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. 2020 Sep 26;13(19):4299.
doi: 10.3390/ma13194299.

The In Vivo Toxicity and Antimicrobial Properties for Electrolyzed Oxidizing (EO) Water-Based Mouthwashes

Yi-Ling Hsieh  1 , Jiun-Cheng Yao  1 , Sung-Chih Hsieh  2 , Nai-Chia Teng  2 , You-Tai Chu  1 , Wen-Xin Yu  1 , Chung-He Chen  1 , Liang-Yu Chang  1 , Ching-Shuan Huang  2 , Tzu-Hsin Lee  1 , Aivaras Kareiva  3 , Jen-Chang Yang  1   4   5   6
Affiliations

The In Vivo Toxicity and Antimicrobial Properties for Electrolyzed Oxidizing (EO) Water-Based Mouthwashes

Yi-Ling Hsieh et al. Materials (Basel). .

Abstract

The objective of this study was to verify the feasibility of electrolyzed oxidizing (EO) water as a mouthwash through the evaluation of its in vivo toxicity by embryonic zebrafish and antimicrobial efficacy against Streptococcus mutans (S. mutans).

Methodology: Each 1.5-3.0 g of sodium chloride (NaCl), sodium bromide (NaBr), or calcium chloride (CaCl2) were added into an electrolyzer with 300 mL of DD water to produce electrolyzed oxidizing (EO) water. A zebrafish embryo assay was used to evaluate acute toxicity of specimens. Antimicrobial property was conducted with 100 μL microbial count of 1 × 108 cfu/mL S. mutans to blend with each 10 mL specimen of chlorhexidine (CHX) gluconate or hypochlorous acid (HOCl) for various time points. The concentration of viable microorganisms was assessed according to individually standardized inoculum by a plate-count method.

Results: Among the EO water produced from NaCl, NaBr, and CaCl2, the EO water from NaCl showed a relatively low mortality rate of zebrafish embryos and was chosen for a detailed investigation. The mortality rates for the groups treated with EO water containing 0.0125% and 0.0250% HOCl were not statically different from those of a negative control, however the mortality rate was 66.7 ± 26.2% in 0.2% CHX gluconate for the same treatment time of 0.5 min. All of the HOCl or 2.0% CHX gluconate groups showed >99.9% antimicrobial effectiveness against S. mutans; while the 0.2% CHX gluconate group showed a bacterial reduction rate of 87.5% and 97.1% for treatment times of 0.5 min and 1.0 min, respectively.

Conclusions: Except for the 0.2% CHX gluconate, all the HOCl specimens and 2.0% CHX gluconate revealed similar antimicrobial properties (>99.9%) against S. mutans. The EO water comprised of both 0.0125% and 0.0250% HOCl showed >99.9% antimicrobial efficacy but with little in vivo toxicity, illuminating the possibility as an alternative mouthwash for dental and oral care.

Keywords: Streptococcus mutans (S. mutans); chlorhexidine (CHX) gluconate; electrolyzed oxidizing (EO) water; in vivo toxicity; zebrafish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The pH dependence of the ORP for EO waters comprising various HOCl contents.
Figure 2
Figure 2
The hatch rate and mortality rate of zebrafish embryos in E3 medium (negative control) and various EO waters with 0.018% hypohalous acid produced from various salts at different time periods, up to 72 hph.
Figure 3
Figure 3
Photomicrographs of zebrafish embryos. (a) In E3 medium (negative control); (b) In EO water prepared from NaCl with 0.018% HOCl; (c) In EO water prepared from NaBr with 0.018% HOBr; (d) In EO water prepared from CaCl2 with 0.018% HOCl for soaking time of 3 min.
Figure 4
Figure 4
The hatch rate and mortality rate of zebrafish embryos in E3 medium (negative control), 0.0125% HOCl, 0.0250% HOCl, 0.2% chlorhexidine (CHX) gluconate, and 2.0% CHX gluconate for soaking of 0.5 min.
Figure 5
Figure 5
Photomicrographs of zebrafish embryos in (a) E3 medium; (b) 0.0125% HOCl; (c) 0.0250% HOCl; (d) 0.2% CHX gluconate and (e) 2.0% CHX gluconate, for a soaking time of 0.5 min.
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