EMA-funded project to predict in-vivo mutagenicity / mutagenicity classification

Fraunhofer ITEM leading an EMA-funded research project

Nitrosamines (NAs) are a class of organic chemical compounds that humans may be exposed to by tobacco smoking or consuming certain foods. N-nitrosamines have been classified as probable human carcinogens and are categorized in the ICH M7 guideline as belonging to the “cohort of concern” group of high-potency mutagenic carcinogens. Some active pharmaceutical ingredients (APIs) carry NAs as impurities from production and/or storage or may cause their formation in the gastrointestinal tract.

Comet assay analysis is one of the key methods to be evaluated in the present project regarding the predictivity of in-vivo mutagenicity. Cells were incubated with the standard methodological positive control ethyl methanesulfonate.

A joint research project funded by the European Medicines Agency (EMA) and led by Fraunhofer ITEM will shed light on the mutagenicity of different classes of NAs to distinguish highly potent from less potent carcinogens. Besides classical nitrosamine structures, API-like nitrosamine derivatives will also be investigated. It will be of particular importance to look for their ability to undergo metabolic activation and to form different DNA adducts. Additionally, corresponding DNA repair mechanisms will be addressed for the first time. By developing novel in-silico as well as in-vitro test systems, the consortium aims to improve risk assessment and to derive reference doses such as acceptable intake (AI) values.

Initially, the focus will be placed on nitrosamine metabolites and their potential to damage DNA when not adequately repaired. The obtained data will be used to correlate the structure of NAs to their potential toxic/mutagenic effects. By means of a quantitative structure-activity relationships (QSAR) approach, this will allow derivation of acceptable intake values for compounds lacking appropriate in-vivo cancer studies, i.e. following a read-across approach.

Secondly, the effect of the physiological environment of the gastrointestinal tract (including the microbiome) on the formation of NAs from drugs or their degradation products will be studied. Due to the lack of knowledge about endogenous nitrosamine formation, it is of utmost importance to elucidate potential mechanisms in order to reduce the carcinogenic risks for patients. Research findings of these laboratory studies will be integrated and generalized in order to develop predictive QSAR models for the susceptibility of drugs to be nitrosated.

Finally, current bacterial mutagenicity test systems as well as novel in-vitro genotoxicity tests, such as the comet assay in liver cell models (primary human and rat hepatocytes, human liver cell lines), will be evaluated, optimized, and validated. Metabolic competence will represent one key topic, to reach the final aim of reliably detecting mutagenicity of different NAs.


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Beneath Fraunhofer, Leadscope is participating in this EMA funded research project as well as the Federal Institute for Drugs and Medical Devices, Technical University of Kaiserslautern,
University Medical Center of the JGU Mainz, Saarland University,
Toxys, ICCR-Roßdorf, and Swansea Medical School.


@kpcross Thanks for adding additional detail. I reached out to Fraunhofer to have them do a short presentation on the project for the community.


I found European Pharmaceutical Review referred to the great “MutaMind project”.

In the project four main streams cover the following:

(i) Formation of NDSRIs endogenously from the reaction of API and nitrite under realistic conditions in different parts of the gastrointestinal tract. ICH M7 indicates that, “Higher acceptable intakes may be justified when human exposure to the impurity will be much greater from other sources eg, food, or endogenous metabolism.”
(ii) Metabolic activation of NDSRIs and identification of the enzyme systems involved
(iii) Types of DNA adducts, kinetics and repair mechanisms
(iv) Optimisation of in vitro mutagenicity assays, eg, the Ames test.

@kpcross explains the details in the video, N-nitrosamine Impurities in Drugs - Introducing the EMA-Mutamind Project (Instem - Video - N-nitrosamine Impurities in Drugs).

And 40NAs were selected for study in the following subprojects:

  1. Quantitative Structure Activity Relationships(QSAR project)
  2. In vitro mutagenicity methodologies for N-nitrosamines(in vitro project)
  3. Endogenous formation of N-nitrosamines from a drug substance(endogenous project)

It is helpful to understand the genotoxicity of NDSRIs. I’m looking forward to hearing the updates in the future.


Thanks, I was not aware of Instem’s video!

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We look forward for a session by Fraunhofer institute @Naiffer_Host…Please try for this. It would be helpful.

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I’m trying to coordinate with them, but please watch the video shared by @Yosukemino because it highlights the project’s goals and work plan.