EMA/2020/46/TDA, Lot 1 – ROC25 Refinement of enhanced Ames test conditions for N-nitrosamines MUTAMIND2

Study type

• Study topic:
  Other
• Study topic, other:
  Refinement of enhanced Ames test conditions for nitrosamines
• Study type:
  Not applicable
• Scope of the study:
  Method development or testing
• If ‘Not applicable’, further details on the study type:
  In-vitro mutagenicity study

Non-interventional study

No information provided.

Study drug and medical condition

No information provided.

Population studied

No information provided.

Study design details

• Study design:
  N-Nitrosamines (NAs) are classified based on their core structure as Cohort of Concern (CoC) compounds in the ICH M7 guideline. NAs represent a class of highly potent mutagenic carcinogens that requires strict controls to limit their amounts. Risk assessment of new NAs or NAs lacking robust in vitro
• Main study objective:
  Objective 1 will focus on the selection of NDSRI positive controls for the EAT and the evaluation of assay performance and sensitivity. ; The selected NDSRI substances will be tested with 3 batches of hamster S9 at 5 concentrations and in compliance with the regulatory approach according to TG OECD471, five bacterial strains (TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA) will be included in the test design. ; Phenotyping of NDSRI activation will be achieved by employing high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), to follow the formation of drug and NDSRI metabolites. CYP dependent metabolism will be studied by incubations which S9 fraction, using CYP specific inhibitors and with recombinant human bactosomes®.; Objective 2 will evaluate the effect of solvent concentrations on the NDSRI activity on the Ames test comparing the preincubation protocol and the plate incorporation protocol.; Benchmark dose (BMD) modelling will be utilized for sensitivity analyses and to compare different study conditions.; This project finally aims to define a sensitive Ames test design for NDSRIs that will lower the risk for false negative outcomes.;
• Summary results:
  Key Findings:; NDSRI metabolic activation is CYP450-dependent but compound-specific. Several human CYP isoforms are involved, including CYP1A1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. N-nitroso-varenicline was not proficiently metabolised by the tested enzymes, indicating that some NDSRIs may require other metabolic pathways.; The three tester strains TA1535, TA100 and E. coli WP2 uvrA were the most broadly responsive strains for detecting NDSRI mutagenicity. TA98 and TA1537 may add value for certain NDSRIs and can help interpret equivocal results.; DMSO concentration influenced both NDSRI metabolism and assay sensitivity; solvent levels therefore need to be carefully controlled and standardised.; The comparison of induced vs. non-induced hamster S9, and of plate incorporation vs. preincubation, provided the basis for defining the recommended EAT conditions.; Strain-specific NDSRI positive controls are proposed, based on reproducible positive responses and adequate fold changes under hamster S9 activating conditions.;