ANTI-AGEING *p<0.05


115 110 105 100 95 90


0 0.04 * * * 0.08 Repista (%) *Significantly different from the control (p < 0.05; Student’s t-test)


Figure 6: Cell viability of the AOH active ingredient on NHEK, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell viability percentage was calculated relative to the untreated control


Skin sensitization is a complex systemic


response of the immune system, which is difficult to evaluate using only in vitro methods. Therefore, in addition to the three in vitro tests, a cumulative skin irritation and sensitization test, namely the repeated insult patch test (RIPT), was conducted to verify the presence or absence of skin sensitization. Since AOH absorbs in the ultraviolet region,


it was determined that the effects of light should be taken into consideration, therefore, a human phototoxicity and photosensitization study was conducted in addition to the in vitro studies. A human patch test was also conducted. The Ames test revealed that AOH was not


mutagenic to any of the tested bacterial strains (> 5000 mg/plate). In vitro skin irritation tests using reconstructed human epidermis showed that AOH was not a skin irritant (cell viability > 50%). AOH did not induce skin sensitization in any of the in vitro tests (DPRA, h-CLAT, or KeratinoSens). Moreover, in RIPT, AOH did not induce any human skin reactions. AOH did not exhibit cytotoxicity under ultraviolet or sham irradiation in the alternative phototoxicity test using BALB/c 3T3 cells (mean photoeffect <0.1). AOH did not induce human dermal phototoxicity or photosensitivity. No adverse reactions to AOH were observed


in any of the safety studies conducted proving its safety (Table 2).7,8


No adverse events were


observed in clinical trials either, and AOH was judged as a safe substance.11 As mentioned above, AOH is found in edible


plants and has also been shown to be a substance present in the foods we consume eat every day.


Guideline 0.15 0.3


2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80


*p<0.05


55


Control ■ 0.3% ■ 1% ■ 3%■ *


* * * * * * * * * * TGM1 IVL CLDN1 DSC1 * * * * *


KRT1 KRT10 HAS3 *Significantly different from the control (p < 0.05; Student’s t-test)


Figure 7: Up-regulated genes involved in skin barrier functioning in normal human epidermal keratinocyte cells treated with the AOH active ingredient.


Thus, the AOH active ingredient can be considered a safe and reliable cosmetic ingredient.


Conclusion The AOH active ingredient showed cell- activating effects on NHEK, and a comprehensive analysis using DNA microarrays suggested it acts on a wide range of epidermal functions, such as promoting epidermal turnover, differentiation, maturation, and the metabolism of old skin cells. A safety evaluation of AOH contained in the


AOH active ingredient was conducted to ensure that it was safe to apply on human skin. No adverse reactions were observed in any of the in vitro studies, indicating that the product was safe. The evaluation of the efficacy of the AOH active ingredient on the skin is ongoing, and we are confident that further positive findings will be obtained. The AOH in the AOH active ingredient is a


plant growth factor that promotes plant growth and increases grain yield. It also maintains plant homeostasis, as it enables stable growth in plants, even under stress conditions, such as drought. Therefore, AOH has the potential to save the world from a possible global food crisis in the future owing to global warming and population explosion. Hence, AOH may not only save our skin, but also the people of the world from a food crisis.


Acknowledgements We would like to express our sincere gratitude to Distinguished University Professor Kawagishi of Shizuoka University, who provided guidance


TABLE 2: COMPARATIVE RESULTS OF VARIOUS AOH SAFETY TESTS Safety test


Ames test


In vitro skin irritation in vitro eye irritation


In vitro phototoxicity (Skinethic Skin Irritation) In vitro sensitization (DPRA)


In vitro skin sensitization (KeratinoSens) In vitro skin sensitization (h-CLAT) In vitro phototoxicity


Human skin patch test (10% Repista)


Human phototoxicity and photosensitization tests (10% Repista)


RIPT (10%Repista) www.personalcaremagazine.com


OECD TG471 OECD TG439 OECD TG492 OECD TG432 OECD TG442C OECD TG442D OECD TG442E OECD TG432 –


– –


Result


No mutagenicity9 No irritation7 No irritation


No phototoxicity7 No sensitization7 No sensitization8 No sensitization8 No toxicity7 Negative7


Negative 8 Negative8


during the implementation and development of this research. We also thank Nikoderm Research Inc. (Osaka, Japan) for performing skin irritation, skin sensitization (DPRA and h-CLAT assays), and alternative phototoxicity tests in vitro, as well as phototoxicity and photosensitization studies on human skin. We are also grateful to DRC Co., Ltd (Osaka, Japan), BoZo Research Center Inc. (Kanagawa, Japan), and the Food and Drug Safety Center, Hatano Research Institute (Tokyo, Japan) for conducting the human patch test, Ames test, and in vitro skin sensitization (KeratinoSens™), respectively. We would also like to thank Editage for English language editing.


References 1. Choi J-H et al. The source of “fairy rings”: 2-azahypoxanthine and its metabolite found in a novel purine metabolic pathway in plants. Angew. Chem. Int. Ed. 53, 1552 (2014)


2. Kawagishi H et al. Are fairy chemicals a new family of plant hormones? Proc. Jpn. Acad. Ser. B. 95, 29 (2019)


3. Ramsbottom J. Rate of growth of fungus rings. Nature. 117, 158 (1926)


4. Smith JD, Fungi and turf diseases. 7: Fairy rings. J. Sports Turf. Res. Inst. 10, 324 (1957)


5. Shantz HL, Piemeisel RL. Fungus fairy rings in Eastern Colorado and their effect on vegetation. J. Agric. Res. 11, 191 (1917)


6. Evershed H. Fairy rings. Nature. 29, 384 (1884) 7. Aoshima H et al. Safety evaluation of 2-aza- 8-oxohypoxanthine based on in vitro and human patch tests. Fundam. Toxicol. Sci. 7(5), 207 (2020)


8. Aoshima H et al. Safety evaluation of 2-aza-8- oxohypoxanthine by in vitro skin sensitization and human tests. Fundam. Toxicol. Sci. 8(4), 123 (2021)


9. Aoshima H et al. The Potential of 2-aza-8- Oxohypoxanthine as a Cosmetic Ingredient. Cosmetics. 8(3), 60 (2021)


10. Roth W. Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18. J. Cell Sci. 125, 5269 (2012)


(skin irritation index: 3.7)


11. Aoshima H et al. Clinical Evaluation of Topical Lotion Containing 2-Aza-8-oxohypoxanthine on Skin Barrier Function against Water Loss. Cosmetics. 8(3), 83 (2021)


January 2023 PERSONAL CARE


PC


Cell viability (%)


Cell viability (%)


INCREASE


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