Redefining Nitrosamine Risks: A Fresh Perspective on Pharmaceuticals -Pub

In the study conducted by @MichaelBurns @David @Naiffer_Host @AndyTeasdale @schlinjo1975, Robert S. Foster, Benjamin P. Thornton, Graham F. Smith and Ian W. Ashworth, a comprehensive re-analysis of the available data has been undertaken, shedding light on a discernible pattern concerning the potential risks associated with complex nitrosamines derived directly from secondary amines.

Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals

The findings underscore a substantial reduction in concern regarding these particular compounds.

Throughout the examination of various datasets, it becomes evident that the prevalence of theoretical nitrosamines categorized as either โ€œCategory 1โ€ or โ€œCategory 2โ€ is relatively minimal in relation to the total population of compounds examined. To illustrate this point, within the USP dataset comprising 8611 APIs, a mere 99 molecular structures containing secondary amines are identified as potentially posing a risk for nitrosamine formation, necessitating control measures down to an alarmingly low threshold of 18 ng. Furthermore, a substantial majority of the datasets fall under the highest AI categories.

This re-analysis serves as a noteworthy contribution to the field, as it not only highlights the potential for a more nuanced and risk-stratified approach to nitrosamine regulation but also underscores the evolving nature of our comprehension of these chemical entities.

Great work!


#nitrosamines โ€ฆ The word is out! Our 2nd publication, โ€œ๐‘๐ž๐ฏ๐ข๐ฌ๐ข๐ญ๐ข๐ง๐  ๐ญ๐ก๐ž ๐‹๐š๐ง๐๐ฌ๐œ๐š๐ฉ๐ž ๐จ๐Ÿ ๐๐จ๐ญ๐ž๐ง๐ญ๐ข๐š๐ฅ ๐’๐ฆ๐š๐ฅ๐ฅ ๐š๐ง๐ ๐ƒ๐ซ๐ฎ๐  ๐’๐ฎ๐›๐ฌ๐ญ๐š๐ง๐œ๐ž ๐‘๐ž๐ฅ๐š๐ญ๐ž๐ ๐๐ข๐ญ๐ซ๐จ๐ฌ๐š๐ฆ๐ข๐ง๐ž ๐ข๐ง ๐๐ก๐š๐ซ๐ฆ๐š๐œ๐ž๐ฎ๐ญ๐ข๐œ๐š๐ฅ๐ฌโ€ has been published.

Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMAโ€™s Carcinogenic Potency Categorization Approach (CPCA)

๐Š๐ž๐ฒ ๐…๐ข๐ง๐๐ข๐ง๐ ๐ฌ:

:one: The majority of potential nitrosamines from secondary amine precursors belong to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng/day

:two: Tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2

:three: The analytical sensitivity required for the quantification of high-potency nitrosamines can be problematic, especially for high-dose APIs

Free access:

[Jรถrg Schlingemann] @schlinjo1975 [Andrew Teasdale] @AndyTeasdale [David Ponting] @David [Robert Foster] [Stephanie Simon] [Ian Ashworth] [Naiffer Romero] @Naiffer_Host

@trust_user_a @trust_user_b @trust_user_c @trust_user_d


Thank you Naiffer for this update!
We appreciate USPโ€™s contribution to this sequence of articles and most of all for organising this nitrosamine exchange group, invaluable for keeping the community informed!


Thanks a lot, Naiffer, we also think that 1500 ng/day is a overkill for many of the nitrosamines. Even N-nitrosopyrrolidine has a AI of 1700 ng/day, NDELA has a AI of 1900 ng/day. But good that we can move out of the 26.5 ng/day myth.


Just to add on Point 2- Might not be for all tertiary amines but only for structures featuring electron-rich (hetero)aromatic substituents that are susceptible to direct nitrosation.

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Great work, my concern would be, the probability of forming potent Nitrosamine (NA) out of API with tertiary amine (TA) higher , while we know formation NA out of TA goes much slower . Any idea in normal Oral Solid Dosage forms (as a tablet) the likeliness that during Shelf-life this NDSRI cat 1 or 2 may form. Any general indication?

Iโ€™m not sure if I fully understood your question, but letโ€™s discuss some considerations based on the published information, and I hope to provide you with a comprehensive response.

:one: Based on the information provided by the authors, among the top 200 drugs by sales, only N-Nitrosovonoprazan was identified as a category 1 nitrosamine derived from a secondary amine. When evaluating the essential medicines list, no category 1 nitrosamines were found to originate from secondary amines within the API structures. However, three structures, including N-nitrososertraline and N-nitrosoepinephrine, were identified in category 2.

:point_right: Additionally, the authors highlight that, in contrast, NDSRIs derived from tertiary amines make up the majority of potential structures, and they exhibit significant categorization in both category 1 and category 2. This phenomenon can be attributed to the presence of small units like NMe2 and NEt2, which theoretically have the capacity to lose one of these groups. This loss results in a secondary amine with an additional methyl/ethyl group alongside the remaining group.

Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals

:two: Regarding your inquiry about Shelf-life, nitrites, peroxides, and formaldehyde are known impurities found in a wide range of excipients. They may contribute individually or in combination to the formation of nitrosamines during the formulation and storage of finished products.

:point_right: The proposed mechanism involves an aldehyde catalyst undergoing a reaction with the secondary amine to form an iminium species. The addition of nitrite to the iminium intermediate forms a nitrosyl donor, which could react with another equivalent of the secondary amine to form the nitrosamine and regenerate the aldehyde catalyst. This pathway could potentially allow for nitrosation even in neutral or alkaline media.

:white_check_mark: As recommended by the authors, conducting thorough drug-excipient compatibility studies and accelerated stability studies can effectively identify potential risks related to the formation of nitrosamines and their precursors. This valuable information can then be used to inform the selection of appropriate storage conditions and container closures, ultimately ensuring the safety and quality of the product.

Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals


A few recently published articles addressing the reactivity of tertiary amines in pharmaceuticals:

  • A Consideration of the Extent That Tertiary Amines Can Form N-Nitroso Dialkylamines, Ashworth et al., OPR&D, 2023
  • Formation of Dialkylโ€‘Nโ€‘nitrosamines in Aqueous Solutionโ€ฆComparison sec and trialkyl Amine Nitrosation, Ashworth et al., OPR&D, 2023
    -N-Nitrosamine Formation in Pharmaceutical Solid Drug Products: Experimental Observations, Moser et al., J. Pharm., Sci, 2023 Redirecting
    cited within the article

Itโ€™s a fantastic paper!!

Yes, you are right. I picked up โ€œdesmethyl NDSRIsโ€ from FDA NDSRI lists. The results showed lots of them are in Category 1 and 2 due to low hydrogen score of 1 or 2. They may have high potency for activation.

Category 1: 39 compounds
Category 2: 12 compounds
Category 3: 11 compounds
Category 4: 4 compounds

NDSRI list by FDA Category desmethyl.pdf (2.4 MB)


Are a lot of these actually the nitrosamines of the secondary amine impurity of the tertiary amine parent compound?
Which then leads to the question of if these are seen in a finished product is it due to the presence of the secondary amine as a low level impurity (or degradation product, and so forming throughout the shelf-life), or due to the tertiary amine but at a much slower rate. Or both?!?!

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I think my PhD advisor Dr. Loeppky turns in his grave everytime it is said that the source of nitrosamine in tertiary amines is due to secondary amine impurities. It was the topic of his PhD and he spent his life dispelling this myth. Apparently he wasnโ€™t successful. Please understand that tertiary amines, at least some of them can undergo facile nitrosation. If there are secondary amine impurities, they can definitley form nitrosamines, but if you do benchtop nitrosation studies with tertiary amines, you may see much more nitrosamine than what can be justified by traces of secondary amine impurities.


I think a distinction is indeed needed between nitrosation on the tertiary amine (dealkylative nitrosation sensu stricto), nitrosation on the secondary amine impurity which is already there and subsequent demethylation and nitrosation of the tertiary amine. I believe Industry is doing this, if not in the risk assessment phase than in the root cause evaluation phase, but I would have expected updates in this context to Industry guidance as well, like more explicit distinctions in the EMA root cause list (checklist CMDh/439/2022).

For some nitrosamines from tertiary amines (stoichiometric conditions) historically, literature debates on the mechanism: first nitrosylation then demethylation or vice versa. By having to study such cases now as impurity chemistry (preferably taking into account moisture, oxygen and metal exposure as risk factors), a lot of new information is coming available, and in my experience the most important pathway for the exogenous nitrosamine formation does not always align with the literature reported most important pathway for endogenous nitrosamine formation linked to the same tertiary amine API. I personally recommend studying the non-classic Polonovski reaction in the context of dimethyl tertiary amines where the third N-substituent also starts with a CH2. Even in cases where the N-nitroso-demethylated API reference standard can be easily made from the API itself (which does not proof the order of the reaction steps of course), direct nitrosation is not per se the root cause on drug product. So besides 3 reaction scenarios there are also 3 cases (exogenous stoichiometrically, exogenous impurity chemistry (with matrix distinctions), endogenous) with possible different trends in mechanism prevalence.

I personally was surprised to see that the FDA guidance update (August and October 2023) didnโ€™t make notes on distinct pathways for nitrosamine formation linked to tertiary amines nor explain their focus on tertiary amines with an N-methyl. Especially because compared to EMA, TGA and HC, FDA lists mostly N-nitroso-APIs and N-nitroso-desmethyl-APIs (linked to tertiary amine APIs) in the limit lists (exceptions are linked to Rifampicin, Rifapentine, Sitagliptin, Nicotine and mostly MAH submission driven (and of course not per se tertiary amine API based)), whereas the other regulators have much more โ€œN-nitroso-API impurities beyond demethylated APIโ€ (cases include Alogliptin, Arpraziquantel, Chloropyramine, Ciprofloxacin, Gliclazide, Hydroxyzine, Levofloxacin, Lidocaine, Quetiapine, Ranolazine, Rivaroxaban, Ropivacaine, Rotigotine, Terbinafine, Valacyclovir, Valsartan (and of course not per se tertiary amine API based)). If the FDA limit list would be partially pushed to industry based on FDA available API lists (could the fact that originally (prior to October 2023 revision) some non-commercialised APIs were mentioned be a hint, together with the increased entries compared to other territories and the recommendation to use the list for re-assessing risks?) and theoretic assumptions rather than per se step 2 submissions or real detections, I would have expected some clarifications on the increased attention to demethylated API nitrosated compared to nitrosated API impurities in general. At first glance this suggests a confidence in the direct nitrosation on the tertiary amine, but in the context of impurity chemistry often N-oxide formation on the tertiary amine and subsequent non-classic Polonovski reaction to the demethylated API and subsequent nitrosation is possible as most relevant pathway towards the nitrosamine, whereas this chemistry is not per se more relevant or faster than dealkylative, deacylative,โ€ฆ chemistry on API towards API impurities in general and whereas API stability in this context can be highly matrix dependent.

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In the AAM meeting, FDAโ€™s Andre Raw mentioned that tertiary amines should be addressed. But as a reason, he talked about secondary amine impurities. I think that is where this perception is coming from. The issue is that a finished dosage form acts like a reaction vessel that can have a varied environment and while tertiary amine nitrosation may be slower than secondary amine nitrosation, it has enough time to happen.

Totally agree, the prevalence of tertiary amine direct nitrosation over nitrosation via the secondary amine API is matrix specific, this is the point I tried to the make as well. I just shifted focus to secondary amine formation as a study focus because I would argue that there is more variability across formulas on the nitrosation via secondary amine kinetics than via the tertiary amine.

I agree with your FDA citation that the biggest concern in general with tertiary amines is the secondary amine impurities rather than the direct nitrosation on the tertiary amine.
What I donโ€™t understand though: if the motivation for FDA would be the prevalence of secondary amine impurities linked to tertiary amines rather than direct nitrosation, why do they narrow down to N-nitroso-desmethylAPIs specifically (which the limit lists seems to suggest).

I think initially this list was made as they thought that these could produce NDMA, which was one of the nitrosamines in the first guidance and of course the nitrosamine that won the popularity contest. Slowly it became obvious that just because a drug has dimethylamino group, it does not produce NDMA as the major nitrosamine. I guess that is where were are now.

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The list in question is the FDA NDSRI limit table of August-October 2023, so not an initial list, but published in a time where a broad interest in N-nitroso API impurities exists and recalls linked to N-nitroso-desmethylAPI have been reported. That list specifically lists N-nitroso-desmethylAPIs, seemingly more arbitrarily than the other agencies and also with less attention for the N-nitroso-API impurities beyond demethylated.

Evaluation methods for the degradation of dimethylamine tertiary APIs towards nitrosamines in historic literature were often indirect (evaluation of the biological response on the mixture before and after attempt towards chemical degradation to a nitrosamine (mutagenicity testing) or evaluating nitroso content after stoichiometric tests and assuming NDMA but no direct proof beyond at most an indication of mutagenicity), leading to presumptions of NDMA risks based on literature evaluation alone, simply because the N-nitroso-desmethylAPI was not (always) directly studied in literature.

But this is not the topic anymore, the topic is the FDA publication selectivity towards N-nitroso-desmethylAPI limits today. Not saying that N-nitroso-desmethylAPIs and NDMA are not belonging in the limit list, but I would like to understand what data and root cause information sits behind the more expressed focus on N-nitroso-desmethylAPIs in the list (if it is not arbitrarily or coincidental) to optimize the derisking or prioritisation of N-nitroso-API impurities beyond demethylated. I didnโ€™t think it was a reflection of the applicant focus per sรฉ, as this trend is not in the limit list of other regulators as much.

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