Ethylene oxide (EO) is a widely used chemical intermediate also formed endogenously from ethylene metabolism. Despite conflicting epidemiological evidence, EO is classified by the IARC as a human carcinogen. The mutagenicity and carcinogenicity of EO is attributed to direct reaction with DNA and formation of multiple 2-hydroxyethyl (HE) DNA adducts. However, the actual lesions responsible for the reported mutagenicity of EO have not been established. This study used the supF mutation assay to investigate the biological relevance of low levels of EO-induced DNA adducts in human Ad293 cells, with respect to the type and level of each HE adduct present. Initial experiments were conducted using pSP189 plasmid containing up to 290 N7-HEGuanine (N7-HEG) adducts/10(6) nucleotides, which far exceeds that typically detected in human DNA. No other HE-lesions were detectable using our validated LC-MS/MS assay. Replication in cells failed to produce a statistically significant increase in relative mutation frequency, above background rates in the solvent control. Furthermore, the mutation spectrum compiled for EO-treated plasmid (10-2000muM) did not differ significantly from the spontaneous distribution, suggesting EO is not strongly mutagenic in this system. Under refined reaction conditions using higher EO concentrations capable of inducing detectable levels of N1-HEdA, O(6)-HEdG and N3-HEdU along with N7-HEG, there was a significant dose-related increase in relative mutation frequency above background (3.76- and 5.30-fold at 10 and 30mM, respectively). EO treatment appeared associated with an elevated frequency of GC-->CG mutations and the occurrence of substitutions at AT base pairs. Additionally, there was a distinct GC-->TA mutational hotspot in the 10mM EO spectrum. Overall, the results suggest a certain level of promutagenic adducts must be attained before mutations become detectable above background, indicating that N7-HEG is not a promutagenic lesion, and support a role for the minor products of DNA hydroxyethylation in the generation of base substitutions by EO.