Safflower (Carthamus tinctorius) petals have been used for centuries as a spice, in tea blends and in traditional Asian medicine. Aqueous extracts of Safflower petals have been used as a colouring food over the last 30 years due to their bright colour. Publications in the past raised concerns about fertility impairing, maternal toxicity, fetotoxic and teratogenic properties in rodents. As the tested extracts were poorly characterized and the studies were not performed according to guidelines, a need for further evaluation was seen. In silico predictions for the main pigments provided negative results for bacterial mutagenicity. Further, in vitro genotoxicity and in vivo reproductive toxicity studies of a well-characterized aqueous safflower concentrate generated more relevant data for risk assessment of its use in food. In vitro AMES tests and a mouse lymphoma cell assay were negative. An OECD guideline 421 screening study was performed in rats with oral daily doses of up to 1000 mg/kg bodyweight, applied via gavage to simulate a bolus effect. The highest dose reflected a toxicological limit test. The study did not give indications of general toxicity, did not show any effect on fertility and reproduction nor any effect on prenatal development and, also in contrast to previous results, treatment did not affect estradiol and FSH levels. Furthermore, pups raised until PND 14-16, developed normally with no adverse effects observed. With the established NOAEL of at least 1000 mg/kg/d, a considerable margin of exposure is achieved when compared with human intake estimates.