title: GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests.
abstract: A recent ECVAM workshop considered how to reduce falsely predictive positive results when undertaking in vitro genotoxicity testing, and thus to avoid unnecessary follow-up with tests involving animals. As it was anticipated that modified versions of existing assays as well as new assays might contribute to a solution, an expert panel was asked to identify a list of chemicals that could be used in the evaluation of such assays. Three categories of test chemicals were chosen comprising a total of 62 compounds. This paper provides test results for these chemicals using the GreenScreen HC assay. All tests were carried out in triplicate, by multiple operators, with and without S9, using invariant protocols. Group 1 chemicals should be detected as positive in in vitro mammalian cell genotoxicity tests: 18/20 (90%) were reproducibly positive in GreenScreen HC. Group 2 chemicals should give negative results in in vitro genotoxicity tests: 22/23 (96%) were reproducibly negative in GreenScreen HC. Overall concordance for Groups 1 and 2 is 93%. Group 3 chemicals should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce chromosomal aberrations or Tk mutations in mouse lymphoma cells, often at high concentrations or at high levels of cytotoxicity: 13/17 (76%) were reproducibly negative in GreenScreen HC. Of the four positive compounds in Group 3, p-nitrophenol was only positive at the top dose (10mM), 2,4-DCP is an in vivo genotoxin, and two chemicals are antioxidant compounds that may be acting as pro-oxidants in the hyperoxic conditions of cell culture. Overall, these predictive figures are similar to those from other studies with the GreenScreen HC assay and confirm its high specificity, which in turn minimizes the generation of falsely predictive positive results.
authors.affiliation: Gentronix Ltd, 46 Grafton Street, Manchester M13 9NT, UK. louise.birrell@gentronix.co.uk
authors.forename: Louise
authors.initials: L
authors.lastname: Birrell
authors.forename: Paul
authors.initials: P
authors.lastname: Cahill
authors.forename: Chris
authors.initials: C
authors.lastname: Hughes
authors.forename: Matthew
authors.initials: M
authors.lastname: Tate
authors.forename: Richard M
authors.initials: RM
authors.lastname: Walmsley
mesh_terms: D001681:Biological Assay; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D005189:False Positive Reactions; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects*; D009152:Mutagenicity Tests / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D000097642:GADD45 Proteins
chemical_list: D018797:Cell Cycle Proteins; D049452:Green Fluorescent Proteins; D009687:Nuclear Proteins; D004364:Pharmaceutical Preparations; D011993:Recombinant Fusion Proteins; C439070:GADD45A protein, human; D000097642:GADD45 Proteins