The degradation of phosphorothioate oligonucleotides (PS-ONDs) and the release of potentially genotoxic modified mononucleotides raise a safety concern for OND-based therapeutics. Deoxyadenosine monophosphorothioate (dAMPαS), a PS nucleotide analog, has been reported to be a potent in vitro mutagen at the thymidine kinase (TK) locus in human TK6 lymphoblastoid cells. This led us to explore the mechanism behind the apparent positive response induced by dAMPαS in the TK gene-mutation assay in TK6 cells. In this work, treatment of TK6 cells with dAMPαS produced a dose-dependent increase in cytotoxicity and mutant frequency at the TK locus. Surprisingly, when the colonies from dAMPαS were re-challenged with the selective agent trifluorothymidine (TFT), the TFT-resistant phenotype was lost. Moreover, dAMPαS-induced colonies displayed distinct growth kinetics and required longer incubation time than 4-nitroquinoline-1-oxide-induced colonies to start growing. Treatment of TK6 cells with dAMPαS induced cell cycle arrest at the G1 phase, enabling cells to grow, and form a colony after the efficacy of TFT in the culture medium was lost. Our findings suggest that a fraction of parental "nonmutant" TK6 cells escaped the toxicity of TFT, possibly via G1 arrest, and resumed growth after the degradation of TFT. We conclude that dAMPαS did not induce real TFT-resistant mutants and caution should be taken with interpretation of mutation data from TK gene-mutation assay in TK6 cells when assessing modified nucleotides.