Nitrosamines are organic compounds, long known to be probable carcinogens, yet are found in low level concentrations in air, water, soil, and food.  Both FDA and EMA have outlined a three step framework for mitigating or preventing the presence of nitrosamines in medicinal products which includes the following:

• Risk assessment
• Testing
• Risk Mitigation Measures

Water has been identified as a potential source of contamination of nitrosamines in pharmaceutical synthesis.  FDA’s publication Control of Nitrosamine Impurities in Human Drugs Guidance for Industry Rev 1, FEB 2021 raises the following concern regarding API manufacturing:

“API manufacturers should be aware that potable water used in API manufacture may contain low levels of nitrite and even nitrosamines from environmental contamination. The existence of nitrites in processing water may lead to nitrosamine contamination in API manufacture. Therefore, to avoid unacceptable levels of nitrosamine impurities in APIs, API manufacturers should analyze nitrite and nitrosamine levels in water and use water that has been purified to remove unacceptable impurities.”

Sources of Contamination

Nitrosamines are formed through a series of mechanisms but most generally from the reaction of amines with nitrites.  Several forms of nitrosamines exist with the most common being N-nitrosodimethylamine (NDMA) which can be found in drinking waters treated with chlorine or chloramines.  There are two most likely sources of nitrosamines contamination in pharmaceutical product waters.  They may be present in the feed water to the treatment system (potable water) and not subsequently removed by a treatment process.  Second, they may be unfavorably generated as a by-product of the treatment process itself when nitrogen containing compounds such as amines are present.  The recent release of EMA’s 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 Rev 21DEC2022 outlines one potential source of nitrosamine contamination when ion-exchange technology is used as part of the water treatment process.

“ Reaction of amines leaching from quaternary ammonium anion exchange resins (e.g. used for purification steps) with nitrosating agents present in the liquid phase. A recent example of this was in the production of water for injections where residual chloramine used to disinfect incoming water reacted with dimethylamine leaching from the anion exchange resin used in the demineralisation step to form NDMA. In addition, disinfection procedures such as e.g. chlorination, chloro-amination and ozonisation can lead to significant N-nitrosamine generation as by-products in case vulnerable amines are present.”

Establishing a Nitrosamine Specification for Pharmaceutical Waters

It is the responsibility of the drug manufacturer to ensure that the product does not contain impurities that may alter the strength, identity, purity or efficacy of the drug product.  This logic extends to pharmaceutical waters.  In addition, any foreign impurities, even if their testing are not mandated by the pharmacopoeiae, are not permitted above acceptable limits.  An acceptable limit is based on the product and risk to patient and may well be different for different drugs or classes of pharmaceutical preparations.  Hence, acceptable limits of nitrosamines may vary for different grades of waters based on their intended purpose.  A risk assessment should be used to to determine if nitrosamine detection is required for pharmaceutical waters, and if appropriate, the acceptable concentration limit, sampling location(s) and frequency

The EU trade organization recently commented on the presence of nitrosamines in WFI.  EFPIA’s N-nitrosamine Impurities in Biological Medicinal Products, dated 11 November 2020 states the following:

Throughout this paper, reference is made to the negligible level of nitrite in the water for injection (WFI) used to manufacture and store biological medicinal products. When WFI is manufactured by distillation of Purified Water, it is expected that there would be essentially no nitrite or other known nitrosating agent present. Since the source Potable Water used to manufacture WFI can vary in nitrite/nitrate content [2], any subsequent Purified Water or WFI manufactured from Purified Water by methods other than distillation (e.g. reverse osmosis combined with ultrafiltration or deionisation), should be evaluated to assure that it too is essentially free of nitrosating agents. 

The concentration of nitrite in Purified Water of WFI is expected to be far below 2×10-7 M (approximately 0.01 mg/L) as this level is typically seen in potable water.

This suggests that testing, to a maximum specification of 10 ppb, is required for the presence of nitrosating compounds in final product water when the WFI is generated by two specific methods; either by distillation when not fed from Purified Water, or when generated using non-distillation technologies.

Removal of Nitrosamines in Pharmaceutical Water Systems

Removal or reduction of nitrosamines to acceptable concentrations in pharmaceutical water systems can be accomplished by several methods or a combination of methods.  Due to the light molecular weight of the compounds, reverse osmosis (RO) will likely reduce concentrations but may not be the most efficient process.  Adsorption via actuated carbon is even less effective.  High dose UV light and advanced oxidation techniques have been proven effective for both water and wastewater treatment applications.

Removal of nitrosamine precursors such as nitrite and nitrates prior to ozonation, and controlling the leaching of amines from ion-exchange resins are also effective techniques.  The latter may be accomplished by avoiding stagnant DI tanks and periodic rinsing of anion or mixed bed DI systems when not in use.

Path Forward

Future guidance from industry organizations and regulatory agencies regarding the presence of nitrosamines in pharmaceutical water preparations is expected.  Although a specific limit for these compounds may never be adopted for compendial waters, all drug manufacturers are responsible that water used in the manufacture of drug compounds or drug components contain no added substances, nor any foreign substances or impurities.  Regardless of the source of contamination, it is likely that the presence of nitrosamines in pharmaceutical waters will be more rigorously scrutinized in the future.