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Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment / ABBIE WILLIAMS

Swansea University Author: ABBIE WILLIAMS

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Abstract

Nitrosamines are a chemical class considered to have mutagenic and carcinogenic potential, primarily exerting their mechanism through DNA alkylation. Under new guidance from the Food and Drug Administration (FDA), it is recommended that nitrosamine impurities are tested in both the enhanced Ames tes...

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Published: Swansea 2026
Institution: Swansea University
Degree level: Master of Research
Degree name: MSc by Research
Supervisor: Johnson, George
URI: https://cronfa.swan.ac.uk/Record/cronfa71911
first_indexed 2026-05-15T10:06:49Z
last_indexed 2026-05-16T05:23:14Z
id cronfa71911
recordtype RisThesis
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spelling 2026-05-15T11:17:05.2055600 v2 71911 2026-05-15 Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment b6dd9e27245d7a6df3a9015a53b8dd0b ABBIE WILLIAMS ABBIE WILLIAMS true false 2026-05-15 Nitrosamines are a chemical class considered to have mutagenic and carcinogenic potential, primarily exerting their mechanism through DNA alkylation. Under new guidance from the Food and Drug Administration (FDA), it is recommended that nitrosamine impurities are tested in both the enhanced Ames test as well as a second in vitro mammalian-cell mutation assay in order to support an acceptable intake of 1500 nanograms/day. Here, we report set-up and validation of the hypoxanthine-guanine phosphoribosyl transferase (hprt) mutagenicity assay in-house using L5178Y cells to establish its application domain within the pharmaceutical industry to support robust nitrosamine mutagenicity assessments. Four Ames-positive nitrosamines were evaluated: N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA) and 1-cyclopentyl-4-nitrosopiperazine (CPNP). The results demonstrated concordance with parallel mouse lymphoma assay (MLA) data for three of the four compounds, with two showing positive results (NDMA/NDEA) and one yielding a negative outcome (NDIPA). It was noted that the Lowest Observed Genotoxic Effect Levels (LOGELs) for the positive nitrosamines were significantly higher than those observed in the MLA, suggesting differences in the sensitivity of the two assays. Due to significant inter-replicate variability, a statistically significant increase in mutation frequency for CPNP was not detected using the hprt assay whereas CPNP was positive by MLA. This project identified hprt assay sensitivity, response data variability and experiment duration as potential drawbacks for effective nitrosamine hazard and risk assessment in the industry setting. However, the project also identified opportunities to address these problems through proof-of-concept development of an automated, image-based, machine learning scoring approach and employment of combined benchmark dose (BMD) modelling to leverage all generated data during point-of-departure estimation. E-Thesis Swansea Nitrosamines, Genetic Toxicology, Mutation 28 4 2026 2026-04-28 COLLEGE NANME COLLEGE CODE Swansea University Johnson, George Master of Research MSc by Research GSK GSK 2026-05-15T11:17:05.2055600 2026-05-15T11:03:22.4938608 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science ABBIE WILLIAMS 1 71911__36740__2eef47cb05fb4ae0bc651550e0768ce8.pdf Williams_Abbie_MSc_Research_Thesis_Final_Cronfa.pdf 2026-05-15T11:10:26.7769825 Output 2221092 application/pdf E-Thesis – open access true Copyright: The Author, Abbie Williams, 2026. true eng
title Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
spellingShingle Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
ABBIE WILLIAMS
title_short Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
title_full Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
title_fullStr Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
title_full_unstemmed Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
title_sort Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment
author_id_str_mv b6dd9e27245d7a6df3a9015a53b8dd0b
author_id_fullname_str_mv b6dd9e27245d7a6df3a9015a53b8dd0b_***_ABBIE WILLIAMS
author ABBIE WILLIAMS
author2 ABBIE WILLIAMS
format E-Thesis
publishDate 2026
institution Swansea University
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 Nitrosamines are a chemical class considered to have mutagenic and carcinogenic potential, primarily exerting their mechanism through DNA alkylation. Under new guidance from the Food and Drug Administration (FDA), it is recommended that nitrosamine impurities are tested in both the enhanced Ames test as well as a second in vitro mammalian-cell mutation assay in order to support an acceptable intake of 1500 nanograms/day. Here, we report set-up and validation of the hypoxanthine-guanine phosphoribosyl transferase (hprt) mutagenicity assay in-house using L5178Y cells to establish its application domain within the pharmaceutical industry to support robust nitrosamine mutagenicity assessments. Four Ames-positive nitrosamines were evaluated: N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA) and 1-cyclopentyl-4-nitrosopiperazine (CPNP). The results demonstrated concordance with parallel mouse lymphoma assay (MLA) data for three of the four compounds, with two showing positive results (NDMA/NDEA) and one yielding a negative outcome (NDIPA). It was noted that the Lowest Observed Genotoxic Effect Levels (LOGELs) for the positive nitrosamines were significantly higher than those observed in the MLA, suggesting differences in the sensitivity of the two assays. Due to significant inter-replicate variability, a statistically significant increase in mutation frequency for CPNP was not detected using the hprt assay whereas CPNP was positive by MLA. This project identified hprt assay sensitivity, response data variability and experiment duration as potential drawbacks for effective nitrosamine hazard and risk assessment in the industry setting. However, the project also identified opportunities to address these problems through proof-of-concept development of an automated, image-based, machine learning scoring approach and employment of combined benchmark dose (BMD) modelling to leverage all generated data during point-of-departure estimation.
published_date 2026-04-28T06:25:30Z
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score 11.106612

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