A pre-validation transferability study of the GreenAScreen HC GADD45a-GFP assay with a metabolic activation system (S9)

Billinton N, Bruce S, Hansen J R, Hastwell P W, Jagger C, McComb C, Klug M L, Pant K, Rabinowitz A, Rees R, Tate M, Vinggaard A M, Walmsley R M

Mut Res Genet Toxicol Environ Mutagen; 700(1-2): 44-50

http://dx.doi.org/10.1016/j.mrgentox.2010.05.001

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.