title: A pre-validation transferability study of the GreenScreen HC GADD45a-GFP assay with a metabolic activation system (S9).
abstract: A new protocol has recently been developed and validated for the GreenScreen HC GADD45a-GFP genotoxicity reporter assay, enabling the incorporation of an S9 metabolic activation system into the assay. The S9 protocol employs flow-cytometric methodology for the detection of both reporter GFP fluorescence and propidium iodide fluorescence for the estimation of cellular viability. In the spirit of assay validation by bodies such as the European Centre for the Validation of Alternative Methods (ECVAM), the adapted metabolic activation protocol for the GADD45a-GFP assay has been undergoing 'pre-validation'. Results of phases I and II of this pre-validation, namely protocol refinement and protocol transfer, respectively, are presented here. In phase I the protocol was transferred to a second laboratory for initial assessment of method portability and subsequent refinement of the protocol. In phase II, the protocol was then transferred to two further laboratories along with the elaborated standard operating-procedure (SOP) for further assessment of transferability. The three transfer sites then undertook an assessment of the method's reproducibility by testing eight compounds. The outcome of the study was a refined protocol that was found to be highly transferable. It yielded 100% agreement in results between all four laboratories.
authors.affiliation: Gentronix Ltd, CTF Building, 46 Grafton Street, Manchester M13 9NT, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Billinton
authors.forename: Shannon
authors.initials: S
authors.lastname: Bruce
authors.forename: Julia Rytter
authors.initials: JR
authors.lastname: Hansen
authors.forename: Paul W
authors.initials: PW
authors.lastname: Hastwell
authors.forename: Christopher
authors.initials: C
authors.lastname: Jagger
authors.forename: Catriona
authors.initials: C
authors.lastname: McComb
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Klug
authors.forename: Kamala
authors.initials: K
authors.lastname: Pant
authors.forename: Adam
authors.initials: A
authors.lastname: Rabinowitz
authors.forename: Robert
authors.initials: R
authors.lastname: Rees
authors.forename: Matthew
authors.initials: M
authors.lastname: Tate
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Vinggaard
authors.forename: Richard M
authors.initials: RM
authors.lastname: Walmsley
mesh_terms: D001711:Biotransformation; D018797:Cell Cycle Proteins; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods*; D009687:Nuclear Proteins; D015203:Reproducibility of Results; D000097642:GADD45 Proteins
chemical_list: D018797:Cell Cycle Proteins; D049452:Green Fluorescent Proteins; D009687:Nuclear Proteins; C439070:GADD45A protein, human; D000097642:GADD45 Proteins