Toxys scientists recently published an article in Environmental and Molecular Mutagenesis entitled “An Enhanced Metabolization Protocol for In Vitro Genotoxicity Assessment of N-Nitrosamines in Mammalian Cells”. In this article, we established a proof of concept for an exogenous metabolization protocol that activates N-nitrosamines (NAs) and is compatible with mammalian cell culture. With this enhanced metabolization protocol (EMP), we detected N-nitrosodimethylamine (NDMA) induced genotoxicity at ~200-fold lower concentrations in comparison to the standard hamster S9 exposure protocol.

Current status on nitrosamine impurities

N-Nitrosamines (NAs) are probable human carcinogens and are part of the cohort of concern in the ICH M7(R2) guidance on impurities in pharmaceuticals. The genotoxic potency of nitrosamine drug substance-related impurities (NDSRIs) is often unknown, and about 40% of all drugs are at risk for NA formation.

To detect this class of metabolically activated mutagens in vitro, an enhanced bacterial mutagenicity protocol (i.e., the enhanced Ames test, or “EAT”) was recently standardized. However, until now, there has been no such standard experimental design for detecting NA induced mutagenicity in mammalian cells. In the current study, we therefore focused on optimizing a protocol for the metabolization of N-nitrosamines utilizing the ToxTracker assay, a mammalian cell-based GFP reporter assay for predicting genotoxicity.

Enhanced metabolization protocol (EMP) for the ToxTracker assay

The standard metabolization protocol in ToxTracker uses exogenous rat S9 liver extract, with a 3-4 hour exposure window, which is inefficient at biotransforming N-nitrosamines. Similar to EAT, we optimized a pre-incubation exposure protocol using hamster S9 and concentrated exposures, followed by a 4-hour exposure with 3% v/v hamster S9 at final N-nitrosamine concentrations. Furthermore, we introduced an additional cofactor into the S9 mix, which taken together, enhanced detection of N-nitrosamines in this mammalian cell-based genotoxicity assay. We believe that this EMP can be extended to other mammalian cell-based assays.

Qualification of EMP

We qualified EMP further by assessing 7 additional mutagenic N-nitrosamines to which humans are commonly exposed: N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDELA), N-nitrosodibutylamine (NDBA), 1-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), 1-cyclopentyl-4-nitrosopiperazine (CPNP), and N-nitrosofluoxetine (NF). We confirmed genotoxicity for 7 NAs (except for NDELA) using the EMP. In addition, we tested two N-nitrosamines that were previously shown to be non-mutagenic. Using the ToxTracker-EMP, we classified both N-nitrosbuproprion (NBuPRO) and N-nitrosoproline (NPRO) as non-genotoxic.

To conclude, our work demonstrates that the new approach methodology (NAM), ToxTracker, or the EMP itself, can become a new standard when evaluating the safety of N-nitrosamines and may assist in justifying daily human intake when managing N-nitrosamine impurities in pharmaceuticals.

If you would like to apply this approach to your project(s), please contact us. You can also learn more about ToxTracker EMP on our website or access the full publication below.