Lac dye is a natural colorant derived mainly from the insect Kerria lacca (Kerr) and has been used in food and beverage as a red-coloring additive. Despite its increasing use for human consumption as an alternative for allergy-associated cochineal, its toxicity profile remained incomplete to sufficiently assess its safety for the intended use. In this study, we evaluated systemic and genetic toxicity by performing acute and subacute oral toxicity studies in Sprague–Dawley (SD) rats using highly purified lac dye (LD) formulated in water and a battery of genotoxicity tests, respectively. To assess antigenic potentials, we carried out an in vivo passive cutaneous anaphylaxis test. A single dose of LD did not cause mortality at 5000 mg/kg body weight (BW), setting oral LD50 of >5000 mg/kg BW in SD rats. In the 90-day study, transient salivation without accompanying histopathological lesions in the salivary glands in 200 and 500 mg/kg BW groups and red-purple pigmentation on the surface of femora and skulls in 500 mg/kg groups were observed as nonadverse effects associated with LD, and no adverse effect was detected in all of the parameters examined, establishing a 500 mg/kg BW as no-observed-adverse-effect-level (NOAEL). Furthermore, LD was not mutagenic nor clastogenic in the genotoxicity tests. When tested for antigenicity, LD did not induce anaphylactic skin responses as opposed to the positive reaction by ovalbumin, suggesting a lack of antigenicity. Taken together, these findings provide extended toxicity information on LD with direct evidence supporting the lack of antigenicity, providing essential guidance for its safe use in humans.