The utility of the in vitro micronucleus test for evaluating the genotoxicity of natural and manmade nano-scale fibres

Our plain-English summary of The utility of the in vitro micronucleus test for evaluating the genotoxicity of natural and manmade nano-scale fibres, by P. Fowler, A. Homan, D. Atkins, et al (Mutation Research. Genetic Toxicology and Environmental Mutagenesis, 2016).

The study

• Authors: P. Fowler, A. Homan, D. Atkins, J. Whitwell, M. Lloyd, R. Bradford
• Journal: Mutation Research. Genetic Toxicology and Environmental Mutagenesis
• Published: 2016;809:33-42
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

A range of fibrous materials, including several types of asbestos and carbon fibres with nano scale diameters that had reported positive genotoxicity data (predominantly clastogenicity), were tested in the in vitro micronucleus test (OECD 487) in GLP-compliant studies in Chinese Hamster Ovary cells. Out of eight materials tested, only one (crocidolite, an asbestos fibre) gave a positive response either in the presence or absence of metabolic activation (S9) and at short (3h) or extended (24h) exposure times (p≤0.001). Our data suggest that the commonly used tests for clastogenicity in mammalian cells require extensive modification before fibrous materials are detected as positive, raising questions about the validity of these tests for detecting clastogenic and aneugenic fibrous materials.

Copyright © 2016 Elsevier B.V. All rights reserved.

In plain English

This laboratory study tested whether a standard regulatory test for chromosome damage, the in vitro micronucleus test (OECD guideline 487), detects fibrous materials. Chinese hamster ovary cells were exposed to eight materials. Three were asbestos types: chrysotile, crocidolite and amosite, traced to UICC standard samples. Five were carbon nanotube and carbon nanofibre products. Cells were treated for 3 hours with and without a rat liver enzyme mix (S9), and for 24 hours without it. Only crocidolite raised the share of micronucleated cells to a statistically significant level above the laboratory's historical control range. This happened at the three lowest doses in the 3-hour test without S9, and at the highest dose (201.8 µg/mL) in the 24-hour test. There was no increase with S9. The authors described the crocidolite response as relatively low, not well reproduced and not concentration related. The other seven materials stayed within the historical range. Most carbon material formed clumps, and fibre uptake into cells was not measured. The authors concluded the test, as designed, may not suit many fibrous materials without extensive modification.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture genotoxicity experiment: micronucleus test run to OECD guideline 487 under Good Laboratory Practice, plus electron microscopy of the fibre suspensions
• Who was studied: Chinese hamster ovary (CHO) cells, WBL subclone, maintained at Covance Laboratories Ltd. in Harrogate, UK. No people or animals were studied.
• Exposure: Eight materials. The asbestos samples were chrysotile, crocidolite and amosite, supplied by the UK Health and Safety Executive and traced to UICC standard batches. The carbon materials were multi-walled carbon nanotubes, carbon nanofibres (low iron and high iron), and short and long single-walled carbon nanotubes. All were suspended in 1% methyl cellulose, with a top concentration of 5 mg/mL unless limited by toxicity or heavy precipitation. Treatments were 3 h plus 21 h recovery with and without S9, and 24 h without S9.
• Main results:
  • Crocidolite, 3 h treatment without S9: micronucleated binucleate cells increased to a statistically significant level that exceeded the historical (negative) control range at the three lowest doses, but not at the three higher doses.
  • Crocidolite, 3 h treatment with S9: no increase in micronucleated binucleate cells over background.
  • Crocidolite, 24 h continuous treatment: the highest concentration tested (201.8 µg/mL) gave micronucleated binucleate cell frequencies statistically significantly above the historical range.
  • The authors judged the crocidolite response to be relatively low in size, not well reproduced and not concentration related. They considered the 24 h increases to be of questionable biological importance.
  • Amosite, chrysotile and the five carbon materials, in all three treatment conditions: micronucleated binucleate cell frequencies did not exceed the historical control range.
  • For these seven materials, some scattered increases were statistically significant against the same-day vehicle control. The authors judged these of little or no biological relevance because they were not concentration related and stayed within the historical range.
  • Electron microscopy of carbon nanofibre and multi-walled carbon nanotube suspensions showed most of the material clumped together in large aggregates.
• The authors' conclusion: The authors concluded that, under the conditions for which they were designed (OECD 487), in vitro regulatory micronucleus tests may not be suitable for assessing the chromosome-damaging potential of many fibrous materials without extensive modification.
• Limitations the authors state:
  • Uptake of fibres into the cells was not measured. The authors say this leaves open whether the negative results reflect no genotoxic potential or no exposure inside the cells.
  • Electron micrographs showed very little free fibre available to interact directly with the cells, because the fibres aggregated in liquid.
  • The highest crocidolite concentration was hard to score under the microscope because of heavy precipitate on the slides.
  • Asbestos purity was assumed to be 100%, with no corrections for lower purity.

Topics

Medical terms in this study, explained in our Lexicon: Genotoxicity, Crocidolite.

Related studies

• A study of the effect of chrysotile fiber surface composition on genotoxicity in vitro — M.J. Keane, J.W. Stephens, et al. (1999)
• Appropriate in vitro test conditions for genotoxicity testing of fibers — G. Speit (2002)
• Validity of the Lymphocyte Cytokinesis-Block Micronucleus Assay (L-CBMN) as biomarker for human exposure to chemicals with different modes of action: A synthesis of systematic reviews — M. Kirsch-Volders, M. Fenech, et al. (2018)
• Aspect ratio has no effect on genotoxicity of multi-wall carbon nanotubes — J.S. Kim, K. Lee, et al. (2011)
• In vitro genotoxicity of asbestos substitutes induced by coupled stimulation of dissolved high-valence ions and oxide radicals — T. Huo, F. Dong, et al. (2018)
• Genotoxicity and cytotoxicity of multi-wall carbon nanotubes in cultured Chinese hamster lung cells in comparison with chrysotile A fibers — M. Asakura, T. Sasaki, et al. (2010)

Browse return to medical research.