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The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines

M A Djuari Orcid Logo, A Londenberg Orcid Logo, A Bassan Orcid Logo, K Cross Orcid Logo, L Elenschneider Orcid Logo, S E Escher Orcid Logo, J Fahrer Orcid Logo, J Fangmann Orcid Logo, C Felske Orcid Logo, R Frötschl Orcid Logo, B Haas Orcid Logo, George Johnson Orcid Logo, M Vogel Orcid Logo, R Whomsley Orcid Logo, C Ziemann Orcid Logo

Archives of Toxicology

Swansea University Author: George Johnson Orcid Logo

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DOI (Published version): 10.1007/s00204-026-04457-1

Abstract

N-nitrosamine (NA) impurities in pharmaceuticals represent "cohort of concern" compounds under ICH M7(R2), due to their mutagenic/carcinogenic potential, involving cytochrome P450 (CYP)-mediated metabolic activation. Increasing interest in mammalian cell-based genotoxicity/mutagenicity ass...

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Published in: Archives of Toxicology
ISSN: 0340-5761 1432-0738
Published: Springer Nature 2026
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URI: https://cronfa.swan.ac.uk/Record/cronfa72078
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Increasing interest in mammalian cell-based genotoxicity/mutagenicity assays prompted our assessment of the in vitro alkaline comet assay regarding its predictive power for NAs. Here, precision-cut liver slices (PCLiS), primary human hepatocytes&#xA0;(PHH), primary rat hepatocytes (PRH), and HepG2 cells with rat or hamster S9-mix were investigated as in vitro model systems. Metabolic competence was characterized beforehand. For performance evaluation, a panel of known-mutagenic [N-nitroso-dimethylamine (NDMA), N-nitroso-diethanolamine, N-nitroso-methylaniline, S-N-nitroso-nornicotine, N-methyl-N-nitroso-2-propanamine]&#xA0;and reported non-mutagenic (methyl-t-butylnitrosamine, N-nitrosoproline) was tested, together with Nitrosamine Drug Substance-Related Impurities [N-nitrosodesloratadine, N-nitrosofolic acid, N-nitrosofluoxetine (NFluo)] at a concentration range of 0.005-10&#xA0;mM. After 2&#xA0;h (PCLiS, PHH and PRH) or 4&#xA0;h (HepG2), NDMA concentration-dependently induced DNA strand breaks in all in vitro models. Sensitivity/specificity of the various liver cell models for prediction of carcinogenic NAs were 100%/50% (HepG2 with hamster S9-mix), 50%/100% (PHH, PRH), and 50%/50% (HepG2 with rat S9-mix), respectively. Benchmark dose modeling indicated a higher relative in vitro comet assay response for NFluo compared to NDMA in all cell systems. In conclusion, the in vitro comet assay represents a sensitive and/or specific tool for complementing regulatory in vitro tests in prediction of mutagenic NAs. 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spelling 2026-06-15T11:54:48.2428793 v2 72078 2026-06-15 The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines 37d0f121db69fd09f364df89e4405e31 0000-0001-5643-9942 George Johnson George Johnson true false 2026-06-15 MEDS N-nitrosamine (NA) impurities in pharmaceuticals represent "cohort of concern" compounds under ICH M7(R2), due to their mutagenic/carcinogenic potential, involving cytochrome P450 (CYP)-mediated metabolic activation. Increasing interest in mammalian cell-based genotoxicity/mutagenicity assays prompted our assessment of the in vitro alkaline comet assay regarding its predictive power for NAs. Here, precision-cut liver slices (PCLiS), primary human hepatocytes (PHH), primary rat hepatocytes (PRH), and HepG2 cells with rat or hamster S9-mix were investigated as in vitro model systems. Metabolic competence was characterized beforehand. For performance evaluation, a panel of known-mutagenic [N-nitroso-dimethylamine (NDMA), N-nitroso-diethanolamine, N-nitroso-methylaniline, S-N-nitroso-nornicotine, N-methyl-N-nitroso-2-propanamine] and reported non-mutagenic (methyl-t-butylnitrosamine, N-nitrosoproline) was tested, together with Nitrosamine Drug Substance-Related Impurities [N-nitrosodesloratadine, N-nitrosofolic acid, N-nitrosofluoxetine (NFluo)] at a concentration range of 0.005-10 mM. After 2 h (PCLiS, PHH and PRH) or 4 h (HepG2), NDMA concentration-dependently induced DNA strand breaks in all in vitro models. Sensitivity/specificity of the various liver cell models for prediction of carcinogenic NAs were 100%/50% (HepG2 with hamster S9-mix), 50%/100% (PHH, PRH), and 50%/50% (HepG2 with rat S9-mix), respectively. Benchmark dose modeling indicated a higher relative in vitro comet assay response for NFluo compared to NDMA in all cell systems. In conclusion, the in vitro comet assay represents a sensitive and/or specific tool for complementing regulatory in vitro tests in prediction of mutagenic NAs. However, further optimization work is needed, using expanded training sets of compounds and thorough validation of liver cell models, before the in vitro comet assay could be incorporated in the standard battery for genotoxicity testing. Journal Article Archives of Toxicology 0 Springer Nature 0340-5761 1432-0738 Alkaline comet assay; In vitro; Liver; N-nitrosamines; Benchmark dose modeling 2 6 2026 2026-06-02 10.1007/s00204-026-04457-1 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee Open Access funding enabled and organized by Projekt DEAL. This study was funded by the European Medicines Agency (EMA) under the framework contract EMA/2020/46/L1.02. 2026-06-15T11:54:48.2428793 2026-06-15T11:43:29.2692105 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science M A Djuari 0009-0004-4493-1031 1 A Londenberg 0009-0007-8268-8601 2 A Bassan 0000-0003-1748-3144 3 K Cross 0000-0002-2462-0533 4 L Elenschneider 0000-0003-4714-2156 5 S E Escher 0000-0001-9616-527X 6 J Fahrer 0000-0001-5225-2718 7 J Fangmann 0000-0002-0108-1346 8 C Felske 0009-0008-8806-7387 9 R Frötschl 0000-0001-8721-7509 10 B Haas 0000-0002-1213-3527 11 George Johnson 0000-0001-5643-9942 12 M Vogel 0000-0003-4814-3900 13 R Whomsley 0000-0001-6108-9908 14 C Ziemann 0000-0002-3238-5385 15 72078__36967__7f1b0ed62dcb4e4aaee2ab89d55f517f.pdf 72078.VOR.pdf 2026-06-15T11:48:06.8090739 Output 2745462 application/pdf Version of Record true © The Author(s) 2026. This article is licensed under a Creative Commons Attribution 4.0 International License. true eng http://creativecommons.org/licenses/by/4.0/
title The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
spellingShingle The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
George Johnson
title_short The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
title_full The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
title_fullStr The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
title_full_unstemmed The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
title_sort The in vitro alkaline comet assay with liver models as a complementary tool for genotoxicity assessment of N-nitrosamines
author_id_str_mv 37d0f121db69fd09f364df89e4405e31
author_id_fullname_str_mv 37d0f121db69fd09f364df89e4405e31_***_George Johnson
author George Johnson
author2 M A Djuari
A Londenberg
A Bassan
K Cross
L Elenschneider
S E Escher
J Fahrer
J Fangmann
C Felske
R Frötschl
B Haas
George Johnson
M Vogel
R Whomsley
C Ziemann
format Journal article
container_title Archives of Toxicology
container_volume 0
publishDate 2026
institution Swansea University
issn 0340-5761
1432-0738
doi_str_mv 10.1007/s00204-026-04457-1
publisher Springer Nature
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
active_str 0
description N-nitrosamine (NA) impurities in pharmaceuticals represent "cohort of concern" compounds under ICH M7(R2), due to their mutagenic/carcinogenic potential, involving cytochrome P450 (CYP)-mediated metabolic activation. Increasing interest in mammalian cell-based genotoxicity/mutagenicity assays prompted our assessment of the in vitro alkaline comet assay regarding its predictive power for NAs. Here, precision-cut liver slices (PCLiS), primary human hepatocytes (PHH), primary rat hepatocytes (PRH), and HepG2 cells with rat or hamster S9-mix were investigated as in vitro model systems. Metabolic competence was characterized beforehand. For performance evaluation, a panel of known-mutagenic [N-nitroso-dimethylamine (NDMA), N-nitroso-diethanolamine, N-nitroso-methylaniline, S-N-nitroso-nornicotine, N-methyl-N-nitroso-2-propanamine] and reported non-mutagenic (methyl-t-butylnitrosamine, N-nitrosoproline) was tested, together with Nitrosamine Drug Substance-Related Impurities [N-nitrosodesloratadine, N-nitrosofolic acid, N-nitrosofluoxetine (NFluo)] at a concentration range of 0.005-10 mM. After 2 h (PCLiS, PHH and PRH) or 4 h (HepG2), NDMA concentration-dependently induced DNA strand breaks in all in vitro models. Sensitivity/specificity of the various liver cell models for prediction of carcinogenic NAs were 100%/50% (HepG2 with hamster S9-mix), 50%/100% (PHH, PRH), and 50%/50% (HepG2 with rat S9-mix), respectively. Benchmark dose modeling indicated a higher relative in vitro comet assay response for NFluo compared to NDMA in all cell systems. In conclusion, the in vitro comet assay represents a sensitive and/or specific tool for complementing regulatory in vitro tests in prediction of mutagenic NAs. However, further optimization work is needed, using expanded training sets of compounds and thorough validation of liver cell models, before the in vitro comet assay could be incorporated in the standard battery for genotoxicity testing.
published_date 2026-06-02T06:03:02Z
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