What Are N-Nitrosamines?

N-Nitrosamines

N-nitrosamines, represented by the structural formula R1(R2)N-N=O, are formed through various mechanisms, including reactions between secondary amines and nitrous acid, tertiary amines and monochloramine, oxidation of N-dimethylformamide (DMF), and exposure to rubber vulcanizing agents and nitrogen oxides. These compounds are widely present in the environment and food sources such as air, water, soil, bacon, and alcoholic beverages. Additionally, they can be generated during drug manufacturing.

Carcinogenic Potential of N-nitrosamines

N-Nitrosamines exhibit genotoxic properties, causing DNA damage and cancer, with their risk being independent of dose—meaning even very low exposures can be harmful. The International Agency for Research on Cancer (IARC) has classified several N-nitrosamines as carcinogens, placing N-dimethylnitrosamine (NDMA) and N-diethylnitrosamine (NDEA) in Group 2A and others in Group 2B. According to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) M7 guideline, highly carcinogenic NDSRIs fall under Class I impurities, necessitating strict control.

The carcinogenic nature of N-Nitrosamines stems from their metabolites. Based on the α-hydroxylation hypothesis, enzymatic hydroxylation activates NDSRIs, leading to the formation of carcinogenic intermediates that result in DNA base alkylation and cancer development.

Mechanism of NDMA's carcinogenicity:

NDSRIs

NDSRIs are a class of nitrosamines sharing structural similarity to the API (having the API or an API fragment in the chemical structure) and are generally unique to each API. NDSRIs form through nitrosation of APIs (or API fragments) that have secondary, tertiary, or quaternary amines when exposed to nitrosating compounds such as nitrite impurities in excipients. Figure 3 shows the representative reaction of an API containing a secondary amine functional group in its structure with nitrite under acidic conditions.

Regulatory and Analytical Advances

Regulatory bodies like EFSA emphasize reducing N-nitrosamine levels in food. Analytical methods such as gas chromatography with chemiluminescence detection (GC-TEA) enable precise measurement, even at trace levels (e.g., 0.1–0.5 μg/kg in meats) 3. Notably, N-nitrosamine levels in Dutch cured meats have declined since the 1970s, reflecting improved processing practices.

The organizations regulate N-Nitrosamine levels includes:

• Food and Drug Administration (FDA): Limits in pharmaceuticals and food products
• Environmental Protection Agency (EPA): Drinking water standards for nitrosamine contamination
• European Medicines Agency (EMA): Strict controls on drug formulations to minimize impurities

Conclusion

N-Nitrosamines remain a critical public health challenge due to their potent carcinogenicity and pervasive presence. Ongoing research and regulatory efforts aim to reduce exposure through improved food processing, analytical monitoring, and consumer awareness. Collaborative action across industries and governments is essential to mitigate their risks effectively.

Keywords: N-Nitrosamines, NDSRIs, OECD 471, Enhanced Ames test, Hamster liver S9, Cytochrome P450 enzymes, Mutation Test