Nitrosamines Research Program

The Nitrosamines research activities exist under the umbrella of the HESI GTTC Mechanism-based Genotoxicity Risk Assessment (MGRA) working group as a “case study” to demonstrate the usefulness of innovative, mechanism-based testing approaches.

GTTC: The mission of the Genetic Toxicology Technical Committee (GTTC) is to improve the scientific basis of the interpretation of results from genetic toxicology tests for purposes of more accurate hazard identification and assessment of human risk; to develop follow-up strategies for determining the relevance of test results to human health; to provide a framework for integration of testing results into a risk-based assessment of the effects of chemical exposures on human health; to promote the integration and use of new techniques and scientific knowledge in the evaluation of genetic toxicology; and to monitor and promote the development of innovative tests and testing strategies.

MGRA working group: The Mechanism-based Genotoxicity Risk Assessment (MGRA) working group aimed to develop a new mechanism-based risk assessment paradigm for genotoxicity that uses a genetic toxicology testing strategy designed from a “clean slate”, incorporating new science and technology to allow for a greater diversity of genomic damage to be addressed, and uses genotoxicity Adverse Outcome Pathways (AOP)s.

Subteams & Goals

  • Ames Optimization Working Group (Goal 1)

    Goal: Develop an optimized Ames protocol that is predictive for the carcinogenic potential of nitrosamines (NAs).

    Phase I Highlights:

    • Conducted a ring-trial on 32 nitrosamines across 16 laboratories under FDA grant NoA: 3U01FD006676-03S2
    • Collected Ames test data to assess sensitivity for nitrosamine hazard detection
    • Evaluated specificity for predicting the carcinogenicity of known nitrosamines
    • Performed benchmark dose (BMD) modeling to support potency ranking and optimization
    • Published findings in Regulatory Toxicology and Pharmacology: https://doi.org/10.1016/j.yrtph.2025.105835
    • Characterized rodent S9 CYP enzymes in collaboration with MOLTOX
    • Focused on CYP enzymes 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4
    • Generated kinetic enzyme assay data with concentration-dependent metabolism curves
    • Published metabolic data in Mutation Research/Genetic Toxicology: https://doi.org/10.1016/j.mrgentox.2025.503855

    Phase II Initiatives:

    • Refining the Ames protocol using nitrosamine-specific positive controls (PCs)
    • Project leads include Alejandra Trejo-Martin (Gilead) and Rhys Whomsley (EMA)
    • Identifying appropriate nitrosamine-related positive controls
    • Conducting interlaboratory testing with at least three labs per control
    • Aiming to standardize the protocol through a defined list of positive controls
    • Preparing for publication of findings from positive control studies
    • Initiating strain condition analysis to evaluate strain sensitivity
    • Assessing the impact of assay conditions on mutagenic outcomes
    • Applying spider plots and BMD modeling to inform strain selection and refine the protocol
    • Preparing for publication of strain condition findings

    Working Group Leadership:
    Anthony Lynch (GSK)
    Joel Bercu (Gilead)

  • In Vitro Test Strategy Working Group (Goal 2)

    Goal: Identification and verification of in vitro assays with alternative cell models supporting nitrosamines risk identification.

    • Identifying alternative in vitro approaches to the Ames test for detecting nitrosamines
    • Identifying in vitro assays that can serve as follow-up tools to the Ames test

    Sub-Group 1: HepaRG Protocol Development & Experimental Testing

    • 2D HepaRG protocol drafted and proof-of-concept (POC) testing completed
    • Experimental design includes 5–7 nitrosamines tested across 6–8 concentrations
    • Key endpoints measured include micronucleus (MN), comet assay, MultiFlow, and γH2AX
    • Focused on evaluating assay sensitivity

    Sub-Group 2: SWOT / Landscape Analysis

    • Conducting a comprehensive landscape analysis of alternative cell lines and systems for nitrosamine hazard detection
    • Building an evidence base to support future assay phases and refine in vitro testing strategies for nitrosamines
    • Focusing on models with improved metabolic competence, DNA damage sensitivity, and relevance to human exposure

    Working Group Leadership:
    Xiaowen Sun (Pfizer)
    Xiaoqing (Carol) Guo (US FDA/NCTR)

  • In Vivo Ames Follow-Up Working Group (Goal 3)

    Goal: Develop an in vivo strategy to verify Ames data within the framework of ICH M7.

    Phase I:

    Assessed in vitro/in vivo concordance for exemplar nitrosamines and NDSRIs
    Project Leads: Alejandra Trejo-Martin (Gilead), Bob Jolly (Eli Lilly)
    Anonymous data provided from drug manufacturers covering 33 nitrosamines
    Conducted dose-response testing of in vivo mutagenicity for 7 exemplar nitrosamines
    Outcomes:

    • Determined effectiveness of in vitro tests to predict in vivo outcomes
    • Generated in vivo BMDL values for comparison with carcinogenic BMDL10 values
    • Publication: Pending internal review before journal submission

    Completed literature review and analysis on less-than-lifetime (LTL) exposure relevance
    Project Lead: Susan Felter (P&G)
    Literature search to determine the applicability of LTL to NAs in general
    Eight nitrosamines met inclusion criteria for the review
    Outcomes:

    • Found stronger correlation between tumor incidence and Lifetime Cumulative Dose (LCD) versus dose rate or exposure duration
    • Supported using LCD as the preferred dose metric for cancer risk assessment
    • Publication: Approved with minor revisions (May 2025), Regulatory Toxicology & Pharmacology

    Phase II:

    Advancing testing of additional N-nitroso compounds, including ureas and carbamates
    Project Leads: Wen Sun (Pfizer) and TBD
    Goals:

    • Evaluate correlation between carcinogenicity data and in vivo Pig-a assay results
    • Develop a process for setting acceptable intakes (AIs)
    • Expand testing to include Comet assay and ecNGS as resources permit

    Carcinogenicity Potency Analysis
    Goal:

    • Analyze relationship between mutagenic potency (TGR data) and carcinogenic potency
    • Work to proceed under the HESI GTTC Quantitative Analysis Workgroup

    Less-than-lifetime (LTL) exposure literature follow-up
    Project Lead: Susan Felter (P&G)
    Goals:

    • Evaluate relevance of single, intermittent, and LTL exposures in nitrosamine risk assessment
    • Refine ICH M7 adjustment factors as needed

    Initiating LTL mutagenicity study design and experimental work
    Project Leads: Zhanna Sobel (Merck & Co., Inc.), Bob Jolly (Eli Lilly)
    Goal: Prospectively evaluate the effect of exposure duration on mutagenic outcomes

    Working Group Leadership:
    Joel Bercu (Gilead)
    Robert Jolly (Eli Lilly)
    Sheroy Minocherhomji (Eli Lilly)
    Timothy McGovern (White Oak Regulatory Tox, LLC)

  • (Q)SAR-QM Working Group (Goal 4)

    Goal: Refine and extend the CPCA using (Q)SAR and QM for more predictive and accurate predictions

    Development of (Q)SAR/SAR models sufficiently robust for prediction of NAs/NDSRI mutagenicity hazards.

    Refinement/enhancement of the CPCA with more structural features based on emerging data.

    Interrogate and validate existing CPCA features by (Q)SAR and QM.

    Provide a “confidence score”/metric per feature.

    • To be applied for internal use/prioritization
    • Determine whether the feature can be included /modified in the CPCA

    Four sub-groups:

    1)Validate electron donating and withdrawing groups in CPCA (decouple from sterics) and their effect on potency categorization | Project Lead: Kevin Cross (Instem)

    • Michael acceptor
    • Use of/applying EWG as activating/deactivating group
    • Using QM/probability schemes to model EWG/EDG effects

    2)Refine the beta-methyl feature in relation to the diazonium ion stability | Project Lead: Suman Chakravarti (MultiCASE)

    • Investigate effect of beta-alkyl on diazonium ion stability
    • Need for additional refinement (e.g., heteroatoms next to the beta carbon in a longer chain)
    • Investigate which transition stages are most important in diazonium ion stability

    3)Explore electronic and experimental data to support an amendment of the ring sizes in CPCA guidance | Project Lead: David Ponting (Lhasa)

    • 4-member rings, 8+ member rings, larger rings (macromolecules, rigid/floppy rings, large akyl rings)
    • N-piperidine
    • Confirming sterics, sampling

    4)Explore the role of different cytochrome P450 (CYP) metabolism on the activation of NA derivatives | Project Lead: Clarence Wybon (Perrigo)

    • Interrogate and validate new CPCA features by (Q)SAR and QM
    • Quantify the domain of applicability
    • Including ranges of phys/chem properties
    • New models to extend beyond the current domain

    Working Group Leadership:
    David Ponting (Lhasa)
    Jakub Kostal (George Washington University)
    Kevin Cross (Instem)

 

Participating Organizations

 

Government/Regulatory 
ANSM (France) BfArM (Germany) CBG-MEB (Netherlands) Danish Medicines Agency
EMA Health Canada INRA (France) NIHS (Japan)
RIVM (Netherlands) Swissmedic US FDA/CDER US FDA/NTR
Academia
Colorado State University George Washington University NY Medical College St. George’s Medical School, University of London
Swansea University University of Milan University of Minnesota
Industry
AbbVie AstraZeneca Bayer BASF
Boehringer Ingelheim Bristol Myers Squibb Charles River Laboratories Corteva
Eli Lilly Gentronix Gilead GSK
Haleon Inotiv Janssen Labcorp
Leadscope Litron Laboratories Merck kGA MultiCASE
Novartis Perrigo Pfizer Proctor & Gamble
Roche Sanofi Teva Pharmaceuticals Vividion
Xenometrix
Non-profit
American Chemistry Society Lhasa Limited US Pharmacopeia
Consulting
FSTox Consulting Nudelman ChemTox Consulting White Oak Regulatory Tox, LLC

 

HESI Staff

Leadership Team

  • Tetyana Cheairs, PhD

    New York Medical College

  • Joel Bercu, PhD

    Gilead

Committee Events

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Publications

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Evaluating the lifetime cumulative dose as a basis for carcinogenic potency of nitrosamines – a key tenet underpinning less-than-lifetime approaches for establishing acceptable intake limits

Regulatory Toxicology and Pharmacology, 2025

Felter et al.

HESI Global Nitrosamines Research Program published a paper on evaluating lifetime cumulative dose as a basis of carcinogenic potency specifically for nitrosamines (NAs). This is an important topic moving forward as we assess the less-than-lifetime acceptable intakes for NAs.

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A comparative analysis of select P450 enzymes in uninduced and PB/BNF-induced hamster and rat liver S9

Mutation Research – Genetic Toxicology and Environmental Mutagenesis, 2025

Evans et al.

As part of its Nitrosamine Research Program (NRP), HESI’s Genetic Toxicology Technical Committee (GTTC) has supported the publication of a new study evaluating the relative CYP enzyme activities in uninduced and induced hamster and rat liver S9 preparations. This research is critical for assessing the metabolic ...

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