What are nitrosamine compounds? Nitrosamines (also known as N-nitrosamines) are a class of compounds that contain a nitroso group (NO) bonded to a deprotonated amine. Most nitrosamines are carcinogenic; however, they are widely found in air, water, cosmetics, tobacco, packaging materials, as well as in foods. In foods, they arise from the reaction of preservatives, such as nitrites, in cured meats, such as bacon and sausages. Food processing at high temperatures or involving fermentation generate nitrogen oxides which react with amines in the food to produce nitrosamines.
Some N-nitrosamines, such as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), are mutagens with such extremely high carcinogenic potency that they are members of a “cohort of concern”. Compounds are mutagenic if they react with DNA (causing DNA damage). Mutagenic compounds may also be carcinogenic.
Ensuring the absence or reduction of these compounds to acceptably low levels in pharmaceuticals is of primary concern, evidenced by the release of guidances on this topic by both the FDA and the EMA over the last two years. ICH M7[1]defines a Threshold of Toxicological Concern (TTC; 1.5 µg/day) for compounds to define an acceptable intake for any unstudied chemical that poses negligible carcinogenicity risk. The intakes of highly potent carcinogens, such as N-nitrosodimethylamine (NDMA), and N-nitrosodiethylamine (NDEA), even below the TTC may be associated with a potential for significant carcinogenic risk.
Drugs recalled due to nitrosamine impurities. Regulatory agencies were initially informed of nitrosamine impurities in the APIs of marketed angiotensin II receptor blockers (ARB), valsartan (NDMA), irbesartan (NDEA), and losartan (NDEA, NMBA), which led to recalls and temporary drug shortages of these drugs. Soon afterward, nitrosamine impurities were reported in other drugs such as ranitidine (NDMA), metformin (NDMA), nizatidine (NDMA), rifampin (1-methyl-4-nitrosopiperazine, MNP) and rifapentine (1-cyclopentyl-4-nitrosopiperazine, CPNP).
Call for review. Because nitrosamines are probable or possible human carcinogens, a risk assessment of nitrosamine contamination or formation in APIs and drug products is required. Marketing authorization holders (MAHs) and applicants of all human medicinal products should ensure control of nitrosamine levels at the lowest levels possible, irrespective of marketing status or type of product (e.g. generics, over-the-counter products).
When a risk for the presence of nitrosamine impurities is identified, confirmatory analysis is conducted, and changes made to prevent or reduce nitrosamine impurities in APIs and drug products with pending applications or approved/marketed drug products are reported to the regulatory authorities. Initially, the risk assessment of nitrosamine impurities was limited to medicinal products containing chemically synthesized APIs, but the scope has recently been expanded to include biological medicinal products, particularly those containing chemically synthesized fragments, employing processes where nitrosating reagents are deliberately added, or packaged in certain primary packaging material, such as blister packs containing nitrocellulose.2
The call for review consists of 3 steps:
(1) risk evaluation to identify risk of nitrosamines in APIs and/or finished drug products;
(2) if risk is identified, confirmatory testing is conducted to either confirm or refute the presence of nitrosamines. MAHs/sponsors should report outcomes immediately; and
(3) if the presence of nitrosamine(s) is confirmed, MAHs/sponsors should implement effective risk mitigation measures through submission of a variation.
Nitrosamine formation. Nitrosamines may be formed in the presence of amines and nitrosating agents such as nitrous acid or nitrite salts (e.g. sodium nitrite) under acidic conditions. Primary, secondary and tertiary amines all undergo nitrosation, but secondary amines generally are the most reactive to nitrosating agents, producing nitrosamines. Primary alkyl amines react with nitrosating agents to give short-lived, highly reactive diazonium ions, which decompose to give molecular nitrogen (N2) by substitution, elimination or molecular rearrangement.
Potential sources of nitrosamine impurities in pharmaceutical drugs are listed by the European Medicine Agency (EMA)[2] and FDA.3
· Use of sodium nitrite (NaNO2), or other nitrosating agents, in the presence of secondary, tertiary or quaternary amines within the same or different steps (carryover) of the manufacturing process. For example, nitrous acid used for quenching residual azide, may react with residual amine in raw materials used in the manufacturing process to give nitrosamines.
· Use of sodium nitrite (NaNO2), or other nitrosating agents, in combination with reagents, solvents (e.g. DMF, DMAc and NMP), and catalysts, which are susceptible to degradation to secondary or tertiary amines, within the same or different process steps. Amide solvents (e.g. DMF) degrade under high temperatures for prolonged periods to give dimethylamine. Secondary and tertiary amines may be present as impurities or degradants formed by dealkylation of quaternary amines (e.g. tetrabutylammonium bromide).
· Use of contaminated raw materials in the API manufacturing process (e.g. potable water, solvents, reagents and catalysts).
· Use of contaminated recovered or recycled materials (e.g. solvents, reagents and catalysts), including recovery outsourced to third parties who are not aware of the content of the materials they are processing and recovery processes carried out in non-dedicated equipment.
· Use of contaminated starting materials and intermediates supplied by vendors who use processes or raw materials which may contain residual nitrosamines or nitrosating agents.
· Carryover of nitrosamines deliberately generated (e.g. as intermediates) during the manufacturing process.
· Cross-contamination due to different processes being run successively on the same manufacturing line.
· Carry-over of impurities between process steps due to operator-related errors or insufficiently detailed batch records such as inadequate phase separations during work-up procedures.
· Degradation processes of starting materials, intermediates and active substances, including those induced by inherent reactivity (e.g. presence of nitro, oxime, or other functionality) or by the presence of an exogenous nitrosating agent. This could potentially occur also during finished product formulation or storage and could be influenced by crystal structure, crystal habit and storage conditions (temperature, humidity etc.).
· Use of certain packaging materials. Nitrosamine contamination has been observed in finished products stored in blister packs with lidding foil containing nitrocellulose. Nitrosamines have been shown to form from nitrocellulose degradation products and low molecular weight amines present either in printing ink or in the finished product during the blister heat-sealing process and to transfer to the product within the blister.
· Reaction of nitrosatable nitrogen functionality in APIs or their impurities with nitrosating agents present in components of the finished product during formulation or storage.
Limits for nitrosamines in medicinal products. Numerous regulatory authorities, including the US FDA have set internationally recognized acceptable daily intake limits for single known nitrosamines.2,[3] The FDA recommends recalls if nitrosamine levels in an approved drug exceed the allowable daily limits.
For new N-nitrosamine impurities with sufficient substance specific animal carcinogenicity, a TD50 should be calculated and use to derive a substance specific limit for lifetime exposure, as recommended in ICH M7(R1).1 For new N-nitrosamines without sufficient substance specific data to derive a limit for lifetime exposure, a class-specific threshold-of-therapeutic-concern (TTC) can be derived as a default option, or an acceptable intake limit based on SAR considerations may be used, if appropriately justified.
When more than one nitrosamine is identified, two approaches are considered acceptable to not exceed the acceptable risk level of 1:100,000 as outlined in ICH M7(R1):1,2
(1) the total daily intake of all identified N-nitrosamines should not exceed the acceptable intake of the most potent N-nitrosamine identified; or
(2) total risk level calculated for all identified N-nitrosamines does not exceed 1 in 100,000.
Sensitive methods with limits of quantitation (LOQ) in the parts-per-billion (ppb) range are needed to meet the low acceptable intake limits for nitrosamines.2,3
Mitigation the presence of nitrosamines in APIs. When there is a potential for nitrosamine impurities, the FDA recommends actions by API manufacturers, including route of synthesis development to minimize or prevent formation of nitrosamine impurities. Specific actions are recommended in the FDA guidance.3
Recommended timelines. Both the FDA and EMA have recommended timelines for completion of the risk assessment, confirmatory testing and submission of required changes, depending on the regulatory status of the drug product.2,3 For example, the risk assessment should be completed for approved or marketed drug products in the US by March 2021, and confirmatory testing – if a nitrosamine risk is identified from the risk assessment – and submission of required changes implemented to prevent or reduce nitrosamine impurities by September 2023.3
[1]. ICH Guideline M7(R1) - Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, August 25, 2015 [2]. EMA – Questions and Answers for Marketing Authorisation Holders/Applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 Referral on Nitrosamine Impurities in Human Medicinal Products, EMA/409815/2020, 03 August 2020. [3]. FDA Guidance for Industry - Control of Nitrosamine Impurities in Human Drugs, September 2020