FAQ

Yes, we regularly screen test compounds for our customers in our laboratories as contracted service. We have a standard protocol for how we test compounds, but custom assay design is possible, we try to work as flexible as possible to accommodate your need.

Yes, we can provide the cells and install the assay in your laboratory under license. Please contact us via the contact form or via info@toxys.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.

Over 450 compounds have been tested in the assay. We have screened the ECVAM library and demonstrated the  sensitivity and specificity  of the ToxTracker assay. Please see our publications and ToxTracker information page.

Example  report is available, please contact us via the contact form or info@toxys.com 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 info@toxys.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:

1. 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
2. protocols to keep stem cells untransformed are not easily available for human stem cells
3. the major difference between human cells and mouse cells is the metabolising system, however in human stem cells this is also not present
4. 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
5. should we develop a human cell system it is expected that this 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 if 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 a 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.

The 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-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.