ToxTracker EMP
ToxTracker EMP (enhanced metabolization protocol) is a dedicated ToxTracker protocol to investigate the genotoxic potential of N-nitrosamines. This enhanced protocol, which may be used across mammalian cell systems, greatly improves bioactivation of N-nitrosamines and increases the sensitivity of ToxTracker to detect their genotoxic potential.
Key Features
- Genotoxicity prediction of N-nitrosamines in mammalian cells
- Optimized metabolization protocol using high levels of hamster S9 liver fraction and enhanced cofactor mix
- Low amount of compound required
ToxTracker EMP employs a unique enhanced metabolization protocol to improve the metabolic activation of N-nitrosamines. This EMP protocol is used with the ToxTracker genotoxicity reporters to predict the genotoxic potential of N-nitrosamines in mammalian cells.
The metabolization protocol that is used in most of the in vitro genotoxicity assays is based on rat S9 liver fraction and has a limited ability to metabolize N-Nitrosamines. The EMP was specifically developed to enhance the metabolization of N-nitrosamines by using hamster S9 liver fraction and optimizing the S9 concentration, cofactors, and incubation times. Hamster S9 is used in the enhanced Ames test (EAT) that was recently developed for the mutagenicity assessment of N-nitrosamines and can more effectively metabolize N-nitrosamines compared to rat S9 liver fraction. The EMP protocol improved the genotoxicity detection limits of N-nitrosamines by 200-fold compared to the standard ToxTracker metabolization protocol.
Assay readouts
- Cytotoxicity profile of each test substance
- Assessment of genotoxicity reporter activation in the absence and presence of the enhanced metabolization protocol
- BMD analysis of each substance
The Enhanced Metabolization Protocol (EMP) was developed to detect the genotoxic properties of N-nitrosamines, like N-nitrosodimethylamine (NDMA). In the absence of S9 liver fraction, no genotoxicity induced by NDMA can be detected. After exposure of the ToxTracker reporter cell lines to NDMA in the presence of hamster S9, the genotoxic properties of NDMA can be detected at high concentrations. With the new EMP protocol, using a high concentrations of hamster S9 liver fraction, enhanced co-factor mix, and an improved exposure regime, the genotoxicity of NDMA can be detected at a 200-fold lower concentration compared to the standard metabolization protocol.
Bscl2-GFP and Rtkn-GFP induction after NDMA exposure in the absence of S9 (A), in the presence of hamster S9 and the standard cofactor mix (B) or with the ToxTracker EMP protocol (C). GFP reporter responses are measured using flow cytometry and shown as the GFP induction compared to the vehicle. Black line shows relative survival as determined by cell count using flow cytometry.
ToxTracker EMP protocol
ToxTracker EMP uses the two genotoxicity GFP reporter cell lines from the standard ToxTracker assay in combination with the enhanced metabolization protocol to predict genotoxic potential of N-nitrosamines. First, a concentration range finding will be performed using the EMP exposure protocol. With the appropriately selected concentrations, the two GFP reporter cell lines (Bscl2-GFP and Rtkn-GFP) will be exposed to seven concentrations, typically in 2-fold dilution steps, of the test substance with and without hamster S9 liver fraction. In the presence of hamster S9 liver fraction, cells are exposed for 30 minutes to a concentrated mix containing the test substance, enhanced cofactor mix, and 10% hamster S9 liver fraction. Next, the mix will be diluted 3-fold and cells are exposed for another 4 hours. Finally, after a wash step, the cells are incubated for 20 hours with fresh cell culture medium before measuring GFP induction and cellular survival using flow cytometry.
ToxTracker EMP validation
ToxTracker EMP was validated by testing a subset of well-established genotoxic and non-genotoxic N-nitrosamines based on the Ames bacterial mutation test. Reporter responses of the tested N-nitrosamines were compared to the test results from the recently optimized enhanced Ames test (EAT). ToxTracker EMP correctly identified the Ames positive N-nitrosamines as genotoxic and the Ames negative N-nitrosamines as non-genotoxic.
Bscl2-GFP and Rtkn-GFP induction after exposure to indicated N-nitrosamines using the ToxTracker EMP protocol. GFP reporter responses are measured using flow cytometry and shown as the GFP induction compared to the vehicle. Black line shows relative survival as determined by cell count using flow cytometry.
ToxTracker EMP can predict genotoxicity for N-nitrosamines that have been tested positive in the EAT.
Table showing the N-nitrosamines with the test results from the enhanced Ames test (EAT) compared to the ToxTracker assay. N.D. = not determined; P = positive; N = negative; E = equivocal.
Publications
A full description of the ToxTracker EMP protocol development can be found here: Geijer et al., 2025.
Flyers
What type of vehicle controls can you use?
Several vehicle controls are compatible with ToxTracker EMP. If solubility allows, we recommend using PBS or water as solvent to prevent any CYP450 inhibition by DMSO. We can always discuss any specific requirements.
Why do you incubate the cells for 4 h?
We use a short incubation period with high levels of hamster S9, enhanced cofactor mix, and N-nitrosamine to stimulate metabolic conversion of the N-nitrosamine and enhance genotoxicity prediction. Cells do not cope well with longer exposure times with these high concentrations of S9 and cofactor mix, and we have shown that 4 h exposure works well for predicting genotoxicity.
How much compound do you require?
We require 5 mg of the test substance.
Can you perform the EMP in parallel to the ToxTracker assay?
Yes, you can run the ToxTracker assay with our standard 24 h exposure with and without rat S9 liver fraction in addition to ToxTracker EMP.
Why do you test up to 1 mM for N-nitrosamines?
N-nitrosamines or NDSRIs are typically impurities in pharmaceuticals. As the maximum test concentration for pharmaceuticals is 1 mM, the concentration of N-nitrosamine will also never exceed 1 mM.
Can the EMP protocol also be applied to detect gene mutations?
Yes, the EMP protocol can be used in the MutaTracker assay. MutaTracker is a mammalian gene mutation assay that combines the ToxTracker reporter assay with error-corrected next-generation sequencing (SMM-seq).
Practical information
- ToxTracker EMP is available as a service from Toxys
- Turnaround time: 2-3 weeks
- Substance requirement: 5 mg
- Type of solvents compatible with the assay: PBS, Water, DMSO
Project report
The project report contains the protocol, cytotoxicity profile of each test substance, reporter activation in the absence and presence of the enhanced metabolization protocol, and BMD analysis of each substance.
We are happy to share a sample report with you. Please contact us to request a sample report.
Dissemination meeting
Upon sharing the draft report, we will invite you for a dissemination meeting where the study director will go over the conclusions in the report. We will make sure you understand the results and provide our expertise on the results and potential next steps.
Meet the study director for ToxTracker EMP
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 EMP in understanding where and how ToxTracker EMP 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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Mathyn Rijkers
Study director for ToxTracker
Mathyn obtained his Master’s degree in Biomedical Sciences at Utrecht University. During his major internship, he studied the effects of micro- and nanoplastics on macrophages at the Institute for Risk Assessment Sciences in Utrecht. In February 2022, Mathyn joined Toxys as an intern for his minor internship, where he investigated the applicability of human skin S9 in the ToxTracker assay. Mathyn is currently working as the study director for ToxTracker.
Can you help with a study design for difficult to dissolve substances?
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