EMA - Update Appendix 3: Enhanced Ames Test conditions for N-nitrosamines

EMA has published today, 26th March 2024 updated Appendix 3: Enhanced Ames Test Conditions for N-nitrosamines

Link → https://www.ema.europa.eu/en/documents/other/appendix-3-enhanced-ames-test-conditions-n-nitrosamines_en.pdf

The following information was added:


This sentence is not a guidance update.

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Hi Lucas, thanks for the remark!
This was already on previous version (EMA/451666/2023, 12 October 2023)

We can refer to previous related post :slight_smile:
:eu: EMA - Enhanced AMES test conditions - Guidance, Documents, Resources - Nitrosamines Exchange (usp.org)

There are some difference in the wording across text e.g.

Last paragraph of page 2 “The choice of the N-nitrosamine positive controls needs to be justified based on the anticipated metabolism of the N-nitrosamine and the cytochrome P450 enzymes most likely involved. In addition, if an organic solvent is used to dissolve the test compound, it is recommended that the volume of the organic solvent employed to dissolve the N-nitrosamine positive controls, results in a
similar concentration as for the test compound in the pre-incubation mix.”
…previous ending was “pre-incubation mix, if applicable.” ("if applicable was deleted).

Any case, from my perspective “main update” is the image I shared above.

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It’s good to have this space for discussion and knowledge building. In fact, the remark I made was on, from the discussion we’re having about “standard Ames vs enhanced Ames,” taking directions towards always seeking the best solution with scientific basis. Thank you @E_da_Silva for the discussion.


I noticed that in the checklist they are asking for an NDSRI positive control as the second positive NA control. Unclear, what NDSRI would be a good model compound for testing. Seems like it will be difficult to find and source an NDSRI specific for routine testing of Nitrosamines.


I agree, I think this is problematic. The recommendations don’t require one of the controls to be an NDSRI in the way they are currently worded. And calling the nitrosamine reference compounds ‘positive controls’ implies the need for historical control data (well, it does to me anyway…).

Nick, totally agree with you. Frankly, I don’t think it was well thought out. Before saying we need a “positive control” we should have data on what would be a good positive control.

@jbercu are you refering to this right?


Hi Naiffer,
Sure, it’s there as an option to use an NDSRI as a control but it is not mandated to use an NDSRI. In the new addition to EMA Appendix 3, the checklist table refers to nitrosamine pos control 1 and NDSRI pos control 2. Seems to imply that the 2nd additional nitrosamine control should be an NDSRI.