Safety evaluation of the excessive intake of Bacillus subtilis C-3102 in healthy Japanese adults: A randomized, placebo-controlled, double-blind, parallel-group, comparison trial

Highlights • Excessive intake of B. subtilis C-3102 leads to unchanged in the medical condition.• Excessive intake of B. subtilis C-3102 does not worsen bone mineral density.• Excessive intake of B. subtilis C-3102 was safe under the study condition.


Introduction
Approximately 100 trillion bacteria and microbes reside in the intestines of humans, forming diverse colonies [1]; these are referred to as gut microbiota. The composition and metabolism of gut microbiota are influenced by dietary habitation [2], stress [3], aging [4], and other factors, and are also affected by several diseases [5], which lowers the quality of life. Therefore, improving the intestinal environment is essential for health maintenance and promotion.
A previous clinical study demonstrated that an intake of 9.0 × 10 8 cfu of Bacillus subtilis C-3102 (hereinafter referred to as C-3102 1 ) for 8 days significantly reduced para-cresol concentration and coliform bacterial counts in feces and significantly increased the relative abundance of genera Bifidobacterium [6]. Moreover, the intake of 2.2 × 10 9 cfu of C-3102 per day for 8 weeks in healthy subjects having loose stools significantly lowered the Bristol scale score and stool frequency and modulated the gut microbiota [7]. Thus, the consumption of C-3102 is considered to enhance human health by improving the intestinal environment.
Recently, postmenopausal women who took 3.4 × 10 9 cfu of C-3102 per day for 24 weeks had improved bone mineral density (BMD 2 ) in the femur [8]. In addition, the relative enrichment of Bifidobacterium significantly increased at 12 weeks of treatment compared with that at the baseline in the C-3102 group [8]. Furthermore, the relative abundance of Fusobacterium was significantly lower in the C-3102 group at 12 and 24 weeks of treatment compared with that at the baseline [8]. These data suggested that C-3102 modulated the gut microbiota and improved BMD by inhibiting bone resorption in healthy postmenopausal women. In the same study, safety problems associated with the intake of 3.4 × 10 9 cfu of C-3102 per day were not observed for 24 weeks; however, studies investigating the safety of excessive intakes of C-3102 were limited [8]. Hence, this study was conducted to investigate the safety of consuming 4.8 × 10 10 cfu of C-3102 per day and its influence on BMD.

Study design
A randomized, double-blind, placebo-controlled, parallel-group study was conducted at Takara Clinic (Medical Corporation Seishinkai, Tokyo Japan). The study protocol was approved by the ethical committee at Takara Clinic (Tokyo, Japan) on February 5, 2018 (approval ID: 1802-1712-AK01-01-TC). We conducted this study in accordance with the principles of the Declaration of Helsinki (2013) and the ethical guidelines for medical and health research involving human subjects of Japan and broader medical ethics. The study protocol was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN000031218).

Subjects
This study included healthy Japanese subjects. The exclusion criteria were as follows: (1) having any medical history of malignant tumor, heart failure, or myocardial infarction; (2) undergoing treatment for arrhythmia, liver disease, kidney disease, cerebrovascular disease, rheumatism, diabetes mellitus, hyperlipidemia, hypertension, irritable bowel syndrome, osteoporosis, or any other chronic diseases; (3) consuming "Food for Specified Health Uses" and/or "Foods with Function Claims" daily; (4) regularly using medications such as herbal medicines and/or supplements, particularly anticoagulants, such as warfarin; (5) having histories of allergic reactions to medications and/or products associated with the study substances, particularly soybeans and fermented soybeans; (6) being lactose intolerant; (7) being pregnant, lactating, or expecting/planning to be pregnant during the study period; (8) participating in another clinical study within the last 3 months prior to signing the study's informed consent form; and (9) identified as ineligible to participate in this study by the primary physician.
All subjects were recruited through the website (https://www. go106.jp/) managed by ORTHOMEDICO Inc. (Tokyo, Japan) between February and April 2018 and enrolled in this study. The study protocol was comprehensively explained to the subjects, who provided written informed consent prior to participation in the study at ORTHOMEDICO Inc. office. Women with increased BMD aged 50-69 years were recruited to evaluate the safety of C-3102 [8]. No subject was part of the sponsors or funding companies. The intervention period was from April to May 2018.

Sample size determination
The number of subjects required to identify at least one or more adverse events with a frequency of 10 % and detection rate of > 90 % from each group was calculated using the following equation: n = log (1− p) / log (1 − r); n: number of subjects, r: frequent of adverse event, p: detection rate From Eq. (1), the required sample size per group was calculated to be 20 subjects. Additionally, two extra subjects were added to each group (22 subjects each) with consideration of dropouts to satisfy a randomized, double-blind, placebo-controlled study as described below.

Enrollment, randomization, and blinding
Among the 63 subjects who submitted signed informed consent, 44 were selected and included in this study. After confirming the indistinguishability between the test foods (C-3102 tablet and placebo), a code was given by the person in charge of shipping from the contract research organization to an allocation controller, who was not directly involved in this study. The allocation controller randomly assigned the subjects to either the C-3102 (n = 22) or P group (n = 22), whose compositions were nearly equivalent in terms of gender and age. The allocation was performed using StatLight #11 Version 2.10 (Yukms Co., Ltd., Kanagawa, Japan), a computerized random-number generator. The sponsors, principal investigator, sub-investigators, entire staff of the contract research organization (such as the study director, an operation director, monitoring staff, statistical analysis director and staff, and the person in charge of shipping), medical institution staff, institutional review board members, contract laboratory, and other personnel involved in this study were completely blinded to the allocation procedure.

Intervention
All subjects were asked to consume either 18 C-3102 tablets or placebo tablets without chewing every day with water for 4 weeks. The test foods comprised uncoated tablets (8 mm Φ), in which the basic composition was fermented soybean powder (containing 4.8 × 10 10 cfu of C-3102 per day) and additives, and only placebo food contained additives. Table 1 shows the study's schedule. The safety was evaluated before, 2, and 4 weeks after initiating the test food intake.

Primary outcomes
The subjects' height, weight, body mass index, body fat percentage, systolic and diastolic blood pressures, pulse rate, and body temperature were measured during physical examination. Height was only measured at baseline to calculate the body mass index.
Urine samples were collected to evaluate protein, glucose, urobilinogen, bilirubin, ketone bodies, pH, and occult blood levels, which were also performed by the LSI Medience Corporation.
Subjects were requested to complete a medical questionnaire to determine their health status at each assessment point. Additionally, they were required to report the medication dosage and any changes in Table 1 Enrollment, intervention, and assessment schedule. their physical condition daily.

Secondary outcome
BMD was calculated using an ultrasound bone densitometer CM-200 (Canon Lifecare Solutions Inc., Tokyo, Japan).

Statistical analysis
Physical examination, urinalysis, blood analysis, and BMD examination data were statically assessed before, 2 weeks after, and 4 weeks after intake (three-time assessment points). The values obtained before intake were considered as baseline values.
The background and demographic data were aggregated based on gender, age, and physical characteristics and compared with those in the P group using the Student's t-test. Physical examination, blood analysis, and BMD examination data were expressed as mean and standard deviation (SD), and baseline values were analyzed using Student's t-test. Physical examination, blood analysis, and BMD examination data at 2 and 4 weeks after intake were analyzed using analysis of covariance (ANCOVA), with covariates used as baseline values. For urinalysis, data were set to a code where 1 and 0 were within and outside the normal range, respectively, and data were expressed as a matrix of the number of subjects (n) and the code, followed by the chi-squared test. A subgroup analysis on the red blood cell count, hemoglobin, hematocrit, γglutamyltransferase, leucine aminopeptidase, cholinesterase, creatinine, uric acid, creatine kinase, serum iron, and high-density lipoprotein cholesterol was performed due to difference in reference ranges between genders. The subgroup analysis was performed using ANCOVA.  The data were calculated using Student's t-test. SD, Standard deviation. All statistical analyses were two-sided, with a significance level of 5 % with no adjustment for multiple comparisons. Data analysis was performed using Windows SPSS version 23.0 (IBM Japan, Ltd., Tokyo, Japan).

Setting analysis
All subjects completed this study ( Fig.1) without violating the protocol and their rates of consumption were > 90 %. Therefore, 22 subjects (9 men and 13 women) in the C-3102 group and 22 (9 men and 13 women) in the P group were included in the analysis on an intentionto-treat dataset basis.
The subjects' demographic characteristics were not statistically significant different between the groups (Tables 2a and 2b).

Physical examination
Systolic blood pressure was significantly higher but within the reference range (Table 3). At 2 weeks after consumption, the body fat percentage was significantly lower in the C-3102 group than in the P group (P = 0.006, P = 0.002, respectively, Table 3).

All subjects
The mean corpuscular hemoglobin level was significantly higher and cholinesterase, total cholesterol, and triglyceride levels were significantly lower 2 weeks after intake in the C-3102 group than in the P group (P = 0.048, P = 0.010, P = 0.046, and P = 0.005, respectively, Tables 4a-4c). Moreover, at 4 weeks after intake, direct bilirubin was significantly higher and total cholesterol significantly lower in the C-3102 group than in the P group (P = 0.029 and P = 0.019, respectively, Tables 4b and 4c). However, all changes in the C-3102 group were within normal ranges (Tables 4a-4c).

Male subjects
All items of the blood analysis in male subjects did not significantly change between the groups (Table 5).

Female subjects
The cholinesterase levels were significantly higher in female subjects in the C-3102 group than in the P group at 2 weeks after intake (P = 0.036, Table 6).

Urinalysis
There were no significant differences between the groups (Table 7).

Medical questionnaire and daily report
No physical condition change related to test foods was recorded in the medical questionnaire and daily report written by the subjects (data not shown).

BMD
No significant differences were observed in BMD between groups (Table 8).

Discussion
This study investigated the safety of C-3102 tablet intake for 4 weeks in healthy Japanese adult subjects. Both groups took the appropriate 18 tablets of either C-3102 (4.8 × 10 10 cfu) or placebo per day. The safety was evaluated through physical examinations, urinalysis, blood analysis, and BMD measurement.
Regarding the physical examination, differences in pulse rate were observed at baseline. Although systolic blood pressure and body fat percentage were significantly different 2 weeks after intake, the fluctuation in systolic blood pressure was within the normal range prescribed in the Japanese Society of Hypertension Guidelines for the Management of Hypertension [9], and the fluctuation in body fat percentage was minor with no medically problematic changes in physical conditions during the intervention period.
The blood analysis results revealed that total bilirubin and indirect bilirubin were significantly different between the groups at baseline. Further, significant differences in mean corpuscular hemoglobin, cholinesterase, total cholesterol, and triglyceride were observed 2 weeks after intake, and direct bilirubin and total cholesterol were significantly different 4 weeks after intake between the groups. Regarding the  Table 4a Results of blood analysis.    subgroup analyses for gender-dependent measurements, the male group presented significant differences in serum iron and high-density lipoprotein cholesterol at baseline between the groups. In contrast, the blood analysis results in women revealed a significant difference in uric acid at baseline and in cholinesterase 2 weeks after intake between the groups. However, despite significant intergroup differences observed in the blood analysis, all the average values measured were within the reference range in both the C-3102 and P groups; thus, we concluded that no harmful effects were caused by the test food intakes. Urinalysis revealed no significant differences between the groups. Individual data review revealed that protein, pH, occult blood, and ketone body levels were higher/lower than the reference range in some subjects; however, all were identified to have no complication in the following comprehensive consideration: one subject in the P group had pH values outside the reference range at baseline and 2 weeks after intake. However, no complication was found in this subject, including any medical problem in physical condition. Although one subject in each group had ketone body values outside the reference range at baseline, no problem in other results were identified and they continued to participate in this study. Protein data showed positive or falsepositive results in two subjects in the C-3102 group and four subjects in the P group between the baseline and intervention period. A previous study reported that the urinary protein-positive rate represents the average in all ages and that the rate in men is 1.8-fold higher than that in women [10]. Furthermore, the positive rate in urinary protein can be caused by mental stress, bathing in hot water, and orthostatic albuminuria [11]. Based on medical observations and examination results, subjects with positive or false-positive results were permitted to continue participating in this study. Five and three subjects in the C-3102 and P groups showed positive or false-positive results for occult blood, and all were women. Among the eight subjects with positive or falsepositive results for occult blood, two were confirmed to be possibly affected by menstruation, and the remaining six exhibited positive or false-positive results despite menopausal or non-menstrual period. However, women typically have a higher positive rate of urine occult blood than women at any age [12], and urine red blood cell count in women is twice higher than that in men, even without abnormal findings [13]. Additionally, a previous study reported that the occult blood positive rate increases with age [14]. Based on these medical examination results, subjects with positive or false-positive results were permitted to continue to participate in this study, and none of these positive or false-positive results were considered to be caused by test foods.
Regarding BMD, no significant differences were observed during the intervention period. Furthermore, ingestion of the test foods did not decrease BMD.
In summary, although significant differences between the C-3102 and P groups were observed in several measurements, these values remained within the reference ranges and did not indicate any complication in the subjects' conditions. Furthermore, no adverse events were found following physician consultations and review of subjects' self-records during the intervention period. Therefore, consumption of an excessive amount of C-3102 tablets determined to be safe.

Conclusion
This study assessed the safety of administering 4.8 × 10 10 cfu of C-3102 tablets daily for 4 weeks on healthy Japanese adult subjects. The results clearly demonstrated that the intake of C-3102 tablets was safe.

Table 4b
Results of blood analysis.

Item (Unit)
Reference range  Table 4c Results of blood analysis.

Item (Unit)
Reference range   Table 5 Results of blood analysis (men).

Item (Unit)
Reference range Baseline  The data were calculated using a Student's t-test or b ANCOVA.
SD, Standard deviation. *P < 0.05 vs P group.. Table 6 Results of blood analysis (women).  The data were calculated using a Student's t-test or b ANCOVA.

Transparency document
The Transparency document associated with this article can be found in the online version.
This study was sponsored by the following companies: Asahi Calpis Wellness Co., Ltd funded the study implementation and manuscript writing. M. H., H. K., R. T., I. N., and M. Y. are members of Asahi Calpis Wellness Co., Ltd. that entrusted ORTHOMEDICO, Inc. T. H. and S. I., who are members of ORTHOMEDICO Inc., were involved in the planning of the study, the study implementation, and manuscript writing. The study was conducted by both Asahi Calpis Wellness Co., Ltd and ORTHOMEDICO Inc. Furthermore, T. T. (MD) is the principal investigator who monitors all subjects' conditions.

Declaration of Competing Interest
This study was sponsored by the following companies: Asahi Calpis Wellness Co., Ltd funded the study implementation and manuscript writing. M. H., H. K., R. T., I. N., and M. Y. are members of Asahi Calpis Wellness Co., Ltd. that entrusted ORTHOMEDICO, Inc. T. H. and S. I., who are members of ORTHOMEDICO Inc., were involved in the planning of the study, the study implementation, and manuscript writing. The study was conducted by both Asahi Calpis Wellness Co., Ltd and ORTHOMEDICO Inc. Furthermore, T. T. (MD) is the principal investigator who monitors all subjects' conditions. Data are presented as number of participants and was calculated using the chi-squared test. N.A.: not applicable. The data were calculated using a Student's t-test or b ANCOVA. SD, Standard deviation.