customer needs.

Below is a table of compounds tested with ToxTracker AO in the presence and absence of ROS scavengers NAC and GSH.

This table shows compounds tested with ToxTracker AO in the presence and absence of ROS scavengers NAC and GSH and the ToxTracker reporters responses are also shown.

From sample to report

This visualisation illustrates the sequence of events between receiving your test compound,  performing the ToxTracker AO and providing the final results.

The ToxTracker AO assay protocol starts with dose range-finding and then, testing the compounds in the presence and absence of S9 liver extract in the ToxTracker cell lines and also in absence and presence of ROS scavengers. Then, GFP reporter induction is measured by flow cytometry. Finally, data analysis is performed.


We can perform ToxTracker AO 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.

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

Local lab installation

The assay can also be installed in your laboratory under license. We provide full support for integration of ToxTracker AO in your toxicology laboratory. Please contact us via the contact form below or via email:

ToxTracker AO in Japan via Eolas Biosciences

Toxys has partnered with Eolas Biosciences to include ToxTracker AO in their portfolio of genetic toxicology services for the Japanese Market. Visit this page to learn more.

ToxTracker via research marketplaces Science Exchange and

Toxys has made its ToxTracker AO 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 AO offered by other CROs

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

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These are some of the typical application of ToxTracker AO:

  • Genotoxicity testing of compounds when oxidative stress is the expected mode-of-action
  • Follow-up of a positive ToxTracker test 
  • Follow-up of a positive in vitro micronucleus test

Meet the study director for ToxTracker AO

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 AO in understanding where and how ToxTracker AO 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
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.

More information

Please click here for more information on ToxTracker

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