Hydroxyanthracene derivatives are widely distributed in the plant kingdom, mainly in botanicals such as the Hypericum, Rheum, Rhamnus and Aloe genera. For centuries, plants containing hydroxyanthracene derivatives have been used as herbal remedies, mainly as laxatives. The root and underground stem (rhizome) are used to make medicine, primarily for digestive complaints including constipation, diarrhoea, heartburn, stomach pain, gastrointestinal bleeding, and preparation for certain gastrointestinal diagnostic procedures. The use of hydroxyanthracene-containing botanicals has raised the attention of European Food Safety Authority (EFSA) for the potential genotoxicity activity, that in 2018 concluded "[.] and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists". No genotoxic activity has been reported with other constituents such as rhein, physcion and chrysophanol. In the present study, Rhubarb ethanolic extract of ground rhubarb rhizome (hydroxyanthracene total content 1.39 %) was tested in the Ames Assay in Salmonella typhimurium and Escherichia coli, up to 5000 µg/plate and up to 5000 µg/mL in human lymphocytes Micronucleus Test (OECD 471 and 487 respectively) in vitro mutagenic and genotoxic effects. Under the experimental conditions used, the rhubarb rhizome extract showed no genotoxic activity.