Evaluation of the Vitotox and RadarScreen assays for the rapid assessment of genotoxicity in the early research phase of drug development.
The Vitotox and RadarScreen assays were evaluated as early screens for mutagenicity and clastogenicity, respectively. The Vitotox assay is a bacterial reporter assay in Salmonella typhimurium based on the SOS-response, and it contains a luciferase gene under control of the recN promoter. The RadarScreen assay is a RAD54 promoter-linked beta-galactosidase reporter assay in yeast. The expression of this beta-galactosidase can easily be quantified by use of the substrate d-luciferin-o-beta-galactopyranoside, which is converted into galactose and luciferin that can be measured luminometrically. Recently, an ECVAM workgroup defined a list of 20 genotoxic and 42 non-genotoxic compounds [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108.] that can be used for the validation and/or optimization of in vitro genotoxicity assays. In the present study, this compound set was used for the validation of the assays. Moreover, an additional set of 192 compounds was used to broaden this validation study. The compounds of this additional set can be classified as non-genotoxins and genotoxins and consists of both in-house and reference compounds. In case of the ECVAM compound list, the results from the Vitotox and RadarScreen assays were compared to the genotoxic/non-genotoxic classification of the compounds in this list. In case of the additionally tested compounds, the results of the Vitotox and RadarScreen assays were compared, respectively, with bacterial mutagenicity (Ames) results or in vitro clastogenicity data obtained in-house or from the literature. The validation with respect to the ECVAM compound list resulted in a sensitivity for both the Vitotox and RadarScreen assay of 70% (14/20). If both assays were combined the sensitivity increased to 85% (17/20). Both tests also gave a low number of false positive results. The specificity of the Vitotox and RadarScreen assays was 93% (39/42) and 83% (35/42), respectively. This resulted in a predictivity of the Vitotox and RadarScreen assay of 85% (53/62) and 79% (49/62), respectively. In case both tests were combined the specificity and the predictivity of the Vitotox and RadarScreen assay turned out to be 81% (34/42) and 82% (51/62), respectively. The results from the additional list of 192 compounds confirmed the results found with the ECVAM compound list. The results from the Vitotox assay showed a high correlation with Ames test of 91% (132/145). Subsequently, the RadarScreen assay had a correlation with in vitro clastogenicity of 76% (93/123). The specificity of the Vitotox assay was 94% (90/96) for Ames test results and that of the RadarScreen assay was 74% (34/46) for clastogenicity. Moreover, the sensitivities of the Vitotox and RadarScreen assays were 86% (42/49) and 77% (59/77), respectively. Implementation of the Vitotox and RadarScreen assays in the early research phase of drug development can lead to fast de-selection for genotoxicity. It is expected that this application will reduce the number of compounds that have a positive score in the regulatory Ames and clastogenicity tests. Moreover, problems with a complete compound class can be foreseen at an early time point in the research phase, which gives more time for issue resolution than late detection of these problems with the regulatory tests.
Westerink
Walter M A
WM
Department of Pharmacology, Schering-Plough Research Institute, P.O. Box 20, 5342 CC Oss, The Netherlands. Walter.Westerink@spcorp.com
Stevenson
Joe C R
JC
Lauwers
Annick
A
Griffioen
Gerard
G
Horbach
G Jean
GJ
Schoonen
Willem G E J
WG
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D064420:Drug-Related Side Effects and Adverse Reactions; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D015162:Micronucleus Tests / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009152:Mutagenicity Tests / Q000379:methods*; D015203:Reproducibility of Results; D012107:Research Design; D013014:SOS Response, Genetics / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000235:genetics; D012680:Sensitivity and Specificity