title: Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro.
abstract: Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration-response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.
authors.affiliation: Institute for the Environment, Brunel University, Kingston Lane, Uxbridge, Middlesex UB8 3PH, UK. sibylle.ermler@brunel.ac.uk
authors.forename: Sibylle
authors.initials: S
authors.lastname: Ermler
authors.forename: Martin
authors.initials: M
authors.lastname: Scholze
authors.forename: Andreas
authors.initials: A
authors.lastname: Kortenkamp
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000008:administration & dosage / Q000633:toxicity*; D016466:CHO Cells; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D015162:Micronucleus Tests / Q000379:methods; D010575:Pesticides / Q000633:toxicity*; D015203:Reproducibility of Results
chemical_list: D001562:Benzimidazoles; D010575:Pesticides
references.citation: Environ Toxicol Chem. 2001 Feb;20(2):448-57
references.pmid: 11351447
references.citation: Mutat Res. 2000 Nov 20;455(1-2):81-95
references.pmid: 11113469
references.citation: Mutagenesis. 1991 Jul;6(4):319-24
references.pmid: 1719337
references.citation: Mutagenesis. 2009 Jan;24(1):85-93
references.pmid: 18854579
references.citation: Mutat Res. 2011 Mar 18;721(1):27-73
references.pmid: 21238603
references.citation: Mutat Res. 2006 Aug 4;607(1):13-36
references.pmid: 16815079
references.citation: Mutat Res. 2007 Jan 10;626(1-2):191-5
references.pmid: 17055335
references.citation: Environ Mol Mutagen. 1995;26(4):292-304
references.pmid: 8575418
references.citation: Mutagenesis. 2004 Sep;19(5):391-7
references.pmid: 15388812
references.citation: Mutat Res. 2009 Aug;678(2):118-22
references.pmid: 19477296
references.citation: Aquat Toxicol. 2003 Mar 17;63(1):43-63
references.pmid: 12615420
references.citation: Mutat Res. 1996 Dec;372(2):233-45
references.pmid: 9015142
references.citation: Toxicol In Vitro. 2010 Feb;24(1):208-16
references.pmid: 19747535
references.citation: Toxicol Lett. 2003 Apr 11;140-141:63-74
references.pmid: 12676452
references.citation: Environ Health Perspect. 2002 Dec;110 Suppl 6:915-8
references.pmid: 12634119
references.citation: Cell Biol Toxicol. 1993 Oct-Dec;9(4):333-43
references.pmid: 8039010
references.citation: Mutat Res. 2000 Jan 3;464(1):41-51
references.pmid: 10633176
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):55-63
references.pmid: 6606682
references.citation: Antimicrob Agents Chemother. 1998 Sep;42(9):2171-3
references.pmid: 9736529
references.citation: Toxicol Appl Pharmacol. 2011 Dec 1;257(2):189-97
references.pmid: 21945941
references.citation: Mutagenesis. 1992 Mar;7(2):111-4
references.pmid: 1579065
references.citation: Pharmacol Rev. 1989 Jun;41(2):93-141
references.pmid: 2692037
references.citation: Mutat Res. 2007 Jun 15;630(1-2):1-13
references.pmid: 17446119
references.citation: Mutat Res. 2006 Aug 4;607(1):37-60
references.pmid: 16765631
references.citation: Mutagenesis. 2007 Jul;22(4):275-80
references.pmid: 17456508
references.citation: Mutat Res. 2006 Aug 4;607(1):61-87
references.pmid: 16797224
references.citation: Mutagenesis. 1995 Jan;10(1):23-9
references.pmid: 7739397
references.citation: Arch Toxicol. 2011 Jun;85(6):689-93
references.pmid: 20938648
references.citation: Environ Mol Mutagen. 2012 Apr;53(3):173-84
references.pmid: 22389207
references.citation: Biochim Biophys Acta. 1978 Dec 18;544(3):605-14
references.pmid: 728472
references.citation: Mutat Res. 1997 Jan 15;388(1):27-32
references.pmid: 9025789
references.citation: Mutagenesis. 1989 Nov;4(6):425-31
references.pmid: 2695755
references.citation: Mutat Res. 2002 Nov 26;521(1-2):103-12
references.pmid: 12438008
references.citation: Mutagenesis. 1998 Mar;13(2):193-8
references.pmid: 9568594
references.citation: Arch Med Res. 2001 Mar-Apr;32(2):119-22
references.pmid: 11343808
references.citation: Mutat Res. 1995 Jan;341(3):169-84
references.pmid: 7529358
references.citation: Mutagenesis. 1993 Jul;8(4):285-93
references.pmid: 7690875