Nitroso-Diclofenac mutagenicity

Hello everyone,

I would like your opinions if Nitroso-Diclofenac could be a potential mutagenic impurity. It is quite obvious that the Carbons next to nitroso group do not contain hydrogens, thus no α-hydrogens available. Is this statement enough to justify this?

Waiting for your feedback,

@elenaly it all depends on which regulatory agency you are referring to. In the case of EMA, the Q&A documents refer to alpha-hydrogens as a mechanism to establish mutagenic potential, but it does not state that can be utilized as a sole derisk mechanism. I am sure additional toxicological information needs to be submitted and justified.

@conudel @SusanFelter any experience and/or comments


Indeed nitroso-diclofenac has no hydrogens on the alpha-carbons and thus it is not expected to undergo the metabolic activation to form the alkylating diazonium metabolite. As such nitroso-diclofenac can be excluded from the cohort-of-concern class of potent nitrosamines and it can follow the ICH M7 as a “regular” potential mutagenic impurity. Accordingly, if it is predicted by QSAR to be positive then it is a Class 3 impurity that needs to be controlled at the TTC (1500 ng/day).


Thank you very much for the info.

In this case, just to clarify, if we follow the ICH M7 when the N-N=O is not part of the “cohort of concern” but a Class 3 for example. Less than lifetime approach can be used? Or still the comment remains that LTL is not recomendable?

Also looking at the compound, can a read-across be performed with N-nitrosodiphenylamine? I would think it is posible.



As it should be considered a non-cohort-of-concern compound the LTL concept should also be applicable.
Nitrosodiphenylamine could theoretically be used as a surrogate to read-across from, and based on it being a weak carcinogen (Gold TD50=167 mg/kg/day) it would derive a limit of 167,000 ng/day. However, I am not sure that this limit would be accepted and I would prefer assigning it with a TTC limit of 1500 ng/day.


@Naiffer_Host @conudel
Thank you very much for your feedback. I am glad that we agree that Nitroso-Diclofenac is not a CoC impurity at the moment. But also agree with @Naiffer_Host that it depends from the regulatory agencies if they accept this approach or not.

Per FDA, TTC does not apply to nitrosamines?

The nitrosamine is classified as cohort of concern (CoC), so general TTC limit does not apply. Therefore, regulators derived Nitrosamine class specific TTC limit 18 or 26.5 ng/day, which should be used if higher limit based on SAR/read across can not be derived/supported.

In this case of nitroso-diclofenac if suitable surrogate with TD50 available, then AI derived from this TD50 should be used.

For the time being, LTL approach is unlikely to be adopted for any nitrosamine by any regulatory authority following precautionary principle for CoC chemicals.

Please remember that you can not consider a nitrosamine non-carcinogenic without an appropriate study. In a read across, you cant claim somthing to be non-carcinogenic. YOu need to find similar structures and see what the TD50 is and then chose the most conservative one. NDPA or N-nitrosodiphenylamine is a weakly postive material. Please do a read across. Or go to the agencies and get their blessings before you decide that this is not in the cohort of concern.


The topic with specifically the title of this topic N-nitrosodiclofenac is that it should not be part of the “CoC”… Is pretty clear for the ones part of the CoC LTL is not acceptable. But I think is posible to use the TTC as the most conservative approach and even higher limits with further studies/investigation (including the LTL) for this nitrosamines with no alpha hydrogens. Nitrosamines are not direct alkylating agents of the DNA… and not all of them are part of the CoC. We should not avoid using the tools that we know are scientifically sustented and will help go through this crisis and this includes separating “no alpha hydrogens” from the CoC (now as already said, not part of the CoC does not mean non-carcinogenic, but other strategies that are not applicable now will be).

Regarding @Naiffer_Host screenshot on what FDA says, last update was from FEB 2021. I think that as of now with all the learning adquired, going with this argumentation would be at least heard. Generally, I also dont think any agency will say as of now “no alpha hydrogens” LTL acceptable. It is a case by case, when we could go individually asking the agencies. Of course if we dont ask, we have already no as an answer.

In any case the final decisions are up to the authorities. However, what about jurisdictions where no N-N=O regulation is applicable by now? Who to ask then? I think a great part of the info that can help jurisdictions just recently coming into this topic would come from the private sector and initiatives like this forum.


Hi, @Diego_HM. I agree with you. The first step is that AI lists include nitrosamines without alpha-hydrogens. Now EMA and other agencies are positively adding nitrosamines to their AI lists. The highest is 1300 ng/day for N-Nitrosopiperidine and NMPH. It’s almost the same as TTC. And nitrosamines without alpha-hydrogens are not included so far. I hope N-nitrosodiphenylamine will be added in near future.

When I searched nitrosamines without alpha-hydrogens in LCDB, only one aside from Nitrosodiphenylamine was retrieved. I’m worried the number of common nitrosamines without alpha-hydrogens may not be so many.

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I am seeing a lot of comments but as I wrote before, a read across can not be used to justify that a nitrosamine is not carcinogenic. Example, N-nitrosoproline is not carcinogenic. But you cant use that to jusitfy any nitrosamine with an alpha carboxylic acid to be non-carcinogenic. The only way to confirm that a nitrosamine is not carcinogenic is to do a modified Ames and a transgenic mouse assy (both). Agency is not taking the results of Ames as confirmatory. If you want to take the easier read across route, find the most conservative number that matches the structure and use it.


Hello to everyone,

please note that EMA recently established an official Acceptable Intake of 78000 ng/day for Mefenamic acid (Limit derived using structure-activity-relationship (SAR) /read-across approach using the TD50 of N-nitroso diphenylamine as point of departure.):
In my opinion, the same read-across approach may be used for Diclofenac, because both molecular structures share the same skeleton (diphenylamine).


Yes you are right and this aligns well with the initial expert comments by @conudel.
Its important to acknowledge the relevance of α–hydrogens towards carcinogenic potency of N-Nitrosamines.

Lower number of α–hydrogens in alkyl-N-nitrosamines and substitution of the α-hydrogen may reduce the mutagenic potential. This consideration would lead to a ranking of the relative mutagenic potency of alkyl-Nnitrosamines: dimethyl, diethyl…> methyl nitroso…butanoic acid (one α–hydrogen)…>> tertiary-butyl (no α-hydrogen).

The responsibility of convincing HAs using this mechanistic de-risking approach lies with us.