Sufficient AMES test, parameters


does anyone know if negative AMES is still acceptable to justify nitrosamines as non-mutagenc? I’ve heard that DMSO is not an appropriate solvent, but other must be used (which one?). And secondly that a higher concentration of S9 mix should be used.

Does anyone have any regulatory experience - would conducting an AMES test in the above, modified conditions be sufficient for regulators to accept the negative AMES result or is now a priori a transgenic study requested?

Kind Reagrds,


Hi @Filip, Welcome to our Nitrosamine Exchange community. You bring a very vivid question. Under which circumstances is AMES test accepted to derisk a Nitrosamine Impurity?

As @conudel share in his recent webinar ( Calculating Nitrosamine Limits (MasterClass) ), there is still a lot of uncertainty about the acceptance or not of AMES test data among regulatory agencies. The good news is the recent development from EMA where AZIDO impurities were classified as ICH M7 class 5 after investigation.

Thank you for the replay. What I heard from the last confferences I attended was that it was mostly not acceptable. At the moment I think that doing the modified AMES test (hamster S9 and longer perincubation times etc…), might be the best course of action, fow now - at least until new information are available.


Does anyone know if the S9 mix for the nitrosamines AMES test should be induced with phenobarbital or Aroclorom? and which one is better fo rnitrosamines?

Indeed Losartan Azide has been determined to be nonmutagenic in the in vivo comet assay after it was positive in the Ames test. This has now been confirmed also by CMDh and it is now accepted as a Class 5 impurity that can be controlled in accordance with ICH Q3A/B.
However, this is true for the azido impurity and currently this method is not accepted for nitrosamines by the health authorities. Namely, the comet assay is not considered by the HAs as a valid test for nitrosamines, and currently the only method to totally de-risk a nitrosamine (and consider it a Class 5 impurity) is by showing it to be negative in the Ames test followed by an in vivo transgenic gene mutation (TGR) assay. The Ames test, even using modified conditions for nitrosamines (pre-incubation, hamster S9, minimal DMSO), is not currently accepted alone in order to consider a nitrosamine as nonmutagenic.
The hope is that the HAs will eventually be “convinced” that a modified Ames protocol would suffice to de-risk a nitrosamine, or that an in vivo comet assay will be accepted valid for nitrosamines.
In the meantime, hold on tight and enjoy the ride :slight_smile:


Dear Raphael, thank you very much for the response - i figured it was as such. I guess we must wait for future developments in the regulatory area.

Kind Regards,

Sorry for the late reply. I have seen that Agency is quite happy to take the outcome when the modified Ames Test result is positive. However, they are ambivalent when the Ames is negative. It is really sad as I have rarely seen a modified Ames giving false negative. So, as you said, we may have to go to the transgenic if we want to show that a nitrosamine is a non carcinogen. However, most agencies are accepting read across. We have gone with molecular weight adjustments and some have accepted it as an interim limit.

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