Propionibacterium freudenreichii ET-3 culture, a cell-free product of whey fermentation using P. freudenreichii ET-3 (7025), has been shown to promote the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA), and therefore, has potential use in the food and supplement industries. Although currently used as a food ingredient in Japan, the safety of this novel ingredient has not been previously evaluated through traditional toxicity testing. Therefore, here we report the results of standard toxicological testing performed on P. freudenreichii ET-3 culture. In a 4-week oral toxicity study, administration of 6000mg/kg body weight/day P. freudenreichii ET-3 culture was without compound-related adverse effects on clinical signs, body weights, food consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and gross and microscopic findings in male and female Sprague–Dawley rats. Furthermore, in vitro mutagenicity testing demonstrated that P. freudenreichii ET-3 culture was non-mutagenic in the bacterial reverse mutation assay using a standard battery of bacterial strains (Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA) and non-clastogenic in Chinese hamster lung cells in the mammalian chromosome aberration test. Together, the results of these studies support the safety of P. freudenreichii ET-3 culture for use in foods for human consumption.