September 15, 2023 PAO-09-23-CL-07
Nitrosamines (or N-nitrosamines) are chemical compounds with the structure R2N−N=O that include a nitroso group (NO+) bonded to a deprotonated amine, and where R is typically an alkyl group. Many members of this class of compounds have been shown to exhibit carcinogenic and mutagenic effects in animal models at several different tissue sites and by several different routes of exposure. Some have been classified as probable carcinogens, others as possible carcinogens, and at least one as a human carcinogen by different regulatory authorities and the International Agency for Cancer Research (IARC).
Some of the most well-known nitrosamines include N-nitrosodimethylamine (NDMA), N-nitroso-diethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitroso-diethanolamine (NDELA), nitroso morpholine (NMOR), N-nitroso-N-methyl-ethylamine, and N-nitrosopyrrolidine (NPYR). It is believed that the carcinogenicity of N-nitrosamines is due to the formation of diazonium salts following metabolic activation.
Nitrosamines themselves are formed when an amine reacts with a nitrosating agent, such as nitrous acid, nitrites, or nitrogen oxides. Most commonly, secondary amines react with such an agent under acidic conditions. More generally, nitrosamines are formed from “vulnerable amines,” or compounds containing an amino group that is located next to a carbon atom with a proton that can be extracted to generate an alkylating diazonium salt. Examples of vulnerable amines include but are not limited to secondary and tertiary amines, quaternary ammonium salts, N,N-dialkylamines (e.g., N-methyl-2-pyrrolidone, dimethylformamide, dimethylacetamide), N,N-dialkyl carbamates, and N,N-dialkylhydrazines.
Compounds with vulnerable amine groups are ubiquitous in nature. In addition, nitrosation of these compounds readily proceeds under both acidic and neutral pH conditions. As a consequence, nitrosamines are present in many foods, consumer products, and medicines and are generally considered to be problematic in the human environment. Some of the most well-known sources of nitrosamines are tobacco, cosmetics (creams, lotions, shampoos), rubber products, pesticides, and pharmaceuticals, forming either during manufacture or product storage.
Due to their higher potency, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) classifies nitrosamine impurities under the ICH M7(R2) guideline as high-potency mutagenic carcinogens. They are thus part of a “cohort of concern” including several types of Class 1 impurities (mutagenic carcinogens) that must be controlled below compound-specific limits in pharmaceutical products. These limits are often much lower than the 1.5 μg/day acceptable intake (AI) for other potentially mutagenic impurities that lack carcinogenicity data.
Nitrosamines can be generated during the manufacture of pharmaceutical intermediates, APIs, and final drug products. Many APIs and impurities in drug substances and drug products can be nitrosated during manufacturing or upon storage in final product packaging. API-derived complex nitrosamines are referred to as nitrosamine drug substance–related impurities (NDSRIs).
Concern regarding the potential for nitrosamine contamination in pharmaceutical products was initially raised in mid-2018, when trace quantities of certain nitrosamines were detected in sartan drug products, which are angiotensin II receptor blockers. The first case appeared when Zhejiang Huahai Pharmaceutical discovered an unknown impurity in Valsartan following a change in the process from the use of tributyltin azide to sodium nitrite for removal of excess sodium azide. Use of sodium nitrite could have led to the formation of nitrous acid, which then reacted with trace amounts of dimethylamine from DMF to produce NDMA. Shortly thereafter, nitrosamine contaminants were also found in the H2 (histamine-2) blocker drugs ranitidine and nizatidine and the drugs pioglitazone and metformin.
Since then, nitrosamine contamination has become a significant challenge for the pharmaceutical industry. Of more than 12,000 small molecule drugs and drug impurities subjected to in silico analysis, slightly more than 40% of the APIs and nearly 30% of the API impurities were determined to be potential nitrosamine precursors.1 In another study of 249 drug samples, including APIs as well as finished products (e.g., tablets, capsules, solutions, creams) available on the European market that were analyzed for 16 nitrosamines, 2% were found to be contaminated by NDMA, NDEA, or NMOR.2 A third, more recent study of solid dosage forms formulated using common excipients with and without added nitrite found that nitrosamines can form under stressed stability conditions.3
Because nitrosamines can be generated during the production of drug substances and drug products, as well as upon storage, it is essential to understand the root causes of their formation in order to implement preventive measures. The root causes and risk factors for formation of nitrosamines must be considered for APIs, drug products, and other sources (typically artefacts of the design of testing conditions and not real risks).4
For APIs, in addition to intrinsic properties of the molecules involved, process conditions are an important factor, including choice of reagents and solvents, reaction conditions, and potential impurity generation, among others.4 Contaminants in raw materials, such as recycled solvents, can contribute to nitrosamine formation, as can certain raw materials themselves. Cross-contamination in multi-product facilities is another potential source that must be considered. Impurities in process water and some processing aids are others.
Determining root causes and risk factors for nitrosamine formation in drug products is more complex due to the nature of the product formulation process.4 In addition to the structure of the API itself (e.g., if it contains a secondary amine group), excipients are important factors, including their molecular structure as well as impurities with relevant structural or reactive moieties. Certain processes, such as wet granulation, are also more likely to facilitate reactions that generate nitrosamines. Product packaging, such as those that contain nitrocellulose or are printed with ink containing secondary amines, can potentially contribute to nitrosamine formation as well.
Following the detection of nitrosamines in sartan and other drugs, the U.S. Food and Drug Administration (FDA) and the EMA, along with other regulatory authorities around the globe, responded promptly, issuing recalls and Warning Letters; rescinding GMP certifications; developing new, more sensitive analytical methods; publishing new acceptable daily exposure limits; and issuing guidance on how to prevent and control nitrosamine formation.
In addition, in 2019/2020, the FDA,5 the EMA,6 the World Health Organization (WHO),7 and other regulatory agencies around the world issued requirements for the review of existing APIs and drug products for the potential presence of nitrosamine contaminants. Drug manufacturers had to implement risk assessment protocols for any new processes and products and conduct comprehensive retrospective analyses of all approved drug products to identify any with nitrosamine impurities above acceptable levels.
The expected three-step approach to assessment included a risk evaluation to identify those APIs and products likely to have nitrosamine contaminants, confirmatory testing of those found to be at risk, and — for any of those with nitrosamines found to be present — notification of regulatory agencies and implementation of risk-mitigation measures.
In 2020, the EMA and FDA also published a list of the most common volatile nitrosamines deriving from common reagents and solvents. This list has been expanded since then as more information has accumulated about nitrosamines in pharmaceutical products. Similarly, revisions and updates have been published by many regulatory authorities (the EMA in July 20238 and the FDA in August 20239,10) regarding prediction of nitrosamine formation and the setting of AI values for compounds with no toxicity data available, particularly NDSRIs.
It is also worth noting that regulatory agencies around the world have made an effort to share information regarding the nitrosamine issue. Specifically, the Nitrosamines International Strategic Group (NISG) formed in 2018, and the Nitrosamines International Technical Working Group (NITWG) was established in late 2020.4 Indeed, one of the key lessons learned during the past few years is the need for more collaboration, not only between regulatory agencies but also between regulators and manufacturers and as importantly between drug developers and their contract manufacturing partners.8 Better communication of information to the public and patients by regulators when safety issues arise also must achieved.
Olon quickly responded to the nitrosamine issue to address the potential health risks they pose and to comply with the new requirements of regulatory authorities to assess the risks of nitrosamine contamination in existing products. A nitrosamine policy was issued in 2019 and applied by all Olon manufacturing plants. A systematic review of the potential for nitrosamine contamination in its ~300 APIs was initiated by a multidisciplinary team comprising experts from R&D, Quality Assurance, Regulatory Affairs, and site- and corporate-level Quality Control. In Phase 1, priority ranking and risk assessment of the APIs was peformed, while, in Phase 2, confirmatory testing was implemented.
Phase One: Priority Ranking and Risk Assessment
Olon’s drug substances were evaluated by applying the ICH Q9 quality risk management approach, with each Olon manufacturing site defining for its production processes the potential risk of nitrosamines (high or low)
and the priority in which the high-nitrosamine-risk APIs should be subjected to analytical verification by means of confirmatory tests.
The internally developed risk-based approach included consideration of the API structure, starting materials and individual manufacturing steps, packaging, and other relevant factors. The assessment also included the potential for cross-contamination in multipurpose plants. Suppliers are also now expected to provide documentation of the risk aspects of nitrosamines as part of the qualification process.
Importantly, because most of the manufacturing steps are performed at Olon, including for internally produced raw materials, processes are carried out in compliance with GMP requirements. As such, the materials used are sourced only from validated suppliers and are tightly controlled. In addition, information on these materials is readily accessible. Even so, large quantities of data were still needed for the assessment, much of which had to be collected from suppliers. Olon’s network of global offices and philosophy of integrating well with local suppliers both greatly benefited the effort. Cooperative contacts led to prompt retrieval of information and the recovery of valuable data relevant to setting informed and appropriate risk levels (high or low). Where information was lacking, the worst-case scenario was considered.
Olon’s prompt and organized response enabled the company to complete the risk assessments for all APIs in advance of regulatory deadlines. It should also be noted that the policy, risk assessment approach, and priority ranking system are continuously updated as new information about root causes for nitrosamine formation are discovered, including for APIs with the potential to form NDSRIs.
Phase Two: Confirmatory Testing
Those APIs found to have a high risk of nitrosamine contamination were subjected to confirmatory testing. Because the acceptable intake limits for many nitrosamine impurities are very low, extremely sensitive analytical methods must be used for their detection. Olon equipped its quality control corporate (QCC) analytical laboratory (located at its headquarters in Rodano, Italy) with an ultra-high-performance liquid chromatography-high-res mass spectrometry system leveraging a Thermo Scientific™ Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific). An Agilent 6495C triple quadrupole LC/MS system (6495C LC/TQ) was also acquired for the QCC lab to ensure the capability to leverage diverse analytical techniques. In some cases. to ensure timely responses, third-party laboratories certified by the QCC group were used.
Eventually, high-resolution mass spectrometers were installed at R&D analytical laboratories within Olon’s production sites to support the development of highly sensitive analytical methods used in the monitoring and control of production processes.
The risk evaluations of the entire global Olon API portfolio were completed within the required EMA timeline: by March 31, 2021 for chemical APIs and July 1, 2021 for biologic drug substances. Confirmatory testing was also completed by the EMA deadline of September 26, 2022 for small molecule APIs, with the results reported to the regulatory agency. Olon has also been actively evaluating its APIs for the potential to form NDSRIs and has already completed numerous confirmatory tests to exclude the risk for many of its products. For those with confirmed risk of NDSRI contamination, release specifications have been appropriately adjusted.
The keys to successfully mitigating risks to patients of nitrosamine impurities include performing appropriate risk assessments, applying appropriate formulation and manufacturing process design strategies, and leveraging an appropriate pharmaceutical quality system.4
With its rapid and comprehensive response to the nitrosamine contamination issue, Olon has been at the forefront in dealing with the regulatory requirements for risk assessment and analysis of nitrosamines. We have also emphasized collaboration with suppliers, customers, and authorities to ensure that Olon’s APIs meet the highest safety standards set by the EMA, FDA, and other health authorities.
Olon has also recognized that, while a challenge, the growing awareness of the risk of nitrosamine contamination has simultaneously presented companies like Olon with the opportunity to deepen our process knowledge, advance our analytical capabilities, better educate ourselves about the raw materials we use, and strengthen our relationships with our suppliers.
The end result of all of the efforts to reduce the risk of nitrosamine contamination has ultimately been the production of improved products, particularly with respect to control of quality and safety. That translates to long-term gains in global public health.
For more information about Olon’s response to concerns about nitrosamine contamination and details about the rifampicin case study, see the new white paper “Olon case, Management of nitrosamine standards.”11
Nice Insight, established in 2010, is the research division of That’s Nice, A Science Agency, providing data and analysis from proprietary annual surveys, custom primary qualitative and quantitative research as well as extensive secondary research. Current annual surveys include The Nice Insight Contract Development & Manufacturing (CDMO/CMO), Survey The Nice Insight Contract Research - Preclinical and Clinical (CRO) Survey, The Nice Insight Pharmaceutical Equipment Survey, and The Nice Insight Pharmaceutical Excipients Survey.