At present, an inevitable consequence of a chemical's inhibitory activity on key regulators of DNA topology in bacteria, the type II topoisomerases, is a less pronounced effect on their eukaryotic counterparts. In the context of anti-infectives drug development, this may pose a risk to patient safety as inhibition of eukaryotic type II topoisomerases (TOPO II) can result in the generation of DNA double-strand breaks (DSBs), which have the potential to manifest as mutations, chromosome breakage or cell death. The biological effects of several TOPO II inhibitors in mammalian cells are described herein; their modulation of DSB damage response parameters is examined and evidence for the existence of a threshold concept for genotoxicity and its relevance in safety assessment is discussed. The potential utility of gammaH2AX, a promising and highly sensitive molecular marker for DSBs, in a novel genotoxicity 'pre-screen' to conventional assays is also highlighted.