Scientific background
ToxTracker® is a unique in vitro genotoxicity assay that combines multiple biomarkers to get mechanistic insight into the MOA of genotoxic compounds. The assay not only includes markers for DNA damage as predictors for the standard genotoxicity assays but also includes markers for non-genotoxic MOA, including oxidative stress, protein misfolding and general cellular stress. All these types of cellular damage are associated with increased cancer risk.
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.
Unique biomarkers
In ToxTracker, genotoxicity is primarily predicted by the activation of either of the two independent fluorescent reporters Bscl2-GFP and Rtkn-GFP. The Bscl2-GFP reporter is activated upon the formation of DNA adducts that can inhibit progression of DNA replication, which is a potent activator of the DNA damage response (DDR) (1) (17).
These replication-blocking DNA lesions often lead to the induction of gene mutations. Activation of the Bscl2 biomarker gene is dependent on the ATR/Chk1 kinase that plays a central role in the cellular DDR (Figure 1) (1). Induction of the Rtkn-
GFP genotoxicity reporter is associated with induction of DNA double-strand breaks and depends on activation of the ATM/Chk2 kinase pathway (2) (18). The DDR-associated ATM kinase is also responsible for phosphorylation of histone variant H2AX. The phosphorylated form of H2AX (γH2AX) is commonly used as a reporter for clastogenic DNA lesions.
Genotoxicity of chemicals is also indicated by the Btg2-GFP reporter. Btg2 is a component of the p53-dependent DDR and is involved in regulation of the G1/S cell cycle checkpoint, but is also induced by various other cellular stressors, including oxidative stress. Chemicals that cause high levels of oxidative stress in the cells can directly or indirectly induce genotoxicity. Insufficient or faulty repair of oxidative DNA lesions can lead to gene mutations and chromosomal aberrations.
Next to genotoxicity caused by direct DNA reactivity, there are also substances that are genotoxic without directly interacting with the DNA (indirect genotoxicity). Tubulin poisons, which interfere with chromosome segregation during mitosis, are indirect genotoxins and exposure to such agents can lead to the formation of aneuploid or polyploid daughter cells. ToxTracker typically identifies aneugenic substances as genotoxic but does not contain a dedicated reporter gene that is activated upon exposure to aneugenic substances. In general, genotoxic chemicals with an aneugenic mode of action (MoA) selectively activate the Rtkn-GFP reporter in ToxTracker but not the Bscl2-GFP reporter. Indication for the aneugenic MoA can be obtained from the kinetics of Rtkn- GFP reporter activation. For DNA-reactive chemicals, activation of Rtkn-GFP is readily observable after 6-8 hours, whereas for tubulin poisons, the activation of the Rtkn-GFP takes 12-14 hours.
Alternatively, an extended ToxTracker protocol (ToxTracker ACE) was developed to discriminate between clastogens and aneugens and depends on activation of the Rtkn-GFP reporter in combination with cell cycle arrest in mitosis after 4-hour exposure to the chemical. However, the ToxTracker ACE protocol has not been validated and is not the subject of this Test Guideline.
Induction of oxidative stress that can lead to indirect genotoxicity is detected in ToxTracker by the Srxn1-GFP and Blvrb-GFP reporters that are activated by the two major antioxidant pathways in the cell. The Srxn1-GFP reporter is strongly activated upon cellular oxidative stress and is directly controlled by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor. Nrf2 plays a key role in the oxidative stress response and is involved in the activation of various antioxidant gene networks. Blvrb has been shown to play an important role in heme metabolism and has been associated with the cellular antioxidant response. The Blvrb promoter sequence does not contain a Nrf2 binding motif and therefore reflects an oxidative stress response different from Srxn1.
Activation of the unfolded protein response following chemical exposure is a potent trigger of apoptosis, which can lead to DNA breaks and chromosome fragmentation. The Ddit3 biomarker gene is a multifunctional transcription factor that has been associated with cell cycle arrest, apoptosis, and the unfolded protein response in the endoplasmic reticulum (ER). Activation of the unfolded protein response is detected in ToxTracker by the Ddit3-GFP reporter.
Cell system
The ToxTracker reporter assay was created using mESC. These untransformed, non-
cancerous cells were selected as they are genetically stable, have an infinite lifespan, a high cell proliferation rate, and are proficient in major DNA damage signalling and cell cycle regulation pathways, in contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing.
Advantages 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
- Dividing rapidly
Metabolic activation
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 phenobarbital and beta-naphthoflavone can be applied. These 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. This metabolism protocol is included in the transferability study and is part of the 446A guideline.
More information about the optimisation of in vitro metabolism using S9 can be found in our poster.
Next to the standard protocol we have also developed a protocol for detecting nitrosamines which uses a specific optimised Hamster S9 metabolism protocol. More information on this can be found in this publication.
Validation
In 2026 ToxTracker was adopted as OECD TG 446A. The guideline enables organizations around the world to incorporate ToxTracker into regulatory testing strategies and represents an important step toward broader acceptance of next-generation safety assessment methods.
Overall, the assay is well validated against various libraries and in inter-lab validation studies.
Validation against ECVAM and Toxcast libraries
The assay is extensively validated – well over 1500 compounds have been tested, including various types of customer substances as well as reference substances listed by EU’s Reference Laboratory for alternatives to animal testing (ECVAM library) and by the US Environmental Protection Agency (EPA; Toxcast library). The ToxTracker assay showed a very high sensitivity (94%) and specificity (95%) for the detection of genotoxic substances and has a strong correlation with the regulatory assays.
International Validation Study for OECD application and ECVAM
In 2024 an international interlaboratory validation according to OECD guidelines of ToxTracker was successfully completed. The main goals of this validation were to investigate the accuracy of ToxTracker to predict in vivo genotoxicity, to establish the intra- and inter-laboratory reproducibility of the assay and to explore the added value of the genotoxic mode-of-action information from ToxTracker. Seven laboratories with expertise in the field have tested a large selection of genotoxic and non-genotoxic substances in the ToxTracker assay. ToxTracker consistently demonstrated high accuracy and excellent reproducibility of test results, both within and between different laboratories. The results from the validation study were published in Environmental and Molecular Mutagenesis which can be accessed here.
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.
Typical applications:
– Genotoxicity testing of pharmaceutical compounds
– Identification of aneugens
– Genotoxicity testing of kinase inhibitors
ToxTracker AO – 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 the 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.
Typical applications:
– 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
Data Analysis, calling & BMD
ToxTracker results are presented as concentration-response curves, showing the fold-change in GFP reporter expression relative to the concentration of the test chemical. Fold changes are calculated for each GFP reporter gene by dividing the MFI in the treated culture by the MFI in solvent-exposed cultures.
Fold changes are calculated for each ToxTracker reporter separately, with and without metabolic activation. Cytotoxicity of the test chemical is also presented in concentration-response graphs, displaying the relative cell count related to the concentration of the test chemical.
Each ToxTracker assay is deemed positive when a chemical induces at least a 2-fold increase in GFP expression in any of the reporters at one or more acceptable test concentrations. Reporter activations at concentrations thatinduce >75% cytotoxicity are excluded and not considered for data interpretation. If a substance activates the ToxTracker reporters more than 1.5-fold but less than 2-fold at acceptable levels of cytotoxicity (<75% cytotoxicity), this response is considered a weak positive result, noted as ‘(+)’, but only in case of a clear concentration-related increase in GFP signals across all tested concentrations when evaluated with an appropriate trend test. If the reporter activation remains below 1.5-fold, the response is negative, noted as ‘-’.
A chemical is identified as genotoxic in ToxTracker if a positive response is observed for the Bscl2-GFP and/or Rtkn-GFP reporters. A lack of induction of the Bscl2-GFP and Rtkn-GFP reporters (i.e. below the 1.5-fold thresholds) leads to a non-genotoxic result for the test chemical. In this situation, activation of the otherToxTracker reporters do not change the non-genotoxic prediction but may provide information that may help contextualise responses observed in other genotoxicity assays.
Activation of both Bscl2-GFP and Rtkn-GFP genotoxicity reporters indicates a direct DNA reactive genotoxic mechanism of action. A positive result for the Bscl2-GFP reporter often correlates with induction of gene mutations. A positive result for the Rtkn-GFP reporter is indicative of a clastogenic MoA. The direct DNA reactivity prediction from Bscl2-GPF and Rtkn-GFP reporter induction is not affected by the results from the Srxn1-GFP, Blvrb-GFP and Ddit3-GFP reporters.
The selective activation of the Rtkn-GFP reporter without a positive result for the Bscl2-GFP reporter and the Srxn1-GFP/Blvrb-GFP oxidative stress reporters is often indicative of an aneugenic MoA. However, ToxTracker does not provide a definitive discrimination between a clastogenic and aneugenic MoA. For differentiation between clastogens and aneugens further investigation will be needed. For instance, an extended ToxTracker ACE protocol is available.
A positive result for the Btg2-GFP reporter indicates activation of the p53 tumour suppressor response and confirms genotoxicity of the tested chemical, but only when Bscl2-GFP and/or RtknGFP are activated.
A positive result for the Srxn1-GFP and/or Blvrb-GFP reporters indicates induction of oxidative stress. Results from these reporters should be combined with the Rtkn-GFP and Bscl2-GFP reporters to inform about indirect genotoxicity related to oxidative stress. Induction of the Srxn1-GFP and/or Blvrb-GFP reporter together with the Rtkn-GFP reporter is indicative of indirect genotoxicity related to oxidative stress. Typically, the lowest concentration that activates the oxidative stress reporters is lower than the concentration that induces the Rtkn-GFP reporter. An extended ToxTracker-AO protocol has been developed to further investigate the oxidative stress-related genotoxicity. Activation of the Ddit3-GFP reporter indicates induction of the unfolded protein response. The unfolded response is a potent inducer of apoptosis. A positive result for Ddit3-GFP, without activation of Rtkn-GFP and Bscl2-GFP reporters, does not lead to a genotoxicity prediction.
Benchmark Dose Analysis
For hazard identification, a qualitative answer whether a substance is genotoxic or not is often sufficient. To use ToxTracker data for risk assessment, quantification of the dose response of a test substance will be required. Therefore, Toxys has incorporated benchmark dose (BMD) response modeling into the standard ToxTracker assay data analysis pipeline.
Benchmark Dose (BMD) analysis is a quantitative method used in toxicology to determine the dose at which a substance begins to produce a measurable biological effect. Instead of relying on traditional NOAEL/LOAEL values, BMD uses dose–response modeling to identify a statistically robust point of departure. This makes it highly valuable for risk assessment, regulatory decision‑making, and comparing potencies across compounds. BMD analysis provides a more objective and data‑driven foundation for evaluating chemical hazards.
The benchmark dose is the concentration of a compound that elicits a pre-defined increase in response when compared to concurrent vehicle controls. Addition of quantitative dose response modeling will determine the benchmark concentration that induces biologically meaningful increases in each of the ToxTracker reporters1. The BMD will be calculated using PROAST software, which provides quantitative information about genotoxic mode of action and facilitates quantitative in vitro to in vivo (qIVIVE) extrapolation2.
A) Typical dose response for the clastogenic chemotherapeutic cisplatin across all ToxTracker reporters. The highest induction is noted in Rtkn-GFP, the marker for DNA double strand breaks. B) Bootstrapping dose response models provide lower and upper estimates of the BMD using a benchmark response of 100 (100% increase over background, 2-fold). C) Lower and upper BMD bounds of the 95% confidence interval (CI) across the positive ToxTracker reporters for cisplatin.
Typical applications of BMD modeling include:
- Potency comparison of related substances (i.e. read-across or weight of evidence (WoE) approach)
- Determining safe exposure levels such as the acceptable daily intake (ADI)
- Protective estimates of in vivo dose using qIVIVE
If you would like to learn more about BMD and how it can help your study, please feel free to reach out via enquiries@toxys.com
REFERENCES
- Boisvert L, Derr R, Osterlund T, Hendriks G, Brandsma I. Quantitative interpretation of ToxTracker dose-response data for potency comparisons and mode-of-action determination. Environ Mol Mutagen. 2023;64(2):132-143. doi:10.1002/em.22525
- Beal MA, Audebert M, Barton-Maclaren T, et al. Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose. Environ Mol Mutagen. 2023;64(2):105-122. doi:10.1002/em.22521
FAQ
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 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:
- 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.
See our resources for more information
Publications
Here you can find an overview of our publications relating to ToxTracker and publications by others on ToxTracker
Video's
We have produced several video's which show how the assay is performed and why ToxTracker has been developed.
Posters
Here you can find posters we and ToxTracker users have published during various toxicology conferences.
Flyers
Please feel free to access the flyers we have prepared on ToxTracker and its extensions.
Use cases
ToxTracker has been applied in various settings, including for regulatory purposes. Here you can find use cases