Evaluation of cytotoxicity, genotoxicity, and zebrafish embryo toxicity of mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts for cosmetic safety assessment

Background Although various natural ingredients and formulated reagents are used as ingredients in cosmetics, it is important to have alternatives to animal tests, such as in vitro tests of toxicity, as animal testing of cosmetics is prohibited in many countries. Objectives Here, cytotoxicity, genotoxicity, and embryotoxicity on zebrafish embryos were evaluated to confirm the safety of ingredients formulated with 30 extracts from natural resources used as cosmetic ingredients. The blended ingredients were tested by dividing them into mixtures of 25 natural products (NMA), 30 natural products (NMB), and concentrated NMB (NMC), depending on the number of blended natural components and their application. Results First, in vitro, HaCaT cells were treated with concentrations of 0 to 20% for 24 h to measure cell viability. DNA damage within the IC 20 value was measured by comet assay. In the comet assay, 10 mM H 2 O 2 was used as a positive control. Additionally, zebrafish embryos were used as a substitute for conventional animal testing to observe natural mixture toxicity. The olive tail moment (OTM) were not affected by NMA, NMB, or NMC in comparison with the positive control versus three representatives within the IC 20 value concentration of each mixture. Zebrafish embryo results were analysed in a manner similar to the cytotoxicity test results. NMA mixtures showed no cytotoxicity, genotoxicity, or toxicity on zebrafish embryos. Other mixtures showed non-toxicity within the representatively concentrated range. Conclusion Therefore, the 3 mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus , and 27 other extracts were considered to be not significantly toxic in HaCaT cell and zebrafish, so it is suggested that they could be used as cosmetic ingredients.