Tackling challenges with nitrosamine risk assessment

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Tackling challenges with nitrosamine risk assessment

When EU regulators were first made aware of nitrosamines in certain blood pressure medicines in mid-2018, they initiated steps to address the issue[i]. Two years later, in July 2020, the human medicines committee (CHMP) of the European Medicines Agency (EMA) issued an opinion by which companies must take steps to limit the presence of nitrosamines in human medicines and ensure levels of these impurities do not exceed set limits[ii].

As companies are discovering, however, defining safe limits is not straightforward. Indeed, as experience has shown companies face many challenges when it comes to addressing the nitrosamine risk.

Data challenges

Nitrosamines are highly potent mutagens and animal carcinogens and as noted by the EMA are considered probable human carcinogens. In the 2020 opinion, EMA noted that limits for nitrosamines in medicines had been set using internationally agreed standards (ICH M7(R1)) based on lifetime exposure. The agency stated that “patients should generally not be exposed to a lifetime risk of cancer exceeding 1 in 100,000 from nitrosamines in their medicines.”

The regulatory expectation is that medicinal products must not exceed an acceptable intake limit. From my experience in the field, the problem is that, for most nitrosamines, there is no carcinogenesis data, and, as such, companies cannot define the safe limit. Where that’s the case, there is a requirement to apply a very stringent limit of 18 nanograms per day[iii]. To detect those levels, and therefore adhere to this limit, companies will have to develop new analytical methods, which would involve considerable investment and time[iv].

Lack of data also presents a challenge when trying to assess a nitrosamine’s carcinogenic potency using structural analogs with known carcinogenic data. From my experience as a toxicologist working in this field, the problem with this approach is the subjective nature of the assessment. One toxicologist might say Compound X is the most relevant while another might point to Compound Y as being more relevant. The assessment, therefore, depends on the perception of the individual assessor at the company, who may hold a different opinion to the toxicology assessor at the regulator.

Test barriers

The type of test that should be performed to ensure a nitrosamine impurity is non-mutagenic presents further challenges. Typically, a toxicological risk assessment is supported by principles described in the ICH M7 guidance, the core of which revolves around the outcome in what is known as an Ames mutagenicity test[v]. An Ames test is a biological assay that determines the mutagenic potential of a compound.

As described in a recently published paper, however, a negative Ames test alone is not sufficient to qualify a nitrosamine impurity as non-mutagenic. In this case, companies would need to perform animal studies in transgenic animals, but the problem is very few laboratories perform these types of advanced toxicological animal studies, and they are very expensive to perform.

The question for companies is how to proceed? If they can’t stick to the extremely low threshold of 18ng per day and they struggle to perform these costly studies, what next? At present, there are no clear-cut answers to this question. However, drawing on regulatory guidances and knowledge of nitrosamine risk, a good approach would be to perform an enhanced Ames test, carry out a toxicological risk assessment using Structure Activity Relationship (SAR) analysis and a read-across approach and derive acceptable intake above the class specific limit of 18 ng/day.

A wider issue

Initially, when the issue of nitrosamines in medicines was discovered, the impurities were largely seen to be coming from excipients or as a byproduct of the manufacturing process [vi]. More recently, however, it has been found that there are certain classes of pharmaceuticals that do have nitrosamines present because of the chemical moieties present in them[vii]. These are referred to as N-nitrosamine drug substance related impurities (NDSRIs). Again, regulators expect companies to follow the stringent 18ng per day default5.

Regulators have established measures to tackle the presence of nitrosamine impurities in medicines[viii], [ix]. However, ensuring these impurities are within toxicologically defined acceptable limits is proving to be complicated and costly for companies.

Current safety evaluation approaches

When the issue of nitrosamines came up, the safety evaluation was mainly focused on ICH M7-based AI limit setting using either compound-specific carcinogenicity data or using carcinogenicity data available for a relevant structural analogue. When the challenge of data gaps became more intense with the discovery of NDSRIs, newer approaches have come into practice in order to ease the process of limit setting. Notable ones in this regard include:

Carcinogenicity potency categorization approach (CPCA)[x]:

CPCAS is based on structure-activity relationship mainly from the point of metabolism of nitrosamines (alpha hydroxylation). Structural features that are known to directly increase or decrease the alpha hydroxylation or that enhance clearance of nitrosamine by other means are evaluated and assigned a predefined score; a net score is then used to place the compound in one of the five potency categories. Each category is assigned a predefined acceptable intake value i.e 18 ng/day for Category 1 to 1500 ng/day for Category 5. CPCA is a quick and easy way to rank the nitrosamines based on their perceived potency.

Enhanced Ames Test (EAT)[xi]:

Another approach to assess nitrosamines is EAT. To determine whether a nitrosamine is mutagenic or not, it can be tested in the Ames test. However, the conventional Ames test is not sensitive enough to detect nitrosamine-related mutagenicity. Therefore, the protocol of the conventional Ames test has been modified and used to test nitrosamine. This modified protocol is called Enhanced Ames test.

Regulatory acceptance of safety assessments

Many regulatory authorities have already established acceptable intake limits for number of NDSRIs based on CPCA or read-across approach. However, higher limits may be possible when experimental data are made available. A negative result in an GLP-compliant enhanced Ames test allows control of the N-nitrosamine at 1.5 μg/day8. Further, for nonmutagenic compounds (negative in enhanced Ames test), higher limits may be possible depending on the maximum daily dose of the drug product, when this can be supported with structure-activity relationship evaluation using data available for a relevant analogue[xii].


About the author:

Dr. Sebastian Joseph is Senior Director, Toxicology and Nonclinical Development, at PharmaLex. Sebastian is a board certified toxicologist with 18 years of experience in preclinical testing, pharmacology/safety pharmacology, experimental toxicology, occupational toxicology, drug discovery and development, toxicological risk assessment and regulatory toxicology, and assessment of cosmetic ingredients and products.


[i] Nitrosamine impurities, EMA. https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/pharmacovigilance-post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities#:~:text=Nitrosamines%20are%20chemical%20compounds%20classified,medicines%20known%20as%20’sartans’

[ii] EMA finalises opinion on presence of nitrosamines in medicines, EMA, July 2020. https://www.ema.europa.eu/en/documents/referral/nitrosamine-impurities-final-outcome-article-53_en.pdf

[iii] EMA, HMA, CMDh, Feb 2021. https://www.ema.europa.eu/en/documents/referral/european-medicines-regulatory-network-approach-implementation-chmp-opinion-pursuant-article-53-regulation-ec-no-726-2004-nitrosamine-impurities-human-medicines_en.pdf

[iv] The Nitrosamine “Saga”: Lessons Learned from Five Years of Scrutiny, Org. Process Res. Dev., Oct 2023. https://pubs.acs.org/doi/10.1021/acs.oprd.3c00100

[v] ICH M7 Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk – Scientific guideline, EMA. https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential-carcinogenic-risk-scientific-guideline

[vi] Nitrosamine impurities in human medicinal products, EMA, 2020. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf

[vii] The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals, Journal of Pharmaceutical Sciences, May 2023. https://www.sciencedirect.com/science/article/pii/S0022354922005251

[viii] Control of Nitrosamine Impurities in Human Drugs, FDA, Feb 2021. https://www.fda.gov/media/141720/download

[ix] Q&A, EMA, CMDh, Jan 2024. https://www.ema.europa.eu/system/files/documents/referral/nitrosamines-emea-h-a53-1490-qa-art-53-implementation_revision-20_en.pdf

[x] Carcinogenic Potency Categorisation Approach for N-nitrosamines, Appendix 2, Q&A, EMA, Oct 2023. https://www.ema.europa.eu/en/documents/other/appendix-2-carcinogenic-potency-categorisation-approach-n-nitrosamines_en.pdf

[xi] Enhanced Ames Test Conditions for N-nitrosamines, Appendix 3, Q&A, EMA, Oct 2023. https://www.ema.europa.eu/system/files/documents/other/appendix_3_enhanced_ames_test_conditions_for_n-nitrosamines_en.pdf

[xii] Updated Information | Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs), FDA, August 2023. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-recommended-acceptable-intake-limits-nitrosamine-drug-substance-related


This blog is intended to communicate PharmaLex’s capabilities which are backed by the author’s expertise. However, PharmaLex US Corporation and its parent, Cencora, Inc., strongly encourage readers to review the references provided with this article and all available information related to the topics mentioned herein and to rely on their own experience and expertise in making decisions related thereto as the article may contain certain marketing statements and does not constitute legal advice. 

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