ToxTracker MoA Extensions

We have developed several extensions to the standard ToxTracker protocol that can be applied to further assess the underlying mode-of-action of genotoxic and non-genotoxic compounds. Protocols are available for separating clastogens from aneugens and identification of kinase inhibitors and microtubule poisoins. ToxTracker can be combined with conventional genotoxicity endpoints by measuring induction of gene mutations and chromosome damage in the reporter cell lines. To assess the mechanisms of oxidative stress, we can analyse the production of ROS and contribution of oxidative stress to the genotoxic and cytotoxicity profile of a compound.

Key Characteristics

  • Unravel the mechanism of toxicity
  • Choose the appropriate Mode-of-Action Extension to follow-up on ToxTracker results
  • Determine Clastogens and Aneugens 
  • Identify kinase inhibitors and microtubule poisons
  • Unravel mechanisms of oxidative stress
  • Identify kinase inhibitors

We can follow up ToxTracker results with a number of experimental protocols to further unravel the mechanisms of action underlying genotoxic compounds. Below is an overview of these protocols,  which we offer as ToxTracker MoA Extensions.

Aneugen Clastogen Evaluation

By integrating ToxTracker reporter data, cell cycle and polyploidy analysis we are able to separate compounds with a clastogenic mode-of-action from compounds with an aneugenic mode-of-action, as well as to identify tubulin poisons and cell cycle kinase inhibitors. Please read more under the tab Aneugen Clastogen Evaluation to learn more details about this MoA extension. The protocol described here is intended as follow up on existing ToxTracker assay data, ToxTracker ACE is our ToxTracker assay that has discrimination of aneugens from clastogens already incorporated.

Oxidative stress

We have various protocols to determine the contribution of oxidative stress in DNA damage as caused by test compounds. Please read more under the tab Oxidative stress.

Kinase inhibitors

We offer a combination of 3 additional protocols that help assess whether the your compound is a  kinase inhibitors which cause DNA damage (and are likely to be positive for cancer hazard) and predict whether they will give a positive micronucleus result. Please read more under the tab Kinase inhibitors.


The mouse ES cells that are applied in the ToxTracker assay have been extensively used to study mutagenesis. The hprt, aprt and tk genes are typically used to study DNA damage-induced mutations. We can extend the ToxTracker assay to allow detection of promutagenic DNA lesions with the quantitative assessment of mutation induction following chemical exposure. Read more

Chromosomal damage

We can use our ToxTracker cell lines for a number of standard genotoxicity assays to identify chromosomal damage. We can perform the following assays:

By combining ToxTracker’s reporter data with cell cycle analysis, polyploidy detection and/or phospho H3 staining, we can rapidly evaluate the aneugenic and clastogenic potential of a compound. The differential activation of these endpoints provide powerful means to separate clastogens from aneugens, as well as to identify potential tubulin poisons and cell cycle kinase inhibitors.

Aneugens and Clastogens example

In the example below, we illustrate our unique approach by comparing clastogenic compound cisplatin (DNA cross linker) with aneugenic compounds taxol (tubulin poison) and AMG900 (kinase inhibitor) for the different endpoints.




  • Activation of both genotoxicity reporters in ToxTracker
  • Cell cycle block in S phase
  • No polyploidy

Aneugens/tubulin poisons

  • Selective activation of the Rtkn genotoxicity reporter
  • Cell cycle block in G2/M phase
  • Polyploidy

Aneugens/kinase inhibitors

  • No activation of the ToxTracker genotoxicity reporters
  • Cell cycle block in G2/M phase
  • Polyploidy
  • Reduction of Histone H3 phosphorylation

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, we can perform the ToxTracker assay 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.

Assessment of oxidative stress as mode of action

In this example, potassium bromate (KBrO3; left panel) and methyl methanesulfate (MMS; right panel) 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 KBrO3only. 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.

Kinase inhibitors

Pharmaceutical compounds that target a specific therapeutic kinase in the cell sometimes also affect the activity of other kinases. The Aurora kinases that play a crucial role in cell division are a known unintended target for many kinase inhibitors. Inhibition of cell cycle checkpoint kinases affect proper progression through mitoses and are thereby inducing the formation of micronuclei and polyploidy in vitro and in vivo, however are generally not carcinogenic.

These (often unanticipated) genotoxic effects are generally seen at much higher concentrations than the therapeutic dosis of the drug. To identify compounds that (unintentionally) affect cell cycle kinases we have developed additional protocols to identify kinase inhibitors.

      1. We start with a standard ToxTracker analysis. Kinase inhibitors are not DNA reactive and do not activate the Bscl2-GFP and Rtkn-GFP genotoxicity reporters in ToxTracker.
      2. We perform staining for PhosphoH3 and propidium Iodine staining to establish inhibition of phosphorylation of histone H3 by the Aurora kinases and polyploidy
      3. We perform a micronucleus assay in the ToxTracker cells to confirm that these compounds give rise to micronuclei

Validation of the protocols for Phospho-H3 and Propidium Iodine staining

We have applied the protocols to 2 known aurora kinase inhibitors: AMG900 and VX680. We exposed the cells to the compounds and controls such as nocodozole (a mitotic spindle poison), cisplatin and etoposide and subsequently stained with an antibody for phospo-H3 positive cells and measured antibody staining by flow-cytometry.

We found a strong decrease of phospho-H3 stained cells when they are exposed to AMG900 and VX680. Subsequently we stained for polyploidy by adding propidium iodide DNA staining and measure staining by flow cytometry.

We found strong induction of Polyploid cell when the cells were exposed to AMG900 and VX680.

Fee-for-service project

We can perform the assay for you at our state-of-the-art laboratory. You can 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

These additional protocols are only relevant when you have accessed the ToxTracker technology for in-house use. Please contact us if you would like more information on using the additional protocols for in-house use.

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