How limiting N-nitrosamine content helps assure patient safety

N-nitrosamines are organic compounds with the chemical structure R2N-N=O. N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were detected in Sartan medicinal products in 2018. Since then, N-nitrosamines have been found in several other treatments, with NDMA reported in the first non-Sartan product in 2019 [1, 2]. The International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) refers to these materials as “cohorts of concern” due to their genotoxic and potentially carcinogenic nature. Therefore, N-nitrosamine impurities in pharmaceutical products pose significant risks to patients.

The toxicity of these compounds is a result of their metabolism within the body. Upon reaching the liver, N-nitrosamines react with DNA base pairs to form unstable -hydroxyalkylnitrosamines and produce alkyldiazonium ions. These materials then transfer an alkyl group onto DNA bases and induce a carcinogenic response [1].

Regulatory agencies impose limitations on acceptable levels of these materials in pharmaceutical products to mitigate the risk to patients. For N-nitrosamines, these limits are defined as the levels where the risk of human cancer is negligible — a class-specific limit of 18 ng/day (EMA class-specific limit) is used where a compound-specific limit cannot be developed [3]. The FDA has a higher-class specific limit of 26.5 ng/day for the most potent nitrosamines. Both the EMA and the FDA allow for a higher acceptable intake (AI) depending on the structure of the nitrosamine. The FDA allows the assignment of differing AI depending on a carcinogenic potency category based on an assessment of activating or deactivating structural features [4]. To ensure that the presence of N-nitrosamine impurities in medicinal products falls below these limitations, several strategies can be adopted.

Understanding N-nitrosamine formation

As N-nitrosamine impurities form unintentionally, it is important to understand this process to prevent it. A non-exhaustive list of common routes to N-nitrosamine formation includes:

• Secondary amines undergo N-nitrosation to form N-nitrosamines.
• Solvent (dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP)) degradation — hydrolytic and/or thermal — produces secondary amines N,N-dimethylamine (DMA) and 4-methylaminobutyric acid (MBA) respectively before subsequent N-nitrosation.
• N-nitrosative de-alkylation of tertiary amines (e.g., trialkyl amines) — such as triethylamine (TEA), N,N-diisopropylethylamine (DIPEA) — yields N-nitrosamines, including N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (DIPNA), N-ntirosoethylisopropylamine (EIPNA)  and N-nitrosomethylphenylamine (NMPA).
• Hydrolytic dissociation of quaternary ammonium salt to produce the tertiary amine, followed by N-nitrosative dealkylation to form N-nitrosamines (e.g., yielding NDEA from tetraethylammonium chloride).

By understanding the pathway to N-nitrosamine formation, measures can be introduced to prevent their formation during drug product synthesis, assuring final product concentrations remain within the threshold of toxicological concern (TTC) limits.

Mitigating risk through reduced N-nitrosamine formation

To ensure patient safety, regulatory agencies demand N-nitrosamine risk assessments to be completed on all pharmaceuticals being brought to market. Through this assessment, all raw materials, processes and procedures are investigated to highlight any potential causes of N-nitrosamine formation. With no control over amine or nitrosating agent presence in raw materials, a risk-based approach minimises the risk of N-nitrosamine formation. From this, the risk can be mitigated by:

• Choosing the best materials: Reducing the likelihood of N-nitrosamine formation by avoiding secondary, tertiary, quaternary amine or amide solvents in the presence of a nitrite or nitrosating agent.

• Preventing unintentional reactions: Assessing and preventing potential reactions between starting materials, intermediates, products and solvents. If N-nitrosamine production risks are found, then processes may be altered to remove these hazards.
• Using purified water: Opting for purified water with a nitrite content estimated at 0.001 mg/L results in negligible risk.
• Employing new solvent every time: Minimising the risk of unknown contaminants within a reaction, solvent recycling is avoided.

As part of this strategy, accurate and robust analytical method development is key to identifying the presence of N-nitrosamines and potential precursor materials (amine and nitrites). Examples include the use of LCMS methods to measure low levels of compounds.

Alternatively, a predictive purge calculation approach can be used. ICH M7 Option 4 permits having a comprehensive understanding of process parameters and their impact on impurities, with confidence that levels will fall below the limit, meaning no analytical testing is required [5]. For this purge calculation, a value is associated with each parameter (reactivity, volatility and solubility), and a score is generated to determine if Option 4 can be applied. Also, proving that the amine source is not present at the same stage as the nitrosating agents mitigates the risk of N-nitrosamine formation.

Reducing risk through lower N-nitrosamine formation

By taking a proactive approach and considering all materials and processes, the formation of N-nitrosamine impurities can be limited to meet regulatory-compliant levels.

Onyx Scientific is a small molecule CDMO with the expertise to support the development and manufacturing of small molecules throughout their life cycle. Our specialist knowledge can help you mitigate N-nitrosamine formation and ensure product quality. Find out more by contacting us today.

References

1. Sedlo, I., Kolonić, T., & Tomić, S. (2021). Presence of nitrosamine impurities in medicinal products. Arhiv za higijenu rada i toksikologiju, 72(1), 1–5. https://doi.org/10.2478/aiht-2021-72-3491
2. Wichitnithad, W., Nantaphol, S., Noppakhunsomboon, K., & Rojsitthisak, P. (2023). An update on the current status and prospects of nitrosation pathways and possible root causes of nitrosamine formation in various pharmaceuticals. Saudi Pharmaceutical Journal: SPJ: the official publication of the Saudi Pharmaceutical Society, 31(2), 295–311. https://doi.org/10.1016/j.jsps.2022.12.010
3. https://www.ema.europa.eu/en/documents/other/appendix-2-carcinogenic-potency-categorisation-approach-n-nitrosamines_en.pdf
4. https://www.regulations.gov/document/FDA-2020-D-1530-0032
5. Barber, C., Antonucci, V., Baumann, J.-C., Brown, R., Covey-Crump, E., Elder, D., … Welch, D. (2017). A consortium-driven framework to guide the implementation of ICH M7 option 4 control strategies. Regulatory Toxicology and Pharmacology, 90, 22–28. https://doi:10.1016/j.yrtph.2017.08.008