Testing for the presence of nitrosamines on pharmaceutical drug products will be a requirement on all prescription drugs, over-the-counter (OTC) drugs, and drug products in clinical development starting on August 1, 2025. This mandate ensures compliance with regulations from the U.S. Food and Drug Administration (FDA) as well as other global regulatory agencies.^1,2 At the time of publication, organizations have less than four months remaining to comply with the new regulations. Organizations that have not yet taken steps to meet the new requirements risk facing significant compliance challenges that could delay the marketing of both new and lead to recalls of existing products. 

Most major regions around the world have already implemented or will soon enforce nitrosamine testing requirements, and it is important for organizations to understand the regulatory requirements of any geographic location where they do business. This article provides background on the approaching FDA nitrosamine requirements, offering guidance on the critical steps organizations should take to ensure compliance, mitigate potential risks, and avoid disruptions to product availability to patients and consumers. 

Growing concerns over nitrosamines in pharmaceuticals

Nitrosamines are compounds that are potentially potent genotoxic agents in several animal species, with some classified as probable or possible human carcinogens by the International Agency of Research on Cancer.³  Nitrosamines form in drug products when a secondary amine within a drug molecule reacts with a free nitrite, or other nitrosating agent.⁴ Nitrosonation risk can be present along different parts of the supply chain due to various factors:

• Synthesis of active pharmaceutical ingredients (APIs)
• Contaminated solvents and/or water
• Cross-contamination in shard facilities
• Packaging
• Storage conditions
• Contaminated excipients⁵

The presence of nitrosamines in pharmaceuticals has become a major concern for regulatory bodies worldwide, resulting in numerous mandatory recalls of pharmaceutical products. One nitrosamine-related recall alone resulted in more than 233,000 bottles of the anti-depressant duloxetine being pulled.⁶

To address the challenges around nitrosamine contamination, the FDA set the upcoming deadline to reduce the number of nitrosamine-related recalls by ensuring that pharmaceutical manufacturers proactively assess and mitigate the risk of contamination. By implementing stringent testing and control measures, regulatory agencies aim to enhance drug safety.

Preparing for compliance

There are two key guidelines included in the FDA-published compliance recommendations that define the framework for nitrosamine assessment and control:

1. Risk assessment and control of nitrosamine formation

The guidance recommends steps manufacturers and sponsors of APIs and drug products should take to detect and prevent unacceptable levels of nitrosamine impurities in two general structural classes:

1. Small-molecule nitrosamine impurities (SMNIs) that do not share structural similarity to the API.
2. Nitrosamine Drug Substance-Related Impurities (NDSRIs), which do share structural similarities to the API and are generally unique to each API.⁷

2. Acceptable daily intake limits

The FDA recommends that drug manufacturers conduct risk assessments (i.e., evaluate whether nitrosamine impurities are likely to be present) followed by appropriate analytical testing reflective of the risk level of nitrosamine contamination if identified.⁸ If testing shows a nitrosamine impurity is present at a level 10% above the acceptable intake limit for that specific nitrosamine, the FDA recommends that a product release specification be developed to ensure nitrosamine levels remain within the recommended acceptable intake limit.⁹

Initial risk assessment

When conducting nitrosamine testing, it is important to ensure a comprehensive approach that includes testing, synthesis, and risk assessment in accordance with United States Pharmacopeia (USP) general chapter 1469.11.¹⁰

Prior to executing any testing, a paper-based initial assessment must be completed to determine the risk level of nitrosamine presence and to identify any specific nitrosamines that could be present and evaluated through confirmatory testing.

This paper-based assessment should include a review of technical (e.g., chemistry) risks associated with the drug substance and drug production chemical processes (solvents, regents, catalysts, etc.), drug product solvents, packaging materials, and excipients used in the drug product formulation to assign an overall level of risk to the product.

Based on the findings, a low-, medium-, or high-risk rating is assigned for nitrosamine formation. Secondary amines represent the highest risk, while primary amines are low risk because the resultant nitrosonated compounds will likely decompose quickly. Low-risk drug product formulations testing focuses on SMNI while “medium” and “high” risk drug product formulations include a requirement for quantification testing for the API related NDSRI.

Tailored testing and advanced analytical expertise

The advanced testing conducted to determine the presence of NDSRIs involves sophisticated mass spectrometry (MS) technology, including Triple Quadrupole Technology, which enables liquid chromatography-tandem mass spectrometry (LC-MS/MS). A high-sensitivity, low-level quantitation is particularly advantageous for detecting nitrosamines (as well as other impurities) that must remain within low permissible levels—typically low parts per million or even parts per billion, depending on maximum daily dose (MDD) of the drug.

With deep expertise and a customizable testing approach, a qualified contract development and manufacturing organization (CDMO) can develop, validate, and optimize highly sensitive methods for detecting genotoxins, impurities, and/or nitrosamines specific to each API or dosage form. Through precise sample preparation and rigorous method validation, they ensure that results are accurate, sensitive, repeatable and specific at every testing stage. This approach ensures the accurate detection of potentially toxic impurities and the reliable detection of contaminants at parts-per-million (ppm) levels—achieving compliant results for developmental and commercial products.

Custom synthesis of NDSRIs

For organizations that do not have internal resources, it’s critical to establish a process that goes beyond standard paper assessments and analytical testing to successfully comply with the new regulations. A qualified CDMO can synthesize NDSRIs as custom reference standards, which is essential for confirmatory testing and meeting regulatory thresholds. Having the standard synthesized also allows for more nimble investigations and more robust method development and method validation procedures. The standard can also be individually tested to confirm potency as well as analytical peak identification. Developing an end-to-end solution ensures compliance, particularly for complex projects that demand expertise in this niche service.

Organizations that opt to partner with a CDMO must understand that compliance responsibility ultimately rests with the regulatory holder—the customer in most cases. If the customer mandates or requests changes to the API or drug product, it is the customer’s responsibility to report those changes to the regulatory agency and evaluate if there is a change in risk associated with nitrosamine contamination. 

For example, if the customer changes the source of water used in production and ensures that no nitrates or nitrites are present, the CDMO may assist in the process, but the customer would be the one to submit this information to the agency.

If a CDMO identifies changes that could potentially impact nitrosamine risk, it is imperative that they inform their customer. The customer must then update their risk assessment documentation and submit it to the appropriate regulatory agency. 

While the responsibility for compliance ultimately lies with the entity holding the regulatory filing, CDMOs also have a significant role to play in ensuring that the risks are appropriately communicated. They must share relevant information, such as details about the water source, and depending on the nature of the relationship, help mitigate nitrosamine risks by recommending changes to processes or other key areas that can prevent nitrosamine introduction. This collaborative approach ensures that the end-to-end solution for nitrosamine risk management is fully addressed.

Risks of delaying regulatory compliance

As of this writing, there are companies that are already prepared for the FDA deadline, but there are others that are working to determine how rigorously the standards will be upheld by regulatory agencies. Hoping for less stringent enforcement, there are some organizations planning to conduct only paper-based, risk assessments without performing the confirmatory testing, despite the above guidance provided by the FDA.

Organizations that are not taking measures to be in full compliance are putting themselves, and potentially patients, at risk. If an NDSRI risk is present, companies know that rectification takes time and money, so by not conducting the confirmatory testing, they are taking a more aggressive risk posture. Relying on paper-based assessments and relying heavily on raw material suppliers (e.g. API suppliers which are solely assessing the API chemical process), may not be sufficient.  For others, it is not worth the risk of harming patients, regulatory deficiencies or action, or a product recall if NDSRIs are found. FDA announced the new guidelines in 2023, giving the industry time to prepare and to ensure there was appropriate time to thoroughly assess products and their compliance.

With the August 1 deadline for nitrosamine compliance approaching, companies face significant hurdles in completing the required testing and reporting. A major challenge is the procurement of NDSRIs testing standards. Unlike other analytical standards available on the open market, many NDSRI standards have never been made before, and must be synthesized and characterized from scratch. Synthesizing NDSRIs is not always straightforward. Because they are not commercially available, they must be custom-made, often requiring trying multiple synthetic conditions to achieve the necessary purity and stability. Unexpected issues with isolation or degradation can further complicate the process, making it unpredictable and potentially time intensive. Only after synthesis and qualification are completed, method development, method validation, and testing start. If you are working with a CDMO or other third party to conduct testing, the available capacity at their testing laboratories could become a choke point.

With all those factors considered, the available time to become compliant is running short for companies that have commercial products already on the market. While clinical-stage products may have more flexibility, those that have not yet started compliance efforts may already be behind schedule as well. Delaying gathering information may result in regulatory questions, particularly in later stage clinical trials. Acting now can help mitigate these risks and ensure a smoother submission process.

Meeting the deadline

Ultimately, compliance with nitrosamine testing requirements is not just about meeting regulatory deadlines, it’s about ensuring patient safety. Companies should take a proactive approach, following the guidelines rather than testing regulatory limits and risking an overly aggressive compliance posture. Such an approach could lead to serious consequences, not only in terms of regulatory pushback, but also in safeguarding public health, and financial setbacks.

It is critical to partner with organizations that can assist with both marketed products and those in your pipeline, ensuring patient safety through comprehensive nitrosamine synthesis, product testing, and risk assessments. Partners with a deep understanding of the chemical reactivity of the products they handle can also help identify any NDSRI risks within a given structure, offering a proactive approach to compliance.

By prioritizing rigorous testing and compliance, manufacturers demonstrate their commitment to patient safety and product integrity. With the August 1 deadline fast approaching, taking decisive action now will help organizations to avoid compliance pitfalls, and reinforce trust with regulators, and most importantly, the patients who rely on these treatments.

References:

1. U.S. Food & Drug Administration, Control of Nitrosamine Impurities in Human Drugs, Guidance for Industry, September 2024, Revision 2, accessed February 24, 2025, https://www.fda.gov/media/141720/download.
2.  U.S. Food & Drug Administration, Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs), Center for Drug Evaluation and Research (CDER), August 2023, accessed February 24, 2025, https://www.fda.gov/media/170794/download.
3. U.S. Food & Drug Administration, Control of Nitrosamine Impurities in Human Drugs, Guidance for Industry, September 2024, Revision 2, accessed February 24, 2025, https://www.fda.gov/media/141720/download.
4.  U.S. Food and Drug Administration, Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs, September 2020, accessed February 24, 2025, https://www.fda.gov/media/141720/download.
5. Pereira, D. M., L. F. De Oliveira, A. S. Alves, and A. R. B. Pereira. “N-Nitrosamines: Carcinogenic Impurities and Pharmaceutical Contamination.” Regulatory Toxicology and Pharmacology 139 (March 2023): 105355. Accessed February 24, 2025. https://doi.org/10.1016/j.yrtph.2023.105372.
6. Ellis, Kiersten. “Duloxetine Delayed-Release Capsules Recalled Due to Potential Nitrosamine Contamination.” Verywell Health, November 27, 2023. Accessed February 24, 2025. https://www.verywellhealth.com/duloxetine-recall-8763375.
7.  U.S. Food & Drug Administration, Control of Nitrosamine Impurities.
8. Kelley Drye & Warren LLP. “FDA Issues Revised Guidance on Control of Nitrosamine Impurities in Human Drugs.” Kelley Drye & Warren LLP Insights. Accessed February 24, 2025. https://www.khlaw.com/insights/fda-issues-revised-guidance-control-nitrosamine-impurities-human-drugs.
9. Kelley Drye & Warren LLP, “FDA Issues Revised Guidance on Control of Nitrosamine Impurities in Human Drugs.”
10. U.S. Pharmacopeia. “USP Standard for Nitrosamine Impurities Becomes Official.” U.S. Pharmacopeia. Last modified January 17, 2023. Accessed February 24, 2025. https://www.usp.org/news/usp-standard-for-nitrosamine-impurities-becomes-official.

Joshua Hoerner, PhD, is the general manager of Purisys, a subsidiary of Noramco, where he heads all operations for the business. Josh also serves as the vice president and head of Noramco R&D. During his tenure at Noramco/Purisys over the last 10-plus years, Josh has held a variety of technical and business roles, significantly expanding the business’ product portfolio and leading modernization of its key manufacturing technologies. 

Amber Burch, PhD, is the senior manager of technical business development at Purisys. She manages customer relationships and contractual negotiations for CDMO, clinical, and custom manufacturing projects. Amber started with Purisys in 2017 and has held various technical and customer-facing roles within the organization.