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AbstractMolnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography—mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use. [ABSTRACT FROM AUTHOR]