5-Fluorouracil, colchicine, benzo[a]pyrene and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster V79 cells at Covance Laboratories, Harrogate, UK in support of OECD draft Test Guideline 487

Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, D.J.; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

doi:10.1016/j.mrgentox.2010.04.022

Cited by 3 publications

(1 citation statement)

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“…There are several important caveats worth noting regarding these results. First, 5FU has been represented as a weak/difficult clastogen to detect with in vitro MN assays [Lorge, ; Whitwell et al, ]. Second, recommendations for studying nucleoside analogs generally indicate the need for alternate treatment/harvest schedules owing to their severe effect on the cell cycle [Kirsch‐Volders et al, ].…”

Section: Resultsmentioning

confidence: 99%

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents

Bryce

Bemis

Mereness

et al. 2014

Environ and Mol Mutagen

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The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results.

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Section: Resultsmentioning

confidence: 99%

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents

Bryce

Bemis

Mereness

et al. 2014

Environ and Mol Mutagen

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Micronucleus Experiments with Non-human Mammalian Cells

Thybaud

2019

The Micronucleus Assay in Toxicology

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Hamster ovary (CHO) and lung (CHL and V79) cells and the mouse lymphoma cell line L5178Y are the non-human mammalian cells most frequently used for in vitro micronucleus experiments, and are those recommended in regulatory test guidelines (e.g. OECD test guideline 487). Because these cells are readily available and relatively easy to handle, they have been used for more than 25 years for the detection of both clastogens and aneugens. This chapter summarizes their main characteristics, the key steps of the in vitro micronucleus test when conducted with these non-human mammalian cells, the parameters and criteria required for a valid study, and the points to consider when interpreting the results.

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Flow cytometric micronucleus assay and TGx-DDI transcriptomic biomarker analysis of ten genotoxic and non-genotoxic chemicals in human HepaRG™ cells

et al. 2020

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Background Modern testing paradigms seek to apply human-relevant cell culture models and integrate data from multiple test systems to accurately inform potential hazards and modes of action for chemical toxicology. In genetic toxicology, the use of metabolically competent human hepatocyte cell culture models provides clear advantages over other more commonly used cell lines that require the use of external metabolic activation systems, such as rat liver S9. HepaRG™ cells are metabolically competent cells that express Phase I and II metabolic enzymes and differentiate into mature hepatocyte-like cells, making them ideal for toxicity testing. We assessed the performance of the flow cytometry in vitro micronucleus (MN) test and the TGx-DDI transcriptomic biomarker to detect DNA damage-inducing (DDI) chemicals in human HepaRG™ cells after a 3-day repeat exposure. The biomarker, developed for use in human TK6 cells, is a panel of 64 genes that accurately classifies chemicals as DDI or non-DDI. Herein, the TGx-DDI biomarker was analyzed by Ion AmpliSeq whole transcriptome sequencing to assess its classification accuracy using this more modern gene expression technology as a secondary objective. Methods HepaRG™ cells were exposed to increasing concentrations of 10 test chemicals (six genotoxic chemicals, including one aneugen, and four non-genotoxic chemicals). Cytotoxicity and genotoxicity were measured using the In Vitro MicroFlow® kit, which was run in parallel with the TGx-DDI biomarker. Results A concentration-related decrease in relative survival and a concomitant increase in MN frequency were observed for genotoxic chemicals in HepaRG™ cells. All five DDI and five non-DDI agents were correctly classified (as genotoxic/non-genotoxic and DDI/non-DDI) by pairing the test methods. The aneugenic agent (colchicine) yielded the expected positive result in the MN test and negative (non-DDI) result by TGx-DDI. Conclusions This next generation genotoxicity testing strategy is aligned with the paradigm shift occurring in the field of genetic toxicology. It provides mechanistic insight in a human-relevant cell-model, paired with measurement of a conventional endpoint, to inform the potential for adverse health effects. This work provides support for combining these assays in an integrated test strategy for accurate, higher throughput genetic toxicology testing in this metabolically competent human progenitor cell line.

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5-Fluorouracil, colchicine, benzo[a]pyrene and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster V79 cells at Covance Laboratories, Harrogate, UK in support of OECD draft Test Guideline 487

Cited by 3 publications

References 5 publications

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents

Micronucleus Experiments with Non-human Mammalian Cells

Flow cytometric micronucleus assay and TGx-DDI transcriptomic biomarker analysis of ten genotoxic and non-genotoxic chemicals in human HepaRG™ cells

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