E-Thesis 111 views 15 downloads
Validation of the hprt Mutagenicity Assay for Nitrosamine Assessment / ABBIE WILLIAMS
Swansea University Author: ABBIE WILLIAMS
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...
| 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 |
| 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 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. |
|---|---|
| Keywords: |
Nitrosamines, Genetic Toxicology, Mutation |
| College: |
Faculty of Medicine, Health and Life Sciences |
| Funders: |
GSK |