Germline mutagenicity of molnupiravir and its active form, β-d-N4-hydroxycytidine, in Caenorhabditis elegans evaluated using whole-genome sequencing

Molnupiravir is a medication used to treat COVID-19 by introducing errors into the SARS-CoV-2 virus's genetic code, thereby preventing its replication. Previous studies, both in vitro and in vivo, have yielded conflicting results regarding its mutagenic potential. While most genotoxicity and mutagenicity tests for molnupiravir and its active form, β-d-N4-hydroxycytidine (NHC), were negative, a few in vitro tests showed positive results. Consequently, further investigation is necessary to evaluate various mutagenic endpoints of molnupiravir. In this study, acute toxicity was assessed by measuring the locomotive activity of Caenorhabditis elegans using a WMicroTracker to determine an appropriate dose range for the germline mutagenicity study. The C. elegans worms were treated with different concentrations of molnupiravir and NHC, along with vehicle controls and ethyl methanesulfonate (EMS) as a positive control. To assess germline mutagenicity, P0 worms from a single clone were exposed to selected concentrations of molnupiravir and NHC, as well as vehicle and positive controls, for 4 h. Molnupiravir and NHC treatments had no significant effect on the locomotion of C. elegans worms after 1-, 2-, 3-, and 4-h exposures, compared to the vehicle control group. In contrast, EMS significantly reduced the worms' locomotive activity. Subsequent whole-genome sequencing of the F1 progeny from the treated P0 worms revealed that neither molnupiravir nor NHC increased the germline mutation frequency or altered mutation types, compared to the vehicle control. In contrast, EMS treatment significantly increased mutation frequency over the vehicle control, with a EMS-specific mutational signature observed. These results suggest that molnupiravir and NHC are not mutagenic in C. elegans germ cells, aligning with previous findings that demonstrate the low mutagenicity of molnupiravir in clinical settings. Additionally, these findings highlight the utility of C. elegans as an alternative animal model for accelerating toxicity assessments and reducing the use of experimental animals.