title: The automated micronucleus assay for early assessment of genotoxicity in drug discovery.
abstract: Recent publications on the automated in vitro micronucleus assay show predictive values higher than 85% for the classification of in vitro aneugens, clastogens and non-genotoxic compounds. In the present work, the CHO-k1 micronucleus assay in combination with cellular imaging was further evaluated. Firstly, the effect of a range of S9 concentrations on micronucleus formation and cytotoxicity was investigated. Subsequently, the reproducibility and predictivity of the micronucleus assay on CHO-k1 cells was investigated with a set of four compounds. Then, a larger set of compounds (n=44) was tested on CHO-k1 cells and inter-laboratory correlation was calculated. Finally, cellular imaging was compared with flow cytometry for in vivo assessment of micronucleus formation. The concentration of S9 had a significant impact on micronucleus formation and cytotoxicity. In addition, calculations of relative cell count (RCC) and cytokinesis-block proliferation index (CBPI) showed to be complementary to cytotoxicity assessment. The CHO-k1 micronucleus assay correctly classified the four reference compounds, with a dose-response relationship and low variability. Based on a larger set of compounds, the assay proved to be reliable with a sensitivity of 94% (n=31) and a specificity of 85% (n=13). A correlation coefficient of 97% was obtained when the lowest observable adverse effect levels (LOAELs) from our study were compared with those published by Diaz et al. (2007) [10]. In conclusion, the in vitro CHO-k1 micronucleus assay combined with cellular imaging is a predictive assay appropriate for genotoxicity screening at early stages of drug development. In addition, for in vivo assessment of micronucleus formation, we preferred to use flow cytometry rather than cell imaging.
authors.affiliation: UCB Pharma, Investigative, Chemical and Environmental Safety, Chemin du Foriest, 1420 Braine l'Alleud, Belgium. Karen.Tilmant@ucb.com
authors.forename: K
authors.initials: K
authors.lastname: Tilmant
authors.forename: H H J
authors.initials: HH
authors.lastname: Gerets
authors.forename: P
authors.initials: P
authors.lastname: De Ron
authors.forename: C
authors.initials: C
authors.lastname: Cossu-Leguille
authors.forename: P
authors.initials: P
authors.lastname: Vasseur
authors.forename: S
authors.initials: S
authors.lastname: Dhalluin
authors.forename: F A
authors.initials: FA
authors.lastname: Atienzar
mesh_terms: D024422:Aneugens / Q000633:toxicity*; D000818:Animals; D001711:Biotransformation; D016466:CHO Cells; D002452:Cell Count; D006224:Cricetinae; D003412:Cricetulus; D048749:Cytokinesis; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D005434:Flow Cytometry; D007091:Image Processing, Computer-Assisted; D015162:Micronucleus Tests / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D024422:Aneugens