🇪🇺 EMA Q&A Rev. 16 EMA/409815/2020 - MAJOR UPDATE

Dear all,

Today EMA released the new major revision for EMA/409815/2020. It is a major update on how to set up limits based on several clasification and would be extraordinary helpful for NDSRI. In particular Q&A 10 and Annex 2 & 3.

Appendix one for limits for + 40 new nitrosamines Appendix 1 Nitrosamine AIs (europa.eu)

I highly recommend to check it as well as the community to share their opinions.

Review on-going…

Nitrosamines EMEA-H-A5(3)-1490 - QA Art. 5(3) Implementation_ QA10_revision 16 (europa.eu)



Dear @Diego_HM,

Thank you for sharing the information. I’m so excited to read it. It’s amazing!!

According to the revision history, the updates are as follows;

Amendment to Q&A 10 to include the Carcinogenic Potency Categorization Approach (CPCA) and the enhanced Ames test (EAT) for establishing AIs for N-nitrosamines. Addition of Appendix 1, listing the nitrosamines for which AI have been established by the Non-clinical Working Party (NcWP), including new AIs for N-nitrosamines determined using the CPCA. Addition of Annex 2, describing the Carcinogenic Potency Categorization Approach for N-nitrosamines. Addition of Annex 3, describing the Enhanced Ames Test Conditions for N-nitrosamines.

Now we can classify nitrosamines into 5 categories. Here we can count alfa-hydrogen and calculate the “Potency score” from the “alfa-Hydrogen Score”, “Deactivating Feature Score” and “Activating Feature Score”.


And the enhanced Ames test condition is also available. A negative result in an GLP-compliant enhanced Ames test (EAT, Annex 3) allows control of the N-nitrosamine at 1.5 μg/day. For substances testing positive, the AI should be established using options 1(category approach) or 3(read-across method).

These approaches will help all patients, pharmaceutical companies, and regulators. Let’s enjoy the journey to great science!!


Thanks a lot Yosukemino for the summary on the changes. I am still astonished of the guideline for the good.

What I could also share is that for the modified Ames test, a major change is the need to use positive control with 2 nitrosamines. From experience and surveys, I understand most Ames that were performed where not GLP compliant and/or used at max. 1 nitrosamine as positive control. Industry will need to recheck several Ames already performed.

Additionally, using 30% S9 Hamster and Rat. That is a bit too much I suppose.

Nevertheless, finally there is clear guidance on the Ames test :slight_smile:


Certainly, it is truly a watershed moment in the discussion on nitrosamines to include a nitrosamine score. It is wonderful to witness this development!


Yes, you are right. I revised the image of the Enhanced Ames Test in my previous post to avoid misunderstanding. Thank you for pointing this out.

And I want to add example 2. It looks confusing. One alfa-hydrogen on each side of N-nitroso group is less concerning.



Thanks for sharing. It is really a great update!

I think entire globe should thank EMA and NcWP team who consistently keeping all of us updated with evolving science and providing guidance in ocean of uncertainty!!

Hi USP Nitrosamines Exchange Team.

I have observed one ambiguity for few nitrosamines such as
1-Methyl 4-Nitroso piperazine-New AI is 400 ng/day but the footnote referred indicates NDEA is surrogate which has AI of 26.5 ng/day in line with previous versions of same EMA Q&A.

Similarly for N-nitroso Rasagiline and N-nitroso Dabigatran the new AI values published were 100 and 400 ng/day, respectively. However again as per their footnote indications, they shall be TTC of 18 ng/day which are in line with previous versions.

Any clarity team on this…

If you allow me, request for help in understanding this @conudel @AndyTeasdale as you could have better idea being part of some key discussions in Europe.


Dear all,

Thank you for sharing this exciting update regarding the new major revision released by EMA (EMA/409815/2020). I am grateful for the notification and appreciate the effort put into revising the guidelines. It is wonderful to see the inclusion of the Carcinogenic Potency Categorization Approach (CPCA) and the enhanced Ames test (EAT) for establishing AI’s.

I am delighted to have the opportunity to review these updates, and it is inspiring to witness the development and progress made in the understanding and handling of nitrosamines. This serves as a testament to the effectiveness of the collaboration and communication between our community and regulatory agencies. It reinforces the importance of our collective efforts in raising awareness and addressing the concerns associated with nitrosamine impurities.

Once again, thank you for sharing this significant information, and I look forward to diving into the details of the revised guidelines.

Best regards,

Christian Romo


Hi, @Pradpharma

Yes, you are right. Footnote 10 looks incorrect. I compared the previous Q&A with the current one.

rev.15: 27 compounds
rev.16(Appendix-1): 83 compounds

The compounds with changed AI(ng/day);
MeNP: from 26.5 to 400
N-nitroso-rasagiline: from 18 to 100
N-Nitrosodabigatran: from 18 to 400

Read-across method is still available and EMA may not want to revise the AI to avoid confusion.

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A great relief to some but questions still remain.


I manually sorted 86 compounds in appendix-1 with the CPCA category. Four compounds are in Category 1. Four compounds are in Category 2. 15 compounds are in Category 3. 17 compounds are in Category 4. And 12 compounds are in Category 5. And four compounds are considered NMI due to in vivo mutagenicity study. Others are calculated from carcinogenicity data or the read-across method.

The total number of Category 4, Category 5, five compounds with AI which is more than 1500ng/day without classification, and four compounds of NMI are 38. 44% of the 86 compounds are not CoC. It is interesting!!


This is fantastic news, everybody!!

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Hi, @Pradpharma

N-nitroso-propanolol is considered a typo of N-nitroso-propranolol. Please add it, too.


Yes @Yosukemino - Agree

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Thanks for sharing this example.

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Hi, @Pradpharma

Thank you for asking. Yes, you are right. I posted the example of N-nitroso-propranolol in the case study thread. Please check it.

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I am not able to figure out how the alpha-hydrogen scoring is done based on the example provided in the EMA guideline.

Why is 0,3 = 2; 1,3 = 3; 2,2=1 and 2,3 =1

I am not a chemist, I hope someone can explain this part

Thank You



Dear @sushantkamath,

Thank you for asking good questions. You asked why is 0,3 = 2; 1,3 = 3; 2,2=1 and 2,3 =1. And I’m not sure of the answer, but I know compounds with less score are vulnerable to metabolic activation by CYP. The alfa-hydroxylation step is the key to nitrosamines’ carcinogenicity. I add the slide and link to Ponting’s presentation.

I guess the score was determined from the results of TD50 of lots of nitrosamines. In other words, the scoring system was built through validation with nitrosamines’ toxicological data. Compounds with low scores have a tendency to be potent in carcinogenicity. And visualization thread may be helpful to understand the scoring system.

If you need further help, do not hesitate to ask us.

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Hi @Yosukemino,

Thank You.

I have gone through the presentation by Ponting as well as the Cross and Ponting publication. EMA has not included all the deactivating features from their publication in the Table, although they have cited the publication.

The problem with the scoring system is when we start to apply it for other possible NDSRIs for which no AI limits have been established. For eg: few NDSRIs have a score of (0,2) with two hydrogen atoms on only one alpha-carbon. These compounds may not have the methylene alpha-carbon or the ethyl group to provide a score of 3 or even 2. How do we then score (0,2)?



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I’m sorry, I couldn’t understand the structure you meant. If you kindly illustrate these structures, I may understand.

And I think compounds of (0,2) score with two hydrogen atoms on only one alpha-carbon are as follows;


Hi @sushantkamath,

Please note that the EMA has itself mentioned the list is not exhaustive.

Coming to question on (0,2)- it would have been better, if the structure is known- that can be discussed well. Nevertheless, Thanks @Yosukemino - He brought in few structures, that may fit to your question, Where i am in opinion we need to assign scores based on availability of alpha-carbon hydrogens and also the reactivity of alpha-carbon hydrogens which depends on the surrounding environment and type of molecule.

Overall, it is fortunate we got some guidance through this EMA update, at the same time-“Yes” it may not address all complex NDSRIS right now-Hope it will do in future soon.