Recent studies have highlighted the poor specificity of a battery of in vitro genotoxicity tests when predicting rodent carcinogenicity, in some cases leading to misleading predictions of genotoxic carcinogenicity. GreenScreen HC (GSHC) is a highly sensitive human cell-based genotoxicity assay that, in contrast to the battery in vitro mammalian cell tests, also exhibits high specificity. GSHC employs the response of the human DNA damage-inducible gene GADD45a as a marker of genotoxic stress, using a GFP reporter construct hosted by the TK6 cell line. In order to better understand the biological basis for the high specificity of GSHC, three approaches were taken. Firstly, the relevance of the choice of a p53-competent host cell line, TK6, was investigated. A database was compiled consisting of published genotoxicity data from the in vitro battery tests performed in TK6 cells, and comparative GSHC data generated. This work revealed that discordance existed between the tests analysed, therefore the specificity of GSHC was not dictated by the choice of cell line alone. To confirm that misleading positive results could still be generated in the GSHC TK6 cell line, mutation assays were performed for compounds with pre-existing misleading positive battery test data, and a dose-dependent increase in mutation frequency was observed for 1 of 3 compounds. Secondly, the importance of wild-type p53 to the specificity of GSHC was investigated by generating p53-deficient GADD45a-GFP reporter cell lines. This work identified wild-type p53 as a key regulator of GADD45a in the context of GSHC, with reduced expression levels observed in response to genotoxic stimuli. Finally NF-kB, a putative direct regulator of GADD45a in response to DNA damage, was shown to contribute to the expression of GADD45a at high doses of 4-nitroquinoline-N-oxide via mutational analysis of two NF-kB binding sites in the promoter and intron 3 of the GADD45a-GFP reporter. This implicates NF-kB in the direct regulation of GADD45a in response to genotoxic stress in the context of GSHC. The work presented here demonstrates that the high specificity of GSHC is not dictated solely by the use of the p53-competent TK6 cell line, but is due to the use of a unique biosensor of genotoxic stress; GADD45a expression. Interference with the function of two key stress response signalling nodes, p53 and NF-kB, revealed that these regulators dictate the magnitude of expression of GADD45a, directly impacting upon the specificity of GSHC.