Improvement of in vivo genotoxicity assessment: Combination of acute tests and integration into standard toxicity testing

Rothfuß, Andreas; Honma, Masamitu; Czich, Andreas; Aardema, Marilyn J.; Burlinson, Brian; Galloway, Sheila M.; Hamada, Shuichi; Kirkland, David; Heflich, Robert H.; Howe, Jonathan; Nakajima, Masahiro; O’Donovan, Mike; Plappert‐Helbig, Ulla; Priestley, Catherine; Recio, Leslie; Schuler, Maik; Uno, Yoshifumi; Martus, Hans‐Jörg

doi:10.1016/j.mrgentox.2010.12.005

Cited by 96 publications

(59 citation statements)

References 55 publications

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“…Recent work has, however, suggested that, at least in the liver, tissue cytotoxicity does not lead to increased DNA migration in the in vivo Comet assay [29,38]. Additionally, work by Rothfuss et al [29] demonstrated an increase in DNA damage in the in vivo liver Comet assay for rats exposed to 100 mg/kg-day of 2,6-DNT for 3 days or 33 mg/kg-day for 29 days [28,29].…”

Section: Discussionmentioning

confidence: 99%

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Lent

Crouse

Quinn

et al. 2012

Mutation Research/Genetic Toxicology and Environmental Mutagene

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a b s t r a c t Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4-and 2,6-DNT) and four minor isomers (2,2,3,and 3,. DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans." In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5-and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.Published by Elsevier B.V.

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Section: Discussionmentioning

confidence: 99%

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Lent

Crouse

Quinn

et al. 2012

Mutation Research/Genetic Toxicology and Environmental Mutagene

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“…Nevertheless, since tissue-toxicity is more likely to be induced during an RDT study than an acute-dose study this issue might be more critical when performing the Comet assay integrated into an RDT study (Vasquez, 2010). A review of published (and some unpublished) in vivo Comet assay data with concurrent cytotoxicity analysis data of mainly non-genotoxic carcinogens recently performed by an IWGT Working Group did not provide evidence that cytotoxicity, by itself, generates increases in DNA migration resulting in false-positive findings (Rothfuss et al, 2011). Anyway, it is imperative to describe in study reports any confounding factors that may have an influence on the induction of comets, such as cytotoxicity or cell division.…”

Section: Impact Of Toxicitymentioning

confidence: 99%

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

2011

EFS2

287

136

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The Scientific Committee reviewed the current state-of-the-science on genotoxicity testing and provided a commentary and recommendations on genotoxicity testing strategies. A step-wise approach is recommended for the generation and evaluation of data on genotoxic potential, beginning with a basic battery of in vitro tests, comprising a bacterial reverse mutation assay and an in vitro micronucleus assay. Consideration should be given to whether specific features of the test substance might require substitution of one or more of the recommended in vitro tests by other in vitro or in vivo tests in the basic battery. In the event of negative in vitro results, it can be concluded that the substance has no genotoxic potential. In case of inconclusive, contradictory or equivocal results, it may be appropriate to conduct further testing in vitro. In case of positive in vitro results, review of the available relevant data on the test substance and, where necessary, an appropriate in vivo study to assess whether the genotoxic potential observed in vitro is expressed in vivo is recommended. Suitable in vivo tests are the mammalian erythrocyte micronucleus test, transgenic rodent assay, and Comet assay. The approach to in vivo testing should be step-wise. If the first in vivo test is positive, no further testing is necessary and the substance should be considered as an in vivo genotoxin. If the test is negative, it may be possible to conclude that the substance is not an in vivo genotoxin. However, in some cases, a second in vivo test may be necessary (e.g. if the first test is negative but more than one endpoint in the in vitro tests are positive, an in vivo test on a second endpoint may be necessary). The combination of assessing different endpoints in different tissues in the same animal in vivo should also be considered.

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“…Several designs for administration of substances in the in vivo Comet assay are described in the literature (Hartmann et al, 2003(Hartmann et al, , 2004Burlinson et al, 2007;Vasquez, 2010;Rothfuss et al, 2010Rothfuss et al, , 2011.…”

Section: Administration Of Dosesmentioning

confidence: 99%

Minimum Criteria for the acceptance ofin vivoalkaline Comet Assay Reports

2012

EFSA Journal

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The in vivo alkaline Comet assay detects primary DNA damage in various organs and tissues of exposed animals and can be used to assess the genotoxicity of a great variety of chemical compounds, which include food additives, flavourings, food contact materials, foodborne by-products, pesticides, contaminants, etc. Among the various versions of the assay, the alkaline method (pH of DNA unwinding and electrophoresis buffer > 13) identifies the broadest spectrum of DNA damage and is, therefore, recommended for regulatory purposes. It can detect double-and single-strand breaks, alkalilabile lesions that are expressed as single-strand breaks and single-strand breaks arising as DNA repair intermediates. No OECD Test Guideline yet exists for the Comet assay but internationally agreed protocols are available for performing this test. Since establishing of an OECD Test Guideline for this assay will require some further time, there is a need for an agreed approach on the minimum requirements on conduction and reporting of the in vivo Comet assay, which should be fulfilled for the purposes of EFSA during this transition period.

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Improvement of in vivo genotoxicity assessment: Combination of acute tests and integration into standard toxicity testing

Cited by 96 publications

References 55 publications

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

Minimum Criteria for the acceptance ofin vivoalkaline Comet Assay Reports

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