Nitrosamines in Pharmaceuticals: Toxicity, Risk Analysis, Chemistry, and Test Methods

Introduction

Nitrosamines are a well-known group of highly potent, mutagenic impurities formed by the reaction of secondary amines with nitrite under acidic conditions. Nitrosamines have been studied for many years due to their presence in foods, cosmetics, tobacco products, industrial solvents, and alcoholic beverages^1-4. Nitrosamines are described in the International Conference for Harmonisation (ICH) M7(R1) Guideline⁵ as Class 1 impurities which are high-potency, mutagenic carcinogens6. In addition, nitrosamines are part of the infamous “cohort of concern”⁵ along with aflatoxin-like structures and alkyl-azoxy compounds⁵.

Nitrosamine Toxicity

The primary toxicity concern associated with nitrosamines found in the environment, human diet, tobacco products, cosmetics, and as an impurity in marketed drug products has been that these structures are genotoxic chemical carcinogens^2,6-12. Evidence of carcinogenicity associated with nitrosamines was succinctly summarized by the National Toxicology Program in the 14th Report on Carcinogens¹¹.

In particular, N-nitrosodimethylamine (NDMA) produced tumors in a variety of nonclinical species ranging from fish, amphibians, rodents, and other mammals after exposure via several different routes¹¹. Benign and malignant tumors after exposure to NDMA were identified in the respiratory tract, kidney, digestive tract, liver and bile duct, hematopoietic system, and female reproductive tract¹¹. Similarly, tumors were identified after exposure of N-nitrosodiethylamine (NDEA) in numerous nonclinical species including dogs and pigs in liver, kidney, respiratory tract, and the digestive tract¹¹.

Although no epidemiological studies evaluating the relationship of exposure to NDEA and human cancer have been conducted, several population-based case-control studies and ecological studies were conducted in order to assess the relationship between dietary sources of NDMA and cancer¹¹. Dose-related associations of colorectal, stomach, esophageal, and oropharyngeal cancers with estimated NDMA exposure were identified in several case-control studies.

Furthermore, an increased risk associated with lung cancer was identified with dose-related increases in estimated dietary exposure to NDMA¹¹.

Therefore, NDMA, NDEA, and other structurally-related nitrosamines are suspected by regulatory authorities to act as carcinogens in humans based on the evidence of carcinogenicity in a variety of nonclinical species and a small number of case-control studies with humans.

Current Regulatory Climate and Risk Assessment for Nitrosamines

Recently, nitrosamines have become an important topic for pharmaceutical manufacturers and health authorities. In June 2018, NDMA was discovered as an impurity in several lots of valsartan, an angiotensin-II receptor antagonist and important member of the sartan class of high blood pressure products^7-10. Shortly thereafter, NDMA was detected as an impurity in certain lots of several other drug products in the sartan class of molecules. The source of the NDMA impurity was eventually traced to a change in the synthetic process for the active pharmaceutical ingredient (API)^7-10. A thorough discussion of the NDMA impurity in valsartan was recently published by Snodin and Elder⁶.

After discovery of the NDMA impurity in several drug products, health authorities reacted to the findings quickly and implemented actions that carefully balanced medical benefit from these important products with patient safety. Some lots of product were formally recalled, and both the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) issued guidances to sartan manufacturers and then to Marketing Authorization Holders (MAH) for all commercial products. In the United States, interim limits for sartans were developed and published by the FDA. The FDA’s recommendations are provided in Table 1.7

In the European Union (EU), EMA established temporary interim limits for NDMA, NDEA, NMBA, N-nitrosodiisopropylamine (NDIPA), and N-nitrosoethylisopropylamine (NEIPA) for sartan products based on the maximum daily dose authorized in the EU as shown in Table 2.^8,9,13 EMA has similar expectations for NDMA, NDEA and NMBA as the FDA.

Since issuance of these temporary limits, nitrosamines have been found in several products outside the sartan class of drugs, including pioglitazone, a member of the thiazolidinedione class that is a Type 2 diabetes mellitus product, and ranitidine, a histamine H2 antagonist that is a gastric acid-reducing product¹⁴. As a result, the FDA and EMA are requiring a risk evaluation and appropriate testing of nitrosamine impurities in all marketed products.

In late 2019, EMA issued a communication requesting MAHs, along with API and drug product manufacturers, to conduct a risk assessment for their marketed medicinal products to be completed by March 2020^9,10. A similar risk assessment for marketed products has been requested by the FDA. According to the requests, MAHs should perform a risk evaluation of their medicinal products that contain chemically-synthesized API. These risk assessments should be conducted using the concepts of quality risk management principles as outlined in ICH Q9. Also relevant are the principles relating to risk characterization, control strategies, and changes to manufacturing processes as described in the ICH M7(R1) guideline. A summary of the recommended risk assessment components is provided below.

What factors should be considered in prioritizing the risk evaluation?

According to EMA, MAHs should establish the sequence in which products are to be evaluated. For the purposes of prioritization, MAHs should consider factors such as the maximum daily dose, duration of treatment, therapeutic indication, and number of patients treated.

Questions to Consider During the Risk Evaluation^9,10

• Is there a risk of nitrosamines forming in the API synthetic process, taking into consideration the combination of reagents, solvents, catalysts, and starting materials used, intermediates formed, and impurities and degradants?
• Is there a potential risk of nitrosamine contamination (e.g. from recovered materials such as solvents, reagents and catalysts, equipment, degradation, starting materials, or intermediates)?
• Is there any potential of nitrosamine formation during the manufacture of the finished product and/or during storage throughout its shelf life?

If the evaluation identifies a potential risk for nitrosamines, confirmatory testing should be conducted using methods that are sensitive and validated. EMA is requiring that products identified as high risk complete confirmatory testing by September 2022¹⁰ using appropriately sensitive and specific validated analytical methods.

A reasonable approach to an assessment might include the following:

• Evaluate synthetic route including regulatory starting materials and solvents
• Identify highest risk active pharmaceutical ingredient and chemical steps
• Identify most likely nitrosamines to form
• Evaluate excipients and packaging
• Establish permitted daily exposures (PDEs)
• Develop tests where needed

Chemistry of Nitrosamines – Formation and Destruction

Formation of Nitrosamines

The chemistry of nitrosamine formation can be quite complex. In order for a nitrosamine to form, both an amine source and a nitrosating agent need to be present. Amines are generally categorized as primary, secondary, and tertiary. Primary amines can react with nitrosating agents to form highly-reactive, unstable diazonium ions, which often decompose to release molecular nitrogen. It is also possible for the resulting diazonium ion to react with the starting primary amine to form a secondary amine, which can then undergo nitrosamine formation (Figure 1)¹⁵. In the case of a molecule with two primary amines that are separated by four to five carbons, a cyclic nitrosamine can form. However, indirect nitrosation of primary amines is low yielding due to the instability of the diazonium ion and the requirement for two consecutive reactions to take place^15,16.

Secondary amines are the most likely amines to react and form nitrosamines, though the rate of reaction is dependent on the reactivity and concentration of both starting materials. A representative chemical reaction scheme for secondary amines to form nitrosamines is provided in Figure 2^15,16.

Tertiary amines cannot directly react with nitrosating agents, but they can first undergo nitrosative cleavage to secondary amines, which can then subsequently form nitrosamines (Figure 3)¹⁷. While this is chemically possible, the reaction is slow and typically requires large excess of the nitrosating agent and high temperatures. It is important to note, however, that tertiary amines (such as the commonly used triethylamine and diisopropylethylamine) can contain secondary amines as impurities and/or can decompose into secondary amines that can then proceed to more readily form nitrosamines. Other compounds that may contain secondary amines as impurities or degradants include amide solvents such as dimethylformamide (DMF), dimethylacetamide (DMAC) and N-methyl-2-pyrrolidone (NMP), quaternary ammonium salts such as tetra-n-butylammonium fluoride (TBAF) and tetra-n-butylammonium bromide (TBAB), and primary amines^16-18.

Common nitrosating agents are nitrites such as sodium nitrite (NaNO2) and tert-butyl nitrite (t-BuONO), nitrous acid (HNO2), nitric oxide (NO), nitrosyl halides (XNO, X=halogen), dinitrogen trioxide (N2O3), and dinitrogen tetroxide (N2O4). However, nitrosation can occur even if these reagents are not introduced into a reaction. Nitric acid (HNO3) can contain nitric oxide as an impurity and/or can convert into nitrous acid if exposed to reducing agents. Hydroxylamine (NH2OH), chloramines (e.g. NH2Cl), ozone (O3), and nitrates such as sodium nitrate (NaNO3) can act as indirect nitrosating agents under some conditions. Additionally, azides are typically quenched with nitrous acid or nitrites and these quenching reagents can produce nitrosate amines. Finally, hydrazines can form nitrosamines under oxidative conditions (in the absence of nitrosating agents)^18-21.

Acidic conditions are typically required for nitrosation to take place, though neutral or basic conditions can lead to nitrosation if a catalyst such as an aldehyde (especially formaldehyde) is present. An aldehyde can act as a catalyst by forming an iminium ion intermediate with the amine first, and the iminium ion can undergo nitrosation more readily than the amine¹⁶.

Destruction of Nitrosamines

The destruction of nitrosamines requires strong reaction conditions. Strong acids such as hydrochloric acid can convert nitrosamines into the corresponding amines and nitrous acid, which, if trapped with a nucleophile such as a thiol, can result in an irreversible reaction. Nitrosamines can be reduced by metals such as zinc (with acetic acid) and aluminum (with potassium hydroxide). Hydrogenation of nitrosamines is possible in the presence of palladium, nickel, and iron catalysts. Organometallic reagents such as Grignards, phenyl lithium, and tert-butyl lithium can also destroy nitrosamines. Finally, nitrosamines can be oxidized by strong oxidants such as hydrogen peroxide and potassium permanganate in sulfuric acid^{18, 22-24}.

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Chemical Risk Assessment

A questionnaire was developed by the IPEC (International Pharmaceutical Excipients Council Europe)¹⁸ to standardize the format for assessment of key nitrosamine formation risk factors.

While the questionnaire was developed for use with excipients, the concepts are also applicable to evaluations of API. The logic from the questionnaire is abstracted and summarized in Table 3 and is a good guide for chemical risk assessments for the presence of or risk to form nitrosamines.

Methods for Nitrosamine Determination

Analytical methods reported in the literature for the detection of nitrosamines in water, tobacco, cosmetics, baby nipples, and food products as shown in Table 4.^25-28 The primary analytical methodology has been to separate thermally labile nitrosamine impurities using gas chromatography (GC) coupled with detection by thermal energy analysis (TEA) or mass spectrometry (MS). Liquid chromatography (LC) coupled with detection by TEA, MS, or ultraviolet light (UV) provides an alternative analytical methodology applicable to both volatile and non-volatile nitrosamines. TEA has been widely used for analysis of nitrosamines in tobacco, cosmetic, and food products due to its sensitivity, and ability to be coupled with both GC and LC for selectivity. MS has similar sensitivity as TEA and can be coupled with both GC and LC. High performance liquid chromatography (HPLC) with UV detection, more commonly available in analytical laboratories, has the least sensitivity but is adequate for analysis of low dose drugs with lower limits.

The analytical methodologies shown in Table 5 have been developed and validated by the FDA and OMCL of the General European Network for analysis of the sartan class of drugs. Because the nitrosamines of interest are volatile, analytical methodology using GC with MS detection has provided the best selectivity and sensitivity.

Conclusion

Nitrosamines are highly potent carcinogens that have been observed as impurities in foods and beverages for many years. However, the recent detection of nitrosamines in widely prescribed pharmaceutical drugs has raised concerns about patient safety and has prompted health authorities to mandate risk assessments and confirmatory testing in marketed products. In particular, the EMA has required risk assessments in accordance with ICH Q9 and ICH M7(R1) guidelines for all chemically synthesized APIs by March 2020 and for completion of confirmatory testing by Sept 2022. This testing will be aided by sensitive analytical methods that have already been established for some of the most commonly occurring nitrosamines. While the pharmaceutical industry and health authorities manage the risk to patients, regulations are expected to rapidly evolve.

References

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