How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop

Kirkland, David; Pfuhler, Stefan; Tweats, David; Aardema, Marilyn J.; Corvi, Raffaella; Darroudi, F.; Elhajouji, Azeddine; Glatt, Hansruedi; Hastwell, Paul W.; Hayashi, Makoto; Kasper, P.; Kirchner, Stephan; Lynch, Anthony M.; Marzin, Daniel; Maurici, Daniela; Meunier, Jean-Roc; Müller, Lutz; Nohynek, Gerhard J.; Parry, James M.; Parry, Elizabeth M.; Thybaud, Véronique; Tice, R.R.; Benthem, Jan van; Vanparys, Philippe; White, Paul A.

doi:10.1016/j.mrgentox.2006.11.008

Cited by 398 publications

(206 citation statements)

References 34 publications

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“…Thus, with this system in place, three genotoxicants and three presumed non-genotoxicants were characterized correctly. Collectively, these results support the concept that guidelines regarding cytotoxicity limits, as well as the methods used to accomplish these measurements, should be reconsidered [19,22]. It seems likely that the implementation of such improvements could increase the meaningfulness of mammalian cellbased genotoxicity data.…”

Section: Discussionmentioning

confidence: 53%

“…The poor assay specificity of mammalian cell genotoxicity tests has become increasingly well documented [18][19]. One of the chief reasons cited for the high rate of false positive results is the confounding effects of overtly cytotoxic concentrations [18][19][20][21].…”

Section: Discussionmentioning

confidence: 99%

“…One of the chief reasons cited for the high rate of false positive results is the confounding effects of overtly cytotoxic concentrations [18][19][20][21]. Furthermore, when unoptimized automated scoring methods are utilized, further erosion of assay specificity can result.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

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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“…CYP2C6 and CYP2C11 have been shown to be the major enzymes responsible for the bioactivation of CP in uninduced male rat liver microsomes (25), and nitro group-containing aromatic compounds (AF-2 and 4NQO) are reported to be activated by bacterial nitroreductase enzymes (26)(27)(28). It is important that each promutagen is not merely activated by a single speciˆc metabolizing enzyme, but also activated or detoxiˆed by some other metabolizing enzymes, including enzymes of the CYP subfamily, and that the magnitude of the mutagenicity is determined by the metabolic balance between the activation and detoxiˆcation of promutagens (12,13,(18)(19)(20). These lines of evidence and knowledge suggest that the inhibitory eŠect of DMSO on the mutagenicity observed in this study may be attributable to its inhibitory eŠect on the exogenous or endogenous drug-metabolizing enzymes involved in the parallel or sequential metabolic activation/detoxiˆca-tion pathways.…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand some studies have demonstrated that DMSO inhibits several kinds of CYP enzymes involved in the metabolic activation or detoxiˆca-tion of chemical mutagens, even at concentrations of 1 z or less (7)(8)(9)(10)(11). Although the issue of poisoning of enzymes by organic vehicles, including DMSO, in genotoxicity tests has been debated (12,13), data on the eŠect of DMSO on the mutagenicity of promutagens that require metabolic activation are still limited in terms of the number of compounds tested, the kinds of chemical classes studied, and comparative data for mutagenicity obtained at low and high concentrations (14z) of DMSO (5,7,(14)(15)(16)(17). Therefore, in the present study, we examined the mutagenicity of 14 promutagens belonging to several chemical classes and a direct mutagen (as a reference compound) in the presence of DMSO at concentrations of 1z and 14z in the Ames preincubation test, and compared the results, to clarify how much the results are aŠected by DMSO.…”

Section: Introductionmentioning

confidence: 99%

Inhibitory Effect of Dimethyl Sulfoxide on the Mutagenicity of Promutagens in the Ames Test

Hakura

Hori²,

Uchida

et al. 2010

Genes and Environment

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The eŠect of dimethyl sulfoxide (DMSO) on the mutagenicity of 14 promutagens belonging to several chemical classes and one direct mutagen as a reference compound was examined in the Ames preincubation test, to clarify how much the test results were aŠected by its inhibitory activity on metabolic enzymes. The mutagens were assayed by the preincubation method using the TA100 or WP2uvrA(pKM101) bacterial test strains in the presence of 1% and 14% DMSO (concentrations in treatment mixture) with S9 mix for 12 promutagens that are known to be activated by CYP enzymes or without S9 mix for 2 promutagens that are known to be activated by bacterial nitroreductase enzymes, and the direct-mutagen. The data indicate that the mutagenicity of 11 of the 14 promutagens was signiˆcantly reduced in the presence of 14% DMSO as compared with that in the presence of 1% DMSO, while the 3 remaining promutagens and the direct mutagen exhibited mutagenicity of equal degree at both concentrations. The largest inhibitory eŠect of DMSO was found on the nitrosamines in WP2uvrA(pKM101) and TA100, and no cytotoxicity was detected by survival test, with 14% DMSO in WP2uvrA(pKM101), at any amounts of dimethylnitrosamine. Further equivalent or slightly lower cytotoxicity in the presence of 14% DMSO than in the presence of 1% DMSO was detected by decrease in the bacterial background-lawn density, as a whole. These observations suggest that the reduction in the yield of revertant colonies in the presence of 14% DMSO with the 11 chemicals was not due to cytotoxicity of DMSO. The inhibitory eŠect of DMSO on the mutagenicity of the promutagens can be explained by its inhibitory eŠect on the drug-metabolizing enzymes involved in the activation/detoxiˆcation pathways. Use of DMSO at a low concentration, such as 1%, may be suggested for the Ames test, as for other in vitro genotoxicity tests.

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Structural Alerts for Toxicity

Blagg

2010

Burger's Medicinal Chemistry and Drug Discovery

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This chapter summarizes structural alerts where there is a significant weight of evidence that their incorporation into a drug candidate molecule is likely to result in a higher than average incidence of attrition due to adverse safety/or toxicity findings. The mechanism of formation of reactive metabolites from each structural alert is summarized. The structure–toxicity relationships and the boundary of definition for each structural alert are discussed. The benefits of low exposure and alternative routes of metabolism are highlighted along with screening methods to manage the risks associated with structural alerts.

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How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop

Cited by 398 publications

References 34 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

Inhibitory Effect of Dimethyl Sulfoxide on the Mutagenicity of Promutagens in the Ames Test

Structural Alerts for Toxicity

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