N-nitrosoamides - a horse of a different color

Of late, I am seeing an alarming trend where people are mislabeling impurities as nitrosamines, when they are really nitrosamides/carbamates/urea/guanidine. Let us understand thatthe N-nitroso compounds can be divided into two categories, the M-nitrosamines and N-nitrosamides** (Figure 1). N -nitrosamines are formally derived by the reaction of secondary and tertiary amines with nitrous acid or other nitrosating agents. These can be formally considered as “amides” of nitrous acid and are stable. N-nitrosamines need to be metabolized in order to elicit their mutagenic or in many cases, carcinogenic behavior and it is believed that most of these compounds are activated by alpha hydroxylation in presence of Cytochrome P450 and NADPH (Figure 2).

Figure 1: N-nitroso compounds

The N-nitrosamides are compounds where there is a carbonyl group (C=O or C=N) attached to the nitrogen bearing the “NO” group. Members of this class include N-nitrosoamides, N-nitrosocarbamates, N-nitrosoureas, N-nitrosoguanidines. This class of N-nitroso compounds are known to be unstable compared to the N-nitrosamines and do not need to be metabolized to produce the electrophile carbocation. Please do not treat them the same way as nitrosamines, where the NNO group is flanked by alkyl groups.
Figure 2: Activation of N-nitroso compounds

Adapted from Loeppky, R., N.; “Nitrosamine and N-Nitroso Compound Chemistry and Biochemistry: Advances and Perspectives”; ACS Symposium Series, 1993, 553 , Chapter 1, pp. 1-18.


Thanks for the info is really useful. However, this could be understood as nitrosoamides are even more “dangerous” of course if present (and that is the key I think “if”), as its formation is way more complicated because of the carbonyl group, etc.

just because something is a direct acting mutagen does not mean it is more dangerous. N-nitrosamines are very stable and can travel to the liver with ease, where they are metabolized. Many of the direct acting mutagens like alkyl halides are not carcinogents.

My purpose is not to create panic but to make participants understand that these class of Nitrosamides/carbamates/ureas/guanidines are different from nitrosamines. Nitrosamides are usually unstable and while they form the carbocation, it is usually quenched fast in presence of water or other nucleophiles like glutathione. With N-alkyl-N-nitroso ureas, there are anti cancer drugs like carmustine and lomustine which are of this structure.

There are several of these, which are known to be non-carcinogenic or have quite high AI.

When it comes to N-nitroso guanidine, there are also studies with some smaller ones which shows these to be less potent than nitrosamines of similar MW range.

What I am seeing is people making collosal error with the chemistry and understanding of what is really a nitrosamine and what is not a nitrosamine.
Please identify if your product is a nitrosamine or a nitroso amide/urea/carbamate/guanidine becase the later are metabolized very differently.


And we really appreciate your comment, what you have just said is the explanation that is needed to avoid any misreading and is greatly appreciated and part of what this community is for. We got a lot of members recently coming into the topic where this information will be really useful. Personally, I have also seen quite a bit of declarations where the nitrosamide topic is mentioned but with lack of information.


The comment is of course correct-nitrosamides etc., are not nitrosamines. However this does not mean that we can ignore them because the ICH7 cause of concern refers to nitroso compounds which embraces all these compounds.

Totally agree. It is just that one has to remember they work very differently than nitrosamines. Their mode of acitivation is different. They are direct actiing mutagens. Do not need the S9 for activation. Also, they dont work by the alpha hydroxylation process. This should be remembered and the correct mechanism of activation should be considered. Also, while doing a surrogate study, I am seeing people use nitrosamines as surrogates. This should be avoided as in a surrogate study “like should be treated alike”. If you have a nitroso guanidine, find a surrogate with that structure. For a nitroso urea, do the same.


This topic is fantastic!!
And I found AAM’s white paper(2020) included alkylated amides, carbamates, and ureas in scope.

  • N-alkylated amides, carbamates, and urea can be nitrosated. So, for reagents—
    starting materials with these structures—there is a possibility of nitrosamines
    being formed based on reaction conditions. In case these are used as reagents,
    the incoming lot(s) will be tested for the corresponding nitrosamines.

  • When secondary and tertiary amines, N-alkyl derivatives of amides, carbamates, or urea generated as intermediates in the synthetic scheme of the API, these will be tested for the corresponding nitrosamines.

I couldn’t find other guidances describing nitrosamides/carbamates/urea.
And Q2 in “Nitrosamines as Impurities in Drugs—Health Risk Assessment and Mitigation Public Workshop” refers to nitrosourea as an example of direct alkylating agents.

Anyway, we should be careful to avoid misclassifying those compounds.

I wrote that white paper on behalf of AAM, should I say LOL!
We submitted it to FDA before the guidance was written. They decided not to include the "nitroso amides/carbamates/urea/guanidine.
Please remember that these are direct acting mutagens and in some cases carcinogens. But their mode of activation is very different from “nitrosamines”. They should not be in the “cohort of concern” but they definitely need to be addressed.

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Thank you for the clarification, @ASrinivasan. I thought you might be related to the document. :smile: :smile: :smile:

We can treat these compounds, not as the “cohort of concern”, but as class2 in ICH M7. We should keep it in mind.

That is my opinon as of now. Some of these are carcinogenic, some are not carcinogenic at all. But the issue is that, so are alkyl halides, sulfonates etc. I think we should seriously think about these.
Agencies have been quite till now. Lets see how this story develops.

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Agreed - these worry me somewhat, especially as there are some that are comparably potent to nitrosamines (the distributions of known potencies for the nitrosamines and non-amine nitroso compounds are similar), but I suspect that given their nature as direct-acting carcinogens/mutagens some aspects that have typified the nitrosamine crisis are less significant:

  • amides etc are probably harder to nitrosate than amines (since amide lone pairs are less reactive than amine), so risk of formation is lower and I’d expect them to be rather less abundant than we are seeing for nitrosamines.
  • they are direct-acting mutagens, so the concern that is currently being raised about S9 activity in the Ames, whether solvent choice to avoid inhibition, or the choice of S9 source, should not be relevant and M7 can be followed fully with confidence that an Ames or in silico positive is positive and a negative is negative.
  • since some are potent, the compound-specific limits for some class 1 nitrosoamides etc may be significantly lower than TTC… but the same applies to some other classes, and other nitrosoamides/ureas/etc can be of low potency.

Read-across wise, I agree the two classes should not be mixed; however, there is still valuable information that can be learned from the nitrosoureas and applied to the nitrosamines; in particular, the formed diazonium ions from nitrosamines and nitrosoureas can be the same, and the DNA alkylation rates due to these would be expected to be very similar (e.g. between methylnitrosourea and nitrosodimethylamine). The EMA article 5(3) guidance cite some interesting kinetic studies by Manso et al (2008), showing significant variation in alkylation rates with different nitrosourea-formed diazonium ions.


Agree David, some nitrosoureas are as carcinogenic as potent nitrosamines and MNNG related work has been happening since 1981 and possible before that. My PhD advisor, Dr. Loepkky always told us not to mix the “amines” with the “amides” and I had actually raised this question when I commented on the FDA guidance in the Federal Register on behalf of AAM (Association of Accessible Medicine). All we need to understand is that if unmodified Ames shows these to be negative, would the agencies should accept that? I hope they do as the burden is becoming unbearable.


I agree totally about different mechanisms for nitroso-amides however as long as ICH M7 CoC uses the term nitroso the regulators will continue to pursue the issue and I don’t see much chance of getting a change in the ICH guidance.

As pointed out, although via a different mechanism, some of these compounds are high risk materials. Does anyone know of a possible read across SAR for nitrosamides. I have not been able to find anything in the databases such as Derek.


With a substructure search, I searched the following compounds on LCDB.

Smiles: N(N(C)C=O)=O

More than thirty N-nitroso amides, carbamates, and ureas are on the list. And many of them are N-nitroso ureas. Here, to discuss simply, I focus on GOLD TD50(Harmonic mean). Please see the attached list.
Nitrosoureas.pdf (75.9 KB)

It seems rough, but the class-specific limit for N-nitroso ureas can justify similarly. These nitroso compounds are less harmful than nitrosamines, but 1.5ug/day may be insufficient.


MNNG is possibly the best surrogate here. But we are seeing nitroso ureas with no carcinogenicity to being quite potent. These being direct acting mutagens do not need modified Ames and the Ames is very dependable. If something is negative in Ames, it should be considered non-mutagenic and treated as a ICH Q3 impurity. I hope that regulatory authorities accept this or they are effectivley throwing out ICH M7


Thank you for your insight, @ASrinivasan. I keep MNNG(N-Methyl-N’-nitro-N-nitrosoguanidine) as the best in my mind. Here is the link to the data in LCDB.


As discussed in this thread, we should distinguish nitrosamines from other similar nitroso compounds and treat them appropriately.

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As there is a probability of forming following 2 NDSRI impurities (Nitrosamine A & B) of Flecainide in its formulation due to presence of Nitrite/Nitrate in the excipients used, can anyone guide whether following Dinitrosamine NDSRI impurity (Nitrosamine C) can be formed in formulation.



the N-nitroso compound B is a nitrosamide. Read my document here, “horse of a different color”.



I suggest if you can read this topic, where this API was already discussed:

Nitroso Flecainide - Limits of Nitrosamines - Nitrosamines Exchange (usp.org)


Hi @JaideepJ : “Flecainide Nitrosamine A” is quite possible in formulation and you have to perform confirmatory testing for the same if formulation is having nitrite, as far as “flecainide nitrosamine b” is concerned, it is quite difficult to synthesize as electron negative group carbonyl is attached near by secondary amine and as “flecainide nitrosamine b” is difficult “flecainide nitrosamine c” is also difficult to synthesize, moreover “Nitroso flecainide EP impurity B” is also possible and you need to evaluate in your formulation if formulation is having nitrite.