HESI GTTC ring trial: Concordance between Ames and rodent carcinogenicity outcomes for N-nitrosamines (NAs) with rat and hamster metabolic conditions
Joel P. Bercu, Alejandra Trejo‐Martin, Connie L. Chen, Maik Schuler, Jennifer Cheung, Tetyana Cheairs, Anthony M. Lynch, D. Thomas, Andreas Czich, Aisar Atrakchi, Timothy J. McGovern, Robert H. Heflich, Alisa Vespa, Roland Froetschl, Yi Yan Yang, Raj D. Gandhi, Joanne Elloway, Verena Ziegler, Anna Hellmann, Michelle Schaefer, Rachael E. Tennant, Walter M.A. Westerink, Roy Hoffmans, Robert A. Jolly, Jessica Noteboom, Pavan Gollapudi, Zhanna Sobol, Katerine K McGettigan, Joel S Christensen, Stéphanie Simon, J Dieckhoff, Andreas Zeller, C Marchand, Kerstin Waese, Michelle E. Bishop, Penny Leavitt, Victoria V. Hargreaves, Connor Glick, Yang Liao, Rosalie K. Elespuru, Raechel Puglisi
Abstract
A multi-sector study (i.e., Ring Trial) was designed to improve the in vitro detection of N -nitrosamine (NA)-associated mutagenicity by optimizing the bacterial reverse mutation (i.e., Ames) assay protocol and testing various conditions on the sensitivity and specificity for the prediction of rodent carcinogenicity. A total of 29 NAs and 3 N -nitroso drug-like compounds from different structural classes and carcinogenicity outcomes were tested (two independent laboratories per compound) across 5 bacterial strains using a 30-min pre-incubation protocol. To evaluate the impact of different metabolic activating systems (MASs), testing conditions included the use of 10 or 30 % liver S9 fractions prepared from rats or hamsters pretreated with inducers of enzymatic activity. Results indicate that E. coli and Salmonella typhimurium strains detecting single base pair mutations, coupled with MASs containing 30 % hamster S9s were the most sensitive (90 %) for identifying NAs that are rodent carcinogens. Regarding MAS combinations, the highest sensitivity was 30 % rat and 30 % hamster (93 %), but has low specificity (45 %), with good laboratory agreement for the Ames calls (91 %). DMSO and water were considered suitable solvents, except for small-molecular weight alkyl NAs. These results will support harmonized Ames testing of NAs, giving high confidence for a negative result. • 30 % hamster S9 had the highest sensitivity of all individual MASs. • 30 % S9 (rat or hamster) had a higher sensitivity than 10 % S9 (rat or hamster). • -S9 and 30 % rat and hamster S9 had high sensitivity (93 %) but low specificity (45 %). • Specificity was similar amongst S9 species or concentration. • DMSO and water were suitable solvents for NA Ames testing.