What Does the FDA's Recent Update of the Nitrosamine Guidance Convey?

In September 2024, the much-anticipated update to FDA’s Guidance for Industry, Control of Nitrosamine Impurities in Human Drugs was published (1). This update is being called Revision 2 by the agency; the original guidance was published in 2020 and updated in February 2021. The most significant change in Revision 2 is the differentiation of nitrosamines into two classes: common, smaller nitrosamines originating from reagents, solvents, etc., and the larger and more complex nitrosamine drug substance-related impurities, referred to as nitroso drug substance related impurities (NDSRIs). This differentiation, when originally mentioned by FDA a few years back, had led to a conundrum in the pharmaceutical industry regarding how and when NDSRIs should be controlled in drugs. Revision 2 of the guidance (September 2024) could be considered a paradigm shift from Revision 1 (February 2021), which implicated the major cause of concern as smaller nitrosamines originating from reagents, solvents, and other extraneous sources and mentioned six nitrosamines with their proposed acceptable intakes (AIs). The current update (Revision 2) has a detailed discussion on several topics related to nitrosamines in pharmaceuticals, the smaller and more ubiquitous ones such as N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), as well as the larger, previously unknown NDSRIs. While not all inclusive, Revision 2 is a beneficial road map to evaluate and control the risk related to nitrosamines in pharmaceuticals.

In this article, the author has attempted to highlight some of the salient features that were not in the original guidance and have been included in Revision 2 and their impact on the industry. The author has also tried to point out some of the gaps in the guidance, which the industry should be cognizant of and address, hopefully in the foreseeable future.

Overview of guidance Section III: Nitrosamine Impurities and Root Causes of Formation

Sections I and II of Revision 2 are general information, and the actual content starts from Section III.

In Section III of Revision 2, FDA has demarcated the difference between the smaller and more common nitrosamines such as NDMA, NDEA, N-Nitrosodiisopropylamine (NDIPA), N-Nitrosodibutylamine (NDBA), N-nitrosoethylisopropylamine ( NIEPA), and N-Nitrosomethylphenylamine (NMPA), which could arise from reagents, solvents, and other sources containing secondary, tertiary, or quaternary amines or amides of secondary amines (e.g., dimethylformamide) from the larger, more complex nitrosamines, which can form based on the inherent structure of the API itself or intermediates, impurities that resemble the API. The latter have been labeled by FDA as NDSRIs(nitroso drug substance related impurities), a terminology that FDA has been using for a few years now.

Additionally, in Section III, FDA has also outlined the root causes of presence of small nitrosamines and NDSRIs in drug substance and drug products. The agency has clarified that even if no nitrite is used in a route of synthesis of an API or the manufacturing process of a drug product, due to its ubiquitous nature, nitrite may be present in small quantities in reagents, excipients, packaging material, and equipment. Thus, nitrosamine risk is high whenever vulnerable amines such as secondary, tertiary, and quaternary amines or amides of secondary amines are present either as a reagent, solvent, or by virtue of the structure of the API, intermediates, or impurities/degradants, irrespective of use of nitrite in the process. Henceforth, in this article, the term, “vulnerable amine/s” will be used to describe the secondary, tertiary, quaternary amines, or amides of secondary amines.

Author’s observation. This clarification by FDA is especially significant because the previous version of the guidance was not clear about the ubiquitous nature of nitrite. Thus, many cases, where sponsors and manufacturers had concluded the absence of nitrosamines even if the process or product included vulnerable amines, based on absence of nitrites or nitrosating agents in their risk evaluation, will need to be revisited. The current update has reiterated the well-known fact that nitrite is everywhere, and thus, the absence of nitrosamine should be verified if API and/or drug product have vulnerable amines.

Revision 2 has an interesting footnote (#28) regarding nitrosamines being formed by tertiary amines and amides of secondary amines. It is well known in the public domain (2) that tertiary amines nitrosate at a much slower rate than secondary amines. However, as the nitrosamine saga unfolded, it was obvious that several drugs with APIs that are tertiary amines showed the presence of the corresponding NDSRIs in the API itself as well as the drug product, over shelf life. In this footnote, the agency has confirmed that tertiary amines are a viable risk of nitrosamines. Amides of secondary amines, which are prone to degrade by hydrolysis to secondary amines, are significant risk nitrosamines and should be considered nitrosamine risks.

Author’s observation. The agency clarified their stance related to tertiary amines and amides of secondary amines and indicated that they should be considered as vulnerable as secondary amines, when it comes to nitrosation. This clarification would help in eliminating the discrepancies in nitrosamine risk evaluation of the same product by different sponsors, some of whom took the tertiary amines in consideration and some who did not. This guidance levels the playing field where nitrosamines of all vulnerable amines should be considered.

In Revision 2, FDA put significant responsibility on the drug product manufacturers to make sure that the supplier/vendor qualifications are adequate when it comes to excipients, water, and packaging materials. In Section IIIC, FDA mentions that, in addition to evaluating the presence of nitrosamines, the supplier qualification program should also take into consideration the presence of nitrites. During the leachable and extractable studies, the possibility of nitrosamines as well as nitrites leaching into the drug product should be taken into consideration.

Author’s observation. This advice from FDA has the potential of increasing the workload of the manufacturers and sponsors of drug products, as, in addition to nitrosamines, they may need to test for the nitrite content in the excipients.

The possibility of studying the nitrite as an extractable and leachable from packaging configurations is relatively new and thus should be noted by the industry.

Overview of Section IV: Recommendations for Control and Mitigation of Nitrosamines in APIs and Drug Products

Section IV is the most significant part of the current guidance, which discusses the controls and mitigation strategies for nitrosamines in drugs. The International Council for Harmonisation (ICH) document, Q9(R1) Quality Risk Management, has been mentioned as a roadmap to risk management (3).

To start with, FDA has provided advice regarding the prioritization of risk from the regulatory perspective. Example, chronic drugs with high maximum daily dose (MDD) would take priority over low-dose drugs or drugs with short-term use.

Author’s observation. The agency has not included market share as one of the critical prioritization criteria that the pharmaceutical industry should pay attention to. If a sponsor has a significant market share of a product that has low dose or short-term use, the priority should be to work on that product over others with high MDD and high dose, to keep the market share intact and to avoid drug shortages.

The author notes that FDA has information regarding prioritization of nitrosamine risk based on short-term use in footnote #29 of the guidance. It is noteworthy that FDA has until now refused to accept the less than lifetime (LTL) approach of ICH Q7(R2) in setting specifications of nitrosamines (4). There seems to be a contradiction to what is being recommended in Revision 2 and not accepting LTL for nitrosamines. In Revision 2, FDA indicates that when two drugs have comparable amounts of a nitrosamine, the drug with short term use is of lower risk. As an extension of this conjecture, and following the recommendations of ICH M7, allowing higher limits of nitrosamines drugs with short-term use should then be considered low risk and health protective. FDA, however, has repeatedly resisted the LTL approach with nitrosamines. Hopefully, this fallacy of nitrosamine and LTL will be addressed in the near future by FDA as well as ICH M7 updates.

In Revision 2, FDA established the much needed bridge between this guidance and the FDA guidance for industry, Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) (5), which has been referred to as RAIL guidance, and the information on nitrosamines on FDA’s website (6). It is clear from the current version that FDA would like the sponsors to take a holistic view of controlling nitrosamines based on all the information provided. While the previous version of this guidance had provided the AIs of a limited number of small and common nitrosamines, and, given a blanket comment that all other nitrosamines should be controlled at 26.5 ng/day unless other information are available, Revision 2 has merged the requirements with the RAIL guidance and subsequent communication of the agency regarding the process of setting AIs of nitrosamines with the carcinogenic potency categorization approach (CPCA) and, in special cases using surrogates, in-vitro or in-vivo methods, mentioned website (6). It has also been suggested that if the total nitrosamines in a drug product is below 26.5 ng/day, additional safety data are not needed.

Author’s observation. FDA has steered the industry to CPCA to determine the AI of NDSRIs that are data poor, based on the RAIL guidance. While the agency has mentioned that other in-vivo and in-vitro approaches may be used to set limits above what CPCA recommends and to derisk the nitrosamines, it is apparent from the concurrent updates provided on FDA’s website (6) that derisking a specific nitrosamine and confirming an AI above 1500 ng/day would be an uphill battle that pharmaceutical companies with limited resources would find expensive and time consuming. The section, “Recommended Safety Testing Methods for Nitrosamine Impurities” in the webpage (6) states the following, “In addition to the enhanced Ames test, the Agency is currently requesting a second in vitro mammalian cell mutation assay and in vitro metabolism data (including human hepatocyte or microsome) to support an AI limit of 1500 ng/day” (6). Thus, proposing an AI of 1500 ng/day for a nitrosamine based on the negative enhanced ames assay is no longer valid. While the agency has mentioned that higher limits based on “read-across” may be acceptable, it has been a trend in FDA to resist an AI that is higher than 1500 ng/day based on read-across, no matter how good the match is and how well studied the surrogate is.

FDA has indicated that it may not be open to accepting limits based on ICH Q3A (7) or ICH Q3B (8) founded on negative in-vivo studies, irrespective of the fact that other agencies may have accepted the above. This decision is confusing and may be the cause of hardship to the pharmaceutical industry, which is global in nature; however, it does help dispel the notion that a nitrosamine has been derisked by the European Medicines Agency (EMA) or Health Canada (HC), is derisked by FDA.

From the combined recommendations in the RAIL Guidance, the webpage (6) and the Revision 2, it is amply clear that FDA’s favored approach for setting AIs for NDSRIs is the use of CPCA, followed by a read-across, as long as adequate information is available for the surrogate and the AI proposed does not exceed 1500 ng/day.

The agency has merged the criteria for justifying limits of multiple nitrosamines in a drug product with the EMA and HC recommendations. FDA has completely adopted the flexible approach that EMA, HC, and other agencies have been using for some time now, to establish a specification when multiple nitrosamines are present (information Appendix C of Revision 2).

Author observation. The updates to the recommendations related to control of multiple nitrosamines are logical and aligned with other agencies and are a welcome change compared to what was provided in the original guidance of 2020 and 2021. Industry should remember that the control of total nitrosamines should include the smaller nitrosamines as well as NDSRIs arising from the APIs, intermediates, and/or impurities.

In Section IV.C.2 of this version of the guidance, FDA has stated that if the level of nitrosamine is above 10% of the proposed AI, then each drug product batch should be tested at release, stability, and retest dates. This version also states that any lot of API with nitrosamine above the proposed AI should not be released.

Author observation. This recommendation varies from the recommendations of EMA and HC, in that these later guidance documents state that an amount of nitrosamines less than10% of AI can justify omission of specification, and an amount less than 30% of AI may justify skip testing. FDA has no option of reduced testing when the nitrosamine is consistently within the range of 10% to 30% of the AI. Instead, FDA has proposed that if the amount of nitrosamine is above 10% of AI, each and every batch should be tested at release and stability, which could lead to significant increase in workload of the pharmaceutical manufacturers.

In Section IV.D.2.a, FDA placed the responsibility of establishing the reliability of API suppliers on the drug product manufacturers and applicants. The guidance indicates that the information regarding the nitrosamine levels provided by the API suppliers should not be accepted by the drug product (DP) manufacturers without testing significant number of lots and repeating the verification testing from time to time.

Author observation. FDA’s opinions indicate that they hold the DP manufacturers and applicants to be responsible for all aspects of controlling the nitrosamine risk. While this could be overwhelming to the drug product manufacturers, they need to take note that not testing the APIs based on the evaluation provided by the API supplier may not be acceptable when the API has vulnerable amines. In addition to being a review issue, this topic could be a compliance-related issue, where the API and raw materials-related vendor validation may need testing of several lots for possible nitrosamines and in some cases, nitrites (see comments in Section III). However, this in no way reduces the responsibility of the API manufacturers, who need to do their own due diligence and propose controls of nitrosamines wherever needed.

When it comes to root cause analysis, in the current guidance, FDA has provided a significant amount of information to cover NDSRIs, which were not discussed in the previous versions. This version clarifies that API control strategies of nitrosamines based on upstream controls could be justified only if there is clear understanding of the process and should be submitted as Prior Approval Supplements (not changes being effected [CBE] 30 or other lower-level supplements).

A signification update in the current version of the guidance is the recommendation regarding reformulation of drug products with nitrosamine scavengers. FDA has named some excipients as antioxidants, which could be used to reformulate products (e.g., ascorbic acid, alpha tocopherol, propyl gallate, cysteine hydrochloride, and sodium bicarbonate [pH modifier]). Interestingly, all these excipients appear in the FDA Inactive Ingredient Database (IID). FDA has referred to the publication Shakleya et al. 2023 regarding reformulation (9).

Author’s observation. The Shakleya paper includes nitrite scavengers such as caffeic acid and ferulic acid in the study of nitroso bumetanide impurity formation and its inhibition in bumetanide tablets. However, these effective nitrite scavengers are not mentioned in the IID and thus omitted in this guidance. In future, it may be helpful for the industry to appeal to FDA to allow the use of these excipients in new drugs as well as generic formulations, based on sufficient literature available in the public domain to demonstrate their safety of these scavengers, when used in small quantities

A helpful addition in this update are the details of reporting categories of reformulation, based on SUPAC (Level 3) (10–12). It has been stated that for reformulation of Biopharmaceutics Classification System (BCS) Class 1, BCS Class 2, and BCS Class 3 drugs, performing in-vitro dissolution comparison may be adequate to justify formulation change with nitrite scavengers. For the justification of reformulation, FDA has reference to the publication from Shakleya et al. 2023 (6). FDA in this guidance has mentioned that for new drugs, bioequivalence (BE) proposals should be submitted by either Type C meeting or for generics, via pre-abbreviated new drug application (ANDA) meeting (not a controlled correspondence).

The guidance refers to the nitrite scavengers as antioxidants and, in some cases, pH adjusters. This information is especially helpful for development of generics of parenteral dosage forms where antioxidants are considered as exception excipients which may differ quantitatively and qualitatively from the reference listed drug (RLD).

Author’s observation. The Shakleya 2023 paper deals with oral immediate-release dosage form related reformulation. There are numerous other dosage forms which may need reformulation and are not addressed. FDA has given a valuable hint that labeling nitrite scavengers as antioxidants may be able to resolve the Q1/Q2-related issues for generics of many formulations. But, how generics are expected to be Q1/Q2 to the reference listed drug (RLD) with respect to all inactive ingredients with no exception, especially otic and ophthalmic products, may be reformulated is not clear.

This process of formulating or reformulating Q1/Q2 generics may become a challenge if there are patents introduced for products with pH ranges that deter nitrosation or inclusion of nitrite scavengers that the agency has mentioned in this guidance.

Overview of Section V

Section V of the revised guidance provides advice regarding how the nitrosamine controls may be implemented in drug substance and drug products and the pathways of communication with FDA. The agency has stated that confirmatory testing of three commercial lots of the drug product at different stages of lifecycle or targeted forced degradation studies may be performed to justify the omission of controls of nitrosamines. However, the testing should be repeated during every process validation throughout the life cycle of the product. This information may be either provided to FDA upon request or submitted in the next annual report.

Control of the nitrosamine is required by this guidance when the nitrosamine limit exceeds 10% of the AI limit but within the AI limit. This may be done by a CBE-30 supplement.

The general advice by FDA is to consider the reformulation of approved products when the levels of nitrosamines exceed the AI limit and submit the changes as prior approval supplement (PAS).

The agency has also recommended the supplementation of the application of approved products when an alternative AI (other than CPCA) is being proposed or a AI limit based on CPCA is being proposed for a NDSRI that is not addressed in the FDA webpage (6).

For over-the-counter (OTC) products and products that are not subject to approved applications, the information should be retained in the manufacturing facility.

The updated guidance encourages sponsors to work with the Center for Drug Evaluation and Research’s Drug Shortage Staff at drugshortages@fda.hhs.gov when a distributed product is found to have nitrosamines above the FDA proposed AI. The agency has indicated that if the amount of nitrosamine in the API or distributed drug product are above AI already acceptable by FDA (either based on CPCA or through PAS), a field alert should be filed. For some of these products, interim higher limits may be allowed on a case-by-case basis. It is noteworthy that the FDA has recommended that an API manufacturer should also file a field alert if they find out that the distributed API lots may have nitrosamines above AI.

Throughout the revised guidance document, FDA has stated that when faced by dilemma regarding the marketing of the products,manufacturers and applicants can contact the Drug Shortage Staff in CDER at drugshortages@fda.hhs.gov. To discuss issues involving recalls, manufacturers and applicants can contact the recall coordinator assigned to the product type and location.

Conclusion

The Revision 2 of the Guidance for Industry, Control of Nitrosamine Impurities in Human Drugs published in September of 2024 could be considered a quantum leap from the original version of 2020 and the update of 2021. In this version, FDA has taken into consideration a significant amount information that it has accrued in the past four years regarding the quality issues that may affect the presence of nitrosamines, especially the NDSRIs, in drugs. The guidance is clear in its directives and has been converged with other contemporary FDA documents such as the RAIL guidance (5) and information on FDA’s website (6). While these clarifications indicate that in some cases the industry members may have to reconsider their risk assessment, confirmatory testing criteria, the information in the guidance would be helpful performing these activities. The disappointing aspect is the non-alignment of AIs of NDSRIs with the EMA and/or HC, which is expected to cause hardship to the industry. Overall, the update shows that FDA has come a long way since NDMA was first detected in valsartan, but also tells us that we, as the pharmaceutical industry, have miles to go before we resolve the concerns of nitrosamines in drugs.

References

1. FDA. Guidance for Industry, Control of Nitrosamine Impurities in Human Drugs, Final Guidance (CDER, September 2024). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs
2. Smith, P. A. S.; Loeppky, R.N. Nitrosative Cleavage of Tertiary Amines, J. Am. Chem. Soc. 1967, 89 (5),1147–1157.
3. ICH. Q9(R1) Quality Risk Management (ICH, May 2023).
4. ICH. M7(R2) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (ICH, July 2023).
5. FDA. Guidance for Industry, Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) (CDER, August 2023).
6. FDA. Recommended AI Limits for Certain Nitrosamine Impurities. FDA.gov (accessed Sept. 25, 2024) https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cder-nitrosamine-impurity-acceptable-intake-limits#compound
7. ICH. Guidance for Industry Q3A Impurities in New Drug Substances (June 2008)
8. ICH. Guidance for Industry. Q3B(R2) Impurities in New Drug Products (August 2006)
9. Shakleya, D.; Asmelash, B.; Alayoubi, et al. Bumetanide as a Model NDSRI Substrate: N-nitrosobumetanide Impurity Formation and Its Inhibition in Bumetanide Tablets. J. Pharm. Sci. 2023, 112 (12), 3075–3087. DOI: 10.1016/j.xphs.2023.06.013
10. FDA. Guidance for Industry, SUPAC-IR: Immediate Release Solid Oral Dosage Forms: Scale-Up and Post approval Changes: Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation (November 1995).
11. FDA. Guidance for Industry. SUPAC-MR: Modified Release Solid Oral Dosage Forms: Scale-Up and Post approval Changes: Chemistry, Manufacturing, and Controls; In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation (September 1997).
12. FDA. Guidance for Industry. SUPAC-SS: Nonsterile Semisolid Dosage Forms: Scale-Up and Post approval Changes: Chemistry, Manufacturing, and Controls; In Vitro Release Testing and In Vivo Bioequivalence Documentation (May 1997).

About the Author

Aloka Srinivasan, PhD, is Principal and Managing Partner, Raaha LLC.