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Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis

Stephanie Simon Orcid Logo, Jörg Schlingemann Orcid Logo, George Johnson Orcid Logo, Christian Brenneis Orcid Logo, Brunhilde Guessregen Orcid Logo, Jakub Kostal Orcid Logo, Jessica Dieckhoff Orcid Logo

Archives of Toxicology, Volume: 99, Issue: 10, Pages: 3935 - 3962

Swansea University Author: George Johnson Orcid Logo

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DOI (Published version): 10.1007/s00204-025-04103-2

Abstract

N-nitroso-bisoprolol (NBP) is a nitrosamine drug substance-related impurity (NDSRI) of bisoprolol, which is used to treat cardiac diseases since decades. To investigate the mutagenic potential of NBP, in vitro methods such as Enhanced Ames Test (EAT) and a mammalian cell gene mutation (HPRT) assay w...

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Published in: Archives of Toxicology
ISSN: 0340-5761 1432-0738
Published: Springer Nature 2025
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URI: https://cronfa.swan.ac.uk/Record/cronfa69760
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To assess the in vivo mutagenicity, a 28-day repeat-dose study was conducted in wild-type NMRI mice, and liver and bone marrow samples were subjected to error-corrected next-generation sequencing (i.e., duplex sequencing) followed by benchmark dose analysis (BMD). NBP did not show mutagenic effects in Ames tests using 10 % and 30 % induced rat or 30 % uninduced hamster S9. However, relevant increases in mutation frequencies were observed in an EAT in the presence of 30 % induced hamster S9 in strains TA100 and TA1535, confirming that the most stringent conditions of the EAT are appropriate to detect the mutagenic activity of weak mutagens, such as NBP. In the HPRT assay conducted in V79 cells, nitroso-diethylamine (NDEA) relevantly induced the mutation frequency, but not NBP. The highly sensitive error-corrected Next-Generation Sequencing (ecNGS) method to detect mutations across the genome represents an appropriate in vivo mutagenicity investigation equally suitable as a TGR assay to assess the mutagenic potential of nitrosamines. A weak induction of mutation frequencies was detected by ecNGS in the liver and the bone marrow of mice. Using BMD analysis, new safe limits were calculated for NBP, which are higher than the published AI of 1.5 &#xB5;g/person/day. Using the approach to calculate Permissible Daily Exposure (PDE) limits according to ICH Q3C, a lifetime PDE of 400 &#xB5;g/person/day was derived. Based on the ICH M7 framework for derivation of Acceptable Intake (AI) limits, an AI of 64 &#xB5;g/person/day was established. Consistent with regulatory emphasis on mechanistic interpretation, in vivo modeling was further supported by in silico calculations. 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spelling 2025-11-07T14:35:19.4137016 v2 69760 2025-06-18 Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis 37d0f121db69fd09f364df89e4405e31 0000-0001-5643-9942 George Johnson George Johnson true false 2025-06-18 MEDS N-nitroso-bisoprolol (NBP) is a nitrosamine drug substance-related impurity (NDSRI) of bisoprolol, which is used to treat cardiac diseases since decades. To investigate the mutagenic potential of NBP, in vitro methods such as Enhanced Ames Test (EAT) and a mammalian cell gene mutation (HPRT) assay were used. To assess the in vivo mutagenicity, a 28-day repeat-dose study was conducted in wild-type NMRI mice, and liver and bone marrow samples were subjected to error-corrected next-generation sequencing (i.e., duplex sequencing) followed by benchmark dose analysis (BMD). NBP did not show mutagenic effects in Ames tests using 10 % and 30 % induced rat or 30 % uninduced hamster S9. However, relevant increases in mutation frequencies were observed in an EAT in the presence of 30 % induced hamster S9 in strains TA100 and TA1535, confirming that the most stringent conditions of the EAT are appropriate to detect the mutagenic activity of weak mutagens, such as NBP. In the HPRT assay conducted in V79 cells, nitroso-diethylamine (NDEA) relevantly induced the mutation frequency, but not NBP. The highly sensitive error-corrected Next-Generation Sequencing (ecNGS) method to detect mutations across the genome represents an appropriate in vivo mutagenicity investigation equally suitable as a TGR assay to assess the mutagenic potential of nitrosamines. A weak induction of mutation frequencies was detected by ecNGS in the liver and the bone marrow of mice. Using BMD analysis, new safe limits were calculated for NBP, which are higher than the published AI of 1.5 µg/person/day. Using the approach to calculate Permissible Daily Exposure (PDE) limits according to ICH Q3C, a lifetime PDE of 400 µg/person/day was derived. Based on the ICH M7 framework for derivation of Acceptable Intake (AI) limits, an AI of 64 µg/person/day was established. Consistent with regulatory emphasis on mechanistic interpretation, in vivo modeling was further supported by in silico calculations. Specifically, the validated Computer-Aided Discovery and RE-design (CADRE) tool was used to predict the potency of NBP and further differentiate its metabolic activity from the anchor nitrosamine NDEA via quantum mechanics (QM) calculations and CYP-binding predictions. Outcomes of this analysis were consistent with in vivo studies, while offering a deeper understanding of the fundamental biochemistry using a physics-led method. The integrated in vivo–in silico investigation provides a data-based determination of safe limits, suggesting that the AI based on structural considerations solely might be over-conservative and should not be capped at the TTC. Journal Article Archives of Toxicology 99 10 3935 3962 Springer Nature 0340-5761 1432-0738 Nitroso-bisoprolol; Beta-blocker; Nitrosamines; NDSRI; Mutagenicity; ecNGS; Duplex sequencing; Benchmark dose analysis (BMD); Acceptable intake; AI; Permissible (permitted) daily exposure; PDE; In-silico modeling; Quantum mechanics; CYP docking; Predictive toxicology 1 10 2025 2025-10-01 10.1007/s00204-025-04103-2 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee 2025-11-07T14:35:19.4137016 2025-06-18T10:08:11.9961568 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Stephanie Simon 0000-0002-2497-5211 1 Jörg Schlingemann 0000-0003-4799-1086 2 George Johnson 0000-0001-5643-9942 3 Christian Brenneis 0000-0003-1814-9535 4 Brunhilde Guessregen 0000-0003-3248-6247 5 Jakub Kostal 0000-0001-9727-0477 6 Jessica Dieckhoff 0009-0009-1908-5351 7 69760__34510__d09689a42da04a3f85cd56115e4ddefc.pdf 69760.VOR.pdf 2025-06-18T12:44:41.8900200 Output 2294751 application/pdf Version of Record true © The Author(s) 2025. This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). true eng http://creativecommons.org/licenses/by-nc-nd/4.0/
title Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
spellingShingle Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
George Johnson
title_short Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
title_full Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
title_fullStr Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
title_full_unstemmed Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
title_sort Deriving safe limits for N-nitroso-bisoprolol by error-corrected next-generation sequencing (ecNGS) and benchmark dose (BMD) analysis, integrated with QM modeling and CYP-docking analysis
author_id_str_mv 37d0f121db69fd09f364df89e4405e31
author_id_fullname_str_mv 37d0f121db69fd09f364df89e4405e31_***_George Johnson
author George Johnson
author2 Stephanie Simon
Jörg Schlingemann
George Johnson
Christian Brenneis
Brunhilde Guessregen
Jakub Kostal
Jessica Dieckhoff
format Journal article
container_title Archives of Toxicology
container_volume 99
container_issue 10
container_start_page 3935
publishDate 2025
institution Swansea University
issn 0340-5761
1432-0738
doi_str_mv 10.1007/s00204-025-04103-2
publisher Springer Nature
college_str Faculty of Medicine, Health and Life Sciences
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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
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description N-nitroso-bisoprolol (NBP) is a nitrosamine drug substance-related impurity (NDSRI) of bisoprolol, which is used to treat cardiac diseases since decades. To investigate the mutagenic potential of NBP, in vitro methods such as Enhanced Ames Test (EAT) and a mammalian cell gene mutation (HPRT) assay were used. To assess the in vivo mutagenicity, a 28-day repeat-dose study was conducted in wild-type NMRI mice, and liver and bone marrow samples were subjected to error-corrected next-generation sequencing (i.e., duplex sequencing) followed by benchmark dose analysis (BMD). NBP did not show mutagenic effects in Ames tests using 10 % and 30 % induced rat or 30 % uninduced hamster S9. However, relevant increases in mutation frequencies were observed in an EAT in the presence of 30 % induced hamster S9 in strains TA100 and TA1535, confirming that the most stringent conditions of the EAT are appropriate to detect the mutagenic activity of weak mutagens, such as NBP. In the HPRT assay conducted in V79 cells, nitroso-diethylamine (NDEA) relevantly induced the mutation frequency, but not NBP. The highly sensitive error-corrected Next-Generation Sequencing (ecNGS) method to detect mutations across the genome represents an appropriate in vivo mutagenicity investigation equally suitable as a TGR assay to assess the mutagenic potential of nitrosamines. A weak induction of mutation frequencies was detected by ecNGS in the liver and the bone marrow of mice. Using BMD analysis, new safe limits were calculated for NBP, which are higher than the published AI of 1.5 µg/person/day. Using the approach to calculate Permissible Daily Exposure (PDE) limits according to ICH Q3C, a lifetime PDE of 400 µg/person/day was derived. Based on the ICH M7 framework for derivation of Acceptable Intake (AI) limits, an AI of 64 µg/person/day was established. Consistent with regulatory emphasis on mechanistic interpretation, in vivo modeling was further supported by in silico calculations. Specifically, the validated Computer-Aided Discovery and RE-design (CADRE) tool was used to predict the potency of NBP and further differentiate its metabolic activity from the anchor nitrosamine NDEA via quantum mechanics (QM) calculations and CYP-binding predictions. Outcomes of this analysis were consistent with in vivo studies, while offering a deeper understanding of the fundamental biochemistry using a physics-led method. The integrated in vivo–in silico investigation provides a data-based determination of safe limits, suggesting that the AI based on structural considerations solely might be over-conservative and should not be capped at the TTC.
published_date 2025-10-01T05:29:01Z
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