Report from the in vitro micronucleus assay working group

Kirsch‐Volders, Micheline; Sofuni, Toshio; Aardema, Marilyn J.; Albertini, Silvio; Eastmond, David A.; Fenech, Michael; Ishidate, Morizo; Kirchner, Stephan; Lorge, Elisabeth; Morita, Takeshi; Norppa, Hannu; Surrallés, Jordi; Vanhauwaert, Annelies; Wakata, Akihiro

doi:10.1016/j.mrgentox.2003.07.005

Cited by 541 publications

(184 citation statements)

References 42 publications

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“…Stock solutions were prepared such that a consistent volume of solvent was added to each flask (from 100x solutions of SUC or MMC, prepared in water; or from 1000x solutions of DEX, VB, ETOPO, or STS, prepared in dimethyl sulfoxide). In all cases, cells were incubated at 37°C for 24 hrs (the length of time that is approximately 2.5 doubling times for untreated cells, as recommended by an expert working group [1]). …”

Section: Chemical Treatmentmentioning

confidence: 99%

“…Relative survival calculations were made based on the mean concurrent solvent control cell density. As suggested by an expert working group and also draft OECD guidances [1,6], concentrations that resulted in > 60% reduction to relative survival were excluded from MN assessment.…”

Section: Cytotoxicity Measurementsmentioning

confidence: 99%

“…The in vitro micronucleus test is widely used as a screening assay to assess cytogenetic damage in mammalian cells [1][2][3][4][5]. Its popularity is largely due to the fact that it can be executed with less technical expertise relative to chromosome aberration analyses, and because it is capable of detecting aneugens as well as clastogens.…”

Section: Introductionmentioning

confidence: 99%

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Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Section: Chemical Treatmentmentioning

confidence: 99%

Section: Cytotoxicity Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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show abstract

“…Therefore, it is often necessary to perform the assay both in the presence and absence of Cyt‐B, which increases the number of samples that must be evaluated. In recent years, several protocols and exposure schedules to perform the MN assay have been developed and validated in a number of recommended cell types 9, 10, 11, 12. Current forms of the assay are able to detect the presence of clastogens and aneugens in both rodent and human derived cell lines as well as in human peripheral blood lymphocytes 13, 14, 15, 16, 17.…”

Section: Introductionmentioning

confidence: 99%

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Rodrigues¹

2018

Cytometry Pt A

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The in vitro micronucleus (MN) assay is a well‐established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide‐scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide‐scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re‐evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono‐ and bi‐nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well‐known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

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“…Cytochalasin B (final concentration of 6 lg/mL) was added after 44 h of incubation in order to block cytokinesis and obtain binucleated cells. After an additional 24 h incubation at 37°C, cells were harvested by centrifugation and slides were prepared for MN test (Fenech 2000;Kirsch-Volders et al 2003).…”

Section: Test Samples and Chemicalsmentioning

confidence: 99%

The effects of 4-thujanol on chromosome aberrations, sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

Kocaman¹,

Rencüzoğulları²,

Topaktaş³

et al. 2011

Cytotechnology

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4-Thujanol, a bicyclic monoterpene alcohol, is present in the essential oils of many medicinal and aromatic plants. It is commonly used as a fragrance and flavouring ingredient in a lot of different products. The potential genotoxic effects of 4-thujanol on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests. The cells were treated with 13, 26 and 52 lg/mL 4-thujanol in the presence and absence of a metabolic activator (S9 mix). 4-Thujanol induced CA (P \ 0.001) and MN formation (P \ 0.05) at all concentrations (13, 26 and 52 lg/mL) in the presence and absence of the S9 mix without a concentration-dependent manner. However, the treatment of peripheral lymphocytes with 4-thujanol did not produce a statistical difference in the frequency of SCEs when compared with control group. Furthermore, this monoterpene did not significantly decrease the mitotic index (MI), proliferation index (PI), and nuclear division index (NDI). In conclusion, 4-thujanol had a significant clastogenic effect at the tested concentrations (13, 26 and 52 lg/mL) for human PBLs. In addition, no cytotoxic and/or cytostatic effects were observed regardless of the concentrations used. This work presents the first report on genotoxic properties of 4-thujanol.

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Report from the in vitro micronucleus assay working group

Cited by 541 publications

References 42 publications

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

The effects of 4-thujanol on chromosome aberrations, sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

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Report from the in vitro micronucleus assay working group

Cited by 541 publications

References 42 publications

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

The effects of 4-thujanol on chromosome aberrations, sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

Contact Info

Product

Resources

About

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer