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

Aloka Srinivasan

N-Nitroso-diethylamine or NDEA carcinogenic molecule. 3D rendering. | Image Credit: ©Studio Molekuul/Wirestock - stock.adobe.com

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(

mpurities), 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.

 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.

 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.

 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, 

 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.

. 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

 (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, 

 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.

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).

. 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.

. 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.

. 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 

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 

 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 

. 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).

 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 

 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.

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 

. To discuss issues involving recalls, manufacturers and applicants can contact the recall coordinator assigned to the product type and location.

 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.

Guidance for Industry, Control of Nitrosamine Impurities in Human Drugs, Final Guidance 

https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs

Smith, P. A. S.; Loeppky, R.N. Nitrosative Cleavage of Tertiary Amines, 

M7(R2) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk 

Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) 

FDA. Recommended AI Limits for Certain Nitrosamine Impurities. 

 (accessed Sept. 25, 2024) https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cder-nitrosamine-impurity-acceptable-intake-limits#compound

Shakleya, D.; Asmelash, B.; Alayoubi, et al. Bumetanide as a Model NDSRI Substrate: N-nitrosobumetanide Impurity Formation and Its Inhibition in Bumetanide Tablets. 

 (12), 3075–3087. DOI: 10.1016/j.xphs.2023.06.013

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 

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 

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 

Articles

This article presents an overview of FDA’s recent update to the guidance document, Control of Nitrosamine Impurities in Human Drugs.

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

-nitroso compounds (Figure 1) are formed by the reaction of secondary and tertiary amines, amides, carbamates, urea, guanidine with nitrite or other nitrogenous agents (including N2O3 and N2O4). The carcinogenic properties of 

-nitrosamines, have been known for many years (1). Based on numerous studies, International Agency for Research on Cancer (IARC) has categorized 

-nitroso compounds in Class 2a and Class 2b, as probable and possible carcinogens, and International Council for Harmonisation (ICH) M7 has considered them as a “cohort of concern” (2). However, it needs to be remembered that 

-alkylurea, guanidine, carbamates are not in the same category as 

. They are direct acting mutagens that do not need metabolic activation to exhibit their mutagenicity or carcinogenicity as is the case of 

-nitrosamines. Thus, they should not be put in the same category as the 

-nitrosamines (3). The purpose of this paper is to discuss the differences in mechanism of activation of 

-nitrosamines and the fact that they should not be treated alike.

Nitrosamides–Should They Be Treated the Same as Nitrosamines?

Read this article in the Trends in Formulation 2022 eBook

Aloka Srinivasan, PhD is a principal and managing partner of Raaha LLC. Chuck Lambert, PhD, DABT, ERT is principal toxicologist at Intrinsik Ltd.

When referring to this article, please cite it as A. Srinivasan and C. Lambert, " Nitrosamides–Should They Be Treated the Same as Nitrosamines?," 

Pharmaceutical Technology's Trends in Formulation

The authors discuss the differences in mechanism of activation of N-nitrosamides versus N-nitrosamines and the fact that they should not be treated the same.

Control of mutagenic and/or genotoxic impurities in an API has been the topic of deficiencies cited by FDA and other regulatory agencies all over the world for the past few years (1–3). Before delving into what may be the source of these deficiencies and identifying any pro-active steps to avoid them, it is important to understand the relationship between mutagenic and genotoxic impurities. For a long time, these two terms have been used interchangeably by regulatory agencies and the pharmaceutical industry. However, International Council for Harmonisation (ICH) M7(R1) (4) specifically mentions “mutagenic” impurities in its title as well as content rather than the general team, “genotoxic”. Hence, a few words related to the relationship between the mutagenic and genotoxic impurities may be useful prior to commencing discussion on this topic. Based on the definitions in ICH M7(R1), genotoxicity is “a broad term that refers to any deleterious change in the genetic material regardless of the mechanism by which the change is induced” (4). Also, the 

Globally Harmonized System of Classification and Labelling of Chemicals (GHS)

, Fifth Revised Edition, United Nations, Chapter 3 has the following definitions of mutagenic and genotoxic materials (5):

	 “3.5.1.3 The term mutation applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including for example, specific base pair changes and chromosomal translocations). The term mutagenic and mutagen will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.”

	“3.5.1.4 The more general terms genotoxic and genotoxicity apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner (temporarily) alter its replication. Genotoxicity test results are usually taken as indicators for
	mutagenic effects.”

	Thus, mutagenicity implies induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms, while all genotoxic effects are not necessarily associated with mutations. Hence, all mutagens are genotoxic, but, not all genotoxic substances are mutagenic. When genotoxic impurities are mentioned in publications and deficiencies cited by FDA and other regulatory agencies, they include mutagens and also other impurities that can cause DNA damage though different pathways. In this paper, the term genotoxic impurities will be used to refer to mutagenic as well as other genotoxic impurities, unless there is specific need to refer to a material as mutagenic.

	The purpose of this article is to provide a direction for identifying genotoxic impurities early in the drug development process, regulating genotoxic impurities at acceptable levels in the API or drug product, and avoiding negative product regulation late in the development and/or marketing process, including expensive recalls. The focus of this discussion are the dossiers submitted to FDA, especially for generic drugs (e.g., 505(j), abbreviated new drug applications [ANDAs], and 505(b)(2) new drug applications [NDAs], which use APIs that are already approved by FDA). However, most of the principles also apply to new chemical entities and also other regions of the world that follow ICH guidelines.

	The current resurfacing of nitrosamines in the pharmaceutical world due to FDA and other international agencies finding traces of these compounds in the angiotensin II receptor blockers (ARBs), commonly known as the “sartans,” has led to recalls of several drugs of this class (6). As of now, valsartan and losartan are the worst affected, and several lots of these products have been recalled (7). Currently, we have also been informed by FDA about the presence of nitrosodimethylamine, NDMA, in ranitidine hydrochloride (8). While the pharmaceutical industry has been diligently working to address the nitrosamines in ARBs, questions have also arisen as to how a situation of this kind could be anticipated and possibly avoided in the future, not just for nitrosamines but other genotoxic impurities as well.

	Genotoxic impurities can occur in drug products based on the manufacturing of the API, degradation of the API, or in some cases, from the excipients (9). The source of genotoxic impurities in the API and drug product generally is the API manufacturing process, including starting materials and reagents. Reagents used in API synthesis are often highly reactive, and genotoxic impurities can result from leftover reagents carried through the manufacturing process, by-products of the chemical transformations, or the subsequent degradation/interactions of the API (9,10). Although rare, genotoxic impurities occasionally form in the drug product as a result of interaction of the API with excipients. This article, however, focuses on the possible genotoxic impurities rising from the manufacture of the API.

	Inability to apprehend the possibility of formation of impurities with genotoxic potential can significantly impact the approval of an NDA or ANDA. Usually, with new chemical entities related to 505(b)(1) applications, the sponsors perform evaluation of the synthetic pathways and address possible and plausible impurities, including impurities with genotoxic potential early in the investigational new drug (IND) process. In the best-case scenario, the control strategies are shared with, and agreed upon, by FDA prior to submission of the NDA. However, with the 505(j) and some 505(b)(2) applications, where an API used is already present in an FDA-approved drug, the detailed understanding of the risk related to the API manufacturing process often takes a back seat. Irrespective of the fact that the API is present in an FDA-approved product, there can be risk related to genotoxic impurities based on a new synthetic route, origin of the starting materials, sources of reagents, and solvents used. This can also be an issue during the lifecycle management of a 505(b)(1) NDA. It is essential that the manufacturing process of the API is appropriately evaluated and also that the origin and controls of the starting material and reagents are appropriately scrutinized to ensure that there are no surprises in the impurity profile during the FDA’s review or lifecycle management.

	The question often comes up as to where to start the process of identifying plausible genotoxic impurities in an API manufacturing process. The best place to start is by being cognizant of the structural alerts related to genotoxicity as provided in 

	For an API manufacturer to increase the chances of avoiding deficiencies related to controls of genotoxic impurities, several aspects of the manufacturing process and controls should be evaluated. If any starting material, reagent, intermediate, or possible side product has one of the structures in 

, an evaluation should be initiated immediately, and a control strategy put in place. In addition, the following are some other angles from which the possible presence of genotoxic impurities may be addressed.

 This is a good place to start to reduce risk related to genotoxic impurities. A manufacturing option with more steps is favored if it involves lesser use of potentially genotoxic reagents or generation of potentially genotoxic intermediates or side products.

 The selection of starting materials in the synthesis of API is crucial for many reasons, one of them being its impact on the impurity profile of the API. The starting material should not be defined significantly downstream in the process, as this may make it difficult to purge impurities arising from the starting material. Also, there needs to be knowledge related to the manufacturing process of the starting material and a good understanding regarding any of the reagents or intermediates or by-products of the process being a structural alert for genotoxic impurities. If there are potential genotoxic impurities in a late-stage intermediate that is being defined as a regulatory starting material (RSM), the API manufacturer may have an uphill task of adding significant controls to the RSM and final API or convincing FDA that these impurities are purged efficiently by the process and do not pose a risk to the API or drug product. Both options are expensive and time consuming and could be the cause of delay in approval.

If any of the by-products of side reactions in the manufacturing process are structural alerts for genotoxicity, it needs to be evaluated to determine whether they are completely consumed in subsequent reactions or persist throughout the manufacturing process. If needed, control for these by-products should be included at appropriate steps. The best place to control an impurity is close to its origin. However, if the by-product persists in the final API, controls may need to be added at the point of origin as well as the API.

 Many of the reagents are structural alerts for genotoxicity or may generate structural alerts downstream based on the reaction with intermediates or other reagents. Many of the surprises related to genotoxic impurities come from a lack of understanding of the reagents and solvents or their impurity profiles, as we have seen in the case of nitrosamines. The behavior of the solvents used during the manufacturing process should also be understood in the perspective of the synthetic steps. Some solvents are considered reactive and can do more than provide a medium for the reaction to happen. A classic example is acetone, which can undergo aldol condensation followed by dehydration under acidic conditions to product a genotoxic impurity, mesityl oxide (12). The nitrosamines in the “sartans” are believed to have originated from impurities like dimethylamine and diethylamine in upstream solvents like dimethylformamide and diethyl acetamide, reacting with the downstream reagent, sodium nitrite (13).

 If the potentially genotoxic or impurity is a degradant of the API in addition to being generated by the manufacturing process, a control of the impurity will be needed in the API and also in the finished dosage form keeping the maximum daily dose (MDD) of the drug product in mind.

 Once the possible and plausible genotoxic impurities are identified in the API manufacturing process, a strategy is needed related to controlling them. This strategy can start with understanding the acceptable intake of the impurity based on the MDD as well as duration of the treatment as provided in Table 2 of ICH M7(R1) (4). There may be significant difference in acceptable intake based on whether the drug is for chronic use or a short-term use. For drugs with variable MDD based on indications, the most conservative MDD should be used to determine the threshold of toxicological concern (TTC). This determination should be followed by developing analytical method(s) that are sensitive enough to detect and quantitate the potential genotoxic impurities at the proposed levels. For potential genotoxic impurities that are generated upstream, spike and purge studies may be performed to ensure that these impurities are present at lower than 30% of the TTC level in the final API. The control strategy may also be based on the scientific understanding of the nature of the impurity and the measured or predicted purge factor (4,14).

	In cases where the impurity is produced downstream in the API manufacturing process and persists in the API based on spike and purge or purge factor studies and/or is a known degradant, the impurity should be controlled in the drug substance and possibly in the drug product. The limit of the genotoxic impurities to be controlled in the intermediate or the final API can be justified by the TTC and also the spike and purge studies. The ICH M7(R1) guidance provides an approach for both classifying genotoxic impurities as well as determining safe levels. Other guidance, such as ICH S2(R1) (15) on genotoxicity testing, identifies bacterial and mammalian 

 testing options if a genotoxic impurity is identified based on quantitative structure activity relationships (15). The exception being if the compound of interest has the same structural alert as the API itself. In that case, it can be treated as a non-mutagenic impurity and controlled based on ICH Q3A(R2) qualification or identification threshold (4).

	A few common genotoxic impurities and some examples of drugs where they can occur are summarized in the following sections. This is not intended to be a comprehensive list but covers some well-known genotoxic impurities, which may originate from the reagents used or even be present as impurities in reagents.

; where R1–4=alkyl, aryl or H) are highly reactive compounds that can interact with the DNA and are considered structural alert for genotoxicity (10,16). Epoxides can be formed as intermediates in reactions and can also when the synthetic route involves generation or use of alkenes and along with oxidants, such as peroxy acids. Epoxides usually undergo reaction in presence of strong acids and alcohols to produce non-genotoxic glycols.

	Some of the examples of drugs that may have epoxide impurities include the following: acebutolol, atenolol, atorvastatin calcium, betamethasone, bicalutamide, fluocinolone acetonide, loteprednol etabonate, mometazone furoate, mupirocin, nadolol, stavudine, and vincristine.

; where R=alkyl, aryl group, or H and X=iodine, fluoride, chlorine, or bromide and other good leaving groups) are used extensively as alkylating agents in the synthesis of APIs (17,18). Alkyl halides are mostly left as residues at the end of alkylating reactions as they are used as excess. Alkyl halides are ubiquitous and are commonly used in numerous API manufacturing processes. The examples below are a few drugs where these impurities have been definitely identified. They are mostly highly reactive (e.g., alkylating agents) and are known genotoxic and carcinogenic compounds.

	Some examples of drug products that may have alkyl halide impurities are the following: fexofenadine, anastrozole, aripiprazole, latanoprost, capecitabin, sitagliptin, sunitinib maleate, pazopanib, conivaptan hydrochloride, linezolid, and linagliptin.

; where R1–R4 are alkyl, aryl group, or a hydrogen), hydrazides (shown in Figure 4, where R1 = O, R2, R3, R4 = H, alkyl or aryl groups), and hydrazones (shown in 

, where R1, R2 = alkyl or aryl groups, R3 = NH2) are common reagents used in the synthesis of APIs. Hydrazine itself is a well-known base and reducing agent (10,18,19). They are also known genotoxic and carcinogenic compounds. Hydrazines, being strong bases, can be washed out if there are steps downstream which involve strong acids.

	Some examples of drugs that may have hydrazine, hydrazide or hydrazone  impurities are the following: allopurinol, carbidopa, celecoxib, dihydralazine, hydralazine sulfate, isoniazid, mildronate, rifampicin, rizatriptan benzoate, sunitinib maleate, sildenafil citrate, saquinavir mesylate,
	and ziprasidone.

; where R1–2 are alkyl or aryl groups) are well-known genotoxic and carcinogenic chemicals that are widely found in the environment and the food chain (20,21). Nitrosamines usually form in the API or drug product when a secondary amine (either part of the API structure or reagents in the manufacturing process) is exposed to a nitrosating agent such as sodium nitrite in a low pH environment. Nitrosamines can also form when there are secondary amine impurities in reagents and solvents. Thus, if an organic or inorganic nitrite is used in manufacturing of the API, the sponsors should evaluate the possibility of nitrosamines based on amines or amides that can be used or formed during the process.

	Some examples of drug products which may have nitrosamine impurities: valsartan, olmesartan, irbesartan, losartan potassium and other “sartans”, chlorhexidine gluconate, propranolol, metoprolol, piperazine, ethambutol, ephedrine, indomethacin, phenytoin, and ranitidine.

 where R1=alkyl or aryl group; where R2=alkyl groups) (10). Sulfonate esters are one of the most common and well-known genotoxic impurities formed during the API manufacturing process. The reason behind this is that the precursors to alkyl sulfonate esters, alkyl sulfonic acids, and alcohol or also alkyl halides are some of the most widely used in organic reactions. Many APIs are salts of alkyl sulfonic acids like mesylates, tosylates, besylates, and triflates, which lead to the presence of a conjugate base of alkyl sulfonic acids in stochiometric proportions in the API. Also, the alkyl sulfonic acids are used in protection and deprotection of functional groups and also as acid catalysts in several reactions. Alcohols like methanol, ethanol, and isopropanol are common solvents used in API manufacturing process. Also, alkyl halides, which can react with alkyl sulfonic acid to generate potentially genotoxic sulfonate esters, are themselves considered structural alerts for genotoxicity and also commonly used reagents in API manufacturing. Sulfonate ester formation can be controlled based on the levels of water or free base present
	during the reactions (22).

	Some examples of drug products that may have sulfonate ester impurities: nelfinavir mesylate and other drugs which are mesylate salts, glycopyrronium tosylate and other drugs with tosylate salts, clobetasol propionate, halobetasol propionate, mometasone furoate, oseltamivir phosphate, fosinopril sodium, esomeprazole sodium, orlistat, and tolterodine tartrate.

	Genotoxic impurities need to be anticipated and identified early in the drug development process. Late identification of potential genotoxic impurities in API manufacturing can result in delays in submission of dossiers due to disruption in the manufacturing process. Request for evaluation of potential genotoxic impurities during the review process by agencies can lead to delays in agency approvals and loss of market share. Should identification occur post-marketing, this could result in disruption in supply of the drug product and, in some cases, drug shortage. Identification of any structural alerts in the API manufacturing process, along with quantitative structure activity relationship (QSAR)  model positive confirmation (23), indicates the need for running a bacterial reverse mutation assay for confirmation and/or creating control strategies as per ICH M7(R1) (4). A sponsor’s awareness regarding the potential genotoxic structural alerts that may result from the chosen manufacturing process of an API, and early planning regarding how the potential genotoxic impurities may be controlled, would be a giant step toward reducing review cycles which in turn may be the pathway to quick approval. After all, as Benjamin Franklin once said, “An ounce of prevention is worth a pound of cure.”

,” Certification of Substances Department, European Directorate for the Quality of Medicines, May 2018.
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FDA Updates and Press Announcements on Angiotensin II Receptor Blocker (ARB) Recalls (Valsartan, Losartan, and Irbesartan)

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Statement Alerting Patients and Health Care Professionals of NDMA Found in Samples of Ranitidine

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Nitrosamines–How to Address these Unwelcome Guests in The Pharmaceutical World

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Guidance on Genotoxicity Testing And Data Interpretation For Pharmaceuticals Intended For Human Use S2(R1)

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	Supplement: APIs, Excipients, and Manufacturing
	October 2019
	Pages: s6–s10

	When referring to this article, please cite it as A. Srinivasan, “Proactive Evaluation of Possible Genotoxic Impurities During the Early Stages of Drug Development," 

Pharmaceutical Technology APIs, Excipients, and Manufacturing

The author provides a direction for identifying genotoxic impurities early in the drug development process, regulating genotoxic impurities at acceptable levels in the API or drug product, and avoiding negative product regulation late in the development and/or marketing process, including expensive recalls. 

Proactive Evaluation of Possible Genotoxic Impurities During the Early Stages of Drug Development

The selection of excipients is important in generic formulations due to the impact it has on the risk and performance of generic drugs.