ToxTracker MACSQuant X

Can ToxTracker replace the in vitro micronucleus test?

Case Study

ToxTracker

Can ToxTracker replace the in vitro micronucleus test?

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Highlights

Approach

To benchmark ToxTracker and micronucleus assay, 68 test chemicals were selected and classified.

Results

ToxTracker showed a superior accuracy to correctly predict in vivo genotoxicity compared to the in vitro micronucleus assay.

ToxTracker can provide critical insight into the MoA of genotoxic compounds to explain different outcomes from the in vitro and in vivo testing battery.

Background

The need for improved in vitro genotoxicity testing

Substances must be evaluated for their potential to induce genetic damage as part of every chemical safety assessment. Positive outcomes in an initial in vitro test battery in most cases require follow-up in vivo studies. However, certain industries can no longer use this strategy e.g. cosmetics companies registering ingredients in Europe, or they are striving to reduce unnecessary animal assays. Therefore, along with a high sensitivity, the test battery needs a high specificity (i.e., the ability to correctly identify true negative chemicals) to avoid unnecessary follow up in vivo studies (in keeping with the 3R principles), as well as losing promising candidate chemicals.

This raises the question as to whether results from in vitro assays could be relied upon with more confidence so that follow-up in vivo studies were rendered unnecessary. Can we do better than using in vitro tests with a simple “yes-no” outcome of whether a chemical causes genotoxicity? Can we develop assays that reveal the underlying mechanism(s) at the molecular or cellular level to be better able to interpret the outcome? Gaining clearer insight into potential genotoxic Modes of Action (MoAs) is crucial to understand why misleading positives occur.

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NAMs as a solution for animal-free MoA insights

In line with this, in recent years, safety assessments have been moving towards the use of New Approach Methodologies (NAMs) based on Adverse Outcome Pathways (AOPs) and the sequence of molecular and cellular key events therein. One such NAM is the ToxTracker Assay, which measures cellular events occurring upstream of, and prior to, genotoxicity, e.g. gene mutation, micronucleus (MN) formation or aneuploidy induction. True genotoxic events are reported in ToxTracker via activation of two reporter genes in mammalian stem cells:

  • The Bscl2-GFP reporter is activated upon DNA replication stress via the ATR pathway. Mutagenic lesions such as bulky DNA adducts typically induce this response.
  • The Rtkn-GFP reporter is activated by DNA double strand breaks via the ATM pathway. These DNA double strand breaks lead to chromosomal aberrations and aneuploidy.

A test substance is classified as genotoxic if either of these biomarkers are induced ≥ 2-fold over concurrent vehicle controls. Unlike the in vitro MN or other standard genotoxicity assays, the ToxTracker reporter cell lines also detect other toxicological effects which can indirectly lead to MN formation. These include oxidative stress [Blvrb and Srxn1], unfolded protein response [Ddit3] and p53 activity [Btg2], which can be an indication of cytostasis, DNA repair, or apoptosis.

Indeed, analysing the cell cycle and the induction of mitotic arrest and aneuploidy in combination with the Rtkn reporter is essential in detecting aneugens. Thus, not only is the assay able to accurately classify compounds as genotoxic or non-genotoxic, but it can also discriminate between DNA-reactive compounds, aneugens and indirect genotoxicity caused, e.g. by oxidative stress (Brandsma et al., 2020, Wills et al., 2021, Mišík et al., 2022).

This is all well and good but how does the ToxTracker perform compared to standard clastogenicity assays?

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A Case Study Benchmarking ToxTracker and Micronucleus Assay

Our Approach

To make this comparison, test chemicals were selected from the published list of recommended validation chemicals for new and improved genetic toxicity assays (Kirkland et al., 2016). These are divided into three groups:

  • 24 genotoxicants (carcinogens, aneugens, and indirect genotoxicants)
  • 27 non-genotoxicants (carcinogens and noncarcinogens)
  • 17 misleading in vitro positives (e.g., indirect genotoxicants and cytotoxicants).

All chemicals were tested in the ToxTracker assay, and the outcomes compared with those in standard clastogenicity assays (MNvit and Chromosome Aberration assays).

Results

The results from various validation studies showed that the GFP-reporter for DNA double strand breaks (Rtkn) was 100% concordant with the sensitivity of in vitro clastogenic endpoints, meaning both assays were equally able to correctly identify true positive chemicals acting via this mechanism.

In many cases, the discrepancy between the in vitro MN and ToxTracker (and in vivo) assays could be explained by high levels of oxidative stress induced by the test chemical and evident in the ToxTracker panel as an increase in Blvrb and/or Srxn1.

new approach methods in vitro toxicity testing
in vitro micronucleus toxtracker
in vivo micronucleus toxtracker

ToxTracker correctly identified all tested genotoxicants. Approximately 40% of ToxTracker negatives gave a positive result in the in vitro MN assay, reflecting the high frequency of misleading positives in the in vitro MN.

Importantly, there was a very high accuracy (92% sensitivity, 93 specificity) to predict in vivo clastogenicity.

In the case of the misleading positive chemical, p-nitrophenol, which was clastogenic in mammalian cells but negative for MN or tumors in mice, the ToxTracker demonstrated that the MNvit result was predominantly driven by a cytotoxic mechanism (protein stress) and not direct DNA damage.

Significance

This study shows that the Rtkn GFP-reporter has high accuracy when benchmarked to in vivo MN test results without losing sensitivity. Additional information from the assay regarding the MoA is critical when translating in vitro hazards into potential in vivo risks.

This all leads to the conclusion that ToxTracker is already a valuable addition to the in vitro testing battery.

  • Indeed, it is included in the SCCS Notes of Guidance (SCCS, 2023) for safety assessment of cosmetics ingredients and is undergoing formal validation, currently under review by the OECD.
  • The report describing the results of the multi-lab validation study is now available from the OECD website and a recent peer-reviewed publication (see Hendriks et al., 2024). It confirms the accuracy of the assay across different labs and highlights the added value of mechanistic information in interpreting the results.

The MoA information that is provided by the ToxTracker assay could be a valuable expansion of the in vitro genotoxicity testing strategy.

Taking this one step further – can these results and activities be also pointing to the possibility of replacing the in vitro MN with the ToxTracker assay for regulatory submissions? Time will tell…

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You can download this case study as a PDF here.

If you have questions, feel free to reach out to us at info@toxys.com

References

Brandsma I, Moelijker N, Derr R, Hendriks G. Aneugen Versus Clastogen Evaluation and Oxidative Stress-Related Mode-of-Action Assessment of Genotoxic Compounds Using the ToxTracker Reporter Assay. Toxicol Sci. 2020 Sep 1;177(1):202-213. doi: 10.1093/toxsci/kfaa103. PMID: 32617558.

Hendriks G, David Kirkland D, Philippe Vanparys P, Jan van Benthem J, Adriaens E. The ToxTracker assay: a stem cell-based reporter assay for mechanistic carcinogenicity hazard screening Validation report. 2023. Available at: https://www.oecd.org/env/ehs/testing/toxtracker-validation-report-2023.pdf

Hendriks, G., Adriaens, E., Allemang, A., Clements, J., Cole, G., Derr, R. et al. Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment. Environ Mol Mutagen. 2024;1–21. doi: 10.1002/em.22592.

Kirkland D, Reeve L, Gatehouse D, Vanparys P. A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutat Res. 2011 Mar 18;721(1):27-73. doi: 10.1016/j.mrgentox.2010.12.015. Epub 2011 Jan 14. PMID: 21238603.

Kirkland D, Kasper P, Martus HJ, Müller L, van Benthem J, Madia F, Corvi R. Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. Mutat Res Genet Toxicol Environ Mutagen. 2016 Jan 1;795:7-30. doi: 10.1016/j.mrgentox.2015.10.006. Epub 2015 Nov 4. PMID: 26774663.

Mišík M, Nersesyan A, Ferk F, Holzmann K, Krupitza G, Herrera Morales D, Staudinger M, Wultsch G, Knasmueller S. Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems. Mutat Res Genet Toxicol Environ Mutagen. 2022 Sep;881:503524. doi: 10.1016/j.mrgentox.2022.503524. Epub 2022 Jul 3. PMID: 36031336.

SCCS (Scientific Committee on Consumer Safety), SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation 12 th revision, 15 May 2023, corrigendum 1 on 26 October 2023, corrigendum 2 on 21 December 2023, SCCS/1647/22.

Wills JW, Halkes-Wellstead E, Summers HD, Rees P, Johnson GE. Empirical comparison of genotoxic potency estimations: the in vitro DNA-damage ToxTracker endpoints versus the in vivo micronucleus assay. Mutagenesis. 2021 Aug 27;36(4):311-320. doi: 10.1093/mutage/geab020. PMID: 34111295; PMCID: PMC8391785.

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