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An Enhanced Metabolization Protocol for In Vitro Genotoxicity Assessment of N-Nitrosamines in Mammalian Cells

M E Geijer1, A M Gernaat1, N Moelijker1

  • 1Toxys B.V., Oegstgeest, the Netherlands.

Environmental and Molecular Mutagenesis
|March 28, 2025
PubMed

Insights

N-Nitrosamines (NAs) are probable carcinogens found in pharmaceuticals. An enhanced metabolization protocol improved in vitro genotoxicity testing of NAs in mammalian cells using ToxTracker.

Area of Science:

  • Toxicology
  • Genetics
  • Pharmacology

Background:

  • N-Nitrosamines (NAs) are probable human carcinogens and are found as impurities in pharmaceuticals, raising health concerns.
  • Metabolic activation is required for NA mutagenicity, but in vitro metabolization using standard S9 liver extract is often inefficient for mammalian cell genotoxicity assays.

Purpose of the Study:

  • To optimize an in vitro metabolization protocol for N-nitrosamines (NAs) in mammalian cells.
  • To assess the genotoxic potential of various NAs using the optimized protocol and the ToxTracker assay.

Main Methods:

  • Developed and optimized an enhanced metabolization protocol (EMP) using hamster S9 liver extract for in vitro NA exposure.
  • Utilized the ToxTracker assay, a new approach methodology (NAM), to evaluate the genotoxicity of NAs under EMP conditions.
  • Validated the EMP with a range of known mutagenic and non-mutagenic NAs, including NDMA, NDEA, and NPRO.

Main Results:

  • The enhanced metabolization protocol (EMP) increased the genotoxic potency of N-nitrosodimethylamine (NDMA) by approximately 200-fold compared to standard protocols.
  • Genotoxicity was confirmed for six out of seven tested mutagenic NAs using the EMP and ToxTracker.
  • Two NAs, N-nitrosobupropion (NBuPRO) and N-nitrosoproline (NPRO), were confirmed as non-mutagenic.

Conclusions:

  • The optimized enhanced metabolization protocol (EMP) significantly improves the detection of NA genotoxicity in vitro.
  • The combination of EMP with the ToxTracker assay demonstrates potential for robust investigation of NA-related genotoxicity in mammalian cells.
  • This approach aids in assessing the health risks associated with NA impurities in pharmaceuticals.
Keywords:
N‐nitrosaminesToxTrackergenotoxicityimpuritiesin vitro metabolization

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